stem cells andstem cells and regenerative ... · pdf filestem cells andstem cells and...
TRANSCRIPT
STEM CELLS ANDSTEM CELLS AND REGENERATIVE MEDICINEREGENERATIVE MEDICINE
Giulio CossuDepartment of Biology and Division of Regenerative MedicineDepartment of Biology and Division of Regenerative Medicine University of Milan San Raffaele Scientific Institute
Organs and tissues that we may be able to repair or replace
Giulio Bizzozzero: Labile, stable and permanent cells
• Giulio Bizzozzero (1846-1901, Professor of Pathology at the University of Turin) found th t th i i /l k i ithat erythropoiesis/leukopoiesis takes place in bone marrow [1868]; he discoverd plateletshe discoverd platelets. Furthermore, Bizzozzero understood that cell renewal varied in different tissues and classified them as:labile (e.g. bone marrow)
stable (e.g. liver)
permanent (e.g. nervous)
Skeletal and cardiac muscle or brain contain very few mitotic cells while epithelia and bone marrow have many p y
Skeletal muscle Epidermidis
St ll
AStem cells
P it llProgenitor cells
Differentiated cells
B C
Unipotent Multipotentp
Two types of stem cells:
1. ADULT (Including fetal, extra-embryonic and from humbelical cord) No ethical issues but nothumbelical cord). No ethical issues but not available for all tissues
2. EMBRYONICForm the inner cell mass of the blastocyst. Unlimited proliferation potency and p p ypluripotency (ability to form all tissues of the body They raise ethical issuesbody. They raise ethical issues.
Human BlastocystHuman Blastocyst
Pluripotency of embryonic stem cells
Stem cells for the therapy ofStem cells for the therapy of human diseases:human diseases:
When which how?When, which, how?
Cell therapy with stem cells
Healthy cells Sick cells
ViralViralvector
Healthydonor
Patient Genetically corrected cells (“cured”)Cells that are
d b dispared by disease
What are the parameters that stem cells must satisfy t di t ffi i li i l d li i l t i l ?to predict efficacy in pre-clinical and clinical trials?
1 Th t b t i th d ti i1. They must be present in the donor tissue in sufficient number (or may be propagated in it )vitro)
2. They must be able to reach the target tissue in hi h b i t ll t ithhigh numbers, survive, eventually compete with resident cells (but do not proliferate indefinitely
d t ll f t )and eventually form a tumor). 3. They should produce efficiently the needed
differentiated progen and f nctionall integratedifferentiated progeny and functionally integrate in the host tissue.
Which diseases are cured today with stem ycells ?
In clinical practice :
1. Bone marrow transplantation for hematological diseases2. Auto-transplantation of epidermis for large burnsp p g3. Corneal unilateral burns
In clinical experimentation: 1 Congenital immune deficiencies1. Congenital immune deficiencies2. Epidermolysis bullosa3. Severe lesions of the skeleton3. Severe lesions of the skeleton
Gene transfer protocol into autologous bone marrow CD34+ cells
No PEG-ADA
Day -4: PurificationBusulfan2 mg/Kg/day x 2 Day 4: Purification
of BM CD34+ cells(days -3, -2)
Day 0: Infusion
ADA SV NeoMLV LTR
Day-4: Prestimulation (TPO FLT3 ligand
Days -3 to -1: 3 cycles of transduction
i ki
MLV LTR
(TPO, FLT3-ligand, SCF, IL-3)
on retronectin + cytokines
Aiuti et al. Science, 2002
In vitro produced epidermis1 cm2 biopsy Permanent epidermal regeneration in
full-thickness burns covering up to 98%of the body surface.
holocloneautologous cultured
ith li l h t
(O’Connor, Lancet, 1979; Gallico, N. Engl. J. Med., 1984;Pellegrini, Transplantation 1999; Ronfard, Transplantation2000)
epithelial sheets
Permanent regeneration of the urethral
2,000-5,000 stem cells
gepithelium in congenital posteriorhypospadias.(Romagnoli, N. Engl. J. Med., 1990; J. Urol. 1993)
Repigmentation in stable vitiligo usingkeratinocyte/melanocyte co-cultures.(Guerra, 2000, 2003, 2004)
holoclone-daughter colonies
Holoclones isolated from the skin many years after transplantationmany years after transplantation
Duchenne Muscular DystrophyX-linked recessive disease caused
by mutation of dystrophin gene theby mutation of dystrophin gene, the largest known gene (2.4 Mb) coding for a 427 kD cytoskeletal protein
It is he second most common genetic disorder in man and the gmost common muscular dystrophy of childhood (incidence 1:3500 male live births)
Characterized by degeneration of skeletal muscle due to absence of dystrophin on muscle fibre
bmembrane
Classic and new model for muscle regeneratioClassic and new model for muscle regeneratio
Pathophysiology of DMD
fragility
∆dystrophin
g y
necrosisnecrosis
regenerationd fib i
fatinfiltrationgand fibrosis
MesoangioblastsCan be isolated from Mouse, Dog and Human embryonic and post‐Can be isolated from Mouse, Dog and Human embryonic and post
natal vessels, cloned and expanded in vitro for approximately 20 PD (except than in rodents).
Express endothelial markers when isolated from the embryo and pericyte markers when isolated from post‐natal tissues.
Differentiate into different types of solid mesoderm including skeletalDifferentiate into different types of solid mesoderm, including skeletal muscle.
Human post‐natal mesoangioblasts Myofibers from mesoangioblasts
Mesangioblasts for the cell therapy of primary myopathiesMesangioblasts for the cell therapy of primary myopathies
Cells isolated from the patient’s Cells isolated from the patient’s tissues (bone marrow, tissues (bone marrow, vessels )vessels )
In vitro In vitro espansionespansion
Transduction Transduction with therapeutic with therapeutic genegenevessels…)vessels…)
Selection of Selection of stem cellsstem cells
genegene
Viral vector with Viral vector with therapeutic genetherapeutic gene
Dystrophic TransducedDystrophic TransducedDystrophic TransducedDystrophic TransducedFibers FibersFibers Fibers
Or wt Or wt Intravascular Intravascular deliverydelivery
Will it work ?Will it work ?
donor donor cellscells
yy
Mouse: yesMouse: yesDog: Dog: yesyesHH ????Human: Human: ????
wt mesoangioblasts wt mesoangioblasts give rise togive rise to αα sarcoglycan sarcoglycan ggpositive fiberspositive fibers
CTR α-SG KO treated
Sampaolesi et al. Science 301, 487, 2003
Mesoangioblast transplantation g pameliorates motility of dystrophic mice
Dystrophic mouse Dystrophic mouse after Dystrophic mouse yst op c ouse a temesoangioblast transplantation
Dystrophinopathy, Dystrophinopathy, th GRMD dth GRMD d
Dystrophin
the GRMD dogthe GRMD dogDystrophin
• Relevant animal model, homology• Phenotype (simulation of human condition)
GRMD
Phenotype (simulation of human condition)• Genotype known (a point mutation in intron 6)
Muscle BF, HE [Myosin]
Dystrophin
Muscle BF, HE
Calpain 94kDa
α-sarcoglycanα sarcoglycan
ontro
l
ontro
l
Calpain 30kDa
D
Dog GRMD Cani
ne co
Hum
an co
GRM
Complete dystrophin reconstitution
Complete rescue of morphologyComplete rescue of morphologyComplete rescue of morphologyComplete rescue of morphologyDonor cellsDonor cells Autologous, mAutologous, m--dys+ cells Untreateddys+ cells Untreated
Mesoangioblast transplantation ameliorates dog motility
On the basis of this evidence we plan a Phase I Clinical Trial:
Allo-trasplantation of mesoangioblasts in DMD patients
1. 3 patients (age 6-9, good clinical status and HLA-matched donors) will be transplantated with donor mesoangioblasts according to a tri-phase protocol: A) intra-muscular; B) infusion in one femoral artery; C)protocol: A) intra-muscular; B) infusion in one femoral artery; C) multi-district intra-arterial infusion.
2. Clinical, biochemical and functional progress of the disease will be p gfollowed for 12 months preceding treatment.
3. Donor mesoangioblasts will be isolated, expanded and characterized d GMP/GLP ditiunder GMP/GLP conditions.
4. Donor cells will be then transplanted as detailed above and adverse events as well as dystrophin expression and functionalevents as well as dystrophin expression and functional improvements will be monitored.
5. Finally, we will measure the final outcome (24 months) and will follow the patients, planning for changes and implementations.
Allo‐trasplantation of mesoangioblasts in DMD patients
Outcomes:
Primary outcome:
Sh t d l f t f i t l d i t t i lShort and long safety of intra-muscular and intra-arterial transplantation of mesoangioblasts
Secondary outcome:
Efficacy of mesoangioblasts in increasing contractile force oftreated DMD patients.treated DMD patients.
Steps to bedsideSourcingManufacturing
Toxicity:Acute: Chronic:
Funding:GovernmentCh iti700 000700 000 €€ 150 000150 000 €€ You need ≥You need ≥g
BankingStandards
Vascular FibrosisInflammation CancerRejection
CharitiesCompanies
700.000 700.000 €€ 150.000 150.000 €€ You need ≥You need ≥2.000.000 2.000.000 €€More than
Efficacy:In vitro
2,000,000 €In vitroRodentsLarge animalsP i t
Clinical Trial800.000 800.000 €€
400 000400 000 €€
, ,for 3 PatientsPrimates 400.000 400.000 €€for 3 Patients
Regulatory issues:National AuthorityEthi l C itt
International Advisory BoardPeer review
Outcome Measures
150 000150 000 €€ 100 000100 000 €€100 000100 000 €€Ethical CommitteeConflict of interest
Peer reviewData Managing Board
Validation150.000 150.000 €€ 100.000 100.000 €€100.000 100.000 €€
Problems ahead:
1. Costs are excessive and not always justified.j
2. Safety is crucial but cannot become a reason to stop medical progressreason to stop medical progress.
3. New ethical issues arise, such as patient and donor selection, a truly informed consent, etc.consent, etc.
Conclusions:Conclusions:
1 Many severe diseases have already been cured1. Many severe diseases have already been cured with stem cells.
2 Recently lethal and so far “incurable” diseases2. Recently, lethal and so far incurable diseases have been successfully treated.
3 Many other diseases are entering clinical3. Many other diseases are entering clinical experimentation.
4 Each new therapy is the results of many years of4. Each new therapy is the results of many years of pre-clinical works
5 Miracles on the other hand are rare and those5. Miracles, on the other hand are rare and those who promise a cure for any disease should be under very close scrutinyunder very close scrutiny.
Come valutare e non cadere in trappole?pp
Regola n° 1: il sito deve contenere informazioni dettagliate su quali cellule staminali vengono utilizzateg q g
Regola n° 2: Se il sito promette la cura di molte malattie diverse tra loro la cosa è sospetta
Regola n° 3: Il responsabile della clinica deve avere bbli t li t di ll b d i i t di t i i t
diverse tra loro, la cosa è sospetta
pubblicato gli studi alla base dei suoi metodi su note riviste internazionali.
Regola n° 4: i costi devono essere dettagliati.
Antonio PigafettagAssistant and Navigator of Ferdinand Magellan
“Noi non conosciamo la via della ritirata”
Collaborators:Collaborators:S.C.R.I. (Dibit, HSR, Milan)S.C.R.I. (Dibit, HSR, Milan) Ecole Veterinarie Mason AlfortEcole Veterinarie Mason Alfort( , , )( , , )Maurilio Sampaolesi (Maurilio Sampaolesi (αα--SGC ko/GRMD) SGC ko/GRMD) Anna Innocenzi (Histochemistry)Anna Innocenzi (Histochemistry)Rossana Tonlorenzi (Cell culture)Rossana Tonlorenzi (Cell culture)
Stephane Blot (dogs)Stephane Blot (dogs)Nicolas GringerNicolas GringerUniversity of PaviaUniversity of PaviaRossana Tonlorenzi (Cell culture)Rossana Tonlorenzi (Cell culture)
Beatriz Galvez (Homing)Beatriz Galvez (Homing)Arianna Dellavalle (Pericytes)Arianna Dellavalle (Pericytes)Sara Benedetti (Immortalization)Sara Benedetti (Immortalization)
University of PaviaUniversity of PaviaRoberto Bottinelli, (physiology)Roberto Bottinelli, (physiology)Giuseppe D’Antona Giuseppe D’Antona University of MilanUniversity of MilanSara Benedetti (Immortalization)Sara Benedetti (Immortalization)
Saverio Tedesco (DysSaverio Tedesco (Dys--HAC)HAC)Graziella Messina (Lentivector)Graziella Messina (Lentivector)C G i li (Old l )C G i li (Old l )
University of MilanUniversity of MilanYvan Torrente (patients)Yvan Torrente (patients)Tottori UniversityMit O hi (HAC)Mit O hi (HAC)Cesare Gargioli (Old muscle)Cesare Gargioli (Old muscle)
Washington UniversityWashington UniversityJeffrey Chamberlain (microJeffrey Chamberlain (micro--dystrophin)dystrophin)
Mitsuo Oshimura (HAC)Mitsuo Oshimura (HAC)EPFL LausanneEPFL LausanneDidier Trono (floxed lenti)Didier Trono (floxed lenti)
University of ModenaUniversity of ModenaStefano FerrariStefano Ferrari (micro(micro--array)array)Enrico TagliaficoEnrico Tagliafico
UT South Western Medical UT South Western Medical CenterCenterWoody Wright (Tert)Woody Wright (Tert)gg y gy g
CureCure Duchenne