pr7b00322_si_006.xls (10.67 kB)
Low Focal Adhesion Signaling Promotes Ground State Pluripotency of Mouse Embryonic Stem Cells
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posted on 2017-08-29, 00:00 authored by Sara Taleahmad, Mehdi Mirzaei, Azam Samadian, Seyedeh-Nafiseh Hassani, Paul A. Haynes, Ghasem Hosseini Salekdeh, Hossein BaharvandMouse embryonic stem cells (mESCs)
can be maintained in a pluripotent
state when cultured with 2 inhibitors (2i) of extracellular signal-regulated
kinase (MEK) and glycogen synthase kinase-3 (GSK3), and Royan 2 inhibitors
(R2i) of FGF4 and TGFβ. The molecular mechanisms that control
ESC self-renewal and pluripotency are more important for translating
stem cell technologies to clinical applications. In this study, we
used the shotgun proteomics technique to compare the proteome of the
ground state condition (R2i- and 2i-grown cells) to that of serum.
Out of 1749 proteins identified, 171 proteins were differentially
expressed (p < 0.05) in the 2i, R2i, and serum
samples. Gene ontology (GO) analysis of differentially abundant proteins
showed that the focal adhesion signaling pathway significantly down-regulated
under ground state conditions. mESCs had highly adhesive attachment
under the serum condition, whereas in the 2i and R2i culture conditions,
a loss of adhesion was observed and the cells were rounded and grew
in compact colonies on gelatin. Quantitative RT-PCR showed reduced
expression of the integrins family in the 2i and R2i conditions. The
serum culture had more prominent phosphorylation of focal adhesion
kinase (FAK) compared to 2i and R2i cultures. Activity of the extracellular
signal-regulated kinase (ERK)1/2 decreased in the 2i and R2i cultures
compared to serum. Activation of integrins by Mn2+ in the
2i and R2i cultures resulted in reduced Nanog and
increased the expression of lineage marker genes. In this study, we
demonstrated that reduced focal adhesion enabled mESCs to be maintained
in an undifferentiated and pluripotent state.
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lineage marker genesGSKRoyan 2 inhibitorsFAKFGFpluripotent stateglycogen synthase kinase -3adhesionR 2i culture conditionsRT-PCRR 2iextracellular signal-regulated kinasemESCR 2i conditions2 i-grown cellsMEKR 2i culturescontrol ESC self-renewalproteinserumLow Focal Adhesion Signaling Promotes Ground State PluripotencyTGFERKground state conditionMouse Embryonic Stem Cells Mouseshotgun proteomics techniqueground state conditions2 i
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