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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