Derivation of highly enriched cultures of differentiated cells from human parthenogenetic stem cells
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California Institute for Regenerative Medicine (CIRM) & Medical Research Council (MRC) Human SCNT Workshop. 14 June, 2010, San Francisco, CA Workshop report: http://www.cirm.ca.gov/files/PDFs/Publications/Human_SCNT_Workshop_Report.pdf
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Derivation of highly enriched cultures of differentiated cells from human parthenogenetic stem cells
- 1. Derivation of highly enriched cultures of differentiated cells from human parthenogenetic stem cells California Institute for Regenerative Medicine Medical Research Council Human SCNT Workshop 14 June, 2010, San Francisco, CA Workshop report: http://www.cirm.ca.gov/files/PDFs/Publications/Human_SCNT_Workshop_Report.pdf Nikolay Turovets, PhD Director of Research and Therapeutic Development
- 2. Parthenogenetic stem cells are a way to create immune-matching cells for transplantation
- 4. Human parthenogenetic stem cells: hepatocytes to treat liver dysfunctions
- 6. Differentiation days Developmental signaling Derivation of hepatocyte-like cells from hpSC hpSC Definitive endoderm (DE) derivation DE-specification toward hepatocyte fate Hepatocyte development Hepatocyte-like cells maturation 0 3 8 13 18 … Activin A Wnt3A FGF4 BMP2 HGF OSM Dex
- 7. hpSC demonstrate appropriate gene expression dynamics during differentiation into definitive endoderm Differentiation days Developmental signaling hpSC Definitive endoderm (DE) derivation DE-specification toward hepatocyte fate Hepatocyte development Hepatocyte-like cells maturation 0 3 8 13 18 … Activin A Wnt3A FGF4 BMP2 HGF OSM Dex
- 8. hpSC demonstrate appropriate developmental transitions during differentiation into definitive endoderm
- 9. hpSC pretreatment by histone deacetylase inhibitor TSA enhances efficiency of definitive endoderm production
- 10. TSA pretreatment enhances efficiency of definitive endoderm production and decreases the number of remaining OCT4-positive cells
- 11. 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
- 12. Differentiation days Developmental signaling Early hepatocyte progenitors derived from hpSC have appropriate hepatocyte morphology, and start expression of AFP and albumin hpSC Definitive endoderm (DE) derivation DE-specification toward hepatocyte fate Hepatocyte development Hepatocyte-like cells maturation 0 3 8 13 18 … Activin A Wnt3A FGF4 BMP2 HGF OSM Dex
- 13. Marker expression by early hepatocyte progenitors derived from hpSC
- 14. Hepatocyte-like cells (HLC) derived from hpSC express appropriate markers Differentiation days Developmental signaling HLC HepG2 hpSC Definitive endoderm (DE) derivation DE-specification toward hepatocyte fate Hepatocyte development Hepatocyte-like cells maturation 0 3 8 13 18 … Activin A Wnt3A FGF4 BMP2 HGF OSM Dex
- 15. Hepatocyte-like cells derived from hpSC positive in glycogen storage (PAS test) and ICG test ICG test PAS test glycogen storage
- 16. Hepatocyte-like cells derived from hpSC demonstrate P450 activity Pentoxyresorufin O-Dealkylase (PROD) assay CYP7A1 CYP7A1 HLC HepG2
- 17. Human parthenogenetic stem cells: RPE to treat retinal degenerative disease
- 18. Isolation of retina pigment epithelium (RPE) cells derived from human parthenogenetic stem cells (hpSC)
- 19. RPE cells derived from hpSC demonstrate appropriate RPE morphology and produce pigment
- 20. RPE derived form hpSC form “dome” structures in vitro
- 21. Marker expression by RPE derived from hpSC
- 23. ISCO Agapova Larissa Turovets Irina Agapov Vladimir Kochetkova Olga Acknowledgments Definitive endoderm ViaCyte/Novocell Ed Baedge Kevin D’Amour Mark Moorman Hepatocyte-like cells University of Pittsburg Stephen Strom RPE UC Irvine Hans Keirstead Magdalene Seiler Gabriel Nistor Funding Research was funded by ISCO and listed collaborators
Editor's Notes
- The major activity of hepatocyte-like cells derived from hpSC – to restore to an OTCD patient’s missing liver function, specifically functioning urea cycle. Hepatocyte-like cells derived from hpSC could become a valuable source of cells for transplantation therapy of metabolic liver desorders caused by genetic defect: The direct differentiation procedure allow the derivation of unlimited numbers of high-purity hepatocyte-like cells from hpSC. Differentiated derivatives of hpSC may overcome limitations brought by histocompatibility issues because one hpSC line can be HLA matched with significant segments of the human population. Autologous cells cannot be used for treatment OTCD patients because they carry the same genetic defect, allogenetic HLA-matched hpSC-derived cells may overcome this “genetic defect” issue. The direct differentiation procedure allows the derivation of immature cells (progenitors) that have several advantages compared to adult liver cells: an increased proliferative capacity and plasticity; less immunogenicity; potentially superior adaptation and integration capacity; and a greater resistance to cryopreservation and ischemia.