Derivation of highly enriched cultures of differentiated cells from human parthenogenetic stem cells California Institute ...
Parthenogenetic stem cells are a way to create immune-matching cells for transplantation
<ul><li>Typical hESC morphology; </li></ul><ul><li>Diploid karyotype 46, XX </li></ul><ul><li>Express appropriate markers;...
Human parthenogenetic stem cells: hepatocytes to treat liver dysfunctions
<ul><li>Target diseases  (over 100 000 cases):   </li></ul><ul><li>OTC deficiency </li></ul><ul><li>α 1-antitrypsin defici...
Differentiation days Developmental signaling Derivation of hepatocyte-like cells from hpSC hpSC Definitive endoderm (DE) d...
hpSC demonstrate appropriate gene expression dynamics during differentiation into definitive endoderm Differentiation days...
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...
2D culture system/ petri dish 3D-ECM system Other  cell types in population Definitive endoderm Definitive endoderm Other ...
Differentiation days Developmental signaling Early hepatocyte progenitors derived from hpSC have appropriate hepatocyte mo...
Marker expression by  early hepatocyte progenitors derived from  hpSC
Hepatocyte-like cells (HLC) derived from hpSC express appropriate markers Differentiation days Developmental signaling HLC...
Hepatocyte-like cells derived from hpSC positive in glycogen storage  (PAS test) and ICG test ICG test PAS test glycogen s...
Hepatocyte-like cells derived from hpSC demonstrate P450 activity  Pentoxyresorufin O-Dealkylase (PROD) assay CYP7A1 CYP7A...
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
<ul><li>High-purity differentiated cell populations can be derived from hpSC using direct differentiation procedures as we...
ISCO Agapova Larissa Turovets Irina Agapov Vladimir  Kochetkova Olga Acknowledgments Definitive endoderm ViaCyte/Novocell ...
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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

Published in: Health & Medicine
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  • 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.
  • Derivation of highly enriched cultures of differentiated cells from human parthenogenetic stem cells

    1. 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. 2. Parthenogenetic stem cells are a way to create immune-matching cells for transplantation
    3. 3. <ul><li>Typical hESC morphology; </li></ul><ul><li>Diploid karyotype 46, XX </li></ul><ul><li>Express appropriate markers; </li></ul><ul><li>Demonstrate high levels of alkaline phosphatase; </li></ul><ul><li>Demonstrate high levels of telomerase activity; </li></ul><ul><li>Form embryoid bodies in suspension culture; </li></ul><ul><li>Form teratomas after injection to immunodeficient animals; </li></ul><ul><li>Give differentiated derivatives of all three embryonic germ layers; </li></ul>hpSC demonstrate characteristics of hESC
    4. 4. Human parthenogenetic stem cells: hepatocytes to treat liver dysfunctions
    5. 5. <ul><li>Target diseases (over 100 000 cases): </li></ul><ul><li>OTC deficiency </li></ul><ul><li>α 1-antitrypsin deficiency </li></ul><ul><li>Crigler-Naijar syndrome </li></ul><ul><li>… </li></ul><ul><ul><li>Restoration of one liver function/activity </li></ul></ul><ul><ul><li>To rescue patient, FULL restoration of function is NOT NECESSARY </li></ul></ul><ul><ul><li>Immature cells already demonstrate required activities </li></ul></ul><ul><ul><li>Transplantation requires less number of cells than for restoration of whole liver function </li></ul></ul><ul><ul><li>Therapy for one disease can be transferred for other similar diseases </li></ul></ul>Derivation of Hepatocyte-Like Cells (HLC) from hpSC to cure metabolic liver diseases caused by genetic defect: restoration of missing liver function
    6. 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. 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. 8. hpSC demonstrate appropriate developmental transitions during differentiation into definitive endoderm
    9. 9. hpSC pretreatment by histone deacetylase inhibitor TSA enhances efficiency of definitive endoderm production
    10. 10. TSA pretreatment enhances efficiency of definitive endoderm production and decreases the number of remaining OCT4-positive cells
    11. 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. 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. 13. Marker expression by early hepatocyte progenitors derived from hpSC
    14. 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. 15. Hepatocyte-like cells derived from hpSC positive in glycogen storage (PAS test) and ICG test ICG test PAS test glycogen storage
    16. 16. Hepatocyte-like cells derived from hpSC demonstrate P450 activity Pentoxyresorufin O-Dealkylase (PROD) assay CYP7A1 CYP7A1 HLC HepG2
    17. 17. Human parthenogenetic stem cells: RPE to treat retinal degenerative disease
    18. 18. Isolation of retina pigment epithelium (RPE) cells derived from human parthenogenetic stem cells (hpSC)
    19. 19. RPE cells derived from hpSC demonstrate appropriate RPE morphology and produce pigment
    20. 20. RPE derived form hpSC form “dome” structures in vitro
    21. 21. Marker expression by RPE derived from hpSC
    22. 22. <ul><li>High-purity differentiated cell populations can be derived from hpSC using direct differentiation procedures as well as spontaneous differentiation protocols. </li></ul><ul><li>Direct differentiation protocols should be optimized to be applied to hpSC. </li></ul>Conclusions
    23. 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
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