Enhancement of the pluripotency (turning cells to pluripotent stage)

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. Introduction
Stem Cells
Embryonic Stem Cells (ESC) Adult Stem Cells
1-Totipotent Stem Cells
2-Pluripotent Stem Cells

3. Introduction
Totipotent
Pluripotent
http://dels-old.nas.edu/bls/stemcells/images/Embryonic-Download.JPG

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 DNA
sequence "GATA" which linked with embryonic cardiac development.
Ex: GATA4 & GATA6

5. Introduction
*FGF (Fibroblast Growth Factors) Signalling Pathway:
- The FGFs are heparin-binding proteins involved in angiogenesis, wound healing, and
embryonic development.
- FGFs are key players in the processes of proliferation and differentiation of wide variety of
cells and tissues.
*MEK Signalling Pathway:
- MEK is a member of kinases involved in cell growth, cell proliferation and cell
survival.
*Wnt Signalling Pathway:
- Wnt is a network of proteins that controls cell-cell communication in the embryo
and adult.

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 developmental
potential of pluripotent cells (Pluripotency).

7. Materials and Methods

8. Embryo Collection and In Vitro Culture

9. Porcine Fetal Fibroblasts
Isolation, Reprogramming and Cell Culture

10. Immunocytochemistry and Alkaline
Phosphatase Activity

11. Quantification of Total and ICM Cell
Numbers in Embryos
 epifluorescence.

12. RNA Isolation and Polymerase Chain
Reaction

13. In Vitro Differentiation
 hanging drop method

14. Result

15. Culture of Porcine Embryos to the Late Blastocyst
Stage

16. Activin/nodal
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17. Effect of FGF Signalling During Hypoblast
Segregation in the Porcine Embryo

18. Effect of GSK3b, JAK/STAT3 and ALK5 Signalling
inhibition During ICM Segregation in the Porcine
Embryo
MEKi+GSK3βi

20. iPS
Change to N2B27 medium

21. MEK and GSK3β Inhibition +LIF
Promotes Naïve iPS Cells
LIF withdrawal or JAK/STAT3 inhibition of cells grown with FCS resulted in a rapid loss
of their compact morphology. These cells showed overt signs of differentiation, which
was confirmed by the expression of SOX17 and NODAL and the reduction in NANOG
expression.

22. 2i/3i + LIF activated genes indicative of
naive pluripotency
overall the cultures in 3i(MEKi, GSK3βi, FGFRi) were more homogeneous and showed
low levels of differentiation

23. Colonies of cells grown in 3i + LIF
STELLA expression correlated with changes in NANOG, OCT-4 and REX1
expression, but were not correlated with FGF5 expression, however high variability
was detected between different lines

24. Differentiation potential of iPS cells
grown under different conditions

25. Naive piPS have Increased Germline
Differentiation Potential
piPS cells expressing STELLA can be efficiently induced to initiate the germ cell
differentiation program in vitro.

26. Conclusion
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27. • Culture condition optimized
 PZM3  X
- Low total cell count, small ICM
 N2B27  O
- Increasing hatching rate
- Last blastocyst development
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28. Early epiblast cells segregation Hypoblast cells
(NANOG+) (GATA-4+)
Efrat Oron and Natalia Ivanova 2012 Phys. Biol. 9 045002
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29. • What signaling pathway effect hypoblast
segregation (GATA-4 expression)
Others ?
phospholipas C, etc.
http://www.cellsignal.com/reference/pathway/ESC_pluripotency.h 29
tml

30. • What signaling pathway participate in the
maintenance of NANOG expression
http://www.cellsignal.com/reference/pathway/ESC_pluripotency.h
tml 30

31. 31

32. • 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.
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33. Thank you for your
attention!
Any question?
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