Modulation of pluripotency in the porcine embryo and i ps cells (Dec.27,2012)


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Enhancement of the pluripotency (turning cells to pluripotent stage)

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Modulation of pluripotency in the porcine embryo and i ps cells (Dec.27,2012)

  1. 1. Modulation of Pluripotency in the Porcine Embryo and iPS Cells Rodríguez A, Allegrucci C, Alberio R. (2012). PLoS ONE 7(11): e49079. doi:10.1371/journal.pone.0049079 Speakers : Ahmad - Jay – Nan – Fred – Jason 2012-12-27
  2. 2. Introduction Stem CellsEmbryonic Stem Cells (ESC) Adult Stem Cells 1-Totipotent Stem Cells 2-Pluripotent Stem Cells
  3. 3. IntroductionTotipotent Pluripotent
  4. 4. Introduction *NANOG: - NANOG is a gene expressed in embryonic stem cells (ESCs) and is thought to be a key factor in maintaining pluripotency. - Pluripotency marker gene. -NANOG is thought to function in concert with other factors such as: POU5F1 (Oct-4) and SOX2 to establish ESC identity.* GATA family :-A family of transcription factors characterized by their ability to bind to the DNAsequence "GATA" which linked with embryonic cardiac development.Ex: GATA4 & GATA6
  5. 5. Introduction*FGF (Fibroblast Growth Factors) Signalling Pathway:- The FGFs are heparin-binding proteins involved in angiogenesis, wound healing, andembryonic development.- FGFs are key players in the processes of proliferation and differentiation of wide variety ofcells and tissues.*MEK Signalling Pathway:- MEK is a member of kinases involved in cell growth, cell proliferation and cellsurvival.*Wnt Signalling Pathway:- Wnt is a network of proteins that controls cell-cell communication in the embryoand adult.
  6. 6. Introduction Aim of this Study : -Investigation the signalling pathways participating in the formation of the porcine ICM (Inner Cell Mass).- Establishing whether their modulation can be used to increase the developmentalpotential of pluripotent cells (Pluripotency).
  7. 7. Materials and Methods
  8. 8. Embryo Collection and In Vitro Culture
  9. 9. Porcine Fetal FibroblastsIsolation, Reprogramming and Cell Culture
  10. 10. Immunocytochemistry and AlkalinePhosphatase Activity
  11. 11. Quantification of Total and ICM Cell Numbers in Embryos epifluorescence.
  12. 12. RNA Isolation and Polymerase ChainReaction
  13. 13. In Vitro Differentiation hanging drop method
  14. 14. Result
  15. 15. Culture of Porcine Embryos to the Late BlastocystStage
  16. 16. Activin/nodal Copyright © 2000-2012 eBioscience, Inc
  17. 17. Effect of FGF Signalling During HypoblastSegregation in the Porcine Embryo
  18. 18. Effect of GSK3b, JAK/STAT3 and ALK5 Signalling inhibition During ICM Segregation in the Porcine EmbryoMEKi+GSK3βi
  19. 19. iPS Change to N2B27 medium
  20. 20. MEK and GSK3β Inhibition +LIF Promotes Naïve iPS CellsLIF withdrawal or JAK/STAT3 inhibition of cells grown with FCS resulted in a rapid lossof their compact morphology. These cells showed overt signs of differentiation, whichwas confirmed by the expression of SOX17 and NODAL and the reduction in NANOGexpression.
  21. 21. 2i/3i + LIF activated genes indicative of naive pluripotencyoverall the cultures in 3i(MEKi, GSK3βi, FGFRi) were more homogeneous and showed low levels of differentiation
  22. 22. Colonies of cells grown in 3i + LIFSTELLA expression correlated with changes in NANOG, OCT-4 and REX1expression, but were not correlated with FGF5 expression, however high variabilitywas detected between different lines
  23. 23. Differentiation potential of iPS cellsgrown under different conditions
  24. 24. Naive piPS have Increased Germline Differentiation PotentialpiPS cells expressing STELLA can be efficiently induced to initiate the germ celldifferentiation program in vitro.
  25. 25. Conclusion 26
  26. 26. • Culture condition optimized  PZM3  X - Low total cell count, small ICM  N2B27  O - Increasing hatching rate - Last blastocyst development 27
  27. 27. Early epiblast cells segregation Hypoblast cells (NANOG+) (GATA-4+)Efrat Oron and Natalia Ivanova 2012 Phys. Biol. 9 045002 28
  28. 28. • What signaling pathway effect hypoblast segregation (GATA-4 expression) Others ? phospholipas C, etc. 29tml
  29. 29. • What signaling pathway participate in the maintenance of NANOG expression 30
  30. 30. 31
  31. 31. • MEK, GSK3β inhibition + LIF can impose porcine embryos into a naive state, but cannot be used to capture NANOG-only ICM cells.• iPSCs also can be imposed into naive state.• STELLAhigh iPSCs had increased capability to differentiate to VASA-expressing germ cell precursors. 32
  32. 32. Thank you for your attention! Any question? 33