nantes, france - bioregate · 6 12 - 14 december 2018 nantes, france program day 1 - wednesday 12...

12 - 14 december 2018Nantes, France

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Regenerative medicine is one of the most exciting topics of modern medicine because it is a way to fight against aging and degenerative diseases. This topic raises ethical issues about Human in term of capacities and evolution. Success of this emerging field relies on an inter disciplinary and inter professional work for examples it requires

material sciences and robotics and other engineering sciences, health sciences gathering engineers, scientists, biologists, clinicians in human and animals, scientists in humanities. In medicine the technologies involved are cell and gene therapies, nanomedicine, biomaterials and tissue engineering, new frontiers for transplantations and Immunology are more and more involved for example 3D bioprinting or cell grafts immune monitoring. This advanced medicine is now called 4R medicine for Replace, Repair, Regenerate and Reprogram. It also involves personalized medicine tools because of the required adaptation of the treatment to the patient using 3D printing (personalized implantable device shaping...) or biomarkers for examples.

In all the industrial countries the market asks and waits for true and robust applicable strategies in Human. More than 400 clinical trials in cell therapies with allogenic cells are currently in progress in the world. Big pharmas slowly move to the field and a lot of start-up companies are created. New players like Fujifilm or Google invest a lot to be the future majors players and influencers in regenerative medicine.

The Bioregate network was created thanks to the Pays de la Loire region financial support. Bioregate gathers more than 300 people involved in regenerative medicine in the Nantes, Angers and Le Mans areas. Bioregate brings together all regional scientific institutions working in the field: Universities of Nantes, Angers and Le Mans, INSERM, CNRS, IFREMER, INRA, the Nantes and Angers University hospitals, Oniris national vet’school. These academic institutions and SMEs are also members of the biocluster Atlanpole Biotherapies. With the support of the regional innovation ecosystem players, Bioregate aims to boost research, training and innovation in the field of regenerative medicine with an international ambition.

This point explains why Nantes University in collaboration with Atlanpole Biotherapies Cluster joined their forces to set up and organize the second edition of the Bioregate Forum, with a common focus: boost scientists, clinicians and industrials discussions and partnerships. This second edition also wants to be more international with participation of scientists from Canada, Belgium, Ireland and US who are delegates from Bioregate international network and also with industrial clusters from Belgium, from Medicen Paris....

Famous scientists and industry representatives and also young researchers, in the fields of cell and gene therapies, nano medicine, 3 D printing and bioprinting, smart biomaterials will present their latest advances in all these hot topics. One to one meetings are also organized for this edition to stimulate new collaborations and extend and deepen our Bioregate network.

Pierre Weiss

EDITO

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Table of ConTenTs12 - 14 december 2018

Nantes, France

Program

P.6

ABSTRACTS Invited speakers and selected short talks

P.12

POSTER abstracts

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BIOSKETCHES of the invited speakers

P.91

Partners & Organizers

P.117

EXHIBITORS

P.108

BIOSKETCHES of the Organizers

P.100

Useful information

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orGanIZErs

orGanIZInG CommITTEELocal organizing committee

Pr Pierre Weiss, Nantes University, Regenerative Medicine and Skeleton Lab (RMeS)Dr Réjane Bihan, Nantes University, Bioregate Executive Director

Dr Laurent David, Nantes University, IPSC core facility, Center for Research in Transplantation and Immunology (CRTI)

Dr Catherine Le Visage, Regenerative Medicine and Skeleton Lab (RMeS)Pr Frank Boury, Angers University, Center for Research in Cancerology and Immunology

Nantes-Angers (CRCINA)Dr Tuan Huy Nguyen, Goliver Therapeutics company CEO, Nantes

Dr Franck Halary, Center for Research in Transplantation and Immunology (CRTI)Dr Oumeya Adjali, Nantes Gene Therapy Laboratory DirectorChristelle Bervas, Nantes University Communication Advisor

Lynda Guérineau, Atlanpole Biotherapies Cluster Networking ManagerNissrine Mekkaoui, Nantes University, Bioregate AssistantMathis Benzina, Nantes University, Bioregate, IT assistance

International Scientific committee Pr Abhay Pandit (NUI Galway, Ireland), Director of the Centre for Research in Medical

Devices (CÚRAM)Pr Julie Fradette (University of Laval, Quebec, Canada), Director of the ThéCell networkPr Christine Jérôme (Liege University, Belgium), Director of the Center for Education and

Research on Macromolecules (CERM)

[email protected]

[email protected] [email protected]

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PRoGRaM

dAY 1 - Wednesday 12 december 20189 am - 12:30 pm Networking session (ground floor, invited delegates only)

11:30 am - 1:30 pm Bioregate Forum Registration (3rd floor)

12:30 - 1:30 pm One 2 One informal meeting for the networking morning participants (ground floor)

1:30 - 2:00 pm Welcome speech by Organizers & Officials Organizers: Olivier Grasset or Olivier Laboux (University of Nantes), Pierre Weiss (Bioregate), Franck Zal (Atlanpole Biotherapies) Officials: Pays de la Loire Region and Nantes Metropole

2:00 - 4:00 pm SeSSiOn 1

cells as therapy and tools Chairmen: Ross Macdonald and Frank Yates

2:00 - 2:40 pm Clinical Development of an iPSC-derived Therapeutic ProductRoss Macdonald (Cynata Therapeutics, Australia)

2:40 - 3:00 pm Human embryonic stem cells and regenerative medicine: the examples of the eye and the skinCécile Martinat (I-Stem, France)

3:00 - 3:20 pm CAR-Tregs for clinical useNadia Lounnas-Mourey (TxCell, a Sagamo company, Valbonne, France)

3:20 - 4:00 pm Short presentations- How could dental stem cells of the human papilla take up the

challenge of spinal cord injury? Anne des Rieux (Louvain Drug Research Institute, Advanced Drug Delivery and Biomaterials, Université catholique de Louvain, Belgique)

- High Yield Production Of Liver Spheroids For Bio-Artificial Liver Devices. Noushin Dianat (Cyprio, Paris, France)

- Generation of nucleus pulposus progenitor cells from human induced pluripotent stem cells. Anne Camus (RMeS, Nantes, France)

- Development of in vitro assays to evaluate unwanted immunogenicity and other interactions between stem cells and the immune system. Severine Giltaire (Immunxperts, Belgium)

4:00 - 4:30 pm Coffee break in Exhibition & Poster hall

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4:30 - 6:30 pm SeSSiOn 2

3d bioprinting Chairmen: Kimberly Homan and Frank Halary

4:30 - 5:10 pm Bioprinting Vascularized Living TissueKimberly Homan (Wyss Institute for Biologically Inspired Engineering, Harvard University, USA)

5:10 - 5:30 pm Tissue Manufacturing by Bioprinting: Challenges and OpportunitiesFabien Guillemot (Poietis, France)

5:30 - 5:50 pm Engineering complex bioarchitectures to biomimic nature: A step closer to the development of artificial organsMarc Thurner (RegenHU, Switzerland)

5:50 - 6:30 pm Short presentations- A 4D-printed convoluted proximal kidney tubule as an innovative model

to study BK polyomavirus/host interactions. Franck Halary (CRTI, Nantes, France)

- In vivo evaluation of 3D printed BCP scaffolds for maxillofacial bone reconstruction in a critical-size bone defect model of rabbit. Dr Boris Feng Hildebrand (Université de Lille, France)

- 3D bioprinting of cartilage and bone. Joris van Aken (I&L Biosystem / Cellink, Sweden)

- Laser-assisted bioprinting: an advanced tool for cell model creation and for bone tissue repair. Hugo De Oliveira (University of Bordeaux, Tissue Bioengineering, U1026, ART BioPrint, Bordeaux, France)

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DAY 2 - Thursday 13 december 20189:00 - 11:00 am SeSSiOn 3

Smart biomaterialsChairmen: Estelle Collin & Catherine Le Visage

9:00 - 9:40 am Redefining Identity of Disease, Tissues and Cells – a Biomaterials ParadigmAbhay Pandit (Curam, Ireland)

9:40 - 10:00 am Chitosan-based nanofiber mats for tissue engineeringChristine Jérôme (Ulg, Belgium)

10:00 - 10:20 am Gecko Biomedical: a versatile polymer platform for tissue engineeringEstelle Collin (Gecko Biomedical, France)

10:20 - 11:00 am Short presentations- The impact of polymeric microcarriers on the behavior of stem cells

in tissue engineering. Rmaidi Assia (INSERM CRCINA U1232 Centre de Recherche en Cancérologie et Immunologie Nantes-Angers Equipe GLIAD, Université d’Angers, France)

- Designing a Bioadhesive Chitosan-Based Thermosensitive Hydrogel for Cell Therapy with Mussel-Inspired Catechol Grafting. Capucine Guyot (Ecole de Technologie Supérieure, Montréal, Québec, Canada)

- Self-setting and Injectable Hyaluronic Acid Hydrogel with Bioinspired Properties for Tissue Engineering. Killian Flegeau (INSERM UMRS 1229, Regenerative Medicine and Skeleton-RMeS, Team REGOS, Nantes, France)

- Nucleic acid based supramolecular systems as novel smart biomaterials. Philippe Barthélémy (University of Bordeaux, ARNA laboratory, France)

11:00 - 11:30 am Coffee break in Exhibition & Poster hall

11:30 am - 12:20 pm Hot topic Development of reporter systems to study DNA repair mechanisms and optimize genome editing approaches in vitro. Knut Stieger (Justus-Liebig-University Giessen, Germany)

12:20 - 12:30 pm Product compliance Cell therapy and associated quality controls: Which tests should be performed? André Silvestro (Clean Cells, France)

12:30 - 2:00 pm Lunch in Exhibition & Poster hall

2:00 - 2:30 pm Poster session

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2:30 - 6:00 pm PARALLEL SLOTS

2:30 - 4:30 pm SeSSiOn 4

2:30 - 3:00 pm Young researchers pitch competitionbuilding integrated human intestinal tissue from pluripotent stem cellsMaxime Mahé (TENS, France),

2:00 - 6:00 pm OneToOne meeting (4th floor)

9 mini talks by young researchers (10 min each) : 3:00 - 4:00 pm

4:30 - 5:00 pm Organizers private meeting (poster and pitch awards attribution decision)

3:00 - 4:00 pm mini talks by young researchers- mini Talk 1: Heparan sulfate mimetics are neuroprotective and

neuroregenerative agents for ischemic stroke. Khelif Yacine, Normandie Univ, UNICAEN, CNRS, CEA, ISTCT/CERVOxy group, GIP CYCERON, Caen, France

- mini Talk 2: Design of an optimal oxygenation strategy for macro-encapsulated pancreatic islets in the bioartificial pancreas. Anne Mouré, IECM, Oniris, INRA, Université Bretagne Loire, Nantes, France

- mini Talk 3: Towards a better understanding of the mechanical properties recovering of regenerating rat calvaria bones via synchrotron diffraction characterization. Ameni Zaouali, Institut de Recherche en Génie Civil et Mécanique, GeM (UMR CNRS 6183), Université de Nantes, Saint-Nazaire, France

- mini Talk 4: Controlled release of biological factors for progenitor cell-mediated endogenous repair of intervertebral discs. Leslie Frapin, INSERM, UMR1229, RMeS, Regenerative Medicine and Skeleton, Université de Nantes, ONIRIS, Nantes, France

- mini Talk 5: Recapitulation of amyotrophic lateral sclerosis with a 3D tissueen-gineered spinal cord model. Aurélie Louit, Laval University, Quebec, Canada

- mini Talk 6: Improvement of bone regeneration by tissue engineering strategies with a new synthetic Calcium Phosphate scaffold. Cyril d’Arros, INSERM, UMR 1229, RMeS, Regenerative Medicine and Skeleton, Université de Nantes, ONIRIS, Nantes, France & Biomatlante SA, Vigneux-de-Bretagne, France

- mini Talk 7: Regulatory B cell generation from human Induced Pluripotent Stem Cells with a repressible granzyme B expression. Florian Dubois, CRTI UMR 1064 team 4, Nantes, France

- mini Talk 8: Myocardial infarct repair with human adult muscle stem cells MuStem. Alice Rannou, INRA/Oniris UMR 703, Oniris, Nantes, France

- mini Talk 9: Development of collagen/hyaluronic acid-tyramine (coll / tha) composite hydrogels with tunable gelling kinetic for the treatment of nucleus pulposus. Antoine Frayssinet, Sorbonne Université, CNRS UMR 7574, Condensed Matter Chemistry Laboratory (LCMCP), Paris, France

5:00 - 6:30 pm Cocktail and Poster and pitch prizes award

7:00 - 10:00 pm Gala dinner at the nantilus restaurant

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dAY 3 - Friday 14 december 20188:45 - 10:45 am SeSSiOn 5

Gene Therapy and Nano medicineChairmen: Gloria Gonzalez Aseguinolaza and Frank Boury

8:45 - 9:25 am Don’t forget about the delivery system in regenerative medicinesKevin Shakesheff (University of Nottingham, UK)

9:25 - 9:45 am Gene therapy for Oculopharyngeal muscular dystrophyAlberto Malerba (Royal Holloway, University of London, UK)

9:45 - 10:05 am Therapeutic efficacy and safety of VTX-801, an optimized AAV vector for the treatment of Wilson’s diseaseGloria González Aseguinolaza (ViVet Therapeutics)

10:05 - 10:45 am Short presentations- Lipid nanoparticles for the sustained released of miRNA: new insight into

intervertebral disc degenerative medicine. Brian Le Moal (INSERM, UMR 1229, RMeS, Regenerative Medicine and Skeleton, Université de Nantes, ONIRIS, Nantes, France & MINT, Université d’Angers, INSERM 1066, CNRS 6021, Angers, France)

- Demonstration of immunomodulatory properties for human MuStem cell population, a promising candidate for cell therapy of muscular dystrophies. Marine Charrier (PAnTher, INRA, École nationale vétérinaire, agro-alimentaire et de l’alimentation Nantes-Atlantique (Oniris), Université Bretagne Loire (UBL), Nantes, France)

- Generation of CD8+ and CD4+ regulatory T cells from human pluripotent stem cells. Léa Flippe (INSERM UMR1064, Center for Research in Transplantation and Immunology ITUN, Université de Nantes, Nantes, France)

- Gene therapy and tissue engineering: a strategy to treat recessive dystrophic epidermolysis bullosa or RDEB. Martin Barbier (Centre de Recherche en organogénèse expérimentale de l’Université Laval/LOEX, Québec, Canada)

10:45 - 11:05 am Coffee break in Exhibition & Poster hall

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11:05 am - 1:05 pm SeSSiOn 6

Lessons from preclinical and clinical trialsChairmen: Oumeya Adjali and Tuan N Guyen

11:05 - 11:45 am The Era of Organoid Medicine from screen to therapeuticsTakanori Takebe (Cincinnati Children’s Hospital Medical Center, USA)

11:45 am - 12:05 pm 3D-Printed Patient Specific Implants and their Preclinical EvaluationDavid Eglin (AO foundation, Davos, Switzerland)

12:05 - 12:25 pm Clinical Production of an Autologous Ex vivo Gene TherapyAzadeh Golipour (Avrobio, USA)

12:25 - 1:05 pm Short presentations- Therapeutic effect of mesenchymal stem cell-derived extracellular vesicles

in rheumatic diseases. Marie Maumus (INSERM U1183, Montpellier, France & Université Montpellier, France)

- RGTA and Matrix therapy is a new branch of regenerative medicine: from basic science to patients Prove of efficacy in human and veterinary medicine through randomized controlled trials. Denis Barritault (OTR3, Paris, France). (cancelled, to be replaced)

- Safety and clinical efficacy of a single intra-articular injection of allogeneic neonatal mesenchymal stem cells (MSC) for the treatment of osteoarthritis in dogs. Nathalie Saulnier (Vetbiobank, Marcy L’Etoile, France)

- ISOCell PRO workstation for cell therapy product and clinical applications. Severina Franco (Euroclone Spa, Pero (MI), Italy)

1:05 - 1:30 pm conclusion remarks and closing ceremony

absTRaCTsInvited speakers,selected short talks and young researcher mini talks

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INVITED SPEAKERS SESSION 1


ross macdonald

Managing Director and Chief Executive Officer

Cynata Therapeutics Limited, Australia

From the first description of reprogramming methods able to generate pluripotent cells from adult somatic tissues in the middle of the last decade, the science and clinical potential of induced pluripotent stem cells – iPSCs – has taken many important steps forward. The use of iPSCs in modelling and investigating human diseases, as well as for screening drugs, has already been enormously enlightening. But it is perhaps their use in the development of therapeutic products where the greatest opportunity for iPSCs arises. Notably iPSCs share many properties with embryonic stem cells (ESCs) but because iPSCs are not derived from embryos they are not associated with the substantial ethical controversies that have overshadowed ESC research and which will most certainly influence commercial development of ESC-derived therapeutic products. iPSCs are now being used to develop a broad range of cell therapy products, including myocardial tissue for potential use to effect recovery post-myocardial infarction, retinal pigmented epithelium for potential use in age-related macular degeneration and mesenchymal stem cells (MSCs) for a broad range of disease targets. The latter cell type is the most advanced, with Cynata Therapeutics Ltd, an Australian biotechnology company, having recently completed a Phase 1 clinical trial in acute steroid resistant graft-versus-host disease (GvHD) with a unique allogeneic iPSC-derived MSC cell therapy product. This clinical trial, the first in the world using an allogeneic iPSC derived product, demonstrated very encouraging safety and efficacy, paving the way for a Phase 2 trial in this and further indications. The trial confirms a viable and clinically relevant process for large scale manufacture of a highly consistent cell therapy product without the challenges presented by requiring multiple donors and massive expansion of the finished product. Data from the Cynata trial in GvHD and the pathway to trial approval and beyond will be discussed.

CLINICAL DEVELOPMENT OF AN IPSC-DERIVED THERAPEUTIC PRODUCT

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cécile martinat

INSERM UEVE UMR 861 unit, I-STEM, Paris, France

Access to unlimited numbers of specific cell types on demand has been a long-standing goal in regenerative medicine. With the availability of human pluripotent stem cells (hPSCs) and greatly improved protocols for their directed differentiation, this prospect has become a reality for several disease-relevant cell types. In this context, we are interested in I-STEM by developing new cell therapy medicinal product based on our expertise in tissue engineering and in the manipulation of human embryonic stem cells. Two main projects are actually under development concerning the use of novel tissue engineered products for skin ulcers and retinal degenerative diseases. The recent progresses concerning these two programs will be presented and discussed.

HUMAN EMBRYONIC STEM CELLS AND REGENERATIVE MEDICINE : THE EXAMPLES OF THE EYE AND THE SKIN

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nadia Lounnas-mourey

TxCell, a Sangamo company, Valbonne, France

TxCell, now a Sangamo company, is a pioneer in the development of regulatory T cells (Tregs) engineered with a Chimeric Antigen Receptor (CAR) or CAR-Treg cells.

The HLA-A2 CAR-Treg is Sangamo’s most advanced CAR Treg product candidate. It targets HLA A2, a common mismatch antigen in transplantation, and is in development for the prevention of chronic rejection after organ transplantation. HLA-A2 CAR Treg has shown strong efficacy in a preclinical GvHD model and is now on track to start a first-in-man study in transplanted patients.

In 2018, Sangamo has succeeded in developing the industry’s very first CAR Treg Good Manufacturing Practice (GMP) process ready for clinical testing. Sangamo selected a population of Treg cells with the CD4+ CD25+ CD45RA+ phenotype (CD45RA+ Tregs). CD45RA+ Tregs display both strong anti-inflammatory activity and stability. This starting Treg subset is rare, accounting for less than 5% of CD4+ T lymphocytes. Sangamo’s process enables the manufacturing of clinical doses of CD45RA+ based CAR-Tregs in under two weeks, ready for post-production quality control. Key achievements include the stability of the Sangamo Treg phenotype after both expansion of the CAR Treg cells and thawing of the final drug product.

Sangamo has notably conducted several full-scale pilot batches using clinical-grade raw and ancillary materials, as well as equipment to be used by the contract manufacturing organization (CMO) for GMP production. Key findings from these pilot batches are:• Cells extracted from leukapheresis keep their Treg identity after cell transduction

with a CAR and cell expansion throughout the process. This is achieved with limited donor to donor variability.

• A high cell purity of the selected CD45RA+ Treg subset is achieved at the end of the expansion phase.

• Expression of the Foxp3 intracellular cell and TSDR gene hypomethylation level remains constant throughout the process. Foxp3 expression is known to be related to the suppressive capacity of Treg cells.

• Importantly, the drug product could be both frozen and thawed with no change in phenotype and function.

CAR-TREGS FOR CLINICAL USE

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SHORT TALKS SESSION 1

Anne Des Rieux, P. de Berdt

Louvain Drug Research Institute, Advanced Drug Delivery and Biomaterials, Université catholique de Louvain, Brussels, Belgium

Spinal cord injury (SCI) is the major cause of long-term paralysis via traumatic event. Strategies for spinal cord repair after injury are extensively studied over the world. While cell-based therapy holds great hope for recovery after SCI, this area remains challenging for scientists and clinicians. Transplantation of different type of mesenchymal stem cells (MSC) has been investigated as potential therapies for SCI but, despite this advancement no definitive treatment exists and SCI continues to pose clinical and socio-economic problems.

Why after so many years of research, so many pre-clinical studies and so much money and energy invested to this grail quest the translation to patient is still so limited? Could it be due to inadequate animal models, the SCI environment complexity, transplantation issues, cell sources, post-transplantation cell survival, lack in understanding about the mechanisms through MSC promote repair and functional recovery?

The aim of this work was to provide an innovative strategy for spinal cord repair based on a new MSC source: dental stem cells from apical papilla (SCAP). Dental stem cells (DSC) have been largely investigated in dental therapies, especially in regenerative endodontics. DSC hold a not yet exploited potential in regenerative medicine and are particularly becoming attractive for nervous system repair strategies thanks to their neural crest origin. Up to now, no one investigated SCAP potential for spinal cord repair so far.

We first evaluated the impact of hydrogel properties on SCAP and we selected a fibrin hydrogel as the most suitable delivery system to evaluate the influence of SCAP for spinal cord regeneration. Then, we observed that implantation of a whole human apical papilla at the lesion site improved gait of rats with spinal cord injury. We then observed that the implantation in the same spinal cord hemisection model of apical papilla induced a significant decrease of several pro-inflammatory cytokines and of CD68 positive cells at the lesion site. Finally, we demonstrated that SCAP have immunomodulatory properties and can stimulate oligodendrocyte progenitor cell differentiation. This work underlines the potential therapeutic benefits of SCAP for spinal cord repair.

HOW COULD DENTAL STEM CELLS OF THE HUMAN PAPILLA TAKE UP THE CHALLENGE OF SPINAL CORD INJURY?

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noushin dianat

R. Eren Ayata(1,2), J. Caron(1,2), N. Bremond(1), J. Bibette(1), N. Dianat(1,2)

(1) ESPCI Paris, PSL Research University, LCMD, Paris, France

(2) Cyprio, Paris, France

Objectives: Bioartificial livers (BALs) are extracorporeal assistance devices destined to improve the health state of patients suffering from acute liver failure (ALF) and to serve as a bridgingtherapy to liver transplantation or regeneration. However, currently BALs struggle to demonstrate significant improvement on patient survival specially in advanced clinical phases mainly due to poor reproducibility of cell cartridge from batch to batch, the scale-up and logistical complications and finally the poor hepatic function of the cartridge failing to recapitulate the native liver. Although three-dimensional (3D) spheroid models are shown to be promising to represent liver physiology over an extended lifespan, still main issues regarding their handling, scale-up and banking remain to be addressed.

Methods: To circumvent these drawbacks, we have developed the BioPearl technology to fabricate 3D liver spheroids inside miniaturized core-shell capsules (≈350μm of diameter) with the elevated rate of 1500 capsules produced per second. Using this unique technology, we could fabricate micro-tissues of liver from different origins : primary human hepatocytes (HepatoPearls), HepaRG cell line (RGPearls) and finally iPSC-derivedhepatocytes, generally named « Liver Pearls ».

Results:The liver Pearls generated using this technology display vivo-mimicking characteristics such as: a) Polarized epithelial morphology with the presence of bile canaliculi network, b) functional detoxification transporters, c) lifespan of more than 45 days d) high and stable metabolic activity of phase I and II metabolizing enzymes e) synthetic activity and g) CYP inducibility over 6 weeks. We then cryopreserved the liver Pearls generated from different cell origins. Post-thawing, the cells were shown to be capable to form a viable spheroid in each capsule and to preserve a high metabolic and synthetic function over weeks.

Conclusions: BioPearl is a unique ultrahigh throughput technology allowing fabrication of cryopreservable easy-to-handle and metabolically functional liver spheroids and answering multiple needs concerning BAL devices.

HIGH YIELD PRODUCTION OF LIVER SPHEROIDS FOR BIO-ARTIFICIAL LIVER deViceS


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P. Colombier(1,2), B. Halgand(1,2), C. Chédeville(1,2), C. Chariau(5), A. Henry(1,2), V. François-Campion(5,9), S. Kilens(5,9), J. Clouet(1,2,3,4), L. David(5,7,9), J. Guicheux(1,2,8) and Anne camus(1,2)

(1) INSERM, UMR 1229, RMeS, Regenerative Medicine and Skeleton, Université de Nantes, ONIRIS, Nantes, France

(2) Université de Nantes, UFR Odontologie, Nantes, France (3) Université de Nantes, UFR Sciences Biologiques et Pharmaceutiques, Nantes, France(4) CHU Nantes, Pharmacie Centrale, PHU 11, Nantes, france (5) INSERM UMS016, CNRS UMS 3556, SFR santé, Plateforme iPSC, Université de Nantes(6) Université de Nantes, UFR Sciences et Techniques, Nantes, France(7) INSERM UMRS1064-ITUN, Institut de Transplantation en Urologie et Néphrologie,

Université de Nantes, France(8) CHU Nantes, PHU 4 OTONN, Nantes, France(9) ITUN, CHU Nantes, Nantes, France

Objectives: Low back pain is one of the most common musculoskeletal disorder and the most common causes of disability in industrialized countries. Although the aetiology of low back pain is unknown, it is often (40%) associated with degeneration of the intervertebral disc (IVD). There is currently no effective treatment for disc degeneration. This is largely due to a lack of basic knowledge of the molecular and cellular controls of disc development, growth, differentiation, and homeostasis, during embryogenesis and at different stages of life.The nucleus pulposus (NP) is the highly hydrated central part of the IVD that plays a key role in spine kinematic and wheredegenerative changes are thought to initiate. Lineage tracing experiments in the mouse demonstrate that notochord cells (NTCs) are the precursor cells that give rise to the NP. There is also considerable evidence that NTCs have a significant influence on the homeostasis of the IVD and their loss has been correlated with aging and degeneration in human. The generation of NTCs is therefore a promising approach to regenerate IVD.Methods: In this study, we examined how several signaling pathways (Wnt, Activin/Nodal, Fgf and Shh) influence the commitment ofhuman induced pluripotent stem cells (hiPSC) towards mesendoderm progenitors with distinct lineage abilities assessed by RTQPCR, DGE-seq, immunofluorescence detection and western blot analysis.Results and conclusions:Our results established that forced expression of notochord-specific transcription factor NOTO directs hiPSC-derived mesendoderm progenitors differentiation toward notochordal fate. Time course analysis of hiPSC differentiation by DGE-seq showed that NOTO overexpression and Wnt signaling activity are sufficient to induce a stable notochord population.

GENERATION OF NUCLEUS PULPOSUS PROGENITOR CELLS FROM HUMAN INDUCED PLURIPOTENT STEM CELLS


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Séverine Giltaire, S. Pattijn and T. Jonckheere

Immunxperts, Belgium

The field of auto- and allogenic stem cell technologies is rapidly expanding and one of the concerns raised is the potential to induce an unwanted immune response, especially in an allogenic setting. During the last decade, low immunogenicity and potential immunosuppressive capacities were described by several groups. Certain cell types, like mesenchymal stem cells are considered to be immune-privileged because of their low immunogenicity due to low levels of MHC class I and the absence of MHC class II expression. However, recently, studies indicate that certain stem cells, even in an autologous setting, can be immunogenic and that this unwanted immunogenicity can be a major limitation for cell replacement therapy.

The availability of specific in vitro assays can be useful to evaluate the potential immunogenicity of stem cells and document their mechanism of action. This can be evaluated by co-culturing of the stem cells in a Mixed Lymphocyte Reaction (MLR) assay (one-way or two-way).However, due to the ability of allogeneic MSCs to suppress T-cell proliferation, there is not always a good correlation between in vitro and in-vivo immunogenicity of allogeneic MSCs either. Therefore, product-specific assay development is required to evaluate the balance between immunosuppression and potential immunogenicity as MSC can be suppressive at high cell concentrations but can also become immunogenic at lower cell concentrations.

The availability of suitable in vitro assays to monitor the unwanted immunogenicity, mechanism of action and therapeutic potential of stem cell products can accelerate the development of new therapies.

DEVELOPMENT OF IN VITRO ASSAYS TO EVALUATE UNWANTED IMMUNOGENICITY AND OTHER INTERACTIONS BETWEEN STEM CELLS AND THE IMMUNE SYSTEM


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Kimberly Homan, David B. Kolesky, Mark A. Skylar-Scott, and Jennifer A. Lewis

Wyss Institute for Biologically Inspired Engineering, Harvard University, USA

Engineered living tissue constructs enable new in vitro applications in 3D cell studies, drug screening, disease modeling, and, ultimately, therapeutic applications in regenerative medicine. To date, traditional scaffold-based manufacturing processes such as 2D photolithography, bulk casting, gas-forming, and 3D printing are limited in both the scale and complexity of the tissues they can create due to the lack of stable, perfusable 3D vasculature, and the inability to create intricate multicellular configurations and vasculature in 3D. Here, we detail our recent 3D printing developments enabling the creation of vascular networks and the concurrent patterning of cells and vasculature, along with new strategies for achieving active perfusion, long-term stability, and thicknesses exceeding 1cm, all of which are essential for creating a physiologically and therapeutically relevant tissue manufacturing method. Examples including thick vascularized tissue, stem cell printing, vascular network generation, and renal tissue modeling will be discussed. With control over multicellular architecture, the chemo-mechanical microenvironment, and the ability to support thick, developing tissue for long time points, this method could serve as a platform for studying emergent biological functions in complex engineered microenvironments, and, may ultimately, find applications in vivo.

BIOPRINTING VASCULARIZED LIVING TISSUE


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Fabien Guillemot

Founder and CEO of Poietis, France

Despite substantial investments to meet clinical and commercial expectations, and while scientific achievements at the preclinical research stage have sometimes been impressive, scaffold-based Tissue Engineering approaches are struggling to find the way to therapeutic and industrial success. Main challenges for the manufacturing of tissue engineered ATMPs concern the improvement of the standardisation of manufacturing processes, tissue functionality, and cost-effectiveness and profitability of related treatments.

Based on our experience in the field of bioprinting, we discuss how this technology – thanks to its characteristics resulting from the convergence of automation, biology and digital technology – should make it possible to overcome current tissue manufacturing bottlenecks and also provide new opportunities.

TISSUE MANUFACTURING BY BIOPRINTING: CHALLENGES AND OPPORTUNITIES


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Marc Thurner

RegenHU, Switzerland

Our cutting edge biofabrication process creates infinite possibilities in the manufacture of artificial tissues and organs. By engineering Macro & Nano architectures in a single process unit, we have opened the door to a whole new world of possibilities allowing us to mimic biological systems identical to those found in nature.

ENGINEERING COMPLEX BIOARCHITECTURES TO BIOMIMIC NATURE: A STEP CLOSER TO THE DEVELOPMENT OF ARTIFICIAL ORGANS


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Franck Halary(1 2), B. Rosa(3), P. de Villemagne(3), J-Y. Hascoet(3)

(1) Centre de Recherche en Transplantation et Immunologie UMR 1064, INSERM, Université de Nantes, Nantes, France

(2) Institut de Transplantation Urologie Néphrologie (ITUN), CHU Nantes, Nantes, France

(3) GeM Laboratory, UMR CNRS 6183, Centrale Nantes, Nantes, France

- Renal transplantation is the only effective regenerative therapy for many end-stage kidney deficiencies but it is often impaired by transplant-related viral infections. The BK polyomavirus causes a nephropathy in kidney transplant recipients leading eventually to a graft loss of function. No antiviral drug is available yet urging for the search of innovative therapeutic options. This virus is species-specific and to date no experimental model, ie in animals or with 2D cell cultures exists. The overall objective of this project is to take advantage of the 3D bioprinting technology to fabricate phenotypically and functionally relevant human renal tissues to serve as models to understand pathophysiology of the BK polyomavirus infection in humans.

- 3D bioprinting is emerging as a new paradigm in biology allowing to fabricate innovative and complex biological objects. First, a home-made high resolution 3D bioprinter was conceived by an interdisciplinary bio-engineering team. The device uses linear axes and pneumatic control allowing a 0.1μm spatial resolution and is composed of an open CNC (Computer Numerical Control) to dynamically control the operating parameters along a given trajectory. The custom made bioprinter enables to master syringes and table temperature (-25 – 100°C) to control bioink viscosity. Based on this unique device confined under a sterile cabinet, we endeavored to fabricate a perfusable tubule mimicking a native human proximal kidney tubule. Stromal and immune cells have also been added to study complex mechanisms of viral spreading in this 3D environment. Here, we present original results opening the way to the screening of new specific anti-BK polyomavirus drugs and to the biofabrication of more complex kidney tissues.

A 4D-PRINTED CONVOLUTED PROXIMAL KIDNEY TUBULE AS AN INNOVATIVE MODEL TO STUDY BK POLYOMAVIRUS/HOST INTERACTIONS


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Feng Hildebrand(1), M. SCHLUND(1,2), A. DEPEYRE(1,3), V. HORNEZ(4), J-C. HORNEZ(5), T. WOJCIK(1,6), P. MACHANDISE(7), G. PENEL(7), P. GOSSET(8), B. DELAIRE(8), N. BLANCHEMAIN(1), J. FERRI(1,2)

(1) Université de Lille, Inserm U1008, Pôle de Recherche, Faculté Médecine(2) Centre Hospitalier Universitaire de Lille, Service de Chirurgie Maxillo-Faciale(3) Centre Hospitalier Universitaire de Clermont-Ferrand, Service de Chirurgie Maxillo-

Faciale, Stomatologie et Chirurgie Plastique de la Face(4) CryoCeram-Startup, Serre Numérique de Valenciennes, Valenciennes(5) LMCPA-Université de Valenciennes, Campus Mont Houy(6) Centre Oscar Lambret, Lille(7) Université de Lille, EA4490 PMOI Physiopathologie des Maladies Osseuses Inflammatoires,

Faculté de Chirurgie Dentaire(8) Immunohistochimie et Recherche, Laboratoire d’Anatomie et Cytologie Pathologiques,

Pôle hospitalo-universitaire de bio-pathologie, Hôpital Saint Vincent de Paul, Lille

- Biphasic calcium phosphate (BCP) bioceramics have great potential for applications as a bone substitute because of their excellent chemical bone-bonding ability and higher bioresorption property. On the other hand, 3D printing methodology is an excellent approach to support effective and fast patient-specific fabrication of individual complex bone substitutes. Here, we aimed to elaborate a 3D printed BCP scaffold, and evaluate its biocompatibility in vitro / in vivo. - The implantable disk samples were fabricated by a custom-designed CryoCeram 3D printer from synthetized BCP powder (60 wt% HA / β-40 wt% TCP). In vitro biological assessment of 3D printed BCP was carried out with preosteoblast cell lines (MC3T3-E1). In vivo evaluation of printed scaffold was further conducted by the implantation in a developed critical-size bone defect model in the rabbit skull (Ø 10 mm) and mandible (11×6 mm) followed by X ray micro-CT imaging and histological evaluation at 4 and 12 weeks after surgery.- The obtained results confirmed the 3D printed BCP scaffold promotes well the adhesion and the proliferation in vitro of preosteoblast cells, which is comparable with pure β-TCP. For in vivo model, the acquired micro-CT images, in agreement with histological staining, showed that the bone defects in the control group, in which no scaffolds were implanted, still lacked newly formed bone at 12 weeks. The defects implanted with the printed BCP scaffolds formed new bone and better-repaired bone defect (higher BV/TV value) than the control.- This study indicates that our 3D printed BCP scaffolds with desired shapes and internal structures exhibited a good biological activity and biocompatibility in vitro andin vivo, and promoted appropriate new bone formation in vivo. Future study will focus on incorporating bioactive factors (platelet-rich fibrin) and osteoprogenitor cells to enhance the osteoconductive and osteoinductive stimuli for better repair of bone defects.- The authors would like to thank the Plate-forme Ressources Expérimentales, D.H.U.R.E, Faculté de Médecine, University of Lille 2, for their support on animal studies. This work was fully supported by the cross-border INTERREG “North-West Europe» regional development fund BONE 2014-2020.

IN VIVO EVALUATION OF 3D PRINTED BCP SCAFFOLDS FOR MAXILLOFACIAL BONE RECONSTRUCTION IN A CRITICAL-SIZE BONE DEFECT MODEL OF RABBIT


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Joris van Aken

I&L Biosystem / Cellink

3D Bioprinting has gained attention in tissue engineering due to its ability to spatially control the placement of cells, biomaterials and biological molecules. The development of new hydrogel bioinks with good printability and bioactive properties has made it possible to 3D bioprint and accelerate the maturation of complex 3D tissue-like models. In this talk, we will focus on the bioink development for 3D bioprinting for cartilage and bone applications. Recent studies on bioprinting of chondrocytes and iPSCs with our bioinks will be discussed.

3D BIOPRINTING OF CARTILAGE AND BONE


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Hugo De Oliveira(1), V. Keriquel(1), D. Hakobyan(1), N. Dusserre(1), C. Medina(1), M. Rémy(1), F. Guillemot(1), J.C. Fricain(1,2)

(1) University of Bordeaux, Tissue Bioengineering, U1026, ART BioPrint, Bordeaux, France

(2) CHU Bordeaux, Services d’Odontologie et de Santé Buccale, Bordeaux, France

Bottom up approaches, based on a brick-by-brick reconstruction of a tissue or an organoid, offer the opportunity to pattern the individual components according to a predefined pattern, able to guide the maturation of the construct towards a final functional architecture. As a result, cellular and biomaterial distribution can be defined at the micrometer scale, enabling the creation of a proper extracellular matrix (ECM) microenvironment. Among different bottom-up techniques, bioprinting, an emerging advanced biofabrication method, has since the last 5 years become one of the most promising technical approaches to attain control over the geometry of engineered tissues. Here we will focus on one of the three main bioprinting technologies, laser-assisted bioprinting (LAB). We and others have shown that LAB presents unprecedented printing precision, without compromising cell survival. Here we will present two examples on the use of this technology for the creation of complex cellular models and on the regeneration of bone tissue, in situ and in vivo.

LASER-ASSISTED BIOPRINTING: AN ADVANCED TOOL FOR CELL MODEL CREATION AND FOR BONE TISSUE REPAIR


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Abhay Pandit

CÚRAM, Center for Research in Medical Devices, National University of Ireland, Galway, Ireland

Biomaterials are no longer considered innate structures and using functionalisation and biofabrication strategies to modulate a desired response whether it is a host or implant is currently an important focus in current research paradigms. Fundamentally, a thorough understanding the host response will enable us to design proper functionalisation and biofabrication strategies. The input from the host response needs to be weighed in depending on the host disease condition. In addition, biomaterials themselves provide immense therapeutic benefits which needs to be accounted in the design paradigm. Using functionalisation strategies such as enzymatic and hyperbranched linking systems, we have been able to link biomolecules to different structural moieties. The programmed assembly of biomolecules into higher-order self-organized systems is central to innumerable biological processes and development of the next generation of biofabricated scaffolds. Recent design efforts have utilized a developmental biology approach toward both understanding and engineering supramolecular protein assemblies. Structural moieties have taken a variety of different forms such as nanofibers and nanoparticulate. This approach has resulted in functionalisation of micro and nanoparticles with biomolecules that include designed peptide motifs, growth factors and a multitude of gene vector systems. In addition, nature itself has abundant structural complexity that can be biofabricated for harnessing in key targeted clinical applications. referencesThomas, D., Fontana, G., Chen, X., Sanz-Nougés, C., Zeugolis, D., Dockery, P., O’Brien, T. and Pandit, A. ‘A Shape-controlled Tuneable Microgel Platform to Modulate Angiogenic Paracrine Responses in Stem Cells.’ Biomaterials. 2014: 35(31): 8757-66. Lang, Y., del Monte, F., Collins, L., Rodriguez, B.J., Thompson, K., Dockery, P., Finn, D.P. and Pandit, A. ‘Functionalization of the Living Diatom Thalassiosira Weissflogii with Thiol Moieties.’ Nature Communications. 2013: 4: 3683Tapeinos, C. and Pandit, A. ‘Physical, Chemical and Biological Structures based on ROS-sensitive Moieties that are able to respond to Oxidative Microenvironments.’ Advanced Materials. 2016: 28(27): 5553-5585Mohd Isa IL, Abbah SA, Kilcoyne M, Sakai D, Dockery P, Finn DP, Pandit A. Implantation of Hyaluronic Acid Hydrogel Prevents the Pain Phenotype in a Rat Model of Intervertebral Disc Injury. Sci Adv. 2018 Apr 4;4(4):eaaq0597Acknowledgement: This publication has also emanated from research conducted with the financial support of Science Foundation Ireland (SFI) and is co-funded under the European Regional Development Fund under Grant Number 13/RC/2073.

REDEFINING IDENTITY OF DISEASE, TISSUES AND CELLS – A BIOMATERIALS PARADIGM


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Christine Jérôme

Center for Education and Research on Macromolecules (CERM), CESAM RU, University of Liège, Liège, Belgium

Polymer hydrogels resemble the natural living tissue due to their high water content and soft consistency. They find many applications in the design and production of contact and intraocular lenses, biosensors membranes, matrices for repairing and regenerating a wide diversity of tissues and organs. Polysaccharides such as chitosan [1] and hyaluronic acid based hydrogels have shown a great potential for biomedical and pharmaceutical applications, on account of their remarkable compatibility with physiological medium. Besides, it is degraded in a physiological environment into non-toxic products, which make them outstanding candidates for short- to medium-term applications, especially for tissue engineering. In this respect, the preparation of nanometric fibers mats based on this polysaccharide are highly interesting as such structure mimics the one of skin extracellular matrix. Chitosan nanofibres can be prepared by electrospinning but might suffer from weak mechanical resistance if they are used as such. We thus have investigated strategies allowing to generate chitosan based nanofiber mats exhibiting a mechanical resistance strong enough to be easily handled while keeping the peculiar features of chitosan hydrogels favoring the interaction with cells and soft tissues to provide efficient tissue reconstruction.

Chemical crosslinking of the nanofibrous mats of chitosan and polyethylene oxide blends [2-3], was a first strategy which allows adjusting the mechanical resistance of the mats while preserving their biocompatibility. Nevertheless, heat sensitive active ingredient cannot hold such thermally activated process. Therefore, combination of chitosan with poly(ε-caprolactone) (PCL) and structuration of the nanofiber mats have been further investigated. Polysaccharide-based nanofibers with a multilayered structure [4] were prepared by combining electrospinning and layer-by-layer deposition techniques. Elastic nanofibers bearing charges at their surface were firstly prepared by electrospinning PCL with a polyelectrolyte precursor. After activation by adjusting the pH, the layer-by-layer deposition of chitosan and hyaluronic acid, can be used to coat the electrospun fibers. A multilayered structure is then achieved by alternating the deposition of the positively charged chitosan with the deposition of a negatively charged polyelectrolyte. These novel polysaccharide-coated PCL fiber mats remarkably combine the mechanical resistance typical of the core material, particularly in the hydrated state, with the surface properties of chitosan [5-6]. Macroscopic structuration of the fiber mats was also found as a performant strategy to control in one shot both the mat porosity and mechanical resistance.

CHITOSAN-BASED NANOFIBER MATS FOR TISSUE ENGINEERING


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estelle collin

Gecko Biomedical, France

While many biomaterial-based startegies have been developed for tissue repair and regeneration in the recent years, only few were able to be translated from the bench to bedside. No ideal system/material is currently available onto the market. Indeed, biologically-based polymers are often mechanically weak with a fast degradation rate and low adhesion properties. Synthetic polymers are for them often associated with a low biocompatibility and degradation properties making them non-compatible for many applications. Therefore, there is still a very important need to find novel materials with the right mechanical and biocompatible properties to enable the translation of solution in the tissue reconstruction space. Gecko Biomedical is a start-up company developing innovative polymer solutions for tissue reconstruction based on a technology developed in Profs Bob Langer and Jeff Karp in MIT. The company is working on the development of polymers from sealant and adhesive to biodegradable resins for cardiovascular to nerve regenerations applications. These novel fully synthetic light-activated polymers can be “printed” both inside the body (acting as sealant, adhesives, barriers or plugs) or outside the body (3D printing of high-resolution implantable devices). These materials have unique chemical and physical properties, including high viscosity, hydrophobicity and on demand curing making them ideal “printed” polymers. These features allow them to be delivered through minimally invasive procedures to challenging wet environments. Furthermore, once ‘printed’ inside or outside the body, Gecko’s materials are biodegradable, biocompatible and elastic, complying with the softness and dynamics of underlying tissues. The range of properties considering the modularity of design of our polymer platform can be tuned to adapt to the desired application. With our first product, a vascular sealant as adjunct-to-suture, CE marked in 2017, we aim to develop new surgical and tissue engineering solutions for improving patients’ life. We will present here our technology and some of our latest results for our sealants and adhesives and 3D printed scaffolds.

GECKO BIOMEDICAL: A VERSATILE POLYMER PLATFORM FOR TISSUE ENGINEERING


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Assia rmaidi and N. Delorme ; R. Dima ; L. Sindji ; F. Boury ; C. Montero-Menei

INSERM CRCINA U1232 Centre de Recherche en Cancérologie et Immunologie Nantes-Angers Equipe GLIAD, Université d’Angers, France

Institut des Molécules et Matériaux du Mans (IMMM), Le Mans Université, France

PAMs are polymeric microcarriers providing a biomimetic 3-dimensional surface to enhance stem cell response. A subpopulation of mesenchymal stem cells (MSCs), can be differentiated towards a neuronal phenotype on a laminin (LM) surface and secrete many repair factors, making them candidates of choice for cell therapy studies. MSCs on the fibronectin surface of PAMs composed of a polylactide co-glycolide and poloxamer triblock (PLGA-P188-PLGA) better survived/proliferated than on the fibronectin surface of the more hydrophobic PLGA PAMs. It still remains unclear how these surface properties affect the behavior of MIAMI cells. We here investigate the relationship between the physico-chemical surface properties of PAMs and the protein (LM) adsorption, and how each factor may have a direct effect on the adhesion and differentiation of MSCs.PLGA or PLGA-P188-PLGA based PAMs were formulated by a simple emulsion processes. The PAMs were functionalized with LM and poly-D-lysine (PDL), and were characterized for size, zeta potential. Surface topography, roughness and surface morphology were evaluated by atomic force and scanning electron microscopies. The distribution of LM and PDL on the microcarrier surface was studied using fluorescent confocal microscopy and ToF-SIMS. The adhesion of MIAMI cells was evaluated, and differentiation analysis is underway.The PLGA-P188-PLGA microcarriers have a rough surface compared to the PLGA, which does not increase much after LM/PDL coating. However, the surface roughness of PLGA microcarriers increases after coating with the mixture, thus both surfaces may promote cell adhesion. LM adsorbs well to the PLGA microcarriers but adsorption is decreased by the presence of Poloxamer188, although more cells are observed after 7 days on these PAMs. Both PAMs functionalized with LM and PDL exhibit a positive zeta potential allowing cell attachment and diminishing the differences in cell number observed with LM alone. Integrin expression analysis showed an increased expression of LM receptors by MSCs on these PAMs. The MSCs adhered and presented a flattened morphology on PAMs-PLGA LM/PDL surface. In comparison, cells adhered less and maintained a round shape on the surface of PAMs-PLGA-P188-PLGA, which could be explained by the low adsorption of LM/PDL on these surfaces.The chemical composition and the surface roughness of the microcarriers influence the adsorption of LM and PDL, which has an effect on the MIAMI cells adhesion.

THE IMPACT OF POLYMERIC MICROCARRIERS ON THE BEHAVIOR OF STEM CELLS IN TISSUE enGineerinG


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capucine Guyot, S. Samaei, M. Cerruti, S. Lerouge

Ecole de Technologie Supérieure, Montréal, Québec, Canada

Université McGill, Montréal, Québec, Canada

Objectives: Despite chitosan’s inherent cationic properties, hydrogels made with this polysaccharide biopolymer lack adhesiveness to tissues. It has been shown that chitosan’s modification with catechol, a compound found in marine mussel’s feet, drastically increases its adhesive properties. Here, we designed a new catechol-chitosan-based injectable hydrogel that is strong, thermosensitive, and most importantly bioadhesive.

Methods: Modifying chitosan with catechol is achieved by grafting hydrocaffeic acid to the carbonated chain at 3% and 9% catechol content (evaluated through UV-visible spectrometry). To make strong injectable hydrogels without toxic compounds, we chose a thermosensitive physical crosslinking mediated by sodium bicarbonate (SHC). Catechol is very sensitive to oxidation: when the pH goes up, hydroxyl groups oxidise into cetones and react with other cetones available, undesirably crosslinking the polymer. Cat-chitosan is therefore dissolved in either DDW or HCl, and mixed with ranging concentrations of SHC to modulate its pH. Gelation time and gelation temperature are evaluated using a Physica rheometer. Unconfined compression tests are performed on a BOSE bench. Shear bioadhesive tests are performed on a BOSE bench in a dry environment, but wet conditions are also assessed with wash-off tests on porcine mucosa at 37°C. Indirect cytotoxicity of the hydrogels is evaluated using Alamar Blue assay on L929 fibroblasts.

Results: Dissolution in HCl (0,05M) resulted in more stability of Cat-chitosan in terms of oxidation. When increasing HCl concentration prior to gelation, Cat-chitosan hydrogels are less brittle, with longer gelation times. Symmetrically, increasing SHC concentration leads to a higher stiffness and cohesiveness, shown both by compression and wash-off tests. All formulations have a gelation time (G’=G’’) of less than 5 minutes. Bioadhesive properties in dry and wet conditions are increased compared to non-grafted chitosan. A higher catechol percentage leads to significantly higher adhesion, but also impacts mechanical cohesiveness and leads to premature breakage inside the gels. Indirect cytotoxicity tests show no adverse effect on L929 fibroblasts.

Conclusion: This new hydrogel shows very promising preliminary results regarding its injectability, mechanical cohesiveness and adhesiveness to tissue. The latter is especially interesting to secure the cells on the injection site, improving both retention and therapeutic efficiency.

DESIGNING A BIOADHESIVE CHITOSAN-BASED THERMOSENSITIVE HYDROGEL FOR CELL THERAPY WITH MUSSEL-INSPIRED CATECHOL GRAFTING


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Killian Flegeau(1,2,5), H. Gautier(1,2,4), G. Rethore(1,2,3), P. Bordat(5), P. Weiss(1,2,3)

(1) INSERM UMRS 1229, Regenerative Medicine and Skeleton-RMeS, Team REGOS, Nantes, France (2) Nantes University, Regenerative Medicine and Skeleton (RMeS), Nantes, France (3) Nantes University Hospital, Pôle Hospitalo-Universitaire 4 (OTONN), Nantes, France (4) Université de Nantes, School of Pharmacy, Laboratoire de Pharmacie Galénique, Nantes, France(5) HTL S.A.S, Javené, France

Introduction: Tissue engineering is a promising approach to regenerate damaged skeletal tissues. In particular, the use of injectable hydrogels alleviates common issues of poor cell viability and engraftment. However, uncontrolled cell fate, resulting from unphysiological environments and slow degradation rates, still remain a hurdle and impedes tissue regeneration. To overcome these hindrances, we aim at developing a platform of injectable and biodegradable hyaluronic acid hydrogels mimicking the mechanical and physical properties of native extracellular matrices (ECMs). Following addition of calcium phosphate granules, we then aim at investigating the ability of these hybrid materials to trigger bone regeneration.Methods:Si-HA synthesis: HA was functionalized with silylated moieties (Si-HA) following the US 20130004460A1 patent.Characterization: Grafting efficiency was determined by ICP-AES. Mechanical analyses were performed on a Texture Analyzer.Cell viability was studied on L929 and human bone marrow stem cells (hBMSCs) using Live/Dead® and CCK8 assays.Cell differentiation of hBMSCs was assessed using ALP activity assays. In vivo implantation of Si-HA hydrogels was performed in a subcutaneous model of C57BL/6 mice.Results and discussion: Si-HA hydrogels are formed in situ through a sol-gel chemistry occurring at physiological pH. By varying the crosslinking density, molecular weight and concentration, we obtained versatile hydrogels spanning a large range of elastic moduli (E = 0.1-50 kPa), similar to those of native ECMs, with tunable biodegradation rates (from 24 hours to > 50 days) and swelling ratios (500 to 5000% (w/w)) while maintaining a high cell viability (>90%). Ongoing in vivo experiments are evaluating the degradation rates and inflammatory response following hydrogel implantation.In parallel, hybrid HA/biphasic calcium phosphate (BCPs) hydrogels with improved mechanical properties have been developed and fully characterized. We showed that hBMSCs are able to adhere and differentiate into the osteogenic lineage at the surface of BCPs. Lastly, in vivo implantations in a rabbit femur model will assess their ability to trigger bone tissue regeneration.Conclusion: These versatile hydrogels with tunable properties display excellent features for their use as biomimetic scaffolds for cartilage and intervertebral disk regeneration. In association with BCPs, these composites may also offer a simple, yet effective, approach to regenerate bone.

SELF-SETTING AND INJECTABLE HYALURONIC ACID HYDROGEL WITH BIOINSPIRED PROPERTIES FOR TISSUE ENGINEERING


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Philippe Barthélémy

(1) University of Bordeaux, ARNA laboratory, Bordeaux, France

(2) INSERM, U1212, ARNA laboratory

(3) UMR CNRS 5320

Amphiphilic biomconjugates are emerging as promising supramolecular materials for biomedical and technological applications. In this presentation we will highlight the recent progresses in the field of nucleic acid based lipids with an emphasis on their molecular design, supramolecular properties, physicochemical behaviors, and applications in the field of biomaterials.

- Objective. Indeed, there is a critical need for soft materials in the field of regenerative medicine and tissue engineering. However, designing injectable or printable hydrogel scaffolds encompassing both adequate mechanical and biological properties remains a key challenge for applications.

- Methods and results. Here we use a bottom-up approach for synthesizing supramolecular gels to generate novel biomaterial candidates.[1] The self-assembly of nucleic acid based amphiphiles creates unique hydrogel supramolecular structures featuring high elastic moduli, thixotropic, and thermal reversibility properties. Interestingly, this type of soft material, which inhibits recognition by macrophages and fibrous deposition, exhibits long-term stability after in vivo injection and can be used as a polymer free matrix/scaffold.

References [1] - Baillet, J., Desvergnes, V., Hamoud, A., Latxague, L., Barthélémy, P., Adv. Mater.

(2018) Lipid and Nucleic Acid Chemistries: Combining the Best of Both Worlds to Construct Advanced Biomaterials, 1705078. DOI: 10.1002/adma.201705078.

- Ramin MA, Latxague L, Sindhu KR, Chassande O, Barthélémy P, Biomaterials (2017) “Low molecular weight hydrogels derived from urea based-bolaamphiphiles as new injectable biomaterials” doi: 10.1016/j.biomaterials.2017.08.034.

- M. A. Ramin, K. R. Sindhu, A. Appavoo, K. Oumzil, M. W. Grinstaff, O. Chassande, P. Barthélémy, Adv. Mater. (2017) “Cation Tuning of Supramolecular Gel Properties: A New Paradigm for Sustained Drug Delivery” DOI: 10.1002/adma.201605227.

- Latxague, L., Ramin, M.A., Appavoo, A., Berto, P., Maisani, M., Ehret, C., Chassande, O., and Barthélémy, P. (2015) Angewandte Chemie, 54 (15), pp. 4517-4521.

NUCLEIC ACID BASED SUPRAMOLECULAR SYSTEMS AS NOVEL SMART BIOMATERIALS


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Knut Stieger

Justus-Liebig-University Giessen, Germany

Genome editing is based on the cells own capacity to repair DNA double strand breaks (DSB), which have been artificially introduced. To introduce a DSB, highly specific endonucleases based on the CRISPR-Cas9 system, TALEN, ZFN, or homing endonucleases such as Isce-I, have been developed over the years, with the CRISPR-Cas9 system currently being the most important player in the field. Three different pathways exist to repair DSBs: nonhomologous endjoining (NHEJ) in the absence of a template, and homology directed repair (HDR) or microhomology mediated endjoining (MMEJ), depending on whether a template with long or short homologous sequences is present, respectively. While NHEJ is an error-prone repair pathway, HDR and MMEJ have the potential to repair a DSB with high fidelity.

In recent years, it became clear that the way cells repair a given DSB can be modulated by external modifications. This way, the efficacy of genome editing can be improved and unwanted DNA repair mechanisms blocked. To study these mechanisms and to optimize therapeutic applications, reporter systems that allow to quantify the repair pathways are urgently needed.

We have developed several reporter systems based either on genomic gene expression systems or based on episomally transfected biosensors that allow us to study and optimize DNA repair mechanisms: (1) a modified traffic light reporter system that allows differentiation of HDR and NHEJ repair events simultaneously, (2) a luciferase based reporter system to study the replacement of large DNA sequences, and (3) a biosensor based on bioluminescence resonance energy transfer (BRET) to study NHEJ activity in any given cell line of tissue.

The use of these and other reporter systems will greatly improve our knowledge about the DNA repair mechanisms and how to optimize therapeutic approaches.

DEVELOPMENT OF REPORTER SYSTEMS TO STUDY DNA REPAIR MECHANISMS AND OPTIMIZE GENOME EDITING APPROACHES IN VITRO

INVITED SPEAKERS HOT TOPIC

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André Silvestro, Business Development Director and Marc Meichenin, CSO

Clean Cells, France

In the last decade, novel therapeutic approaches involving the use of biologics have multiplied. Recombinant proteins (ie therapeutic monoclonal antibodies for cancer treatment) and virus vaccines are now widely used. Accordingly, number of regulatory guidelines related to these products are in application both in Europe and US.

For Cell and Gene therapy products, the situation is a bit more complicated due to the need for adaptation of the guidelines and for a thorough risk assessment policy in line with the variety of processes. The aim of the presentation will be then providing a review of applicable regulatory requirements and a focus on GMP principles that should be applied on these products. Specific quality controls will be also discussed.

CELL THERAPY AND ASSOCIATED QUALITY CONTROLS: WHICH TESTS SHOULD BE PERFORMED?

SPONSOR SPEAKERS PRODUCT COMPLIANCE

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INTRODUCTION by Dr Maxime Mahé, TENS, Nantes, France

“Building integrated human intestinal tissue from pluripotent stem cells”

The use of human pluripotent stem cells offers great avenues to generate human tissues. The understanding of intestinal development and its translation to human pluripotent stem cells, allowed the field to move forward in understanding intestinal development and gastrointestinal diseases. In this talk, I will highlight our previous work which had focused on generating functional human intestinal organoids (HIOs) from embryonic stem cells and induced pluripotent stem cells. Building on this model, I will highlight the additional complexity we were able to engineer in order to gain insights into intestinal physiology and diseases. In this context, the development of human intestine with an enteric nervous system (ENS) represents a real opportunity to expand our knowledge into the effect of ENS on intestinal development and toward the understanding of pathophysiological processes leading to functional gastrointestinal neuropathies. Finally, I will delineate the forthcoming strategies that could be used to create a fully functional intestine that could pave the way for intestinal regenerative medicine.

YOUNG RESEARCHER PICTH COMPETITION


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12 - 14 december 2018Nantes, FranceMini Talk 1

Khelif Yacine, Normandie Univ, UNICAEN, CNRS, CEA, ISTCT/CERVOxy group, GIP CYCERON, Caen, France

“Heparan sulfate mimetics are neuroprotective and neuroregenerative agents for ischemic stroke”

Introduction: The extracellular matrix (ECM) is altered during ischemic stroke. ECM repair using heparan sulfate mimics such as RGTA® (ReGeneraTing Agent) has been shown to be promising for protection and regeneration of different tissues. RGTA® are synthetic polysaccharides that bind to extracellular structural proteins and act as a reservoir of growth factors. RGTA® protect growth factors from degradation and increase their bioavailability. Moreover, the specific molecular structure of RGTA® prevents their enzymatic degradation during stroke-induced tissue and ECM degradation. Based on these observations, we evaluated a novel therapy based on RGTA®’s ability to protect the brain against ischemic stroke, favor functional recovery and improve brain plasticity.Materials and Methods: Ischemic stroke was induced in Sprague Dawley rats using transient intraluminal (1h) middle cerebral artery occlusion (MCAo). Brain damage was assessed with 7T MRI at d2 and d15 post-occlusion. Functional deficits were evaluated with a battery of sensorimotor behavioral tests for 5 weeks following ischemia. Cellular reactions including were evaluated by immunohistochemistry 35 days following stroke induction.Results: To define the optimal dose of RGTA®, three doses were administered including 0.5 mg/kg, 1.5 mg/kg, or 5 mg/kg, after MCAo. The 0.5 mg/kg dose conferred the best and the most persistent neuroprotection (-46 % and -29% of infarct volume at 2d and 14d respectively) (ANOVA followed by a post hoc Tuckey HSD test p < 0.05). Interestingly, the adhesive test revealed that 0.5 mg/kg dose significantly improved animals’ sensorimotor recovery up to 5 weeks after MCAo (ANOVA followed by a post hoc HSD of Tuckey test, p < 0.05). RGTA®-treated animals showed a significant increase (2 folds) of RECA-1+/Ki-67+ cells, attesting an increase of angiogenesis in the peri-infarct area (Student-t test p < 0.05). Moreover, RGTA® also increased the number of DCX+ cells (1.5 fold) in the subventricular neurogenic zone and around the lateral ventricle, and BrdU+/NeuN+ cells (1.4 fold) in the ipsilateral hemisphere (Student-t test p < 0.05) underlying an increase of post-ischemic neurogenesis.Conclusion: This study shows for the first time, that an ECM-based therapeutic strategy using the RGTA® protect the brain tissue over a large time frame, resulting in enhanced neuroprotection, brain plasticity and significant improvement in sensorimotor recovery.


SELEcTED SPEAKERS SESSION 4


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Anne Mouré, IECM, Oniris, INRA, Université Bretagne Loire, Nantes, France

“Design of an optimal oxygenation strategy for macro-encapsulated pancreatic islets in the bioartificial pancreas”

The bio-artificial pancreas (BAP) encapsulating pancreatic islets in alginate hydrogel is a promising therapeutic alternative for type 1 diabetes. However, as for many other tissue-engineered devices, the main limitation of this approach is O2 supply. The O2 balance in BAP results from a combination of several factors such as the local O2 partial pressure in the transplantation site, the vascularization of the graft surface, the O2 diffusivity through the BAP, and the islet O2 consumption rate, and density. Maintaining O2 supply to encapsulated islets remains a major challenge especially until graft neovascularisation. Silicone-encapsulated calcium peroxide (silicone-CaO2) has been shown to effectively supply O2 to mitigate pancreatic islet hypoxia damage. However, this O2-generating biomaterial led to the generation of detrimental O2 gradients and reactive O2 species. Previously, we described that the encapsulation of an extracellular haemoglobin (Hemoxcell, Hemarina, Morlaix) in alginate improved the O2-diffusivity through the hydrogel and exhibited anti-oxidant properties able to neutralized reactive O2 species produced by silicone-CaO2.In this context, we aimed to (i) screen the best O2-strategy to mitigate hypoxia damage on the viability and function of alginate encapsulated pseudo-islets obtained from the MIN6 beta cell line and (ii) design an optimal O2-regulated BAP based on the use of Hemoxcell and/or silicone-CaO2.Using factorial designs of experiment, a screening of Hemoxcell, silicone-CaO2 and their combination was realized on their ability to preserved pseudo-islets up to 6 days under O2 tension mimicking the extravascular transplantation site. As expected, the best O2-solution was the combination of Hemoxcell and silicone-CaO2. Then, the O2 production rate of silicone-CaO2 disk and the O2 consumption rate of pseudo-islets were characterized. These results allowed determining a pseudo-islets density target of 6500 islet equivalent quantities per silicone-CaO2 disk. Using response surface methodology, the optimal concentration of Hemoxcell and seeding islet density will be determined in order to maximize the number of viable islets in the BAP under low O2 tension.To conclude, we highlighted a synergic effect of an O2 carrier and an O2 generating material to provide O2 to encapsulated islets. Our work should allow designing a more realistic BAP with a maximized islet density based on O2 balance before graft neovascularization.




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Ameni Zaouali, Institut de Recherche en Génie Civil et Mécanique, GeM (UMR CNRS 6183), Université de Nantes, Saint-Nazaire, France

“Towards a better understanding of the mechanical properties recovering of regenerating rat calvaria bones via synchrotron diffraction characterization”

In recent years, research in bone surgery has focused on developing tissue engineering strategies to accelerate bone regeneration. The major challenge of these researches is to repair large bone defects where innovative implants (biomaterials) have been developed to fill and heal the latter. However, the mechanical behavior recovering of the newly formed bone, as the regeneration process takes place, remains still poorly understood.This work aims at mechanically characterize the bone regeneration process in the tissue engineering frameworks related to bone substitute isolated implantation. This study highlights the distribution of mechanical stresses induced by the healing process and the evolution of crystallographic orientation of minerals, along with regeneration, through the mechanical behavior mapping in various regeneration levels by means of rat calvarias samples harvested at different regeneration stages until complete reconstruction. In situ tensile testing under WAXS/SAXS synchrotron m-beam have been achieved to spatially resolve the elastic strain/stress response of mineral crystals and collagen fibers of bone architecture, respectively. Results are correlated with the microbiological processes through histological and micro-tomography observations.The analysis of the mechanical response and microstructural features of bone tissue throughout the healing process shows a strong correlation between the bone mechanical resistance and the crystal organization. Indeed the evolution of crystal size and their distribution in the newly synthesized matrix during regeneration maximize bone tensile strength. Observations also show that the scheme is strongly affected by the substitute isolated implant nature, resulting in significant differences in regenerated tissue quality and healing rate.




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Leslie Frapin, INSERM, UMR1229, RMeS, Regenerative Medicine and Skeleton, Université de Nantes, ONIRIS, Nantes, France

“Controlled release of biological factors for progenitor cell-mediated endogenous repair of intervertebral discs”

The recent description of progenitor/stem-cells in the degenerated intervertebral discs (IVDs), raised the possibility of harnessing their regenerative capacity for IVD endogenous repair. To develop such strategy, it seems essential to recruit these resident stem cells toward inner parts of the IVD (ie.nucleus pulposus (NP)) followed by their differentiation into NP-like cells. For this, specific chemokines (CXCL12 or CCL5), known to modulate the recruitment of stem cells, and synergic growth factors allowing the in vitro NP-differentiation (TGF-β1 and GDF5) are promising candidates, but these molecules present a very short half-life. The aim of this work was to develop an intradiscal polysaccharide microbeads-based delivery system for the sequential release of chemokines and nucleopulpogenic factors. This delivery system would sequentially contribute to 1) the recruitment of resident progenitors (CXCL12 or CCL5), 2) the differentiation of the mobilized progenitors (TGF- β1 and GDF5), and 3) the subsequent regeneration of NP. Chemotaxis assays were performed to determine the in vitro cell migration. Human mesenchymal stem-cells (1250cells/μl) were cultured on transwells for 4h, with or without CXCL12 or CCL5, and migratory cells were stained by crystal violet then quantified by spectrophotometry. In parallel, pullulan microbeads (PMBs) (100μm) were prepared by a simultaneous crosslinking protocol coupled to a water-in-oil emulsification process. Freeze-dried PMBs (20mg) were incubated for 24h with CXCL12, CCL5, TGF-β1 and GDF5 (1, 2 and 4μg/mL) at 4°C. Release assays were performed at 37°C for 21 days and supernatant concentrations were measured by ELISA. Cell migration was improved in presence of each chemokine, with 3.9 (CXCL12) and 7.5 (CCL5) fold increase respectively, as compared to untreated cells. All factors were successfully adsorbed on PMBs and a burst release within the 1st day was observed. At day 7, 27.5% and 83% of the chemokine CXCL12 and CCL5 respectively was released and at day 21, both growth factors were released (20% and 100% for TGF-β1 and GDF5, respectively). We have confirmed that CXCL12 and CCL5 improved in vitro hMSCs migration, and that PMBs are suitable micro-carriers for the controlled loading and release of these different factors. Currently, released cytokine bioactivity is being analyzed and an ex vivo ovine IVD model is developed to determine the repair potential of this controlled release approach.




41


Aurélie Louit, Laval University, Quebec, Canada

“Recapitulation of amyotrophic lateral sclerosis with a 3D tissue-engineered spinal cord model”

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease, causing motor neuron (MN) degeneration and leading to patient death due to respiratory failure. A mutation in the Superoxide Dismutase 1 (SOD1) gene inducing protein misfolding and accumulation of aggregates in MN has been identified as a cause of ALS in 5% of patients. Recently, studies have shown that the combination of MN, astrocytes, microglia and myoblasts could constitute a metabolic unit and that non-neuronal cells could contribute to the development of ALS.Hypothesis / objectives: Our main goal is to develop and characterize by tissue engineering an in vitro murine 3D model of spinal cord reproducing the ALS phenotype, based on the use of MN, glial cells, myoblasts derived from SOD1G93A mice overexpressing the mutated human SOD1 (mice express an ALS phenotype), or SOD1WT mouse overexpressing the nomal human SOD1, serving as control. Then, the objective will be to determine the role of each cell type in the development of the ALS phenotype in vitro, using various combinations of healthy or diseased cells. Methods: Cells were obtained form mutant SOD1G93A or normal SOD1-WT mice: MN have been extracted from spinal cord mouse embryos aged of 14 days of development, while astrocytes and microglia were obtained from adult mouse spinal cords, and myoblasts, from adult mouse muscles. These cells were purified, characterized by immunofluorescence or immunohistochemistry and then seeded onto 3D collagen sponges.Results: The cell extraction method was optimized (> 1 million MN per embryo, purity> 90%). In the 3D model, when SOD1G93A MN were cultured with mutant astrocytes and microglia, there was a 25% reduction in the length of TUJ-1 positive neurites, compared to the control seeded with SOD1WT MN, astrocytes and microglia. In addition, a similar decrease in the length of MN neurites was observed with non-transgenic MN grown in presence of SOD1G93A glial cells, compared to SOD1-WT glial cells.Conclusion: MN are able to organize themselves into nerve fibers in presence of healthy or diseased glial cells, but axonal migration was found 25% shorter in presence of glial cells overexpressing the mutated SOD1, recapitualting in part an ALS phenotype. Such in vitro ALS modelling should lead to a better understanding of the disease mechanisms, and could serve as a platform for drug screening. Moreover, this 3D model would be adaptable to other type of mutations involved in ALS.




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Cyril d’Arros, INSERM, UMR 1229, RMeS, Regenerative Medicine and Skeleton, Université de Nantes, ONIRIS, Nantes, France & Biomatlante SA, Vigneux-de-Bretagne, France

“Improvement of bone regeneration by tissue engineering strategies with a new synthetic Calcium Phosphate scaffold”

Tissue engineering strategy is required for large bone regeneration. The results of European clinical trial REBORNE show a promising future for a use of stem cell associated with Biphasic Calcium Phosphate granules (BCP, MBCP+TM Biomatlante SA).A new ambitious European program ORTHOUNION has just started. Its aim is 1) to determine the appropriate cell dose seeded to the BCP and compare this strategy with current clinical standard such as autograft and 2) to develop a more suitable synthetic bone scaffold for tissue engineering. It appears that the malleability of a paste (called putty) as a bone substitute should improve greatly the usability for surgeons.The present study concerns the evaluation of the new biomaterial developed in the project.A Freeze Dried Bone Scaffold (FDBS) powder composed of Biphasic Calcium Phosphate (MBCP+TM with HA/TCP, 20/80) and polysaccharidic hydrogel carrier was prepared and sterilized by Ethylene Oxide (EtO). The product was characterized by physico-chemical analysis (XRD, FTIR, SEM, cohesion and pH). In vitro assay were conducted with L929 cells (mouse fibroblasts) to evaluate the scaffold cytotoxicity.We showed that FDBS is easy to mix with physiological solution (less than 30 seconds of mixing) and allow to get a cohesive, elastic and malleable putty during more than 30 minutes. Moreover the pH of this biomaterial and the extractible pH in a medium was measured around 7 corresponding to physiological conditions. The chemical analyses demonstrated no modifications of the BCP used for this new scaffold after the production process. The in vitro results showed no cytotoxic effects. Indeed an increase in cell activity for 7 days was measured by MTT assay indicating cell proliferation. In vivo implantation in rat epiphyses during 3 weeks is planned. This new bone substitute should be a perfect platform as tissue engineering tool. Further preclinical tests will be performed to evaluate the potential of bone regeneration in function of strategy associated to the FDBS (cells, bone marrow, blood, PRP...).




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Florian Dubois, CRTI UMR 1064 team 4, Nantes, France

“Regulatory B cell generation from human Induced Pluripotent Stem Cells with a repressible granzyme B expression”

We reported in kidney transplantation that B cells expressing granzyme B (GzmB) are present in a higher number in tolerant patients who do not reject their graft without immunosuppression compared to stable patient under immunosuppression (Chesneau et al., JASN 2015). We showed that they display regulatory properties, they block T cell proliferation and promote Tregs in vitro, supporting the idea that they may play a role in tolerance. Our aim was to generate B-lymphocytes from human Induced Pluripotent Stem (hIPS) cells with a controlled GzmB expression as a tool to better understand their function and ultimately to translate them toward clinical use. hIPS cells represents an attractive tool with self-renewal properties, offering the advantage to select the donor haplotype to efficiently match transplant recipients. Starting with two human Embryonic Stem (hES) cell lines WA01 and WA09 as a proof of concept, and following a well-established protocol (French et al., Stem Cells Dev. 2015), we succeed in generating a small proportion (1,5%) of B cells characterized as pre-B cells with no IgM expression. To optimize this protocol we find that both increasing the number of CD34+ cells and adding cytokines for the 42 days of B cell differentiation, increase the amount of differentiated B cells by 2 fold. Different densities of the MS-5 stroma cells were tested, and better differentiation was obtained with MS-5 stroma set-up with 500 000 MS-5 irradiated cells. In order to perform a Knock Out (KO) of the endogenous GzmB expression using CRISPR/Cas9, we validate the GZMB plasmid composed of the Cas9 and the designed sgRNA on 293T cells, with high cut efficiency (33%) and ready to use on hES cells. An expression cassette composed of the GzmB gene with an expression controlled by a Tet-Off system will then be inserted in the AAV locus of hES GzmB KO. In this system, we expect a constitutive expression of GzmB by B cell differentiated from those hES cells, while addition of doxycycline will abrogate its expression. This tool will allow deeply studying GzmB+ B cell function and future translation toward clinical uses.




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Alice Rannou, INRA/Oniris UMR 703, Oniris, Nantes, France

and INSERM UMR 1087/CNRS UMR 6291, Institut du Thorax, Nantes, France

“Myocardial infarct repair with human adult muscle stem cells MuStem”

Background & Objectives - Heart failure is a major public health and lacks effective cure. Cell therapy represents a promising strategy for heart failure, although none of the cells investigated until now fulfils all the expected requirements. We isolated a stem cell population from healthy dog skeletal muscle, that we named MuStem cells, and established a proof of efficacy of its use in dog model of Duchenne Muscular Dystrophy (Rouger et al., 2011; Robriquet et al., 2015; Lardenois et al., 2016). We also isolated the human counterpart and characterized it by contribution to fiber formation after injection into mouse injured muscle and high secretory activity (Lorant et al., 2018). Overall, these data placed the MuStem cells as a potential advanced therapy medicinal product. In a context of cardiac failure, beneficial tissue remodelling and modulation of contractile function were reported following local administration of murine muscle-derived stem cells (Oshima et al., 2005; Payne et al., 2005). Similar effects mainly attributed to trophic factors were described after mesenchymal stem cells delivery (Sassoli et al., 2012; Nakamura et al., 2015). Considering the high regenerative capacity and paracrine effect of human MuStem cells, we investigated whether they could be an interesting alternative for tissue repair after myocardial infarction.Methods - Coronary ligation and intra-myocardial administration of MuStem cells were performed in a new immunodeficient rat model, Rag1xIl2Rg KO (Ménoret et al., 2018). ECG and ultrasound investigations were used to assess longitudinally the consequences of cell delivery. After 3 weeks, rats were sacrificed and analyses were performed to explore the fate of MuStem in the infarcted heart. Results & Conclusions – Histological and molecular analyses showed that human MuStem cells were implanted into the rat host cardiac tissue without generating arrhythmia 3 weeks post-injection. Echographic analyses highlight their capacity to ameliorate the functional and structural features of infarcted heart towards a less severe phenotype with an improvement of the left ventricle ejection fraction. In fact, as opposed to treated rats, control rats present atelectasis, dilated heart and often damage of the liver. In conclusion, MuStem cells are able to implant into infarcted hearts and generate beneficial functional impact.




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Antoine Frayssinet, Sorbonne Université, CNRS UMR 7574, Condensed Matter Chemistry Laboratory (LCMCP), Paris, France

“Development of collagen/hyaluronic acid-tyramine (coll/tha) composite hydrogels with tunable gelling kinetic for the treatment of nucleus pulposus”

Back pain is considered as the disease of the century as 80% of the world population will be affected during its lifespan. Half of back pains are associated with intervertebral disc degeneration. Current treatments are based on physiotherapy, anti-inflammatory drugs administration or on invasive surgery. Novel treatments, relying on the injection of stem cells embedded in injectable biomaterials have been developed with the aim of regenerating Nucleus Pulposus (NP). As cell behavior depends on the biochemical, mechanical and 3D-structural environment, we hypothesized that a biomimetic hydrogel would promote NP regeneration by providing adequate clues to cells.Collagen and hyaluronic acid are two major components of intervertebral disc (IVD). They give resistance and hydration to Nucleus Pulposus. In this study, we assessed the impact of Collagen (COLL) and Hyaluronic acid-Tyramine (THA) contents on the mechanical properties and the structure of composite hydrogels. For this purpose, a range of composites were obtained using a 4 mg/mL collagen concentration and different COLL: THA ratios from 8:1 to 1:5 (w/w). Composite gelling was performed by pH increase, triggering collagen fibrillogenesis and oxidative coupling of tyramine moieties in THA catalyzed by H2O2 and horseradish peroxidase (HRP). To modulate the THA gelling kinetic, different HRP concentrations were used.Composites with a low THA content exhibited a fibrillar structure and possessed mechanical properties close to those of pure collagen hydrogels (200 Pa). From the ratio 1:1, the storage modulus increased to reach c.a 1200 Pa for the ratio 1:5. From the ration 1:2, sheets characteristic of THA hydrogels, were observed in scanning electron macroscopy. The fibrillar structure of collagen with its characteristic D-banded pattern was also observed regardless of the COLL:THA ratio. The HRP activity dramatically impacted the physical properties. Moreover, a rapid THA gelling associated with a high THA content tended to destabilize collagen hydrogels but promoted the interactions between collagen and THA, thereby increasing the mechanical properties of composites. A high THA content associated with a fast gelling stabilized collagen fibrils and suggested the creation of THA/COLL covalent bonds.Taken together, these results show that a rapid gelling and a high THA concentration (20 mg/mL) are the appropriate conditions to obtain biomimetic biomaterials for the treatment of Nucleus Pulposus.




46


Kevin Shakesheff

Pro-Vice Chancellor of the Faculty of Science

Director of UK Regenerative Medicine Hub in Acellular Materials

University of Nottingham, United Kingdowm

Regenerative medicines act slowly because tissue growth is complex. This creates a problem in the design of effective products because the cells or molecules that we use to stimulate regeneration are either fragile, mobile or both. Therefore, for virtually all regenerative medicines there is a need to consider a sophisticated delivery system that holds the active at the site of repair and releases the active slowly enough to get a persistent effect. There is an added complication in regenerative medicine because the new tissue will need to occupy the same space as the delivery system so very high porosity and biodegradation need to be engineered into the chemistry and structure of the system. This talk will explore new delivery systems that promote cell retention, differentiation, angiogenesis and tissue repair. The clinical and commercial translation of exciting advances in regenerative medicine is dependent on the adoption of effective delivery systems early in the development life-cycle of future products.

DON’T FORGET ABOUT THE DELIVERY SYSTEM IN REGENERATIVE MEDICINES


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Alberto malerba

Centres of Gene and Cell Therapy and Biomedical sciences, School of Biological Sciences, Royal Holloway, University of London, Egham, Surrey, TW20 0EX, United Kingdom

Oculopharyngeal muscular dystrophy (OPMD) is a rare, autosomal dominant, late-onset muscular dystrophy. OPMD is mainly characterized by progressive eyelid drooping (ptosis) and dysphagia, although also muscles of the lower limbs can also be affected later in the progression of the disease. OPMD is due to a trinucleotide repeat expansion in the polyA binding protein nuclear-1 (PABPN1) gene. Patients express an expanded protein (expPABPN1) that is misfolded and prone to form intranuclear aggregates. Currently no cure is available. We recently tested a combination of 2 AAV vectors designed to silence endogenous expPABPN1 and replace it with a wildtype copy of the gene in a mouse model of OPMD. This strategy was effective in improving both pathology and measures of muscle function. Afterwards we collaborated with Benitec Biopharma for the development of BB-301, which is a single “silence and replace” AAV vector designed to allow significant muscle-specific shRNA-mediated endogenous PABPN1 knock-down and simultaneous expression of a codon-optimized shRNA-insensitive wildtype PABPN1. Mid-range doses of BB-301, resulting in 75% inhibition of expPABPN1 and 26% restoration of wildtype PABPN1, produced full phenotypic correction of muscle strength and weight, suggesting a broad therapeutic window. Safety/toxicity studies are currently being performed in large animals to support a first-in-human study.

GENE THERAPY FOR OCULOPHARYNGEAL MUSCULAR DYSTROPHY


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Gloria González Aseguinolaza

CSO of ViVet Therapeutics, France

Wilson disease (WD) is a disorder of copper metabolism that can present with hepatic, neurologic, or psychiatric disturbances, or a combination of these. If untreated WD is a life-threatening condition.

Recently, we have demonstrated that an adeno-associated vector (AAV) serotype 8 carrying the human ATP7B cDNA provides long-term correction of copper metabolism in WD young male mice. However, the size of this vector genome (5.1 Kb) surpasses the optimal size of a packaged AAV genome representing a major drawback for its clinical application. Furthermore, the efficacy of this vector in female mice and in animals with advanced disease (12 weeks) is notably lower than in young male mice. In this work, our main objective was to develop an optimized version of the gene therapy vector for clinical use.

THERAPEUTIC EFFICACY AND SAFETY OF VTX-801, AN OPTIMIZED AAV VECTOR FOR THE TREATMENT OF WILSON’S DISEASE

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brian Le moal(1,6), E. Lepeltier(6), V. Geoffroy(1,4), A. Galvani(1,4), C. Levisage(1,4), C. Passirani(6), J. Guicheux(1,4,5), J. Clouet(1,2,3,4)


(2) CHU Nantes, Pharmacie Centrale, Nantes, France(3) Université de Nantes, UFR Sciences Biologiques et Pharmaceutiques, Nantes, France(4) Université de Nantes, UFR Odontologie, Nantes, France(5) CHU Nantes, PHU4 OTONN, Nantes, France(6) MINT, Université d’Angers, INSERM 1066, CNRS 6021, Angers, France

Introduction-Objectives: Misregulation of miRNAs, notably of miRNA-155, and their target genes have been associated with several diseases including low back pain induced by intervertebral disc degeneration (IDD). However, free miRNAs therapy suffers from disadvantages related to their fast in vivo degradation, requiring iterative injections. Therefore, miRNAs protection against nucleases is considered as a prerequisite for the development of sustainable delivery systems. Nanoparticulate systems, such as lipid nanocapsules (LNCs), offer a suitable strategy thanks to their ability to encapsulate DNA (Vonarbourg et al., Biomaterials, 2009). The purpose of this project was to formulate and fully characterize innovative miRNA-loaded LNCs to determine their efficiency for the IDD treatment.Methods: LNCs were formulated by phase inversion process as previously described (Passirani, C.,2014, FR Patent 3026009). To obtain miRNA loaded LNCs, the quenching water was replaced by lipoplexes containing the miRNA of interest (miR-155). After purification, miRNA LNCs were fully characterized (size, polydispersity index PDI, zeta potential) by Dynamic Light Scattering method and by Nanoparticle Tracking analysis. Encapsulation efficiency (EE) and drug loading (DL) was assessed by fluorescence quantification of miRNA inside and outside LNCs. Then, miRNAs release was studied by dialysis and their protection against degradation by LNCs was investigated using gel electrophoresis.Results: Optimized formulation had an average diameter of 75,0±1.3 nm, PDI of 0,06±0.03 and a positive zeta potential. EE and DL were 75.2±1.2 % and 590 μg/g of LNC, respectively. The release profile of miRNA from LNCs was delayed compared to free miRNAs. After 2 h, 28 % of miRNA was released from LNCs compared to 70 % of free miRNA. Protection of miRNA by LNC against endonuclease degradation was confirmed by electrophoresis.Conclusion: LNCs showed promising properties to encapsulate miRNA and to protect them against degradation, in allowing its delayed release. Thanks to these results, miRNA LNCs shows a good capacity to encapsulate and deliver miRNA for a future use in IVD regenerative medicine. In a foreseeable future, we will study the delivery of miRNAs by LNCs (FACS and confocal microscopy) and evaluate their biological effects in vitro (pathway activity, qPCR, Immunoblotting). Finally, in vivo experiments will be performed using animal models of IDD to validate a first proof of concept.

LIPID NANOPARTICLES FOR THE SUSTAINED RELEASED OF MIRNA: NEW INSIGHT INTO INTERVERTEBRAL DISC DEGENERATIVE medicine


50


marine charrier(1,2,3), J. Lorant(1), R. Contreras Lopez(4), B. Lieubeau(5), C. Schleder(1), I. Leroux(1), C. Babarit(1), G. Téjédor(4), N. Jaulin(6), O. Adjali(6), Y. Pereon(7), F. Djouad(4), G. Lamirault(8) and K. Rouger(1)

(1) PAnTher, INRA, École nationale vétérinaire, agro-alimentaire et de l’alimentation Nantes-Atlantique (Oniris), Université Bretagne Loire (UBL), Nantes, France

(2) INSERM, UMR 1087/CNRS UMR 6291, Institut du Thorax, Nantes, France (3) Université de Nantes, Nantes, France(4) INSERM, UMR 1183, Institut de Médecine Régénératrice et Biothérapies, Hôpital Saint-Eloi,

Montpellier, France(5) IECM, INRA, Oniris, Université de Nantes, UBL, Nantes, France(6) INSERM, UMR 1089, Centre Hospitalier Universitaire Hôtel Dieu, Nantes, France(7) Centre de Référence Maladies Neuromusculaires AOC, Laboratoire d’Explorations Fonctionnelles,

Centre Hospitalier Universitaire Hôtel Dieu, Nantes, France(8) INSERM UMR 1087/CNRS UMR 6291, Institut du Thorax, Nantes, France

Allogeneic cell transplantation protocols are highly limited by graft rejection. To overcome this issue, long-term immunosuppression (IS) is usually used that results in improved cell engraftment but also major adverse effects (Zhu et al., 2013; Bottomley et al., 2013).Many in vitro studies showed pleiotropic immunomodulatory properties for adult stem cells, especially mesenchymal ones (English et al., 2013). These cells have been shown to modulate the behavior of many immune cells through paracrine secretion or direct contact with target cells (Vaithilingam et al., 2017; Baharlou et al., 2017). These features could increase their ability to engraft in allogeneic recipient despite the lack of strong IS. Also, immunomodulatory cell delivery may be beneficial in degenerative disorders to limit chronic inflammation that characterizes tissues and interfere with repair.We showed that systemic delivery of allogeneic muscle-derived stem cells, termed MuStem cells, into dystrophic dogs under IS lead to muscle regeneration and long-term clinical status stabilization (Rouger et al., 2011; Robriquet et al., 2015; Lardenois et al., 2016). Recently, the human MuStem cells were isolated and characterized as exhibiting in vitro/in vivo myogenic potential, positioning them as an attractive candidate for muscle-dedicated regenerative medicine (Lorant et al., 2018a). Interestingly, a transient IS was also shown as sufficient to sustain their transplantation benefits and prevent host immunity response in allogeneic context (Lorant et al., 2018b). These findings suggested that MuStem cells could display immune privilege behavior.Here, we explored modulation of immune effectors by hMuStem cells. Using in vitro assays, we examined the impact of hMuStem cells on the T cell features and the complement system activation. Flow cytometry experiments and Elisa assays were performed to determine the immunophenotype of hMuStem cells.We demonstrated that hMuStem cells inhibit LT proliferation through iNOS expression and PGE2 secretion and induce regulatory cells. We also revealed that they inhibit complement-mediated lysis through factor H secretion. They secrete various immunomodulatory molecules suggesting action on other immune cells.Overall, these findings show that hMuStem cells display an immune regulatory phenotype and modulate in vitro both T cell and complement functions, reinforcing their qualification as promising cell agent for clinical application in muscle diseases.

DEMONSTRATION OF IMMUNOMODULATORY PROPERTIES FOR HUMAN MUSTEM CELL POPULATION, A PROMISING CANDIDATE FOR CELL THERAPY OF MUSCULAR DYSTROPHIES


51


Léa Flippe(1, 2), A. Gaignerie(3), S. Bezie(1, 2), L. David(1,2,3), C. Guillonneau(1, 2)

(1) INSERM UMR1064, Center for Research in Transplantation and Immunology ITUN, Université de Nantes, France

(2) Institut de Transplantation Urologie Néphrologie (ITUN), CHU Nantes, Nantes, France (3) iPSC corefacility, University of Nantes; INSERM UMS 016; CNRS 3556; CHU de

Nantes, France

Organ or cell transplantation is the only therapeutic solution for pathologies causing an irreversible loss of vital organs function. The development of novel specific and non-toxic anti-rejection immunotherapies is a major goal in transplantation. Strategies based on regulatory T cells (Tregs) are promising. However, Tregs cell-based therapies have been hampered by the technical limitation of obtaining large batches of functional Tregs cells. The project aims are to remove technological locks in order to obtain an unlimited number of Tregs cells from human pluripotent stem cells (hPSCs). This very innovative project will also allow us to better understand the development biology of Tregs through their in vitro differentiation.We have developed a new protocol of differentiation that requires two separate steps, the first step is to obtain hematopoietic stem cells (HSCs) from hPSCs and the second step is to differentiate the obtained HSCs to the lymphoid lineage. We have generated HSCs thought embryoid bodies (EBs), which are three dimensional aggregates of hPSCs, formation for 9 days. After 9 days, we obtained 50% of cells expressing CD34+, a key marker of HSCs, which are also CD43+ or CD43-. Digital gene expression sequencing (DGE-RNAseq) in comparison to cord blood cells highlighted that the CD34+CD43+ cells as the best HSCs population. Then, we dissociated EBs and transferred them onto bone marrow stromal mouse cells expressing the delta-like ligand of the Notch pathway (OP9 DLL1) to induce T-lymphoid differentiation in an established co-culture system for about 26 days. The cells were monitored during the 26 days. After 26 days, we obtained CD5+CD7+ cells which are early T type markers. These CD5+CD7+ cells were also CD4+CD8+, CD4+CD8- or CD4-CD8+. However, these cells didn’t express TCRαβ and CD3. DGE-RNAseq showed that our cells expressed gene of lymphoid lineage like IKAROS or IL7R but didn’t express FOXP3. To improve our protocol, we used a lentiviral vector encoding FOXP3, a known master transcription factor of Tregs that can drive the cell fate to the Tregs lineage. Transduction at day 10 of co-culture resulted in significant differentiation of Foxp3+CD3+TCRαβ+ CD8+ or CD4+ Tregs cells until day 26.Altogether, our results demonstrate that human CD4+ and CD8+ Tregs differentiation is under the control of Foxp3 and that for the first time CD4+ and CD8+ Tregs can be differentiated from hPSCs opening new possibilities for cell therapy.

GENERATION OF CD8+ AND CD4+ REGULATORY T CELLS FROM HUMAN PLURIPOTENT STEM CELLS


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martin barbier(1,3), M. Boivin Welch(1,2,3), A. Dakiw Piaceski(1,3), S. Larochelle(1,3), D. Larouche(1,3), K. Ghani(2,3), M. Caruso(2,3), L. Germain(1,3).

(1) Centre de Recherche en organogénèse expérimentale de l’Université Laval/LOEX, Québec, Canada

(2) Centre de Recherche sur le cancer de l’Université Laval, Québec, Canada(3) CHU de Québec-Université Laval Research Center, Québec, Canada

Background: Recessive dystrophic epidermolysis bullosa (RDEB) is a genetic disease in which minor mechanical stress in the skin causes the formation of blisters and erosions. Mutations in the COL7A1 gene are responsible for RDEB. The COL7A1 gene encodes type VII collagen that is normally secreted into the extracellular space by dermal fibroblasts and epidermal keratinocytes. We have generated a high-titer 293Vec retrovirus packaging cell clone to produce a SIN vector (viral enhancer-deleted) that allows the delivery of the COL7A1 gene into normal keratinocytes and fibroblasts.

Hypothesis: We hypothesized that the effective transduction of the normal COL7A1 gene in stem cell keratinocytes is a prerequisite to achieve a long-term therapeutic effect of a transgenic tissue-engineered skin grafted on RDEB patient.

Objective: The objective of this project was to extract and cultivate RDEB fibroblasts and keratinocytes and to transfer the COL7A1 gene using our SIN vector.

Method: Fibroblasts and keratinocytes were extracted from a small skin biopsy harvested in a RDEB patient. Cells were used to reconstruct bilayered tissue-engineered skin substitutes by the self-assembly approach. Amphotropic pseudotyped retroviral vector and the transduction enhancers polybrene or EF-C were used to transduce the COL7A1 gene into RDEB cells. COLVII expression was evaluated by immunofluorescence and flow cytometry.

Results: Overall, RDEB cells normally proliferated and their morphological aspect under phase contrast microscope appeared normal. A biobank containing enough cryovials of cells to perform in vitro and animal experiments was established. Bilayered tissue-engineered skin substitutes were successfully produced and a fragility of the dermal-epidermal junction was noted. We observed that viral particles infect the keratinocytes and fibroblasts of this RDEB patient with a transduction rate of 50% and 65% respectively (single transduction).

Perspectives and conclusions: The next step is to evaluate if stem cell keratinocytes are effectively transduced with this method, and to produce “COL7A1 gene-corrected” tissue-engineered skin substitutes. In conclusion, our results indicated that our clone of cells producing a safe COL7A1 gene vector has the potential to be used for gene therapy for RDEB.

GENE THERAPY AND TISSUE ENGINEERING: A STRATEGY TO TREAT RECESSIVE DYSTROPHIC EPIDERMOLYSIS BULLOSA Or rdeb


53


Takanori Takebe

Cincinnati Children’s Hospital Medical Center, Custom, USA

Organoids hold great promise to revolutionize 21st century healthcare through transforming drug development, precision medicine, and ultimately, transplantation-based therapies for end stage diseases, namely an “organoid medicine”. Recently, we have developed a novel culture principle, named self-condensation culture, whereby mesenchymal progenitors initiated organoid (or organ bud) formation within multiple progenitor cell mixtures. Defining optimal mechanical properties of the substrate promoted formation of 3D, transplantable organ buds from diverse tissues including kidney, pancreas and cartilage (Cell Stem Cell, 2015, Cell Reports, 2018). By using self-condensation principle, we showed iPSC-derived human hepatic endoderm with supportive mesenchymal and endothelial cells results in three dimensional liver bud like organoid that becomes vascularized upon transplantation into mice (Nature, 2017). Transplantation of these organoids is capable of extending life in a mouse model of liver failure (Nature, 2013, Cell Reports, 2017). Furthermore, with the goal of clinical translation of liver bud transplant therapy, we established a massive (108-scale) organoid production platform entirely from human iPSCs, reproducibly demonstrating a metabolically functional property both in vitro and in vivo. Overall the technology outlined in this study sheds light on an exciting therapeutic paradigm based on iPSC-multi-cellular organoids towards organoid medicine (drug testing and regenerative) applications.

THE ERA OF ORGANOID MEDICINE FROM SCREEN TO THERAPEUTICS


54


David Eglin

AO Research Institute Davos, AO foundation, Davos, Switzerland

In orthopaedics and trauma surgeries, three-dimensional (3D) printing and additive manufacturing are fast growing technologies used for the fabrication of clinical guides and advanced medical devices. Developing 3D-printed devices and associated procedures specific to the patient are anticipated to shorten the surgery time, increasing its accuracy, and success. The customizability of 3D-printed devices introduces new complexities with the ability to design patient specific implants and to potentially impart biological functionality. A consequence is that the need for qualitative assessment of the design, and better preclinical evaluation, are also increased. These will be discussed using the example of a patient specific implant workflow in cranio-maxillofacial surgery from the production of the personalized implants (i.e. implant shape accuracy, mechanics) to the assessment of their performance (i.e. implant stability, position, bone formation) in a large preclinical model.

3D-PRINTED PATIENT SPECIFIC IMPLANTS AND THEIR PRECLINICAL EVALUATION


55


Azadeh Golipour

Avrobio, USA

Gene therapy offers permanent genomic integration, with a demonstrated safety record, for a durable and potential life-long curative benefit. The majority of treatments for life-threatening diseases address symptoms rather than root causes. Many simply delay disease progression but are not curative. This is especially true for rare diseases where existing treatments only manage, but do not cure, the patient’s underlying disease. We believe the solution is to address the root cause – the defective gene. AVROBIO’s gene therapy differs from other approaches, as it has the capacity to insert directly into the patient’s genome for a durable and life-long potential curative benefit.

Therapy begins when the patient receives a conditioning agent to stimulate the bone marrow to produce stem cells (CD34+) and release them into the blood. The patient’s peripheral blood stem cells are extracted and genetically modified by adding a new copy of the faulty gene. The modified cells are then delivered back into the patient via a one-time infusion.

CLINICAL PRODUCTION OF AN AUTOLOGOUS EX VIVO GENE THERAPY


56


marie maumus (1,2), S. Cosenza(1,2), K. Toupet(1,2), R. Maxime(1,2), B. Claire(1,2), C. Jorgensen(1,2,3), D. Noël(1,2,3)

(1) INSERM U1183, Montpellier, France(2) Université Montpellier, Montpellier, France(3) CHU, Unité Clinique d’Immuno-Rhumatologie, Montpellier, France

Mesenchymal stem cells (MSC) are multipotent cells that possess regenerative and immunomodulatory functions that are of interest for osteoarticular diseases such as osteoarthritis (OA) and rheumatoid arthritis (RA). These functions are mediated by soluble mediators that can be released within extracellular vesicles (EV). EVs consist of exosomes (Exo), microparticles (MP) and apoptotic bodies that mirror the effect of parental cells but little is known about their respective role. The aim of this study was to compare the effects of Exo and MP secreted by MSCs on OA and RA features.

EV subsets were isolated from murine bone marrow MSCs by differential ultracentrifugation. Size and structure of EV were evaluated by nanotracking analysis (NTA) and electron microscopy. Expression of specific markers was tested by flow cytometry. In vitro, EVs were incubated with OA-like chondrocytes and chondrocyte markers evaluated by RT-qPCR. Proliferation of murine splenocytes was quantified after incubation with EVs. In vivo, EVs were injected in the knee joint in the collagenase-induced osteoarthritis (CIOA) model or systemically in the collagen-induced arthritis (CIA) model. Clinical score and histomorphometric analyses of joints were performed by histology, μCT or confocal microscopy.

Exo were around 120nm in diameter and expressed CD9, CD81, TSG101 markers while MP were 165 to 500nm in diameter and expressed CD29, CD44 and Sca1. In OA-chondrocytes, both MP and Exo could reinduce the expression of chondrocyte markers, type II collagen and aggrecan and they inhibited the catabolic and inflammatory markers MMP-13, ADAMTS5, iNOS. Addition of EV in proliferative assays significantly inhibited the proliferation of CD8+ T cells and increased Treg cell population. Proliferation and activation of B lymphocytes were decreased. In vivo, after injection in the CIOA model, MSCs, MP and Exo equally protected mice from joint damage (similar cartilage and sub-chondral bone parameter values in healthy and treated mice as compared to OA controls). Beneficial effect of EV was also observed on reduced calcification of joint ligaments and menisci. The immunomodulatory function of both types of EVs was also observed in the CIA model with a significant reduction of clinical symptoms.

Our data indicated that both types of EVs reproduced the chondroprotective and immunosuppressive effects of MSCs in vitro and in vivo and could be used as a substitute of MSC in the treatment of OA and RA.

THERAPEUTIC EFFECT OF MESENCHYMAL STEM CELL-DERIVED EXTRACELLULAR VESICLES IN RHEUMATIC DISEASES


57


denis barritault(1), V. Coudry(2), S. Jacquet(2), J. M. Denoix(2), K. Gumus(3), C. Baudouin(4), A. Giocanti-Aurégan(5), P. Desgranges(6), S. Ahmad Roohi(7)

(1) OTR3 Company, France and University Paris Est Creteil (2) CIRALE - Hippolia, INRA USC BPLC 957, ENVA-UPEC, RD 675, Goustranville, France and Université

Paris-Est, Ecole Nationale Vétérinaire d’Alfort, Maisons-Alfort, France(3) Department of Ophthalmology, Erciyes University School of Medicine, Kayseri, Turkey(4) Institut de la Vision, and Centre Hospitalier National d’Opthalmologie des Quinze Vingts, Paris, France(5) A. Giocanti-Aurégan, Ophthalmology Avicenne Hospital, Paris 13 University, Bobigny, France(6) Department of Vascular Surgery, Hopital Henri Mondor, Université Paris-Est Créteil, France(7) University Putra Malaysia, Hand Surgery Serdang 43400, Malaysia

ReGeneraTing Agents (RGTAs) are polysaccharide engineered to mimic heparan sulfate. When introduced into a damage tissue RGTA will replace destroyed heparan sulfate. It will bind and protect matrix proteins (collagens etc...) and communication peptides (growth factors, cytokines, chemokines etc. By restoring the extracellular matrix Scaffold and a proper cellular micro environment this RGTA based matrix therapy approach has considerably improved the quality of healing in various animal models with reduction or absence of fibrosis and resulting in a real regeneration process. The RGTA technology has been validated in clinics with over hundred thousands of patients treated for corneal or skin ulcers with no adverse effect. Today several randomized controlled trials provide evidence based efficacy supports to this technology and products, both in human and veterinary medicine. Hence, re-epithelization of Cornea in the epi-off and stromal cross linking treatment of keratoconus is significantly improved by topical use of a dedicated formulation with RGTAOTR4120, racing sport horses with tendonitis recover quicker, race back as before and have less recurrences after intra tendon injection of RGTAOTR4131. OTR4120 based topical treatment of critical upper or lower limb ischemia wounds have prevented patients from predicable amputation by inducing unexpected closure of the wound while severe persistent corneal ulcers or Sjogren dry eye responded to this treatment. Associated to these regenerative properties, significant pain killing activity was reported, reversing if treatment was not completed. ReGeneraTing Agents (RGTAs) are polysaccharide engineered to mimic heparan sulfate. When introduced into a damage tissue RGTA will replace destroyed heparan sulfate. It will bind and protect matrix proteins (collagens etc...) and communication peptides (growth factors, cytokines, chemokines etc. By restoring the extracellular matrix Scaffold and a proper cellular micro environment this RGTA based matrix therapy approach has considerably improved the quality of healing in various animal models with reduction or absence of fibrosis and resulting in a real regeneration process. The RGTA technology has been validated in clinics with over hundred thousands of patients treated for corneal or skin ulcers with no adverse effect. Today several randomized controlled trials provide evidence based efficacy supports to this technology and products, both in human and veterinary medicine. Hence, re-epithelization of Cornea in the epi-off and stromal cross linking treatment of keratoconus is significantly improved by topical use of a dedicated formulation with RGTAOTR4120, racing sport horses with tendonitis recover quicker, race back as before and have less recurrences after intra tendon injection of RGTAOTR4131. OTR4120 based topical treatment of critical upper or lower limb ischemia wounds have prevented patients from predicable amputation by inducing unexpected closure of the wound while severe persistent corneal ulcers or Sjogren dry eye responded to this treatment. Associated to these regenerative properties, significant pain killing activity was reported, reversing if treatment was not completed. Adapted RGTA are in development for more tissue injuries extending RGTA as a new therapeutic class in the field of regenerative medicine exploiting our natural potential to regenerate without need for exogenous cells. RGTA can combine with cell therapy by constructing a niche to favor homing. The future of regenerative medicine lays in a proper adjustment of the microenvironment to optimize cell colonization, expansion, replacement and recovery of their functions.

RGTA AND MATRIX THERAPY IS A NEW BRANCH OF REGENERATIVE MEDICINE: FROM BASIC SCIENCE TO PATIENTS PROVE OF EFFICACY IN HUMAN AND VETERINARY MEDICINE THROUGH RANDOMIZED CONTROLLED TRIALS


58


E. Viguier(1,2), N. Gomez(1), M. Fèbre(3), V.e Livet(1), N. Plantier(3), P. Pillard(1), nathalie Saulnier(3), T. Cachon(1,2), Stéphane Maddens(3)

(1) Small Animal Surgery Department, VetAgro Sup, Marcy L’Etoile, France(2) UPSP 2016A104, ICE, Interaction Cells Environment, Campus Veterinaire VetAgro

Sup, Université de Lyon, Marcy l’Etoile, France(3) Vetbiobank SAS, Marcy L’Etoile, France

As in human, prevalence of osteoarthritis (OA) is increasing in canine population. First line treatment to manage OA is based on non-steroidal anti-inflammatory drugs (NSAID), nonetheless long-term use may induce severe side effects. New therapeutic approach, such as MSC-based therapy, is gaining increasing interest for the treatment of chronic inflammatory diseases such as OA. The objective of this study was to evaluate the effects of a single IA injection of allogeneic canine neonatal MSCs in dogs suffering from severe naturally-occurring OA.MSCs were prescribed as a compassionate treatment in an open-label, uncontrolled study after other therapeutic approaches being excluded. Dogs enrolled in this study were injected intraarticularly with 10E7 neonatal MSCs, in 1 or 2 joints independently. Outcome measures were veterinary clinical evaluation by scoring at 1, 3, and 6 months post-injection, along with a global owner assessment of their dog’s wellness up to 2 years post-treatment. To complete the understanding of the safety profile and biological response to neonatal allogeneic MSC administration, the presence of alloantibodies against MSC antigens was evaluated in the recipient.Twenty-two client-owned dogs were recruited in the study among whose 16 completed the 6-months study. Three dogs were dropped out from the study because of medical reasons not in relationship with MSC injection, and 3 dogs were withdrawn because of non-compliance of the follow up visits. No systemic adverse effect was observed after MSCs injection during the entire course of the study. Ten dogs were administrated with a single injection of MSCs in one joint, while 6 animals received 1 injection in 2 joints, 6 months apart. For each individual joint, clinical evaluation showed a significant improvement of the clinical score from baseline up to 6 months post-treatment. Long-term safety results collected from a survey completed by 13 owners reported no MSC-related adverse event during the two-years follow up. No MSC-antibodies were detected in dog’s serum following treatment.This study provides additional evidence for the long-lasting effect of allogeneic MSC therapy (up to 6 months) in alleviating pain and lameness in dogs with OA and a stabilization of OA progression over 2 years. The absence of detection of alloantibodies following IA injection of neonatal allogeneic MSCs suggests that this therapeutic approach is well-tolerated and could be repeated if required.

SAFETY AND CLINICAL EFFICACY OF A SINGLE INTRA-ARTICULAR INJECTION OF ALLOGENEIC NEONATAL MESENCHYMAL STEM CELLS (MSC) FOR THE TREATMENT OF OSTEOARTHRITIS IN DOGS


59


Franco Severina, Export Manager

Euroclone Spa, Pero (MI), Italy

Good Manufacturing Practice (GMP) compliant procedures are a prerequisite for cell production in clinical application and clean rooms are ideal zones for cell therapies production.

The clean rooms useful for clinical application require high running and maintenance costs and need trained operators and strict procedures to prepare the rooms and the people involved in the processes.

This requires huge efforts in terms of facilities, personnel training and quality control.

To provide a streamlined workflow environment reducing the set up and running costs of cell therapy products we have used ISOCell PRO, a Cell Therapy Grade A Isolator alternative to the use of GMP class A Biological Safety Cabinet in a class B clean room environment.

Indeed, ISOCell PRO is a Closed System that requires Grade D surrounding environment. The Positive Pressure Isolator guarantees Grade A environment in the working area with aseptic conditions according to GMP. A CO2 incubator is integrated, what makes the system easily validated at affordable costs. Decontamination process is automatic, fast, safe and economically affordable and no need for special operators’ clothes.

Actually, SwissMedic completed the validation of a GMP process involving the use of ISOCell PRO units installed at the Centre de Production Cellulaire (CPC-CHUV - Lausanne).

The CPC in compliance with GMP produces tissues and cells necessary to the treatment of burn patients ensuring the production of autologous skin (the patient is the donor and the recipient of its own cells). With these treatments, CHUV is for many years a center of reference at Swiss, European and international levels for the care of burn patients. The CPC are ongoing the validation of other cell type productions for clinical trial in cardiology and neurosurgery.

Our experience suggests this workstation as a possible alternative to the classic clean room due to its small size and the simplification of the working and maintenance operative procedures. In conclusion this kind of isolators may represent an interesting solution in the perspective of the more and more strong request for costs reduction of GMP in clinical application.

ISOCELL PRO WORKSTATION FOR CELL THERAPY PRODUCT AND CLINICAL APPLICATIONS


PosTeR absTRaCTs

60

61


Sophie domingues*, Masson Y.(1)*, Poulet A.(1), Saïdani M.(1), Polentes J.(1), Lemaître G.(1), Peschanski M.(1), Baldeschi C.(1) (* Equally contributing authors)

(1) I-STEM, INSERM/UEVE U861, CECS, Evry, France

Skin is the largest organ of the body involved in self-protection against external damages. Epidermis, the upper layer of the skin is mainly composed of keratinocytes organized to form a physical barrier at the interface of the environment. Some of diseases associated to genetic mutations or not could weaken this protection and lead to the disruption of skin integrity. Cell therapy approaches using adult keratinocytes are currently envisaged however these cells present limited proliferative capacities and variability in genetic background. Access to an unlimited source of embryonic pluripotent stem cells (hESC) will aim at overcoming these limitations since these cells are available in unlimited quantities thanks to their unlimited proliferation capacity and their pluripotency.

In this context, a protocol allowing the generation of keratinocytes from hESC able to perform functional pluristratified epidermis was developed. In the perspective of a human clinical application, the entire protocol have been optimized and adapted following good manufacturing practice (GMP) conditions from a clinical grade hES cell line (RC9) obtained at the Biotech Company Roslin Cells. A quality control of the keratinocytes was established. These controls include the checking for contaminations, karyology, and viability. Specific controls such as the analyses of the expression of keratinocytes markers and the absence of pluripotency markers were performed to verify the quality of the keratinocytes cells bank. In addition, a clinical grade support was selected for this capacity to allow the formation of a pluristratified epidermis in vivo.

To certify the safety in human therapy using cells derived from hESC, pre-clinical experiments will be performed to analyze the theoretical risk of a cell shedding to distant organs, and tumorigenicity due to residual pluripotent cells.

PRODUCTION OF CLINICAL GRADE TEMPORARY EPIDERMAL SUBSTITUTE OBTAINED FROM HESC DERIVED KERATINOCYTES FOR THE TREATMENT OF SICKLE CELL LEG ULCERS: A CHALLENGE FOR REGENERATIVE MEDICINE

POSTER 1

62


cyprien denoeud(1), J. Paquet(1), J. Boisselier(2), P. Becquart(1), A. Moya(1), A. Diallo(1), S. Marinesco(3), A. Meiller(3), V. Larreta-Garde(2), E. Pauthe(2), E. Potier(1), H. Petite(1)

(1) Laboratory of Osteo-Articular Bioengineering and Bioimaging, UMR 7052, University of Paris Diderot, Paris, France

(2) Biomaterial for Health Group, ERRMECe, EA 1391, Department of Biology, University of Cergy-Pontoise, France

(3) Neuroscience Research Center, AniRA-NeuroChem Platform, UMR 5292, University of Lyon 1, Lyon, France

In the context of cell-based regenerative medicine, exogenously administered mesenchymal stromal cells (MSCs) exhibited a poor survival rate. A possible explanation for this limited cell survival is that, upon implantation, MSCs encounter a harsh ischemic microenvironment characterized by low oxygen tension and nutrient deprivation. This issue can be overcome by in situ supplying glucose that acts as the main metabolic fuel for MSCs in hypoxia and enhances their survival (Deschepper et al.2011 and 2013). The objective of the study is to engineer a tissue-construct that provides sufficient level of glucose to MSCs and enhances their survival when transplanted in vivo.

To this aim, hydrogels containing fibrin, starch (a polymer of glucose) and AMG (an enzyme that release glucose from starch) were formulated. These injectable, self-supported hydrogels released glucose amounts in accordance with that required by hMSCs for their survival. In vitro, under near anoxia, MSCs loaded in fibrin/starch/AMG hydrogels exhibited a survival rate 115 times higher than the one loaded in fibrin hydrogels, after 14 days. Moreover, when ectopically implanted in nude mice, luciferase-labelled hMSCs loaded in fibrin/starch/AMG hydrogels exhibited a significant improvement of their viability (x4 after 14 days) in comparison to hMSCs loaded in fibrin gels as demonstrated by the follow-up of the luciferase activity by bioluminescence imaging. These data were further substantiated by monitoring the number of hMSCs remaining in the hydrogels implanted ectopically in mice. At day 14 days, fibrin / AMG / starch scaffolds contained 7.5 times more viable hMSCs than fibrin hydrogels.

This work establishes for the first time that a construct based on a fibrin/starch/AMG hydrogel delivers glucose over time and enhances the survival of hMSCs. Most interestingly, the data obtained with hMSCs are now extended to adipose-derived MSCs and myoblasts.

GLUCOSE DELIVERY SYSTEM BASED-HYDROGEL COMPOSITE ScAFFOLd FOr enHAncinG MSC SURVIVAL

POSTER 2

63


F. Regent(1,2), L. Lesueur(1, 2, 3), Alexandra Plancheron(1, 2, 3), W. Habeler(1, 2, 3), L. Morizur(1, 2, 3), K. Ben M’barek(1, 2, 3), C. Monville(1, 2)* (*: corresponding author)

(1) INSERM, UMR861, Istem, AFM, France

(2) UEVE, UMR861, Istem, AFM, France

(3) CECS, Istem, AFM, France

Retinal pigment epithelium (RPE), the monolayer of pigmented cells localized between the neural retina and the choroid plays crucial roles in sight mainly by contributing to the blood/retina barrier, absorbing light energy but also by transporting nutrients from the blood to the photoreceptors. Therefore, dysfunction or death of RPE is followed by the death of photoreceptors and is responsible for a group of rare hereditary diseases called retinitis pigmentosa and to some extend for age related macular degeneration (AMD). Since there is currently no treatment for dry AMD and for most of the retinitis pigmentosa, the replacement of RPE cells is an attractive solution. The ability of human pluripotent Stem Cells (hPSCs) to spontaneously differentiate into RPE has allowed their use for clinical applications. We have recently developed a cell therapy product consisting of human embryonic stem cells (hESC) disposed on a biological substrate (Ben M’Barek et al.). RPE cells were differentiated using spontaneous differentiation of hESCs. However, even if this protocol allows obtention of a nearly pure population of RPE, it remains a long and largely inefficient method that requires a fastidious manual enrichment by dissecting pigmented area.

This process is therefore, not compatible with industrial large scale production and marketing which would be the next step to treat larger cohort of patients. We have developed a simple automated protocol with limited exogenous factors and obtained a pure population of RPE. With this protocol the main steps of the retinal development are recapitulated with the generation of retinal progenitors that could be differentiated into all retinal cell types such as retinal precursors. Finally, such protocol should allow cost reduction, large scale and reproducible production of RPE cells. This new protocol could open avenues for AMD clinical trial but although for retinal disease modeling and large high through put drug screening.

DEVELOPMENT OF AN AUTOMATED PROTOCOL FOR LARGE SCALE DIFFERENTIATION OF RETINAL PIGMENT EPITHELIAL CELLS FROM HUMAN PLURIPOTENT STEM CELLS

POSTER 3

64


Audrey Smith(1,3), J. Boulestreau(1), M. Marquis(2), B. Hagland(1,5), D. Renard(2), C. Vinatier(1), A. Des Rieux(3,4)*, J. Guicheux(1,5)*, C. Le Visage(1)* (* equivalent contribution)


(2) UR1268 BIA (Biopolymères Interactions Assemblages), INRA, Nantes, France(3) Louvain Drug Research Institute, Advanced Drug Delivery and Biomaterials, Université

Catholique de Louvain, Bruxelles, Belgium(4) Institute of Condensed Matter and Nanosciences,Université Catholique de Louvain, Louvain-

la-Neuve, Belgium(5) CHU Nantes, PHU 4 OTONN, Nantes, France

Osteoarthritis (OA) is a degenerative and inflammatory joint disease that affects the whole joint tissue. Mesenchymal Stem Cells (MSC) ability to secrete anti-inflammatory and immuno-modulatory factors represents an attractive tool in the treatment of OA (Pers YM, 2016). Considering the risk of cell leakage and the massive cell death upon intra-articular injection (Toupet K, 2013; Aguado BA, 2012), MSC encapsulation appears as a good solution and could also provide a 3D microenvironment supporting MSC biological activity. Previously, we have demonstrated that alginate particles support MSC viability and bioactivity (Hached F, 2017). As these particles were too large to be injected into joints of small animals, we developed a method of cell encapsulation compatible with intra-articular injection (IAI) through a 26G needle.Human adipose stem cells (hASC) were encapsulated via a micromolding method. First, polydimethylsiloxane (PDMS) chips containing circular micromolds (150 μm of diameter) were manufactured and a solution of 2% alginate containing 3M of hASC per mL was deposited on the molds. Cell loading into the micromolds was done either by sedimentation or by centrifugation. Alginate particles were then obtained by crosslinking with Ca2+ and cultured into complete medium. Cell number and metabolic activity were evaluated for 7 days after encapsulation. To study encapsulated cells ability to sense and respond to a pro-inflammatory stimulus, they were stimulated with TNF-α and INF-ϒ and the presence of prostaglandineE2 (PGE2) and indoleamine2,3-dioxygenase (IDO) was evaluated in the supernatant.We obtained alginate particles with a diameter of 150±0.7μm. Using cell quantification, we determined that the number of cells per particles was 5 times higher when the molds were loaded by centrifugation.Cell number and metabolic activity remained stable for 7 days after encapsulation and injection had no impact on cell viability. After stimulation, PGE2 concentration was multiplied by 13 and 7 and IDO activity was 2 and 4 times higher as cell loading was done by sedimentation or centrifugation, respectively.We encapsulated hASC into alginate particles via micromolding and demonstrated in vitro that cell viability was stable even after injection through a 26G needle. We also showed that encapsulated cells were able to sense and respond to an inflammatory stimulus. We will now evaluate encapsulated cells potential in vivo via IAI in a murin model of osteoarthris.

MESENCHYMAL STEM CELL ENCAPSULATION IN ALGINATE MICRO PARTICLES FOR INTRA ARTICULAR INJECTION IN OSTEOARTHRITIS

POSTER 4

65


Anne Gaignerie-ilunga, N. Lefort, M. Rousselle, V. Forest-Choquet, A. Girardeau, L. Flippe, V. Francois - Campion, A. Caillaud, C. Chariau, Q. Francheteau, A. Derevier, F. Chaubron, S. Knöbel, N. Gaborit, K. Si-Tayeb, L. David

Plateforme IPSc, IRS UN, Nantes, France

Over a decade after their discovery, induced pluripotent stem cells (iPSCs) have become a major biological model. The iPSC technology allows generation of pluripotent stem cells from somatic cells bearing any genomic background. The challenge ahead of us is to translate human iPSCs (hiPSCs) protocols into clinical treatment. To do so, we need to improve the quality of hiPSCs produced. In this study we report the reprogramming of multiple patient urine-derived cell lines with mRNA reprogramming, which, to date, is one of the fastest and most faithful reprogramming method. We show that mRNA reprogramming efficiently generates hiPSCs from urine-derived cells. Moreover, we were able to generate feeder-free bulk hiPSCs lines that did not display genomic abnormalities. Altogether, this reprogramming method will contribute to accelerating the translation of hiPSCs to therapeutic applications.

URINE-DERIVED CELLS PROVIDE A READILY ACCESSIBLE CELL TYPE FOR FEEDER-FREE MRNA REPROGRAMMING

POSTER 5

66


B. Rosa(1), Perrine de Villemagne(1), F. Halary(2,3), J. Guicheux(4), C. Le Visage(4), J.-Y. Hascoet(1)

(1) GeM Laboratory, CNRS, UMR6183, Centrale Nantes, Nantes, France

(2) Centre de Recherche en Transplantation et Immunologie, UMR1064, INSERM, Université de Nantes, Nantes, France

(3) Institut de Transplantation Urologie Néphrologie (ITUN), CHU de Nantes, Nantes, France

(4) Regenerative Medicine and Skeleton (RMeS), UMR1229, INSERM, Nantes, France

3D bioprinting is an additive manufacturing technology based on layer-by-layer deposition of a cell-containing bioink to create tissues with complex architectures that evolve over time (4D). Physiological tissues are rarely homogeneous in composition but rather exhibit spatial distribution of different cell types and extracellular matrix proteins. The ability to recapitulate this internal organisation is critical and still remains a challenge in the field of 4D bioprinting. Our interdisciplinary team has expertise in the field of additive manufacturing for metallic applications, and more specifically in functionally graded material (FGM). The aim of this work is to develop a methodology to deposit multiple bioinks of different compositions with controlled percentages in order to generate gradients.

In the context of this interdisciplinary research, a high precision bioprinting device that operates under fully opened programming has been developed which enables total control over the printing process. The device is equipped with temperature controllers for both the printing stage and dispensing syringes. The prototype presented uses linear axes and pneumatic control allowing high spatial resolution (0.1μm). In parallel, a gelatin-alginate based bioink is being developed and shows limited toxicity of embedded human primary embryonic fibroblasts after 3 days. In addition, the bioink presents good printability and long term stability as assessed by the generation of a 1x1x0,5 cm cube with high shape fidelity. Together, these tools will be used to control the multimaterial nozzle that is being conceived in order to deposit the bioinks according to pre-defined gradients. The intervertebral disc (IVD) is composed of a peripheral network of collagen (Annulus fibrosus, AF) that enclose a central gelatinous and hydrated structure (Nucleus Pulposus, NP). A characteristic transitional zone merges these two regions and consitutes an intersting physiological application to validate our ability to generate controlled physiological gradients.

The technologies elaborated in this project will contribute to build a strong knowledge in the field of regenerative medicine with possible applications to various types of tissues.

4D-BIOPRINTED FUNCTIONALLY GRADED MATERIAL

POSTER 6

67


Agata Zykwinska(1), M. Marquis(2), M. Godin(1,2), S. Cuenot(3), L. Marchand(1), C. Sinquin(1), C. Chédeville(4), C. Le Visage(4), J. Guicheux(4), S. Colliec-Jouault(1)

(1) IFREMER, Laboratoire Ecosystèmes Microbiens et Molécules Marines pour les Biotechnologies, Nantes, France

(2) INRA, UR1268 Biopolymères Interactions Assemblages, Nantes, France

(3) Institut des Matériaux Jean Rouxel (IMN), Université de Nantes-CNRS, Nantes, France

(4) INSERM, UMRS 1229, RMeS Regenerative Medicine and Skeleton, Nantes, France

Articular cartilage is an avascular, non-innervated connective tissue with limited ability to regenerate. Articular degenerative processes arising from trauma, inflammation (rheumatoid arthritis) or due to aging (arthrosis) are thus irreversible and may lead to the loss of the articular function. In order to repair cartilaginous defects, several surgical techniques have been developed mainly based on subchondral bone marrow, implantation of healthy cartilage or chondrogenic tissue. However, multiple disadvantages arising from these techniques led to development of a new therapeutic strategy, namely tissue engineering. Indeed, the association of cells (chondrocytes or mesenchymal stem cells, MSC) together with signaling molecules (growth factors) into biocompatible hydrogel matrix could lead to regeneration of the functional tissue. However, in order to guide the MSC differentiation into chondrocytes, the supply of growth factors during the differentiation phase is required. In this context, the encapsulation of growth factors within micro-matrices allowing both their protection against degradation conditions and their release leading to the enhancement of their bioactivity and bioavailability becomes an interesting approach. The aim of the present study was to develop a growth factor delivery system based on a marine exopolysaccharide (EPS) displaying glycosaminoglycan (GAG)-mimetic properties that could further be used for stimulation of MSC chondrogenic differentiation in vitro and in vivo. To preserve its bioactivity, the growth factor was gently encapsulated inside two gelled polysaccharide-based matrices elaborated at micro-scale level using a capillary microfluidic method. The release of the growth factor from both systems was followed under in vitro conditions and the bioactivity of the released protein was assessed. It appears from this study that the micro-matrices based on a marine EPS may become promising candidates as growth factor delivery systems.

Acknowledgment: The financial support was provided by RFI Bioregate program in the frame of “μEncapEPS” project.

ENCAPSULATION OF GROWTH FACTORS INSIDE MICRO-MATRICES BASED ON A MARINE EXOPOLYSACCHARIDE FOR TISSUE REGENERATION

POSTER 7

68


Melody Riaud, L. Perdomo-Gilbert, L. Sindji, M. Carmen Martinez and C. Montero-Menei

INSERM U1063 and INSERM U1232, University of Angers, France

Myocardial infarction (MI) is defined by the destruction of the heart muscle due to the obstruction of a coronary artery that supplies the heart with blood and oxygen. Despite the curative approaches developed, cardiac cell death causes dysfunctional contractions that depend on the extent of the ischemic area and the reperfusion time. Cardiac regeneration offers attractive therapeutic possibilities. Extracellular vesicles, including Microparticles (MPs), are carriers of information involved in intercellular communication in many physiological and pathological conditions,that have immense potential especially for regenerative medicine. Previously, we have found that MPs, carrying the Sonic Hedgehog morphogen (MPsShh+), induce capillary-like structures in vitro, neovascularization in vivo and have a cardioprotective effect in a model of ischemia-reperfusion in rats and pigs. In order to limit the spread of MPsShh+ in the body, a consequence of systemic administration, the use of Pharmacology Active Microcarriers (PAMs), a biodegradable and biocompatible 3D biomimetic (fibronectin or vitronectin) support, could help to control and locally increase the activity of MPsShh+. PAMs are capable of delivering previously encapsulated growth factors that may have cardioprotective properties. Thus, our aim is to evaluate the regenerative potential of the therapeutic association of MPsShh+ and PAMs and the additional potential of a controlled delivery of a cardioprotective growth factor. For this study, the microspheres were formulated by a simple emulsion process using either poly (D,L-lactic-co-glycolic acid) (PLGA) or PLGA-poloxamer188-PLGA and were then coated with fibronectin or vitronectin to obtain the PAMs. The MPs were generated by cell culture from a human T cell line with a combined treatment of phytohaemagglutinin, phorbol 12-myristate 13-acetate and actinomycin D, and isolated by serial centrifugation steps. After a period of co-incubation, attachment of MPsShh+ to PAMs will be evaluated. By confocal microscopy, we confirmed that extracellular matrix coating is necessary for MPsShh+ adhesion to microcarriers. PAMs with a fibronectin surface allowed a better adhesion of MPs on their surface compared to PAMs with a vitronectin coating. Further experiments to verify the functionality of the association of MPsShh+-PAMs are in progress. These results suggest that the association of MPsShh+-PAMs could represent an innovative approach in regenerative medicine.

THE ASSOCIATION OF EXTRACELLULAR VeSicLeS And micrOcArrierS AS An INNOVATIVE APPROACH IN CARDIAC REGENERATIVE MEDICINE

POSTER 8

69


Pierre Tournier(1,2), A. Maltezeanu(1), A. Paré(1), A. Barbeito(2), R. Bardonnet(2), A. Gaudin(1,4), V. Geoffroy(1), P. Corre(1,4), J. Guicheux(1,4),* P. Weiss(1,4),* (* Equally contributing authors)

(1) INSERM, UMRS 1229, RMeS Regenerative Medicine and Skeleton, Nantes, France(2) BIOBank SAS, Presles-en-Brie, France(3) SC3M Plateform, Nantes, France(4) CHU Nantes, PHU4 OTONN, Nantes, France

In the maxillo-facial area, sequels of traumatisms, diseases or surgery, often lead to bone defects that fail to self-repair. Whereas the gold standard of bone reconstruction remains the autologous bone graft (ABG), it exhibits some drawbacks (low amount of available tissue, complication at the donor site and lengthening of the surgical time). To overcome these limitations, synthetic bone materials and allo- or xenografts are used. Despite their clinical success, these bone substitutes still remain far from having the osteogenic capacity of ABG. In this context, this work aims at developing a new injectable allogeneic bone substitute for maxillo-facial reconstruction.An injectable allogeneic bone substitute (AlloBS, WO2015162372) has been produced from sifted decellularized cortico-spongious powders (CSP, 0.5mm in diameter) of crushed human femoral heads. After being partially demineralized, the CSP is heated to obtain AlloBS, an injectable scaffold composed of particles consisting in a mineralized core surrounded by demineralized bone matrix, engulfed in a collagen I gelatin.To assess the in vivo ability of AlloBS to support bone repair, we used a guided bone regeneration model in syngeneic Lewis 1A rat calvaria. Briefly, two defects per rat (5mm in diameter, n=6 per condition) were filled with AlloBS or CSP. In a second set of experiment we performed a comparative study with calvarial defects filled with AlloBS or synthetic bone substitute (biphasic calcium phosphate (BCP) granules, 0.5-1mm), associated or not with syngeneic total bone marrow (TBM).After 7 weeks, the percentage of mineral volume (MV) related to total volume (TV) was measured by μCT and histological analyses were conducted using a Movat’s pentachrome staining. Quantitative analysis by μCT revealed a 1.7(AlloBS), 1.8(CSP) fold increase in MV/TV compared with empty defects for the first set of experiments, and 1.9(BCP), 2.1(BCP+TBM), 1.7(AlloBS), 2.4(AlloBS+TBM) in the second set of experiment. Histological analyses confirmed the presence of a mineralized tissue, exhibiting osteoid and a collagen-rich matrix in all the tested conditions.These data show that AlloBS is a promising candidate for maxillo-facial bone reconstruction with substantial osteogenic capacity as well as injectability and ease of manipulation. To assess whether AlloBS may be a relevant clinical alternative to ABG, further experiments in larger animal models of maxillo-facial bone defects are under consideration.

A NOVEL INJECTABLE BONE ALLOGENIC SUBSTITUTE FOR CRANIO-FACIAL SKELETAL REGENERATIVE MEDICINE

POSTER 9

70


Hilel moussi(1,2), S. Quillard(2), H. Terrisse(2), V. Geoffroy(1), T. Rouillon(1), F. Tancret(3), K. Flégeau(1), G. Réthoré(1), P. Weiss(1), J. Le Bideau(2), H. Gautier(1,4)

(1) INSERM, UMRS 1229, RMeS Regenerative Medicine and Skeleton, Nantes, France(2) CNRS UMR 6502, Institut des Matériaux Jean Rouxel, Nantes, France(3) CNRS UMR 6502, Polytech Nantes, Institut des Matériaux Jean Rouxel, Nantes,

France(4) Université de Nantes, Faculté de Pharmacie, Nantes, France

One of the challenges in the clinic application of calcium phosphate cements (CPCs) is to create macroporous degradable, injectable and functional materials. In this approach, the challenge is to incorporate encapsulated human mesenchymal stem cells (hMSCs) in a degradable CPC to functionalize it. The goal is to maintain cells alive as long as possible to secrete bioactive factors for bone repair. The approach used is the syringe-foaming process1, changing silanizedhydroxypropylmethylcellulose (Si-HPMC)2 hydrogel that is poorly resorbable, by silanized hyaluronic acid (Si-HA) hydrogel. The thus obtained injectable cement foams are studied regarding their porosity, phase identification and mechanical behaviour.For the formulation of the cement, α-TCP and Na2HPO4 are mixed to form the cement’s phase of calcium deficient hydroxyapatite (CDHA). α-TCP is synthetized at high temperature (1365°C) followed by very fast tempering. The hydrogel and the NaH2PO4 (with air) solutions are initially sealed in two commercial syringes, being ready for use. Directly after the mix, these two components (CDHA cement and Si-HA hydrogel) are mixed in the syringe and create the foam material.α-TCP is characterized by X-ray diffraction and by Scanning Electronic Microscopy for granulometry.The behaviour of different formulations of Si-HA are compared to Si-HPMC by rheology with the elastic modulus (G’), the maximum strength fracture and the resistance energy. Then the Si-HA is tested in the composite (Si-HA/CDHA) to find the same mechanical properties than Si-HPMC/CDHA. For this, the strength compression, the strength flexion and the young modulus are compared. Finally, porosity and pores’ interconnection of the composite are observed and ionic interactions are investigated. Additionally, the biocompatibility of the different composite formulations is studied.The present study is a new route towards formulation of different and functional hierarchically porous CPCs using silanized polysaccharides as foaming agent. The next step will be to compare the biological behaviours of the different formulations with and without encapsulated cells.1 J. Zhang et al., Acta Biomater., vol. 31, pp. 326 -338, 2016.2 W. Liu et al., Acta Biomater., vol. 9, no. 3, pp. 5740 -5750, 2013.

FORMULATION AND CHARACTERIZATION OF POROUS INJECTABLE CALCIUM PHOSPHATE CEMENT FOAM FOR BONE REPAIR

POSTER 10

71


erwan nicol, T. Nicolai, A. Klymenko, C. Chassenieux, O. Colombani, J. Zhao, T. Narita

Institut des Molécules et Matériaux du Mans, UMR CNRS 6382, Université du Maine, Le Mans, France.

Poly(ethylene glycol) (PEG) is one of the most widely used synthetic polymers for biomedical applications. Functionalized by acrylate or methacrylate groups (PEGDA or PEGDMA), it allows obtaining hydrogels frequently studied for tissue engineering or the delivery of active ingredients. Nevertheless, these hydrogels have low mechanical properties (especially at low concentration) and a small porosity and controlled mainly by the size of the PEG precursor and the concentration.

To overcome the shortcomings of PEG-based hydrogels, we have developed various strategies to lower the percolation threshold of these hydrogels, improve their mechanical properties and have a well controlled porosity. The use of an amphiphilic triblock copolymer (based on PEG) bearing polymerizable functions on the hydrophobic blocks allows the elaboration of self-assembled hydrogels, photo-cross-linkable very quickly under UV or visible irradiation, leading to covalent hydrogels with mechanical properties superior to those of conventional PEGDA hydrogels. The combination of this copolymer with a very similar but non-crosslinkable triblock copolymer leads to dynamic / covalent hybrid hydrogels (after crosslinking) exhibiting higher moduli and deformation at breakage. Finally, the combination of the photo-cross-linkable triblock copolymer with a homopolymer (Dextran or PEG) leads to a phase separation allowing the formation of PEG hydrogels with well controlled porosity. The lack of cytotoxicity of these hydrogels has already been demonstrated. Their cytocompatibility and biocompatibility are under evaluation.

PHOTO-CROSSLINKABLE SELF-ASSEMBLED BIPHASIC HYDROGELS BASED ON POLY(ETHYLENE GLYCOL) (PEG)

POSTER 11

72


Angélique Fourrier(1,2), F. Delbos(2,4), C. Collet(2,4), R. Rispal(2,4), M. Gantier(2,4), D. Atticus(2,4), S. Kilens(1), L. Tolosa Prado(5), Gómez-Lechón MJ(5), I. Anegon(2,4), T. H. Nguyen(1,2).

(1) GoLiver Therapeutics, Nantes, France

(2) INSERM, UMRS 1064-Center for Research in Transplantation and Immunology, France

(3) CHU Hôtel Dieu, Institut de Transplantation Urologie Néphrologie, Nantes, France

(4) Université de Nantes, Faculté de Médecine, Nantes, France

(5) Unidad de Hepatología Experimental, Instituto de Investigacion La Fe, Valencia, Spain

Cell therapy represents an exciting clinical alternative to liver transplantation. In this context, human pluripotent stem cells have been investigated as an unlimited source of transplantable hepatocytes-like cells (HLC). However, the use of HLC produced according to a cGMP protocol to treat a liver disease has not been demonstrated, yet. Here, we report the production of HLCs from human embryonic stem cells (pStemHep) using a xeno-free, feeder-free protocol based on chemically defined culture media and recombinant laminin as an extracellular matrix.

pStemHep were transplanted intrasplenically in a NOD/SCID mice with acetaminophen-induced acute liver failure to investigate their therapeutic potential (APAP mice). Following transplantation of pStemHep in APAP mice, the animal survival rate is significantly higher than for control animals, which underscores pStemHep therapeutic efficacy. We used the level of alanine aminotransferase (ALAT) as a marker of liver lesions and liver metabolic activity. In APAP mice transplanted with pStemHep, ALAT concentration decreased rapidly and significantly in comparison to control untreated APAP mice, and returned to normal within 15 days post-transplantation.

In conclusion, we establish for the first time the efficacy of HLCs using a cGMP cell production protocol for treating acute liver failure. In regards of our results and the unlimited source of stem cells derived hepatocytes-like cells, pStemHep represent a promising therapeutic solution for liver diseases.

TOWARDS A CLINICAL USE OF STEM CELLS DERIVED HEPATOCYTES FOR TREATING ACUTE LIVER FAILURE

POSTER 12

73


E. Viguier(1,2), Gomez N.(1), Fèbre M.(3), Livet V.(1), Plantier N.(3), Pillard P.(1), nathalie Saulnier(3), Cachon T.(1,2), Maddens S.(3)

(1) Small Animal Surgery Department, VetAgro Sup, Marcy L’Etoile, France(2) UPSP 2016A104, ICE, Interaction Cells Environment, Campus Veterinaire VetAgro Sup,

Université de Lyon, Marcy l’Etoile, France(3) Vetbiobank SAS, Marcy L’Etoile, France

Introduction: As in human, prevalence of osteoarthritis (OA) is increasing in canine population, mainly in relationship with the ageing of the population and dog specie is considered as a good model for human OA. First line treatment to manage OA is based on non-steroidal anti-inflammatory drugs (NSAID), however, frequent dosing or long-term NSAID administration may induce severe side effects, such as gastrointestinal disorders. New therapeutic approach, such as cell therapy with Mesenchymal Stem/stromal Cell (MSCs), is gaining increasing interest in the field of orthopedics for the treatment of chronic inflammatory diseases such as OA. MSCs, through their interaction with cellular microenvironment and through a secretion of a wide array of bioactive molecules may reduce inflammation and limit tissue degradation. The objective of this study was to evaluate the effects of a single IA injection of allogeneic canine neonatal MSCs in dogs suffering from severe naturally-occurring OA.Material & methods: MSCs were prescribed as a compassionate treatment in an open-label, uncontrolled study after other therapeutic approaches being excluded. Following initial examination, dogs enrolled in this study were injected intraarticularly with 10E7 neonatal MSCs, in 1 or 2 joints independently. Outcome measures were veterinary clinical evaluation by scoring at 1, 3, and 6 months post-injection, along with a global owner assessment of their dog’s wellness up to 2 years post-treatment. Furthermore, to complete the understanding of the safety profile and biological response to neonatal allogeneic MSC administration, we evaluated a potential humoral response towards cellular product by measuring the presence in the recipients of alloantibodies against MSC antigens or product contaminant like FCS.Results: Twenty-two client-owned dogs were recruited in the study among whose sixteen completed the 6-months study. Three dogs were dropped out from the study because of medical reasons not in relationship with MSC injection, and 3 dogs were withdrawn because of non-compliance of the follow up visits. No systemic adverse effect was observed after MSCs injection during the entire course of the study. Of the 16 patients included, 10 dogs were administrated with a single injection of MSCs in one joint, whereas 6 animals received 1 injection in 2 joints, 6 months apart. For each individual joint, clinical evaluation showed a significant improvement of the clinical score from baseline up to 6 months post-treatment. Long-term safety results collected from a survey completed by 13 owners reported no MSC-related adverse event during the two-year follow up. No MSC-antibodies were detected in dog’s serum following treatment.Discussion: This study provides additional evidence for the long-lasting effect of allogeneic MSC therapy (up to 6 months) in alleviating pain and lameness in dogs with OA and a stabilization of OA progression over 2 years. The absence of detection of alloantibodies following IA injection of neonatal allogeneic MSCs suggests that this therapeutic approach is well-tolerated and could be repeated if required.

SAFETY AND CLINICAL EFFICACY OF A SINGLE INTRA-ARTICULAR INJECTION OF ALLOGENEIC NEONATAL MESENCHYMAL STEM CELLS FOR THE TREATMENT OF OSTEOARTHRITIS IN DOGS

POSTER 13

74


M. Roudaut(1, 2), A. Caillaud(1), A. Thédrez(1), Wieneke Dijk(1), A. Girardeau(1), M. Pichelin(1), L. Arnaud(1), M. Croyal(1, 3), C. Le May(1), N. Maubon(2), B. Cariou(1), Karim Si-Tayeb(1)

(1) Institut du thorax, INSERM UMR 1087 / CNRS UMR 6291, IRS-UN, CHU Nantes, Nantes, France

(2) HCS Pharma, Lille, France

(3) Plateforme de Spectrométrie de Masse, CRNHO, Inra UMR 1280, Nantes, France

We previously showed that human pluripotent stem cells (hiPSCs) provide a suitable model to study metabolic diseases upon hepatocyte-like cell (HLC) differentiation. In particular, HLCs have been used to model cholesterol metabolism regulation, by mimicking the main disease features in vitro. Human iPSCs can be generated from urine samples of patients with a well-described phenotype and carrying specific genotypes. This non-invasive approach allowed the study of LDLR- and PCSK9-mediated autosomal dominant hypercholesterolemia (ADH) as well as PCSK9-mediated familial hypobetalipoproteinemia (FHBL). While the direct link between hiPSCs and patients, as well as the abundance of HLCs provide promising advantages of such strategy, it is impaired mainly by the neonatal characteristic of HLCs as well as the difficulty to perform high throughput studies for pharmacological investigations.

Therefore, to overcome these burdens, we choose to 1. Differentiate hiPSCs into HLCs in a 3D environment instead of the classical 2D culture systems to enhance their maturation; 2. Adapt our 3D differentiation process to a 96 wells format to make it compatible for drug screening.

To reach our goals, we established a partnership with HCS Pharma, which has an expertise in high content phenotypic screening and produces an innovative 3D scaffold, Biomimesys (TM). This scaffold is composed of hyaluronic acid that can be functionalized with extra cellular matric derivatives, with adjustable stiffness and porosity. We setup conditions for hiPSCs seeding and differentiation to reach a new protocol adapted to a 3D environment. Our preliminary data indicate that our procedure enhanced expression of hepatic markers such as transcription factors (FOXA2, FOXA3, HNF1a, HNF1b, HNF4a), cytochrome P450 (CYP450) family members (CYP3A4, CYP2A6, CYP7A1) or cholesterol metabolism regulators (PCSK9, Lipoprotein(a)). During our presentation, we will discuss our data on gene expression throughout hiPSCs differentiation in 3D, CYP450 activities and induction, as well as response to statin or insulin treatments.

A STRATEGY TO ENHANCE FUNCTIONALITIES OF HUMAN INDUCED PLURIPOTENT STEM CELLS-DERIVED HEPATOCYTES AND ADAPT THEM FOR HIGH CONTENT PHENOTYPIC SCREENING

POSTER 14

75


Julien Pichon(1,2)*, L. Lagalice(1)*, J. Deniaud(1), M. Ledevin(1), C. Babarit(1), V. Maurier(1), L. Dubreil(1), T. Larcher(1), E. Ayuso(2), C. Ciron(1)*, K. Rouger(1)*, and M.-A. Colle(1)* (* Authors contributed equally to this work)

(1) PAnTher, INRA, Ecole Nationale Vétérinaire, Agro-alimentaire et de l’alimentation Nantes-Atlantique (Oniris), Université Bretagne Loire (UBL), Nantes, France

(2) INSERM UMR 1089, University of Nantes, Centre Hospitalier Universitaire, Nantes, France

Pompe disease (PD) is a lysosomal storage disorder caused by the mutation of acid α-glucosidase (GAA), the unique hydrolase degrading glycogen in glucose in lysosome. A massive glycogen overload is described in Pompe patient, mainly in skeletal and cardiac muscle. In addition to accumulation of glycogen-filled lysosomes, a severe impairment of autophagic flux highlighted by autophagic buildup leads to a disruption of the contractile apparatus. Currently, no treatment allows to cure efficiently and durably skeletal muscle in PD. We identified the transcription factor Forkhead box O3 (FoxO3a) as a potential target to alleviate skeletal muscle impairments through its key role on regulation of autophagy-lysosomal pathway and glycogen homeostasis.

The aim of our study was to analyze in-depth i) the pathophysiology of the skeletal muscle with the disease course and ii) the consequences of FoxO3a modulation, to design a new therapeutic approach. First, we realized an exhaustive longitudinal histopathological characterization of skeletal muscles of the murine model of PD to better understand the natural course of the disease. Then, we induced the overexpression of FoxO3a in newborn Pompe mice using a single intravenous administration of an AAV2/9-FoxO3a. All mice were sacrificed at 4 months. Glycogen overload was analyzed using periodic acid-Schiff (PAS) stain and biochemical assay. Autophagic buildup characterization was performed using LC3 and p62, markers of autophagosome and disruption of the autophagic flux, respectively.

We described a progressive establishment of autophagic buildup with the course of the disease in skeletal muscle with a severe accumulation of glycogen. Small-sized muscle fibers are also significantly increased in Pompe mice. Overexpressing FoxO3a, we showed a significative prevention of glycogen overload in treated mice. Furthermore, we exhibited a decrease of the number and the size of autophagic buildup in treated mice. Moreover, p62 accumulation appeared reduced in male treated mice, suggesting a restoration of a functional autophagic flux. Finally, we observed a decrease of small-sized muscle fibers, highlighting the prevention of atrophic phenomenon.

We realized here the proof of concept of the key role of FoxO3a in the prevention of skeletal muscle impairments. Our results suggest that FoxO3a modulation could be used as a therapeutic target to alleviate both glycogen overload and autophagic buildup in PD.

PROTECTIVE ROLE OF FOXO3A IN MUSCLE AGAINST BOTH GLYCOGEN OVERLOAD AND AUTOPHAGIC BUILDUP IN POMPE DISEASE

POSTER 15

76


Franck Zal

K. Chathoth(1), B. Martin(1), E. Saucisse(1), M. Bonnaure-Mallet(1), F. Zal(2), E. Leize-Zal(2), C. Baysse(1)

(1) Université Rennes 1 - Institut NuMeCan - Unité Inserm 1241, Rennes, France

(2) HEMARINA SA, Morlaix, France

Objectives: Periodontitis is characterized by the damage of periodontal tissues causing periodontal pockets and bone loss, leading to tooth loss. The evolution of the disease is linked to a modification of the oral microbiota. The pathogens involved are strictly anaerobic germs, therefore there are potentially sensitive to oxygen. Thus, oxygenation of the oral biofilm by Arenicola marina extracellular hemoglobin (M101), should reduce the number of oxygen-sensitive periodontal pathogens, without altering the balance of the oral microbiota. M101 could therefore be used to treat periodontitis.

Methods: A mixed biofilm composed of 2 periodontal pathogens, Porphyromonas gingivalis and Treponema denticola, together with the primary colonizer Streptococcus gordonii, which is needed to initiate the biofilm, was grown for 24 hours and exposed for 1 hour to 2g/l M101 or stabilisation buffer as negative control. The biofilm parameters (biomass, thickness and dead/live ratio) were measured by confocal microscopy after a staining with Syto40 (live cells) and propidium iodide (dead cells). The sessile and planktonic cells (detached cells) were recovered from the samples and numbered by species-specific qPCR.

Results: The exposition to M101 significantly increased the cells detachment from the biofilm as well as the dead/live ratio as compared with stabilisation buffer alone.

Conclusions: M101 has the potential to reduce the biofilm responsible for periodontitis. Further work will investigate the viability of each species inside the biofilm and the effect of M101 on biofilm formation for preventive application.

ARENICOLA MARINA EXTRACELLULAR HEMOGLOBIN: A NEW PROMISSING TREATMENT IN PERIODONTAL DISEASE

POSTER 16

77


Séverine Bezie(1), Vimond N.(1), Daguin V.(1), Belier-Waast F.(2), Duteille F.(2), Levings M.(3), Charreau B.(1), Anegon I.(1), Guilloneau C.(1)

(1) INSERM U1064 ITUN, Nantes, France(2) Chirurgie Plastique et Reconstructrice, CHU de Nantes, France(3) Department of surgery, University of British Colombia, and Child and Family

Research Institute, Vancouver, Canada

Introduction: Regulatory T cell based immunotherapy is a promising treatment to prevent allograft from rejection. We previously demonstrated the therapeutic potential of polyclonal CD8+Tregs in humanized mice transplantation models (Bézie, Front Immunol, 2018). Conferring donor-antigen specificity through chimeric antigen receptor (CAR) increased therapeutic potential of CD4+Tregs in mice models, suggesting lower dose requirement for treating patients and reduced risk of non-specific immunosuppression (MacDonald, JCI, 2017). This study aims to assess the potential of donor-antigen specific CD8+Treg based therapy in transplantation.Materials and Methods: Tregs were sorted on CD8+CD45RCIow expression markers and expanded with anti-CD3/CD28 mAbs in presence of IL-2 and IL-15. HLA-A2-CAR and irrelevant-CAR plasmids were kindly provided by M. Levings to generate lentiviruses. Suppressive function was assessed on CD4+CD25-T cells stimulated by allogeneic APCs, one or the other expressing the HLA-A2 target. NSG mice were infused with HLA-A2+PBMCs for xenogeneic GVHD model, or with HLA-A2-PBMCs and were grafted with HLA-A2+human skin for allograft model.Results: We demonstrated that CD8+Tregs can be efficiently transduced and stably express A2-CAR, and 100-fold expanded in 14 days while maintaining a Tregs phenotype. Specific interaction with HLA-A2+target cells activated A2-CAR CD8+Tregs through zap-70 phosphorylation, upregulating activation markers and suppressive cytokines such as CD69, CD25, IL-34 and IFNgamma. Interestingly, calcium flux imaging revealed different activation kinetics through the CAR in Tregs vs Teffs. Importantly, A2-CAR CD8+Tregs displayed minimal cytotoxic activity toward HLA-A2+ donor kidney graft endothelial cells after specific contact. Finally, A2-CAR CD8+Tregs were more potent than irrelevant-CAR CD8+Tregs to suppress HLA-A2 specific proliferation in vitro, and in vivo to prevent GVHD development induced by HLAA2+PBMCs and to delay HLA-A2+skin graft rejection in humanized mice models.Conclusions: We have provided a proof of concept for clinical application in a MHC-I incompatible transplantation context, that the CAR CD8+Treg-based therapy had high potential to prevent graft from rejection.

CELL THERAPY WITH ENGINEERED HLA A2 SPECIFIC CAR CD8+ TREGS TO AVOID TRANPLANT REJECTION

POSTER 17

78


caroline chariau, A. Gaignerie, Q. Francheteau, A. Derevier, L. David

SFR-Santé, INSERM, CNRS, UNIV Nantes, CHU Nantes, Nantes, France

Somatic cell reprogramming has become a key tool for disease modelling, cell fate regulation studies and drug screening. Diffusion of knowledge depends on specialized iPSC core facilities. The iPSC core faiclity of Nantes was created in 2012 to provide expertise in France. In this poster we will present our R&D.

First, we will show the difference between reprogramming protocols, using 4 donor cell types (urine, PBMC, fibroblasts and dental pulp, and 2 different methods: mRNA and SeV.

Second, we will show the pros and cons of pluripotency validation strategies: in vitro differentiation, DGE-RNA-seq, teratomas, caryotype and SNP analysis.

Finally we will present our strategy to developp genome editing and provide training for differentiatin protocols.

Altogether, this poster will provide an overview of the state of the art of iPSC cells.

IPSC CORE FACILITY OF NANTES: PAST, PRESENT, FUTUR

POSTER 18

79


M. Safoine, C. Paquette, A. Côté, Julie Fradette

(1) Centre de recherche en organogenèse expérimentale de l’Université Laval/LOEX, CHU de Québec Research Center - Université Laval, Québec, QC, Canada

(2) Department of Surgery, Faculty of Medicine, Université Laval, Québec, QC, Canada

Introduction: Chronic wounds that are refractory to usual wound care regimen represent a major complication of diabetes and an important burden on the healthcare system. Cell-based therapies have the potential to enhance tissue regeneration, and they are actively investigated to enhance skin wound repair. Adipose-derived stromal/stem cells (ASCs) are an attractive cell source of mesenchymal cells for tissue engineering strategies considering their abundancy and potent therapeutic secretome.

Objective: Determine if tissue-engineered biological dressings produced from human ASCs in a serum-free system will have beneficial effects on skin healing in a diabetic murine model.

Methods: ASC-based dressings were produced using the self-assembly approach of tissue engineering. The suitability of a commercially available serum-free medium was compared to standard medium containing 10% fetal bovine serum. Functionality of the dressings in vitro was evaluated by ELISA assays to determine the secreted levels of various growth and proangiogenic factors. These dressings were applied to full thickness 8-mm splinted skin wounds created on the back of polygenic diabetic NONcNZO10/LtJ mice.

Results: Global wound closure kinetics evaluated by macroscopic imaging showed that ASC-based dressings accelerated wound closure by 83% at day 8, 57% at day 12 and 35% at day 16 (p<0.001, n=10) compared to untreated wounds which consistently had a 1-week delay during the healing process. On histological sections, treated wounds exhibited regenerated skin of better quality with a more organized, homogeneous and 1.6 fold thicker granulation tissue (p<0.001, n=7). Neovascularization, assessed by CD31+ labeling, was 1.3 fold higher in the treated wounds (p<0.05, n=8).

Conclusion: Our study showed that ASC-based dressings produced under a serum-free culture system exhibit a therapeutic potential promoting in vivo diabetic cutaneous healing. This is mediated, in part, through enhanced angiogenesis and granulation tissue formation. The entire dressing production was realized under a clinically-compatible serum-free system using the self-assembly approach of tissue engineering, leading to natural tissues rich in human cells and matrix components.

ADIPOSE-DERIVED STROMAL CELLS FORMULATED AS TISSUE-ENGINEERED biOLOGicAL dreSSinGS cAn enHAnce MURINE DIABETIC WOUND HEALING

POSTER 19

80


H. Richter, I. Bleeker, D. Ramirez, F. Will, birgitta Stolze

LLS ROWIAK LaserLabSolutions GmbH, Hannover, Germany

Objectives: Preclinical studies in the field of regenerative medicine become more and more important due to regulatory requirements. Cost and time efficiency are an important factor for competitiveness. The histological analysis of implants or device integration often is a mandatory, yet laborious part of the preclinical study design. The preparation of hard tissue samples or samples containing implants is especially challenging and requires specialized and experienced technicians. Routine methods like rotary or sledge microtomes are limited in the variety of samples they can cut. Samples with implants like stented vessels or undecalcified bone samples damage the blades quite fast. This means ground sections must be prepared as an alternative. Unfortunately, their quality varies, due to high section thickness and uneven laboratory skills. Other limitations include low throughput and kerf/grinding specimen loss. Laser microtomy is a novel method for fast and easy preparation of histology sections of non-decalcified hard tissue and tissue containing implants or biomaterials.

Methods: Resin embedded thin sections of high quality have been generated for non-decalcified hard tissue or implanted tissue (e.g. stented vessels) using laser microtomy. A broad range of tissue and materials from the field of orthopedics, cardiovascular medicine, dental and audio medicine has been tested so far, e.g. stented vessels (swine coronaries), human cardiovascular plaques, different types of hard tissue (e.g. cochlea, dog jaw, sheep spine, rat or mouse femur and tibia). Stainings have been applied as indicated in results.

Results: Nearly serial sectioning at 10 μm is possible. The great material loss associated with ground sectioning is virtually eliminated. Beyond, laser microtomy reduces the overall processing time per sample and also the effective time a technician has to work on it. This increases the efficiency of lab procedures which results in 4 to 6 times higher sample throughput compared to ground sectioning. Common histological stains and histochemistry are available for resin embedded sections produced by laser microtomy. We present a variety of routine stainings (e.g. H&E, Masson Goldner, McNeal, van Gieson Elastica), histochemistry and even immunohistochemistry (IHC).

Conclusion: This contribution shows that laser microtomy saves time and material and opens a new range of possibilities in histological analysis of hard tissue and implant evaluation.

ENHANCEMENT OF PRECLINICS IN REGENERATIVE MEDICINE BY LASER BASED HISTOLOGY FOR HARD TISSUE AND IMPLANTS

POSTER 20

81


Clément Toullec(1,2,3), Buchtová N.(1), Grignard B.(2), Terrisse H.(3), Le Bideau J.(3), Jérôme C.(2), Boury F.(1). (1) Centre de Recherche en Cancérologie et Immunologie Nantes-Angers, Université d’Angers, France(2) Centre d’Etude et de Recherche sur les Macromolécules, Université de Liège, Belgique(3) Institut des Matériaux Jean Rouxel de Nantes, Université de Nantes, France

Introduction: The loss of bone tissues because of age, obesity, trauma or diseases can provoke a significant morbidity and an important socio-economical cost. During the last few decades, tissue engineering and regenerative medicine have become promising strategies for rebuilding bones. The tissue engineering of bones has for objective the functional regeneration of bones through a synergetic combination of biomaterial scaffolds, cells and active biomolecules such as proteins, peptides or growth factors1. The objective of our work was to synthesize a copolymer, curdlan-graft-PLGA, in supercritical CO2 without any organic solvent. This copolymer was then used for the formulation of nanoparticles that are intended to be loaded with bioactive molecules for bone regeneration.Material & methods: Starting with cyclic monomers, glycolide and lactide, poly-lactic-co-glycolic acid (PLGA) was obtained through ring-opening polymerization (ROP) in supercritical carbon dioxide (scCO2)2. Curdlan (1,3-β-glucan), a polysaccharide, was used as an initiator. Organic catalysers, including 1,8-diazabicyclo[5.4.0]undec-7-ene and thio-urea, were also used. The purity, composition and molecular mass of the curdlan-graft-PLGA copolymer were determined with liquid NMR. Nanoparticles were prepared through a phase separation method3. The copolymer was dissolved in dimethyl isosorbide (DMI), then an anti-solvent, an aqueous solution of glycine and NaOH, was progressively added in large excess. The average nanoparticle size was determined by dynamic light scattering. The morphology of nanoparticles was studied with scanning and transmission electronic microscopy. Finally, the zeta potential was also measured.Results and Discussion: In this work, we used curdlan both as an initiator for the ROP and as a backbone for the copolymerization with PLGA. Thus, the resulting curdlan-graft-PLGA copolymer is composed of PLGA chains grafted on the curdlan backbone. scCO2 is considered as a “green” solvent because its critical pressure and temperature are relatively low, it is abundant, inert, nontoxic and non-flammable. Therefore, the ROP in scCO2 allows to obtain the curdlan-graft-PLGA copolymer in one step without any toxic solvent. Moreover, the synthesis parameters were optimised to diminish the amount of organocatalysts used. The rate of conversion of this process was determined to be of 98% and the molar mass of the PLGA grafts synthesised of 10 000g.mol-1.Nanoparticles were formed using this copolymer via a method of nanoprecipitation by phase separation. This method uses the difference in hydrophilicity between PLGA and curdlan. Curdlan is more hydrophilic than PLGA, therefore it will be preferentially oriented toward the aqueous phase on the surface of the nanoparticles while the PLGA will find itself in the core. This configuration was confirmed by modifying the quantity of curdlan in the copolymer which leads to a difference in the zeta potential of nanoparticles. The particles were observed to be spherical with a diameter of 300 nm in average and a polydispersity index of 0.2.

DESIGN OF NANOMEDICINES USING « GREEN » PROCESSES FOR APPLICATION IN BONE REGENERATION

POSTER 21

82


S. Maddens(1), R. Rakic(1,2), B. Bourdon(2), N. Plantier(1), M. Demoor(2), nathalie Saulnier(1) and P. Galéra(2)

(1) VETBIOBANK, 69280 Marcy l’Etoile, France

(2) Caen Normandy University, France ; UNICAEN EA7450 BioTARGen (Biologie, Génétique et Thérapies ostéoArticulaires et Respiratoires), Saint-Contest, France

Introduction: Horse is considered as an accurate and relevant specie to investigate human cartilage biology. MSCs ability to differentiate in vitro in cartilage offer the opportunity to create de novo cartilage-like tissue for tissue engineering. Newborn tissues are attractive sources for MSCs, including umbilical cord blood MSCs (UCB-MSCs) and umbilical cord matrix MSCs (UCM-MSCs). Considered as medical waste, their procurement is non-invasive and raise no ethical concerns for allogeneic use, compared to their counterparts isolated from aged adult tissues. UCB- and UCM-MSCs display both chondrogenic potential and represent an attractive alternative source for cartilage tissue engineering. In order to determine which would be the best source of MSCs for ex-vivo cartilage tissue engineering, a detailed comparative analysis of their chondrogenic potential was realized, using a 3-D model.

Material & method: UCB- and UCM-MSCs were isolated from equine tissues collected after foaling. Cells were characterized regarding standard quality criteria (morphology, growth rate, immunophenotype, and multipotency). Chondrogenic differentiation was induced by incubating UCB- or UCM-MSCs with 3D collagen type I/III scaffolds for 28 days, in the presence of BMP-2 and TGF-ß1 in cultures. In addition, for the first time, the influence of hypoxic condition was evaluated on equine neonatal MSC’s lineage commitment. An exhaustive characterization of the extracellular matrix generated by these two types of MSCs was carried out. Gene expression of hyaline cartilage, hypertrophic chondrocytes and osteogenic markers along with western blot analyses were used as a readout.

Results: Our data showed significant differences in the ability of UCB- and UCM-MSCs to differentiate towards the chondrogenic lineage. UCB-MSCs differentiated into chondrocytes and express an abundant, dense and hyaline-like cartilage matrix. By contrast, despite their expression of cartilage markers, UCM-MSCs failed to differentiate fully into chondrocytes. Furthermore, our results suggest that, unlike bone marrow MSCs, low oxygen tension do not improve the chondrogenic differentiation of neonatal MSCs

Conclusion: These results suggest that UCB-MSCs should be preferred when considering ex-vivo equine cartilage tissue engineering. How those results should be translated to in-vivo direct cartilage regeneration remains to be determined through dedicated study.

NEWBORN TISSUES FOR CARTILAGE REGENERATION: A COMPARATIVE ANALYSIS OF THE CHONDROGENIC POTENTIAL OF UMBILICAL CORD MATRIX AND UMBILICAL CORD-DERIVED MESENCHYMAL STEM/STROMAL CELLS (MSC)

POSTER 22

83


eléana SOmViLLe, Anne des RIEUX, S. DEMOUSTIER-CHAMPAGNE, A. M. JONAS, K. GLINEL

Institute of Condensed Matter & Nanosciences (Bio & Soft Matter), Université catholique de Louvain, Louvain-la-Neuve, Belgium

Louvain Drug Research Institute (Advanced Drug Delivery and Biomaterials), Université catholique de Louvain, Brussels, Belgium

Human mesenchymal stem cells (hMSC) are considered for a wide range of healthcare applications and they are currently under investigation in over 300 clinical trials, which reflects their strong potential. [1] They can be used to treat a variety of diseases including myocardial infarction and liver cirrhosis, or in combination with a scaffold, to regenerate damaged organs such as bones and muscles. [2] However, several challenges remain for the complete development of cell therapies. One major limitation is the large number of cells required to treat a single patient, which can reach up to 5 million cells per kg of patient. [2] Thus, rapid and scalable cell expansion methods are required to meet the clinical demands. The objective of this work was to develop new porous polymeric microcarriers with a bioactive surface for stem cells expansion. These microcarriers (MCs) are made by the controlled spherulitic crystallization of biodegradable poly(L-lactide) (PLLA) and are obtained via an organic solvent-free method. MCs porosity and size can be tuned by adjusting the process parameters. To facilitate the separation of MC from cells at the end of the culture, magnetic MCs were prepared by incorporating supraparamagnetic nanoparticles (SPIO) in the MCs during the elaboration process. To maximize cell adhesion on MCs, a bioadhesive coating was deposited onto the MC surface. It consisted of a crosslinked polyelectrolyte multilayer (Layer-by-layer poly-ornithin/hyaluronic acid) on with was grafted a bioadhesive peptide. Our first short term culture assays showed that RGD-MCs exhibited an enhanced MSC adhesion compared to unmodified carriers, which bodes well for the envisioned application. Further optimization of cell cultures conditions and impact on cell behavior are planned for the next experiments.

[1] T. Lawson et al. “Process development for expansion of human mesenchymal stromal cells in a 50 L single-use stirred tank bioreactor”. Biochemical Engineering Journal 120 (2017) 49–62.

[2] F. dos Santos et al. “Toward a Clinical-Grade Expansion of Mesenchymal Stem Cells from Human Sources: A Microcarrier-Based Culture System Under Xeno-Free Conditions”. Tissue Engineering: Part C, 17 (2011) 1201-1210.

NEW MAGNETIC PLA POROUS MICROCARRIERS FOR MSC EXPANSION IN THE SCOPE OF CELL THERAPY

POSTER 23

84


Gaëtan Le bel(1,2), F. Bisson(2), S. Guérin(1) and L. Germain(1,2)

(1) Département d’ophtalmologie, Université Laval, CUO-recherche/LOEX, Centre de recherche du CHU de Québec - Université Laval, Québec, QC, Canada

(2) Département de chirurgie, Université Laval, Centre de recherche en organogénèse expérimentale de l’Université Laval/LOEX, Centre de recherche du CHU de Québec - Université Laval, Québec, QC, Canada.

Purpose: Because of the worldwide shortage of graftable corneas that are to be used to restore visual impairments, alternative solutions, such as the production of a functional human cornea by tissue engineering, have emerged. Self-renewal of the corneal epithelium through the maintenance of a sub-population of corneal stem cells is required to maintain the functionality of such a reconstructed cornea. We previously reported an association between stem cell differentiation and the level to which they express the transcription factors Sp1 and NFI. Our goal is to characterize the impact of different feeder layers on the proliferative properties of human cornea epithelial cells (HCECs) in monolayer cultures. Methods: HCECs were isolated from the corneal limbus of human donor’s eyes and grown with either irradiated murine (i3T3) or human (iHFL) fibroblasts as feeder layers. The cells were amplified on several passages until they reached replicative senescence. The expression and DNA binding properties of Sp1 and NFI were determined at each passage by microarray, Western Blot and gel shift analyses. Influences of feeder layers on Sp1 and NFI half-life were analysed by immunoprecipitation and by treatment of HCECs with cycloheximide. HCECs co-cultured with both feeder layers were seeded on tissue-engineered human corneal stroma to allow regeneration of the corneal epithelium. After 7 days of treatment with bromodesoxyuridina (BrdU), the impact of the feeder layer on the maintenance and localization of corneal stem cells was assessed at various times (1, 7, 14 and 21 days) by indirect immunofluorescence targeting of BrdU and keratin 19 (K19). Results: HCECs co-cultured with iHFL were maintained for two additional passages in culture relative to HCECs grown with i3T3. Expression and DNA binding of NFI were increased at each subsequent passages when co-cultured along with i3T3. These changes correlated with increased expression of the NFIA and NFIB isoforms that are less important in HCECs grown with iHFL. In both conditions, the positive BrdU and K19 labeling demonstrated the presence of HCECs stem cells in the basal layer of the stratified epithelium of the human reconstructed cornea. Conclusions: Our results suggest that the iHFL feeder layer is the most effective for maintaining the proliferative abilities of HCECs in culture. These cells are able to regenerate human corneal epithelium with a subpopulation of stem cells located in its basal layer.

CONTRIBUTION OF THE FEEDER LAYER TYPE TO THE PRESERVATION OF STEM CELLS IN CULTURED HUMAN CORNEAL EPITHELIAL CELLS

POSTER 24

85


A. Asplund(1), Alex Abadie(1), B. Ulfenborg(2), J. Synnergren(2), B. Küppers-Munther(1)

(1) Takara Bio Europe AB, Gothenburg, Sweden

(2) Systems Biology Research Center, School of Bioscience, University of Skövde, Skövde, Sweden

The liver plays an essential role in metabolic disorders and progression of multiple diseases. Therefore, a physiologically relevant in vitro hepatocyte model with low batch-to-batch variation, conserved genetic inter-individual variations and of unlimited supply is needed. Human pluripotent stem (hPS) cell-derived hepatocytes have a great potential to become a future model for numerous hepatocyte applications if they possess relevant functionality and usage window. Our new version of highly homogenous cryopreserved hPS cellderived hepatocytes with a novel maintenance medium shows multiple mature liver features. They secrete albumin and urea during a 2-weeks window and show mRNA expression levels of albumin and urea cycle enzymes that are comparable to those of human primary hepatocytes (hphep). In addition, genes involved in both glucose and lipid metabolism are expressed on the same levels as in hphep.

The hPS cell-derived hepatocytes respond to insulin by phosphorylation of AKT and show capacity to take up low-density lipoproteins and become steatotic if incubated with fatty acids. Moreover, activity of cytochrome P450 enzymes and expression of genes essential for the drug metabolizing machinery are stably detected during a 2-weeks window and on similar levels as in hphep. The novel maintenance medium developed for hPS cell-derived hepatocytes was tested, with minor modifi cations, on hphep in order to see if similar improvements in long-term viability and functionality could be achieved. Surprisingly, the novel culture medium allowed maintained morphology, viability and functionality of hphep for 4 weeks post-thawing and, thus, could prevent the typically observed rapid loss in functionality and cell viability of hphep in conventional 2D cultures.

This is in sharp contrast to existing hepatocyte maintenance media. Both the novel generation of hPS cell-derived hepatocytes with mature hepatocyte functions and the new maintenance medium for hphep will empower the usage of hepatocytes in the area of metabolic diseases.

NOVEL HUMAN IPSC-DERIVED HEPATOCYTES WITH ADVANCED FUNCTIONALITY AND LONG-TERM 2D CULTURES OF HUMAN PRIMARY HEPATOCYTES FOR METABOLIC DISEASE STUDIES

POSTER 25

86


Lousineh Arakelian(1,2), A. Parouchev(1,2), I. Ben Mosbah(3), R. LI(3), C. Chesne(3), J. Larghero(1,2), V. Vanneaux(1,2)

(1) Cell Therapy Unit, AP-HP, Hopital St Louis, Paris, France

(2) INSERM, CIC-BT and UMR U1160, Institut Universitaire d’Hernatologie, Hôpital St Louis, Paris, France

(3) Biopredic International, Saint-Gregoire, France

Liver failure is one of the main causes of mortality in the world and represents a serious burden for global health system. Currently, the only curative treatment for end stage liver diseases is liver transplantation. However, the number of donors being limited, most of the patients are not able to receive one. Liver is a unique organ in its capacity to regenerate spontaneously, but isolated primary human hepatocytes cultured in-vitro in 2D conditions lose their polarity, their functionality and their capacity to proliferate. Therefore, in order to overcome these barriers, new culture methods are necessary to increase the functionality of hepatocytes. Cell sheet technology consists of culturing cells on a thermoresponsive polymer, the Poly (N-isopropylacrylamide) (pNIPAM), which upon temperature modification, changes its hydrophobicity and allows cell detachment as cell sheets.

Unlike enzymatic treatments, cell sheet technology allows to preserve the extracellular matrix secreted by the cells, as well as the cell polarization. It is therefore an interesting technology to apply to liver tissue engineering. Moreover, this technology could prevent cell death of hepatocytes by anoikis and increase their functionality. The purpose of this study was to prepare cell sheets with HepaRG cells as a hepatic model and to stack them with either other HepaRG cell sheets or endothelial cell sheets. We showed the feasibility of this method by cultivating HepaRG cell line in UpCell thermoresponsive dishes and showed that these cells could be successfully detached and transferred into new non-thermoresponsive dishes coated with collagen type I. Cell sheet stacking with endothelial cell sheets or other HepaRG cell sheets was achieved by addition of components of the extracellular matrix. We further showed that these cell sheets are viable after transfer and can be kept in culture for a long period. They preserve their morphology and functionality, as well as their capacity to secret albumin. Based on these encouraging results, we are currently working on optimizing the stacking methods and to transpose this technology onto primary human hepatocytes.

LIVER TISSUE ENGINEERING USING CELL SHEET TECHNOLOGY

POSTER 26

87


C. Diaz(1)*, C. Hayward(1)*, C. Paquette(1), J. Langevin(2), J. Galarneau(2), L. Archambault(3), N. Pollock(4), Julie Fradette(1)

*co-first authors (1) Centre de recherche en organogénèse expérimentale de l’Université Laval / LOEX, CHU

de Québec Research Center - Université Laval and Department of Surgery, Faculty of Medicine, Université Laval, Québec, QC, Canada

(2) Department of radio-oncology, Cégep de Ste-Foy, Québec, QC, Canada (3) Department of Physics, Université Laval / Centre de Recherche sur le Cancer, Université

Laval / Centre de recherche du CHU de Québec, Québec, QC, Canada(4) Service de médecine hyperbare, Hôtel-Dieu de Lévis, Lévis, QC, / Department of

Kinesiology, Université Laval, Québec, QC, Canada

Radiotherapy for cancer treatment can lead to incapacitating hard-to-heal skin wounds. Such wounds can benefit from hyperbaric oxygen therapy (HBOT), although this requires daily sessions over several weeks. We have engineered biological dressings from human adipose-derived stem cells (ASCs) that secrete high levels of prohealing factors. Our goal is to develop a murine model recreating skin lesions following local irradiation in order to assess the therapeutic outcomes of various treatments. Our hypothesis is that serial HBOT sessions combined with repeated topical application of biological dressings engineered using ASCs would accelerate healing in a murine model of excisional wounds on irradiated tissues. Skin alterations were noticeable 3 to 5 weeks after the delivery of a single-dose of 45 Grays (Gy) to dorsal skin (1 cm2) of CD-1 mice. This included a thickening of the epidermis (5.6 fold) with increased epidermal Ki-67 expression and increased dermal α-smooth muscle actin (α-SMA) protein expression (5.4 fold), indicative of increased myofibroblast content. Doses of 20 or 30 Gy did not result in visible skin lesions or increased epidermal thickness. Full-thickness splinted excisional wounds (8 mm) were then created in irradiated and non-irradiated areas. Global wound closure evaluation from macroscopic images revealed that wounds in irradiated tissues featured delayed healing (60% vs 90% healed surface area, P<0.007). Our preliminary data suggest that a course of HBOT (2 x 45 min daily, 5 days a week, for 4 weeks) could potentially promote wound closure in irradiated tissues, whether administered alone (79% vs 60%, P<0.06) or combined with ASCs dressings (85% vs 60%, P<0.0083). Detailed characterization will determine the impact of HBOT and stem-cell dressing treatments on scar quality (neoepidermis, granulation tissue, neovascularization). For example, irradiation induced a thichkening of the epidermis, which was 1.6 fold lower for the animals treated with HBOT combined with ASCs dressings. Upon completion, these preclinical studies will provide an evidence-based evaluation of the efficacy of two treatments, separately and in combination, to improve healing of incapacitating skin wounds, to speed recovery and return patients to a higher quality of life.

IMPACT OF TISSUE-ENGINEERED DRESSINGS AND HYPERBARIC OXYGEN THERAPY ON WOUND HEALING USING A SKIN IRRADIATION MODEL

POSTER 27

88


Sébastien boni, G. Podevin

Plateforme LentiVec, SFR ICAT, Université d’Angers, France

Objectives : Lentiviruses, RNA viruses from the retrovirus family, are able to integrate genoma of infected cell in order to express their own genes. By deleting the wild pathogenic viral sequences and replacing them with genes of interest, the constrcuction of retroviral vectors, beyond gene therapy trials, provide to researchers tools defective for replication, integrative, which expresses constituvely the DNA sequences of interest into target cells, in the same way as any cellular gene, in vitro and in vivo. Our goal has been to set up academic facilities in order to provides «à façon» retroviral vectors to researchers.

Methods: Our academic facilities, thanks to its experience and equipement in molecular biology, bacteriology and cell biology, provides to researchers services ranging from assistance to project conceptualization to production of high-titer, high-quality lentiviral vectors free of any contaminant. A quality approach has been established for all steps of manufacturing process, validated by the Biogenoust network.

Results: For the past 3 years, we have provided many services, mainly in the west but also in other French regions and in Germany, to academic teams, and for 2 projects to biotechnology companies. The projects were diversified, ranging as example from the tracking of intracellular metabolic processes to the gene editing (CRIPR Cas 9 system), via the blocking of immune reaction through miRNA expression, the selection of enteric neurons in intestinal tissue, or the expression enhancement of messenger RNAs. We can imagine other applications such as the creation of transgenic animals or the production of specific proteins.

Conclusion: Lentiviral vectors are tools for solving many research problems in biology and regenerative medicine. For researchers, the availability of a dedicated platform, combining technical advices and quality approach, improves the efficiency in producing scientific results. Its academic structure allows to provide services at cost price.

LENTIVIRAL VECTORS: THE IDEAL TOOL TO WRITE INTO THE GENOMA OF ALiVe ceLL

POSTER 28

89


maylis Farno(1,2), G. Conzatti(1), R. Bushkalova(1,2), M. Castel-Molières(1), C. Nadal(1), S. Cavalie(1), C. Recoche(1), J. Torrisani(3), S. Girod-Fullana(1), A. Tourrette-Diallo

(1) Université Paul Sabatier, CIRIMAT Institut Carnot Chimie Balard Cirimat, Faculté de Pharmacie, Toulouse, France

(2) Université Paul Sabatier, I2MC, Toulouse, France

(3) RCT, UMR1037 INSERM/Université Toulouse III-Paul Sabatier, Toulouse, France

Summary: Alginate and chitosan are both polymers of choice for biomedical applications. As natural polymers they provide high biocompatibility, low rejection rate from the body and good biodegradability. Thanks to their inherent properties they can be used on their own or mixed together to form polyelectrolyte complexes of opposite charge (PEC). PPB team developed several polysaccharide-based biomaterials for different biomedical applications such as surgical applications and cell therapy. The first system consists of thermosensitive PEC films which were elaborated in order to obtain a thermocontrolled bioadhesion for wound dressing applications. The second system is a polymeric porous scaffold designed for bone marrow mesenchymal stem cells (MSCs) culture. The success of these therapeutic strategies lies on the scaffold’s design, as its biocompatibility and architecture influence host’s response. The main challenges were to control both scaffolds’ structure and porosity according to their aimed application. Here we show how alginate/chitosan ratio as well as the drying and homogenization techniques impact scaffold physicochemical properties. The physicochemical characterizations of these systems will be presented, as well as their in vitro evaluation for cell viability or cellular bioadhesion.

ALGINATE - CHITOSAN SCAFFOLDS FOR SURGICAL APPLICATIONS AND CELL THERAPY

POSTER 29

90


caroline ceccaldis, E. Leroux, A. Guerry

Bioxis Pharmaceuticals

Natural polysaccharides are particularly interesting for the design of dermal fillers since they represent structural analogues of tissues. Currently, crosslinked-hyaluronic acid represents over 80% of dermal fillers available on the market. Despite their demonstrated safety, it is still necessary to improve their long term efficacy. However, increasing their mechanical properties and resorption time while maintaining a reasonable injectability is still challenging (De Boulle, 2013).

Bioxis research team proposed a chitosan-based formulation called MTl-12 that has pH-sensitive properties. MTl-12 is liquid in syringes and able to gelify after injection to recreate volumes. It has been designed to have a slow degradation rate associated with tissue induction properties for a long term efficacy. The goal of this work is to characterize physicochemical and biological properties of MTl-12.

MTl-12 was prepared according to the patented formulation and process (Dupasquier, 2011 ; Guerry, 2015). Its pH, osmolality, viscosity and injectability were measured with a potentiometer, osmometer, rheometer and by compression tests respectively. To evaluate the kinetic of gelation, 0.5 ml was sub-cutaneously injected in rats and explants were macroscopically observed after 1, 2 and 5 hrs post-injection. The biocompatibility of MTl-12 was evaluated following the 150-10993 and tissue induction was quantified after immunostaining of collagen I and Ill content during 52 weeks.

MTl-12 batches presented a pH, an osmolarity, a viscosity and an extrusion force comprised between 5.5 and 6.8, 270-360 mOsm/L, 30-300 Pa.s and 10-25 N (with 27G½”needle) respectively. After sub-cutaneous injection in rats, MTl-12 permitted to rapidly create well defined and malleable volumes. The time of gelation occurred between 1 and 2 h after injection. MTl-12 was well integrated to surrounding tissues and no migration was observed. Histological analyses showed a rapid increase of coll-Ill/coll-I ratio followed by a stabilization until 52 weeks. After 52 weeks of implantation, the MTl-12 coherence declines and presented cracks and fragments showing the beginning of degradation.

MTl-12 is the first chitosan-based dermal filler. Its innovative formulation allows an easy injectablility associated with a rapid volume creation. Its slow kinetic of degradation as well as its ability to induce in situ collagen production is a promise of a long lasting effect based on the regenerative properties of chitosan.

DESIGN AND EVALUATION OF A NEW CHITOSAN-BASED DERMAL FILLER

POSTER 30

bIosKeTCHesof the invited speakers

91

92

dr ross macdonald is the Chief Executive Officer and Managing Director of Cynata Therapeutics Limited. He has 30 years’ experience and a track record of success in pharmaceutical and biotechnology businesses. His career history includes positions as

Vice President of Business Development for Sinclair Pharmaceuticals Ltd (now Sinclair Pharma PLC), a UK-based specialty pharmaceuticals company and Vice President, Corporate Development for Stiefel Laboratories Inc, the largest independent dermatology company in the world and acquired by GlaxoSmithKline in 2009 for £2.25b. He has also served as CEO of Living Cell Technologies Ltd, Vice President of Business Development of Connetics Corporation and Vice President of Research and Development of F H Faulding & Co Ltd. He also has extensive business experience in Europe, USA and Asia.

dr cécile martinat is at the head of the INSERM UEVE UMR 861 unit located in I-STEM, an institute fully dedicated to the development of innovative treatments for monogenic disorders by using human pluripotent stem cells. Two main applications are developed in I-STEM: cell therapy and drug screening. After a PhD on virology at Pasteur Institute, It is during her post doctoral fellowship at Columbia University that C. Martinat started to be interested in the therapeutic potential offered by human pluripotent stem cells.

93

nadia Lounnas-mourey (Senior Scientist Process Development at TxCell, a Sangamo company, Valbonne, France). Nadia is a senior scientist combining 14 years of experience in molecular and cellular biology.

She joined TxCell in 2014 is now responsible for the Company’s production platform for its genetically-modified regulatory T cells (CAR-Treg cells). Nadia has been instrumental in developing the industry’s very first CAR-Treg Good Manufacturing Practice (GMP) process ready for clinical testing. Her team is notably in charge of the isolation and purification of CAR-Treg cells, as well as the final freezing/thawing step.

Before joining TxCell, Nadia was a postdoctoral researcher at the Institut de Biologie Valrose in Nice, France, where she studied the signaling pathways used by healthy and cancer cells to survive, with the objective of identifying new therapeutic targets in oncology. Nadia holds a PhD in molecular and cellular biology from the University of Nice in France. During her PhD, her work included targeting death and survival signaling pathways in hematologic malignancies.

Dr Kimberly Homan is a Research Associate at the Wyss Institute for Biologically Inspired Engineering at Harvard. She started her scientific career at the University of Arizona where she earned a chemical engineering degree. She then took a break from science and was commissioned an officer in the United States Marine Corps where she served for 6 years. After her service, she attended the University of Texas at Austin where she earned a Ph.D. in biomedical engineering. While in Austin, Texas she started a company based on the biomedical imaging contrast agents she developed in her graduate work. Her company, NanoHybrids Inc, is still based in Austin, and has brought several lines of unique gold nanoparticles to market. Kimberly now applies her biomedical engineering expertise to tissue engineering and has worked for the last 5 years to use 3D bioprinting to create functional living tissues. In the laboratory of Jennifer Lewis at Harvard University in Wyss and co-advised by Annie Moisan at Roche, she builds kidney tissue on perfusable chips that can be used for drug screening, mechanistic safety, and ultimately, regenerative medicine.

94

dr Fabien Guillemot is the Poietis Founder and CEO. Fabien has an over 20 years experience in the field of Regenerative Medicine.He holds a PhD in Material Science from the National Institute of Applied Sciences Rennes (2000) and an Habilitation in Health and Life Sciences from Bordeaux University (2010). Fabien was appointed Researcher at INSERM in 2005 and was also Invited Researcher at Harvard University in 2010.He initiated pioneering work in the field of bioprinting and has published over 20 scientific articles and more than 100 invited lectures on this new technology. He also completed its scientific training by an entrepreneurship program provided at HEC, Paris in 2012-2013.

Pr Abhay Pandit is currently the Scientific Director of the Centre for Research in Medical Devices (CÚRAM), and an Established Professor of Biomaterials at the National University of Ireland, Galway. CÚRAM is a multi-disciplinary academic-industry-clinician translational research centre coordinated by the National University of Ireland, Galway. The centre is developing innovative and transformative device-based solutions to treat global chronic diseases. The centre supports industry from basic scientific research, through translational preclinical and clinical development, into regulatory and commercial readiness. The group has designed and developed functionalised biomaterial platform technologies with inbuilt gradients of physical, chemotropic and neuroprotective cues to target injury mechanisms at the molecular and cellular levels. These multi-modal biomaterial scaffolds, facilitate the spatiotemporal delivery of multiple biomolecules targeting different phases (inflammatory, neuroprotective, and angiogenic phases) of the underlying disease pathology resulting in an enhanced therapeutic response. These platforms have been designed to address unmet clinical needs for cardiovascular, musculoskeletal, soft tissue and neural targets and have been validated in pre-clinical models. Phase 1 clinical trials planned to streamline clinical translation.

Marc Thurner is CEO and founder of regenHU Ltd. Active in the advancement of biofabrication for over 15 years he is a leading contributor to the 3D bioprinting and tissue engineering scene.

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Pr Christine Jérôme is full Professor at the University of Liege and director of the Center for Education and Research on Macromolecules. She earned a chemical science degree from Liege University and performed postdoctoral research on the synthesis of magnetic nanoparticles at the University of Ulm under the auspice of an Alexander Von Humboldt grant. She has expertise in polymer chemistry and passion in developing biomaterials to improve health and well-being. She develops green strategies for the synthesis, functionalization and processing of polymer materials, degradable or not, to precisely tailor their properties and design supramolecular structures customised to the targeted biomedical application and needs.

estelle collin is 3D Printing New Concepts Lead at Gecko Biomedical in Paris, France. She started her scientific career at the University of Sciences and Technology (USTL) of Lille where she graduated with a Master degree in Molecular and Cellular Biology. She then graduated with a PhD degree in biomedical engineering with a specialization in tissue engineering specifically on gene and cell therapy for †he intervertebral disc at the National University of Ireland Galway under the supervision of Prof Abhay Pandit. She joined Prof Alvaro Mata’s team as postdoctoral research in Queen Mary University of London working on self-assembled 3D microenvironments for in vitro testing and tissue regeneration. She has joined in 2017 Gecko Biomedical a start-up founded in 2013 in Paris developing synthetic polymers for tissue regeneration from sealants and adhesive to scaffolding using 3D printing technology. The polymers developed in this start-up are based on a technology developed in the labs of Profs Bob Langer and Jeff Karp at MIT, USA. Their first product, a vascular sealant as adjunct to suture, was CE marked in 2017. Since her arrival to Gecko Biomedical, she has taken the position of 3D printing new concepts lead working on the development of 3D printed scaffolds for tissue regeneration.

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dr Knut Stieger (Justus-Liebig-University Giessen, Germany)

Dr. Stieger has more than 15 years of experience in the development of gene therapeutic applications for inherited retinal disorders. His expertise ranges from basic scientific approaches to vectorology and pathology of retinal diseases, the use of animal models to preclinical and clinical application of AAV based gene therapies.

He started his career as PhD student in the INSERM laboratory “laboratoire de thérapie génique” in Nantes under the supervision of Dr. Fabienne Rolling, where he was involved in the development of AAV based gene therapy for the large animal dog model of RPE65 deficiency as well as in studies on the regulated expression of reporter genes in the retina of non-human primates.

In 2007, he moved to Giessen to join the group of Dr. Birgit Lorenz, who is an internationally renowned expert in inherited retinal dystrophies and who follows a large number of patients with all kinds of retinal disorders. He focus moved to therapeutic genome editing applications using the CRISPR-Cas9 system, TALEN and homing endonucleases. In 2013, he received an ERC starting grant from the European Union to study genome editing in the retina and DNA repair mechanisms in post-mitotic cells.

Currently, he is Professor of Experimental Ophthalmology at the Department of Ophthalmology at Justus-Liebig-University Giessen and serves as coordinator of the recently established research priority program SPP2127 “Gene and cell based therapies to overcome neuroretinal degeneration” of the German Research Society, in which over 20 research groups from Germany join forces to develop new therapeutic strategies in the next 6 years.

Dr Maxime Mahé is a member of the Bioregate executive board.

Maxime Mahé’s research program seeks to investigate the molecular and cellular mechanisms underlying the effects of the enteric nervous system on human intestinal development. To address this, he is working to create integrated human gut models derived from embryonic stem cells and inducible pluripotent stem cells. Achieving this research goal will extend our knowledge on gastrointestinal pathophysiology and regenerative medicine.

Dr Mahé is currently a junior research associate at the INSERM UMR 1235 in Nantes and holds an adjunct assistant professor position in the division of Pediatric General and Thoracic Surgery at Cincinnati Children’s Hospital Medical Center (CCHMC), USA.

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Pr Kevin Shakesheff’s independent scientific career began at the Massachusetts Institute of Technology under a NATO fellowship following a PhD and his qualification as a registered pharmacist. He returned to the UK as an EPSRC Advanced Fellow and became Professor of Tissue Engineering and Drug delivery in 2001 at the University of Nottingham. In 2013 he became a Royal Society Wolfson Merit Award Holder. He was a Sub-Panel Member for the Research Excellence Framework (REF) for 2014. In 2011 he was made a Fellow of the Royal Pharmaceutical Society and in 2013 a Fellow of the Royal Society of Biologists. In 2014 he was selected as one of the 10 most inspirational scientists in the UK by the Engineering and Physical Sciences Research Council (RISE Leader Award). Kevin’s research involves the use of materials to control drug release and cell behaviour in 3D.

dr Alberto malerba (Royal Holloway, University of London, UK)Dr Malerba was awarded a PhD in Biotechnologies, describing novel approaches to improve muscle regeneration. He joined Prof George Dickson’s laboratory at Royal Holloway, University of London, in 2007 and since then he contributed to the development of novel gene therapy agents and antisense therapeutics for the treatment of rare diseases. His work was instrumental in optimizing the dosing regimens used for the morpholino exon skipping phase II/III clinical trial for Duchenne muscular dystrophy. Afterwards, he worked for 2 years as independent research fellow at the Royal Veterinary College in London, where he conceived and developed a scientific program based on new splicing-modulating molecules for the treatment of cardiovascular diseases. In 2013, he re-joined Prof Dickson’s group as Research Officer and Project Manager where he has been working on the optimization of a gene therapy medicinal product for Duchenne muscular dystrophy. He is also supervising the work on new drugs and gene therapy approaches for the treatment of Oculopharyngeal Muscular Dystrophy (OPMD) a rare muscle proteinopathy. His work was crucial in the development of a gene therapy AAV vector in collaboration with Benitec Biopharma that has been recently granted orphan drug status by EMA and FDA and will enter a first-in-human phase I/II clinical trial in 2019.

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dr Gloria González Aseguinolaza (ViVet Therapeutics)I have been working on Gene Therapy from the beginning of my scientific career. I did my PhD (Madrid) and postdoc (NY) in the development of genetic vaccines against parasites, that mainly affect developing countries, like Leishmania and Malaria. Them, I joined the Gene Therapy and Hepatology department of the University of Navarra in 2001 to head the lab of gene therapy for liver diseases. Since 2014, I am the director of the Gene Therapy and Regulation of Gene Expression department at the Center for Applied Medical Research (CIMA) of University of Navarra in Pamplona Spain, the department is composed by 7 independent research groups working of gene therapy and regulation of gene expression. Furthermore, I am also the coordinator of the area of advanced therapies and diagnostic innovation of the institute of biomedical of our Region (Navarra), IdiSNA, comprising 19 groups that belong to different research institutes, hospital or universities from Navarra. In 2016, I co-founded a gene therapy company, named Vivet Therapeutics, where I work part time as CSO. I have trained 13 PhD students and five additional PhD thesis are currently ongoing in the laboratory doing projects directly related to gene therapy, moreover a number of undergraduate, graduate and master students have been trained in the laboratory in the gene therapy field.

Dr Takebe is an Associate Director of the Center for Stem Cell and Organoid Medicine (CuSTOM) at the Cincinnati Children’s Hospital Medical Center and Professor at Yokohama City University, and Tokyo Medical and Dental University, Japan. He is elected as Board of Directors at the International Society of Stem Cell Research (ISSCR). His lab investigates the mechanisms of human organogenesis, and develops complex organoid technologies from human stem cells – namely organ bud based approaches. He is applying iPSC-liver buds into drug discovery study as well as transplant application – for patients with a rare congenital metabolic disorder, ultimately expanding the clinical indications to diseases like liver cirrhosis.

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Dr David Eglin is Principal Investigator and leader of the Polymers team at the AO Research Institute Davos in Switzerland. His group is using additive manufacturing technologies for both basic understanding of biomaterials and cells interaction, and for translational research in the orthopaedic field. He has extensive expertise in the synthesis and the processing of responsive materials based on biopolymers such as hyaluronic acid and collagen. In 2011, David Eglin was given the Jean Leray award by the European Society for Biomaterials for outstanding research contributions to the field of biomaterials.

dr Azadeh Golipour: After training as a molecular biologist, Dr. Azadeh Golipour has worked in the cell and gene therapy field. At AVROBIO, Azadeh is the director of manufacturing operations and oversees the process development and manufacturing operations for two gene therapy products.At CCRM, Azadeh’s team evaluated technologies in the field of regenerative medicine and developed technologies related to directed differentiation and translation of bench scale protocols to scalable platforms.Azadeh obtained her PhD in Molecular Genetics from the University of Toronto, where she used a combination of laboratory and bioinformatics approaches to define and interpret gene regulatory networks controlling reprogramming of somatic cells towards pluripotency.

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Pr Pierre Weiss, PhD, DDS, received his dental doctorate in 1989. He receives his Master of Science in biomaterial (Nantes) in 1993, his PhD in Biomaterial (Nantes) in 1997. He is Professor in biomaterials of the University of Nantes in dental surgery department and hospital. He is Head of REGOS team in UMRS 1229 RMeS Unit. His scientific activities are Skeletal tissue engineering, physicochemistry in hydrophilic polymer to make hydrogels for synthetic extra cellular matrix and bone substitutes. His research interests include the chemistry and characterization of macromolecular solutions and hydrogels to prepare synthetic extracellular matrices for tissue engineering of cartilages and bone. His scientific skills are on macromolecular chemistry and characterization like FTIR, Rheology, mechanical experiments and material design with nano particles blended with viscous solution before injection and cross linking into a 3 dimensional scaffold with alive cell encapsulated inside the structure. He also managed clinical research in Odontology. He is currently the scientific director of the “Pays de la Loire” Regenerative medicine cluster named “Bioregate” created in 2015. He is also the president of the society for biohydrogels, the vice dean of the Nantes dental school and in the scientific council of Nantes University.

More than 150 ISI indexed publications, 7 patents and Hirsh index: 42, 4000 citations. Researcher ID : P-1372-2014

dr réjane bihan is the Bioregate Executive Director, she drives the network strategy and its implementation with the support of the scientific director.

Holding a PhD in Genetics and developmental biology complemented by a MBA, she has worked for more than 10 years in health biotechnologies innovation. She participated in the setting up of R&D collaborative projects, of structuring projects to strengthen regional specialized economic sector and initiated a start up company creation. She experienced research, education & training, innovation policies in an international framework as well as their implementation.

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Dr Laurent David received his Ph.D. from University Joseph Fourier, Grenoble, France, in 2007. During his PhD, he discovered that BMP9 and BMP10 are physiological ligands of the receptor ALK1, which stemmed an active field of research in angiogenesis, and led to new therapeutic strategies for HHT, a disease caused by mutations of ALK1.Dr. David started to work on somatic cell reprogramming during his post-doc in Jeff Wrana lab, in Toronto, Canada. His work led to a better understanding of the mechanisms of somatic cell reprogramming, such as the characterization of the mesenchymal-to-epithelial transition that initiates the reprogramming of fibroblasts.In 2013, Dr. David joined the Medical School of University of Nantes as an Associate Professor. His lab is particularly interested in studying the regulation of pluripotency in human pluripotent stem cells and in human embryos. His lab combines stem cells, bioinformatics, developmental biology and clinical approaches to unravel human preimplantation development. Dr David is the director of Nantes iPSC core facility, embedded in the Bioregate cluster. Dr David is also treasurer of the French society for stem cell research (FSSCR).

dr catherine Le Visage, PharmD, PhD, is a Research Director and the Deputy Director of the Regenerative Medicine and Skeleton (RMeS) laboratory at the University of Nantes, France (www.rmes.univ-nantes.fr). She was trained as a Pharmacist, she received her PhD in Pharmaceutical Technologies and then performed a post-doctoral training at the Johns Hopkins School of Medicine, Biomedical Engineering Department (Baltimore, USA) in Prof Kam Leong’s laboratory with a focus on soft tissue engineering projects. In 2007, she joined the French National Institute of Health and Medical Research (INSERM) as a tenured Senior Research Scientist to investigate chemically cross-linked polysaccharides hydrogels as cardiac cell delivery systems and vascular replacement scaffolds. In 2013, she was appointed as a Research Director and joined the Regenerative Medicine and Skeleton laboratory in Nantes. In the «Skeletal Physiopathology and Joint Regenerative Medicine» team headed by Prof J. Guicheux, she investigates hydrogels as i) tools for stem cell-based organogenesis and ii) carriers of cells or bioactive molecules in the context of intervertebral disc disease and osteoarthritis. Her current research interest lies in bioengineering approaches to orchestrate molecular and physical signals that regulate stem cell fate. Her most recent works have focused on the development of self-setting hydrogels for long-term delivery of biochemical cues such as growth factors and chemokines to address intervertebral disc (DDD) and cartilage (osteoarthritis, defects). She co-authored 58 publications in ISI-indexed journals (h-index 23), 11 patents and gave 40 invited lectures.


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Pr Frank Boury, 52 years old, PharmD, obtained his PhD degree at the University of Paris XI in 1995 awarded by a fellowship from GTRV (Thematic group of research on vectors) and by APGI (Industrial pharmaceutical technologies association). He exerts his professional activity at the School of Engineering ISTIA+ (University of Angers) where he teaches formulation sciences and biophysics and he is researcher at CRCINA, INSERM U1232, University of Angers,team Gliad. From 2008, he is also the vice-director of the doctoral school Biology-Health Nantes-Angers and the director of the M.Sc. “Innovative technologies in formulation”. From 2011 he is the coordinator of the Erasmus Mundus Joint Doctorate European Doctorate in Nanomedicine and Pharmaceutical Innovation “NanoFar” (6 partners, 6M€, 47 international PhD students involved). He is the principal investigator of several national (ANR Calcomed, international strategy in nanomedicine) and partner of regional projects (Bioregos, Matières, RFI Bioregate) and of several industrial projects. He is author of 94 scientific publications and of more than 100 papers presented at international conferences. He organized several international summer schools in the field of nanomedicine in collaboration with many academic and industrial partners. He is particularly interested by Supercritical Fluid Technologies (SCF) and developed a platform aiming developing nano-micromedicine for the controlled release of protein and fragile molecules.During 2013-2017, Frank Boury was also Chair of the WG « education & training » European Technology Platform of Nanomedicine (ETPN) and member of the ExBo of ETPN. He is also member of the ExBo of the CCRRDT “Biology health” Région Pays de la Loire)

Dr Tuan Nguyen, Liver cell and gene therapy expert, is a co-founder of GoLiver Therapeutics and was recruited in April 2017 as chairman and chief scientific officer (CSO) to develop an Advanced Therapy Medicinal Product (ATMP) based on human pluripotent stem cells for the treatment of severe liver failures. He previously headed a research group on liver-directed gene and cell therapy (lentiviral vectors, genome editing with CRISPR/cas and ZFNs, pluripotent stem cells) and the GenoCellEdit platform at the INSERM UMR1064 unit - Center of Research in Transplantation & Immunology at Nantes University Hospital (France) from 2012 to 2017. In 2016, he was the chairman of the 21st NAT (Nantes Actualités Transplantation) “When Stem Cells Meet Immunology”. He was Deputy Editor of Current Gene Therapy journal and has published over 64 original publications in the field of cell and gene therapy. The Goliver Therapeutic project was awarded of the Grand Prize “pharmaceuticals and biotechnology” from the French Research Ministry in the national innovation start-up contest i-LAB and of the start West award, a contest for innovative start-ups in the France « Great West » (« Grand Ouest ») in 2016, and of the “Coup de Coeur” award of the Atlanpole forum in 2018.


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dr Franck Halary and his group are interested in understanding how dendritic cells, known to orchestrate the immune response, can participate to the capture in situ and spreading of transplant-related viruses, the human cytomegalovirus and the BK polyomavirus. His work mainly relies on the use of cellular models and cells isolated from human tissues. More recently, in collaboration with the Ecole Centrale de Nantes, he started to implement 3D bioprinting as a way to fabricate biologically-relevant humanized tissues/organs for basic research and eventually replace disabled functions/organs in grafted patients.

Franck Halary is an associate professor at the Centre de Recherche en Transplantation et immunologie (CRTI, Nantes).

dr Oumeya Adjali (MD. PhD) is a senior research scientist at the French Institute of Health and Medical Research INSERM. She leads the «Translational Gene Therapy laboratory» (INSERM UMR 1089) in Nantes University since January 2017. She has been working at the interface of biotherapies (cell and gene therapies), immunotherapy and immunomodulation for more than 16 years. Since 2007, she supervises a research program dedicated to the immunology of Adeno Associated-Virus (AAV)-derived gene therapy products following their in vivo delivery with a special interest to host immunity against the vector and the transgene product using large animal models. In addition, Oumeya Adjali leads Gene Therapy Immunology core (GTI) in Nantes University and has been involved as immunology expert in a large number of gene therapy preclinical studies as well as AAV gene therapy phase I/II clinical trials for the treatment of muscular, liver, CNS and retinal genetic diseases.

Christelle Bervas is a Nantes University Communication Advisor. Her mission is dedicated to strengthen relationships between Nantes University and companies.


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Lynda Guerineau has completed her Food Sciences Engineering degree in 2003. She started her career in research and development department of several companies in food industry. In 2009, she decided to join Atlanpole Biotherapies team. She is in charge of the animation of the network and also event organization and communication.

Atlanpole Biotherapies is one of the 7 French Bioclusters, focused on personalized medicine and innovative therapies. It gathers more than 190 members working in academic lab, companies and platforms.

nissrine mekkaoui is Bioregate Assistant.

Graduated with a Master’s degree in Law and a lawyer by training, she worked on behalf of the European Commission to award grants for large-scale projects. Today, she puts her skills to the benefit of Bioregate.

mathis benzina is Bioregate Education and training engineer.

Graduated with a Master degree in training and education engineering, he has gained an expertise in pedagogy and training engineering in the field of ICTs. He brings his skills to Bioregate developments to improve, create or internationalize university courses in regenerative and repair medicine (on-campus and distance learning courses).


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InTErnaTIonal sCIEnTIFIC CommITTEE

Pr Abhay Pandit is currently the Scientific Director of the Centre for Research in Medical Devices (CÚRAM), and an Established Professor of Biomaterials at the National University of Ireland, Galway. CÚRAM is a multi-disciplinary academic-industry-clinician translational research centre coordinated by the National University of Ireland, Galway. The centre is developing innovative and transformative device-based solutions to treat global chronic diseases. The centre supports industry from basic scientific research, through translational preclinical and clinical development, into regulatory and commercial readiness. The group has designed and developed functionalised biomaterial platform technologies with inbuilt gradients of physical, chemotropic and neuroprotective cues to target injury mechanisms at the molecular and cellular levels. These multi-modal biomaterial scaffolds, facilitate the spatiotemporal delivery of multiple biomolecules targeting different phases (inflammatory, neuroprotective, and angiogenic phases) of the underlying disease pathology resulting in an enhanced therapeutic response. These platforms have been designed to address unmet clinical needs for cardiovascular, musculoskeletal, soft tissue and neural targets and have been validated in pre-clinical models. Phase 1 clinical trials planned to streamline clinical translation.

Pr Julie Fradette (PhD) is a Full Professor at Université Laval, department of Surgery, Faculty of Medicine. She is a researcher at the Centre LOEX de l’Université Laval, at the research center of the CHU de Québec-Université Laval since 2005. Her research activities focus on adipose-derived stem/stromal cells (ASCs) and their use in regenerative medicine. Her expertise encompasses tissue engineering of skin and various microvascularized connective tissues including human adipose and bone-like tissues. During her graduate studies, she studied skin epithelial stem cells for improvement of skin substitutes. Her postdoctoral training at the University of Pittsburgh established that ASCs and adipose tissue can be used for gene delivery using herpes-based viral vectors. Her research program is now focused on how human mesenchymal cell’s potential can be harnessed for tissue/organ reconstruction using natural cell-based, scaffold-free strategies. She is the director of the ThéCell network for cellular and tissular therapies for the province of Québec, Canada.

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InTErnaTIonal sCIEnTIFIC CommITTEE

Pr Christine Jérôme is full Professor at the University of Liege and director of the Center for Education and Research on Macromolecules. She earned a chemical science degree from Liege University and performed postdoctoral research on the synthesis of magnetic nanoparticles at the University of Ulm under the auspice of an Alexander Von Humboldt grant. She has expertise in polymer chemistry and passion in developing biomaterials to improve health and well-being. She develops green strategies for the synthesis, functionalization and processing of polymer materials, degradable or not, to precisely tailor their properties and design supramolecular structures customised to the targeted biomedical application and needs.

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Bioregate is a Western France network dedicated to the development of Regenerative Medicine technologies and skills. It gathers 3 universities, 2 university hospitals, 4 national academic research institutions, a vet school and about 50 SME partners through Atlanpole Biotherapies competitiveness pole. Bioregate cluster is piloted by Nantes University and is supported by local authorities among which the “Pays de la Loire” Region and “Nantes metropole”.Bioregate core activities:• Translational research:

With competences in: - cell and gene therapies, - tissue engineering,- bio and nanomaterials,- engineering sciences.

Bioregate players have been developing products of 1st and 2nd generations in the field of repairing and regenerative medicines, mainly to treat patients with skeletal, muscle, cardio vascular, neurological, skin, metabolic or eye pathologies.More than 20 clinical trials are ongoing thanks to intense collaborations between researchers, veterinarians, clinicians and companies and also the facilitated access to key core facilities such as those focusing on IPS cells, manufacturing of clinical grade batches, housing of small and big animals relevant for innovative therapy testing, biobanking, biotherapy clinical trials.• Education & training:

- Initial training: Bioregate players are currently designing a Master and a European doctoral course specialized in regenerative medicine

- In course training: an hybrid course is being set up on a campus based and distance teaching

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Bioregate is opened to provide research and clinical skills, technology platforms, education and training, innovation opportunities in a collaborative framework. Bioregate players constantly look for:

- public-public and public-private collaborations for research, R&D or education and training programs

- stable partnerships with other European clusters dedicated to regenerative medicine

Bioregate players also enjoy welcoming high level researchers or lecturers for seminars or permanent positions. Aiming at strengthening its value chain, Bioregate is also a facilitator in the establishment of new businesses in collaboration with its local partners.

Bioregate also looks for complementary fund raising through Nantes University Foundation. For more information, please visit this webpage http://www.bioregate.com/.Contacts: Dr Réjane Bihan, Bioregate Executive Director Bioregate, [email protected] & Pr Pierre Weiss, Bioregate Scientific director, [email protected]

A major higher education and research centre in Western France, Université de Nantes has never stopped moving forward. In the last 50 years, Université de Nantes has taken training and research to the highest level and, in 2015 took a spot in the Times Higher Education World University Ranking. Université de Nantes is ranked among the top 25 French universities. Faced with today’s challenges, we reinvent a new university model that places the student at the centre of its attentions and the human being at the heart of its dynamics and its ambitions. Université de Nantes promotes interdisciplinary as a factor of innovation and academic success. Dynamic, competitive, creative and socially responsible, the Université de Nantes possesses the assets to promote local, national and international cooperation.

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Atlanpole Biotherapies is one of the 7 French bioclusters based in Western France and focused on Innovative therapies. It represents over 170 members working on personalized medicine and biotherapies:

- more than 100 companies dedicated to life sciences,- research laboratories, hospitals and universities,- technological platforms, engineering schools.

Within this ecosystem, restorative and regenerative medicine began by transplant technology whose growth has been marked by the development of innovative solutions, as well as alternatives to the organ shortage, and for the immunological monitoring of transplanted patients. In parallel, new therapeutic approaches have developed (biomaterials, tissue engineering, cell therapy, gene therapy) which apply to diseases of the bone and joints, dermatology, oncology, pneumology, as well as rare, neurodegenerative and cardiovascular diseases. Bioregate is the name of the local network on regenerative medicine (located in Pays de la Loire Region, Western France). It brings together more than 3 hundred people working on Cell Therapy, Gene Therapy and Biomaterials.Atlanpole Biotherapies is also well-known for innovative approaches such as:

- Immunotherapies specializing in Transplantation / Oncology / Inflammation and Auto-immune diseases

- Radiopharmaceuticals with the use of radioisotops for diagnostic and therapy.

- Innovative Technologies and e-Healthwww.atlanpolebiotherapies.com

The iPSC core facility of Nantes was created in 2012. Its main activity is to generate hiPSC from patients. We routinely work with labs in Paris, Lyon, Nice, Montpellier, Spain or Germany. We have the IBiSA and Biogenouest French core facilities accreditation. The iPSC core facility of Nantes is part of the Biotherapy core facilities of Nantes, covering fundamental developments to clinical grade phase III cellular and viral batches.

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Goliver Therapeutics is a biotechnology company, spin-off from the Inserm and Nantes University, focused on developing novel stem cell-based therapies to address the high unmet medical need in liver transplantation. Goliver Therapeutics aims to deliver the first ATMP from human pluripotent stem cells that promises to become a live-saving alternative to liver transplantation and to regenerate the own patient’s liver.

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useful InfoRMaTIon


MEETING VENUE

Nantes - Saint-Nazaire Chamber of Commerce - Centre des Salorges16 quai Ernest Renaud - 44105 Nantes

HOW TO GET THERE?

Chamber of Commerce is located alongside the Loire River, approximatively 15 minutes walk from the city center. You can easily get there:

By plane: take the Shuttle Bus to Nantes City Center (Commerce) then take Tramway line 1 and stop at “Gare Maritime” station.

By train: at Nantes railway station, take the north exit and then Tramway line 1 and stop at “Gare Maritime” station.

By Tram: Line 1- “Gare Maritime” station.

By Taxi: Oh Taxi Nantes : +33 (0)2 28 00 00 82 or Allo Taxis : +33 (0)2 40 69 22 22

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Île Beaulieu

PORTE DEL'ESTUAIRE30

RENNES N137

ST-NAZAIRELA BAULE N165

PORNIC D751

BORDEAUX A83

CHOLET N249

ANGERSPARIS A11

GARE

AÉROPORT

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USEFUL PHONE NUMBERS AND ADDRESSES

Medical help/SAMU: 15

Police: 17

Fire & accident: 18

SOS - all services (recommended when calling from a mobile) 112

www.salonsdunantilus.com

Agence Nantes-Saint Nazaire développement

Quai de la Fosse

Qua

i Ern

est R

enau

d

Bd. De Launay

Rue Bisson

PlaceMelinet

Pont Anne de Bretagne

VERS PÉRIPHÉRIQUE

“PORTE DES ESTUAIRES”

VERS CENTRE VILLE

Place René Bouhier

Bd. Allende

Arrêt Gare maritime

LA L

OIR

E

ParkingEsplanade

l’Abeille

LES SALONS DE NANTILUS

ParkingCCI

HOW TO GET THERE FROM THE CONFERENCE CENTER:

GALA dinner

Have a nice networking dinner on the Nantilus boat! Facing the Nefs and the Caroussel des Mondes Marins, you will appreciate this outstanding place with its unique view on the Loire River and the city center. And maybe you will have the chance to see the Elephant of Nantes.

Location:

Parc des chantiers - Pont 3 du Nantilus30, Quai Fernand Crouan44200 Nantes

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