Post on 15-Jan-2015
description
Derivation of highly enriched cultures of
differentiated cells from human parthenogenetic
stem cells
California Institute for Regenerative MedicineMedical Research Council
Human SCNT Workshop14 June, 2010, San Francisco, CA
Workshop report:http://www.cirm.ca.gov/files/PDFs/Publications/Human_SCNT_Workshop_Report.pdf
Nikolay Turovets, PhDDirector of Research and Therapeutic Development
Parthenogenetic stem cells are a way to create immune-matching cells for transplantation
• Typical hESC morphology;• Diploid karyotype 46, XX• Express appropriate markers;• Demonstrate high levels of alkaline phosphatase;• Demonstrate high levels of telomerase activity;• Form embryoid bodies in suspension culture;• Form teratomas after injection to immunodeficient animals; • Give differentiated derivatives of all three embryonic germ layers;
hpSC demonstrate characteristics of hESC
Human parthenogenetic stem cells:
hepatocytes to treat liver dysfunctions
Human parthenogenetic stem cells:
hepatocytes to treat liver dysfunctions
Target diseases (over 100 000 cases): • OTC deficiency• α1-antitrypsin deficiency•Crigler-Naijar syndrome•…
Restoration of one liver function/activity
To rescue patient, FULL restoration of function is NOT NECESSARY
Immature cells already demonstrate required activities
Transplantation requires less number of cells than for restoration of whole liver function
Therapy for one disease can be transferred for other similar diseases
Derivation of Hepatocyte-Like Cells (HLC) from hpSC to cure metabolic liver diseases caused by genetic defect: restoration of missing liver function
hpSC Definitive endoderm (DE) derivation
DE-specification toward hepatocyte fate
Hepatocyte development
Hepatocyte-like cells maturation
0 3 8 13 18 …
Activin AWnt3A
FGF4BMP2
HGF OSM Dex
Differentiation days
Developmental signaling
Derivation of hepatocyte-like cellsfrom hpSC
hpSC demonstrate appropriate gene expression dynamics during differentiation into definitive endoderm
hpSC Definitive endoderm (DE) derivation
DE-specification toward hepatocyte fate
Hepatocyte development
Hepatocyte-like cells maturation
0 3 8 13 18 …
Activin AWnt3A
FGF4BMP2
HGF OSM Dex
Differentiation days
Developmental signaling
hpSC demonstrate appropriate developmental transitions during differentiation into definitive endoderm
hpSC pretreatment by histone deacetylase inhibitor TSA enhances efficiency of definitive endoderm production
TSA pretreatment enhances efficiency of definitive endoderm production and decreases the number of remaining OCT4-positive cells
2D culture system/ petri dish 3D-ECM system
Other cell types in population
Definitive endoderm
Definitive endoderm
Other cell types in population
92%51% 49% 8%
3D-ECM differentiation system enables derivation of high-purity definitive endoderm from hpSC
CXCR4 CXCR4
hpSC Definitive endoderm (DE) derivation
DE-specification toward hepatocyte fate
Hepatocyte development
Hepatocyte-like cells maturation
0 3 8 13 18 …
Activin AWnt3A
FGF4BMP2
HGF OSM Dex
Differentiation days
Developmental signaling
Early hepatocyte progenitors derived from hpSC have appropriate hepatocyte morphology, and start expression of AFP and albumin
Marker expression by Marker expression by early hepatocyte progenitors derived from hpSC hpSC
Hepatocyte-like cells (HLC) derived from hpSC express appropriate markers
hpSC Definitive endoderm (DE) derivation
DE-specification toward hepatocyte fate
Hepatocyte development
Hepatocyte-like cells maturation
0 3 8 13 18 …
Activin AWnt3A
FGF4BMP2
HGF OSM Dex
Differentiation days
Developmental signaling
HLC
HepG2
Hepatocyte-like cells derived from hpSC positive in glycogen storage (PAS test) and ICG test
ICG testPAS test
glycogen storage
Hepatocyte-like cells derived from hpSC demonstrate P450 activity
Pentoxyresorufin O-Dealkylase (PROD) assay
CYP7A1
CYP7A1
HLC
HepG2
Human parthenogenetic stem cells:
RPE to treat retinal degenerative disease
Human parthenogenetic stem cells:
RPE to treat retinal degenerative disease
Isolation of retina pigment epithelium (RPE) cells derived from human parthenogenetic stem cells (hpSC)
RPE cells derived from hpSC demonstrate appropriate RPE morphology and produce pigment
RPE derived form hpSC form “dome” structures in vitro
Marker expression by RPE derived from hpSC
•High-purity differentiated cell populations can be derived from hpSC using direct differentiation procedures as well as spontaneous differentiation protocols.
•Direct differentiation protocols should be optimized to be applied to hpSC.
Conclusions
ISCOAgapova LarissaTurovets IrinaAgapov Vladimir Kochetkova Olga
Acknowledgments
Definitive endoderm
ViaCyte/NovocellEd BaedgeKevin D’AmourMark Moorman
Hepatocyte-like cells
University of PittsburgStephen Strom
RPE
UC IrvineHans KeirsteadMagdalene SeilerGabriel Nistor
Funding
Research was funded by ISCO and listed collaborators