How is Parthenogenesis Done? (ANIMATED)

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San Diego Research Ethics Consortium,
Salk Institute for Biological Studies.
Workshop, Parthenogenetic stem cell lines: Ethical considerations.
8 July 2011, La Jolla, CA

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  • Results of these work were published in two papers of “Cloning and Stem Cells” Journal in this and last years.

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  • 1. Nikolay Turovets, PhD Director of Research and Therapeutic Development How is Parthenogenesis Done? San Diego Research Ethics Consortium Salk Institute for Biological Studies Parthenogenetic stem cell lines: Ethical considerations 8 July 2011, La Jolla, CA parthenogenesis.eventbrite.com www.turovets.com
  • 2. Parthenogenetic activation is a way to create pluripotent stem cells without disruption of the viable embryo Fertilization Parthenogenetic activation Ca 2+ time fertilization Ionomycin 5 min Ca 2+ time parthenogenetic activation ionomycin 6-DMAP or puromycin 4-5 hours 6-DMAP or puromycin Nikolay Turovets, PhD www.turovets.com
  • 3. Parthenogenetic activation Parthenogenetic activation is a way to create pluripotent stem cells without disruption of the viable embryo Fertilization No life possible Embryonic stem cells Parthenogenetic stem cells Nikolay Turovets, PhD www.turovets.com
  • 4. HLA heterozygous hpSC Immune-matching with donor HLA homozygous hpSC Immune-matching with people other than the donor Two types of human parthenogenetic stem cells (hpSC) Nikolay Turovets, PhD www.turovets.com Recipient Transplantation Recipients cells Recipient Donor Transplantation Donor cells
  • 5. 23 23 46 1pb 2pb 23 Embryonic stem cells 46 fertilization Nikolay Turovets, PhD www.turovets.com
  • 6. 46 1pb Ionomycin 6-DMAP 23 23 46 Heterozygous parthenogenetic stem cells 46 parthenogenetic activation: heterozygous stem cells fertilization Nikolay Turovets, PhD www.turovets.com
  • 7. parthenogenetic activation: heterozygous stem cells fertilization parthenogenetic activation: homozygous stem cells 23 46 1pb 2pb 23 Ionomycin puromycin Homozygous parthenogenetic stem cells 23 23 ? + = 46 Nikolay Turovets, PhD www.turovets.com
  • 8.
    • 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 Nikolay Turovets, PhD www.turovets.com
  • 9. HLA-heterozygous hpSC lines are totally immune-matched with the oocyte donors Nikolay Turovets, PhD www.turovets.com
  • 10. One HLA-homozygous hpSC line can be immune-matched with millions of people Nikolay Turovets, PhD www.turovets.com
  • 11. hpSCs are patented and published by ISCO Nikolay Turovets, PhD www.turovets.com
  • 12. hpSCs respond to well characterized differentiation signals and demonstrate appropriate gene expression dynamics and transitions Nikolay Turovets, PhD www.turovets.com
  • 13. Hepatocytes derived from hpSC demonstrate appropriate functions in vitro and in vivo ICG test PAS test glycogen storage PROD assay/P450 activity in vitro in vivo Nikolay Turovets, PhD www.turovets.com
  • 14. Differentiation capacity of hpSC: Retinal pigment epithelium Nikolay Turovets, PhD www.turovets.com
  • 15. Differentiation capacity of hpSC: Cornea-like structures CK 18-19 DAPI ZO-1 DAPI Nikolay Turovets, PhD www.turovets.com
  • 16. Major Collaborators Hepatocytes Cedars-Sinai Medical Center, LA Jeffrey Fair UC San Francisco Holger Willenbring Definitive endoderm ViaCyte/Novocell Ed Baedge Kevin D’Amour RPE UC Irvine Hans Keirstead Magdalene Seiler Methylation and global gene expression The Scripps Research Institute Jeanne Loring Louise Laurent Neurology University of Wurzburg, Germany Albrecht Muller Immunogenicity studies UC San Diego Ewa Carrier Matthias von Herrath University of Berlin, Germany Hans-Dieter Volk Genetic studies Genesis Genetics Institute Marcus Hughes Nikolay Turovets, PhD www.turovets.com