EMAMay, 2010

iPS for regenerative iPS for regenerative medicinemedicine

Hopes, dreams and nightmares…

mpeschanski@istem.fr

Marc PeschanskiI-STEM

Evry (Paris district) France

Producing iPS for regenerative medicineProducing iPS for regenerative medicineKLF4c-Myc

Oct-4 Sox2

fibroblasts

Plating on gelatin

1d

1d

3-5w

Culture in hES medium + VPA

KLF4c-Myc

Oct-4 Sox2

EcoRcMyc

→

From controls or patients fibroblastsEmergence of « ES-like » clones

iPS iPS clonesclones

You name it!

iPS, what do we expect from them in regenerative medicine?

Like ES cellsUnlimited resource at the undifferentiated stage

Industrialization of the production processGMP compliance and comprehensive control

Fully versatile capacity at differentiationAccess to « any » cell type at « any » stage of differentiationCell populations at near homogeneity or enriched cultures

Open ability at genetic engineeringTo provide them with additional phenotypes of interestTo improve protocols of production/differentiationTo address safety issues

Unlike ES cells,A way to create pluripotent stem cell lines expressing any desired genotype

iPS production : methods and potential developments

5

Somatic cells

Reprogramming

Induced pluripotent stem cells (iPS)

Which cells?Which cells?

Which RF? Which way of Which RF? Which way of delivery? delivery?

Optimization of the Optimization of the reprogramming reprogramming process?process?

Selection?Selection?Quality control?Quality control?

FibroblastsKératinocytesBlood cells

Viruses (lenti/rétro)EpisomesPolycistronic vectorProteins

Oct-4/Sox2KLF4/c-MycLIN28/NanoghTERT/SV40

Small moleculesCulture conditions

MorphologyLive markers

Quality control of iPS clones: QC similar to ES

Morphology

Karyotype

Genotype

Pluripotency markers

Differentiation capacitiesEmbryoid Bodies/3 germ layersDirected differentiation

OCT-4/DAPI

PLURIPOTENCY

MESODERME

ECTODERME

ENDODERME

Expression level

PLURIPOTENCY

MESODERME

ECTODERME

ENDODERME

PLURIPOTENCY

MESODERME

ECTODERME

ENDODERME

Expression levelExpression level

Controlling Quality of iPS lines for regenerative medicine: seeking which

specific controls?

Methylation status

Transgenes silencing OCT4 TRANSGENE

1,E-04

1,E-03

1,E-02

1,E-01

1,E+00

1,E+01

2102EP Fibr4603

Fibr4603 J4

MYCGes Cl1

PolA PolF Cl5 Cl27 Cl28 SA01

Pluripotent stem cells differentiation: How we do it with ES cells

DIV0 20 40

Milieu FAD + BMP4 + AA Milieu FAD

Selection ofK-hESC

hESC Epithelial progenitors

Milieu Epilife®

Amplification Banking

Reconstruction of epidermis

(Guenou et al., The Lancet, Nov 2009)

0,E+00

5,E-01

1,E+00

2,E+00

Day 0

Day 10

Day 20

Day 40

OCT4

Expr

essi

onre

lativ

e to

hES

C

*** *** *** 0,E+00

1,E+01

2,E+01

3,E+01

4,E+01

Day 0

Day 10

Day 20

Day 40

KRT8

***

***

0,E+00

8,E+04

2,E+05

2,E+05

Day 0

Day 10

Day 20

Day 4

0

KRT14***

***

ES cells differentiation: how we check for potential in regenerative medicine

In vitro reconstruction of epidermis

In vitro

Keratinocytes derived from

hESCIn vivo

Grafting in immunodeficient mice

In vivo reconstruction of epidermis

iPS for regenerative medicine: is there a limit to their pluripotency?

Fibroblasts iPS

NANOGOCT4

SOX2 LIN28

KLF4OCT4

SOX2 c-MYC

Lentiviral Reprogrammation

Rétrovirale Reprogrammation

iPS-IMR90 / MRC5

iPS-4603 K-iPSC

K-iPSC

BMP4 + AA

BMP4 + AA

40 Jours

40 Jours

iPS derived keratinocytes: identical to ES-derived cells?

1,E-04

1,E-01

1,E+02

1,E+05

1,E+08

18SOCT4

NANOGKRT8KRT18KRT14

KRT5ITGB4ITGA6

COL7A1LAMB3

Expr

essi

on re

lativ

e to

hES

C

hESK-hESCHK

1,E-04

1,E-02

1,E+00

1,E+02

1,E+04

1,E+06

1,E+08

18S

OCT4

NANOG

SOX2

KRT8

KRT18Delt

aNP63

KRT5

KRT14

hESC iPSC K-hESC K-iPSC HK

iPSC

K-iP

SCH

KTRA 1-60 K18 K5 K14

1,E-04

1,E-01

1,E+02

1,E+05

1,E+08

18SOCT4

NANOGKRT8KRT18KRT14

KRT5ITGB4ITGA6

COL7A1LAMB3

Expr

essi

on re

lativ

e to

hES

C

hESK-hESCHK

iPS for regenerative medicine: are all pluripotent stem cell lines alike?

1,E-04

1,E-03

1,E-02

1,E-01

1,E+00

1,E+01

iPSC K-iPSC iPSC K-iPSC iPSC K-iPSC

IMR90 MRC5 PolyF

OCT4 NANOG SOX2

1,E-07

1,E-05

1,E-03

1,E-01

1,E+01

iPSC K-iPSC iPSC K-iPSC iPSC K-iPSC

IMR90 MRC5 PolyF

KRT5 KRT14

Rel

ativ

e ex

pres

sion

to iP

SCR

elat

ive

expr

essi

on to

iPSC

KRT10 / K14

Filaggrin / K14

Organotypic culture

K10

K14

KK--hESChESC

KK--hiPShiPS

Engineering iPS cell lines

For improving protocolsOne major logistic issue: « good » and « bad »clones?Looking for new reprogrammation/differentiation protocols

For improving the end-productIntroducing selection markersCoaxing cells to specific cell phenotypes

For addressing safety Pre-graft actionPost-graft « off-switch »

Real and fantasmatic safety issues with pluripotent stem cells tumorigenicity

Teratomas: actually not that worrysome (and let’s be careful

about terms!)

Post-graft control of normal progenitors

proliferation: the actual problem

What iPS can do for regenerative medicine that ES cells can’t:

the international haplobank prospective

• ObjectivesProviding clinicians with clinically compatible iPS cell lines and,

when relevant, cell therapy productsContributing to an international network of clinically compatible

iPS cell banks for meeting cell therapy needs of the entire world population

• FocusCollecting and banking samples from haplotypically

homozygous donors for HLA-A, -B and –DR (bone marrow registries contain 0.5-0.6%)

Developing and implementing GMP technology for derivation of iPS cell lines, banking and differentiation into specific therapeutically relevant cell phenotypes

Hemi-similarity: a biological basis to make a cell bank foreseeable

Haplotypically homozygous donors in the French registry: altogether >200 haplotypes (broad types)

Haplotypes differ from one population to another in proportions, i.e. initiative for a world cell bank consortium is timely

0

50

100

150

200

250

300

1 2 29 3

1.8.3

2.7.15

2.12.429.12.7

3.7.15

3.35.1

most frequent haplotypes