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Triploid pluripotent stem cells

Triploid pluripotent stem cells

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Triploid pluripotent stem cells

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  1. Triploidpluripotent stem cells Reporter:XueBinghua Date:2011.06.15

  2. Human oocytes reprogram somatic cells to a pluripotent state Scott Noggle & Dieter Egli Nature 1 August 2011

  3. Background • Limit:Legal and social considerations • Different:iPS and blastocyst-derived stem cells (gene expression,DNA methylation and differentiation potential) • Compromise: Genomic integrity (de novo mutations and copy number variations)

  4. Results: 1、Development fails after somatic genome replacement

  5. Schematic of genome exchange in human oocytes

  6. 38/50 43/43 (12 without) Skin cells (♂T1D and ♂WT ) GFP CAGGS promoter:(H2B):GFP MII oocyte & Donor cell

  7. 35/35 Chromosome condensation

  8. Normal cleavage :22/31(71%) Arrests at a stage of 6–10 cells Parthenotes:13/52 ( 25%) IVF:16/21(76%) Developmentalpotential & Development arrests

  9. 6-8h Control for the quality of the oocytes

  10. 32/32 4/7

  11. Results: 2、Development arrests with transcriptional defects

  12. 6-12cells *ZGA starts at the 4–8 cell stage28, or on day 3 of development*Upregulatedtranscripts incomparison to unfertilized oocytes 761 transcripts upregulated at zygotic genome activation 124 (16%) were upregulated after genome exchange 62 (8%) were upregulated after amanitin treatment 536(70%) were upregulated at parthenotes*1,406 genes expressat higher levels in the skin donor fibroblasts than in MIIoocytesGADD45G is involved in stress-induced cell cycle arrest

  13. Results: 3、The oocyte genome rescues development

  14. Schematic of somatic genome transfer

  15. Developmental progression

  16. Developmental potential

  17. Results: 4、Derivation of pluripotent stem cells

  18. Karyotype & Sequence of the mitochondrion

  19. Pluripotency

  20. 463/1327 670/1327 Global gene expression analysis &Up and down *463:Lin28a,Oct4,Sox2, Nanog ,Lefty1 *670:FAP, PAPPA, MMP3,CTHRC1 SNAI2 *28 genes and 24 genes were expressed at higher levels in soPS2 and soPS1 than NYSCF1 neuropeptide galanin, SRY, rex1 , neuronatin, NODAL, Cerberus 1 , Lefty2

  21. Results: 5、Epigenetic reprogramming

  22. Methylation of DNA at the Nanog promoter

  23. RT–PCR andSNP & DMR of PEG3

  24. Schematic of genome-wideSNP capture and allelotyping Use a genome-wide digital allelotyping approach to distinguish gene expression from the somatic cell-derived genome and the oocyte-derived genome in soPS cells

  25. Contribution of the reprogrammed genome

  26. Transcript ratio for the different cell genes

  27. Thank you!