how is parthenogenesis done? (animated)

Nikolay Turovets, PhDDirector of Research and Therapeutic Development

How is Parthenogenesis Done?

San Diego Research Ethics Consortium Salk Institute for Biological Studies

Parthenogenetic stem cell lines: Ethical considerations8 July 2011, La Jolla, CA

parthenogenesis.eventbrite.com

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Parthenogenetic activation is a way to create pluripotent stem cells without disruption of the viable embryo

Fertilization Parthenogenetic activation

Ca2+

time

fertilization

Ionomycin5 min

Ca2+

time

parthenogenetic activation

ionomycin

6-DMAP or puromycin

4-5 hours

6-DMAP or puromycin

Nikolay Turovets, PhD www.turovets.com

Parthenogenetic activation

Parthenogenetic activation is a way to create pluripotent stem cells without disruption of the viable embryo

Fertilization

No life possible

Embryonicstem cells

Parthenogeneticstem cells

Nikolay Turovets, PhD www.turovets.com

HLA heterozygous hpSC

Immune-matching with donor

HLA homozygous hpSC

Immune-matchingwith people other than the

donor

Recipient

Transplantation

Recipients cells

Recipient Donor

TransplantationDonor cells

Two types of human parthenogenetic stem cells (hpSC)

Nikolay Turovets, PhD www.turovets.com

2323

46

1pb2pb

23

Embryonicstem cells

46fertilization

Nikolay Turovets, PhD www.turovets.com

46

1pbIonomycin

6-DMAP

232346

Heterozygousparthenogenetic

stem cells

46

parthenogenetic activation: heterozygous stem cells

fertilization

Nikolay Turovets, PhD www.turovets.com

parthenogenetic activation: heterozygous stem cells

fertilization

parthenogenetic activation: homozygous stem cells

23

46

1pb2pb

23

Ionomycin

puromycin

Homozygousparthenogenetic

stem cells

2323?+ = 46

Nikolay Turovets, PhD www.turovets.com

• 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

HLA-heterozygous hpSC lines are totally immune-matched with the oocyte donors

Nikolay Turovets, PhD www.turovets.com

One HLA-homozygous hpSC line can be immune-matched with millions of people

Nikolay Turovets, PhD www.turovets.com

hpSCs are patented and published by ISCO

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hpSCs respond to well characterized differentiation signals and demonstrate appropriate gene expression dynamics and transitions

Nikolay Turovets, PhD www.turovets.com

Hepatocytes derived from hpSC demonstrate appropriate functions in vitro and in vivo

ICG testPAS testglycogen storage

PROD assay/P450 activity

in vitro

in vivo

Nikolay Turovets, PhD www.turovets.com

Differentiation capacity of hpSC: Retinal pigment epithelium

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Differentiation capacity of hpSC: Cornea-like structures

CK

18-

19 D

API

ZO-1

DA

PI

Nikolay Turovets, PhD www.turovets.com

Major CollaboratorsHepatocytesCedars-Sinai Medical Center, LAJeffrey FairUC San FranciscoHolger Willenbring

Definitive endodermViaCyte/NovocellEd BaedgeKevin D’Amour

RPEUC IrvineHans KeirsteadMagdalene Seiler

Methylation and global gene expressionThe Scripps Research InstituteJeanne LoringLouise Laurent

NeurologyUniversity of Wurzburg, GermanyAlbrecht Muller

Immunogenicity studiesUC San DiegoEwa CarrierMatthias von HerrathUniversity of Berlin, GermanyHans-Dieter Volk

Genetic studiesGenesis Genetics InstituteMarcus Hughes

Nikolay Turovets, PhD www.turovets.com