ips for regenerative medicine...ips, what do we expect from them in regenerative medicine? like es...
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EMAMay, 2010
iPS for regenerative iPS for regenerative medicinemedicine
Hopes, dreams and nightmares…
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