Derivation of highly enriched cultures of differentiated cells from human parthenogenetic stem cells

transcript

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

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

0 3 8 13 18 …

Activin AWnt3A

FGF4BMP2

HGF OSM Dex

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

Other cell types in population

92%51% 49% 8%

3D-ECM differentiation system enables derivation of high-purity definitive endoderm from hpSC

CXCR4 CXCR4

0 3 8 13 18 …

Activin AWnt3A

FGF4BMP2

HGF OSM Dex

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

0 3 8 13 18 …

Activin AWnt3A

FGF4BMP2

HGF OSM Dex

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

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

UC IrvineHans KeirsteadMagdalene SeilerGabriel Nistor

Funding

Research was funded by ISCO and listed collaborators

Derivation of highly enriched cultures of differentiated cells from human parthenogenetic stem cells

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