how is parthenogenesis done? (animated)
TRANSCRIPT
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
www.turovets.com
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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olay
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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