Original article from STEM CELLS

Dual Inhibition of Src and GSK3 Maintains mESCs, Whose Differentiation Is Mechanically Regulated by Src Signaling

mESCs (NIH3T3) were grown on gelatin-coated polyacrylamide gels with a range of different elasticities in differentiation inducing conditions (with DMEM/10% FBS without LIF).   A soft 7.5 kPa (kilopascals) substrate allowed for more extensive spreading of mESCs associated with differentiation and it was noted that cells exhibited the highest level of overall tyrosine phosphorylation on this substrate of all the substrate elasticities tested.    Gene expression analysis of cells grown without LIFon the 7.5 kPa substrate, as compared to the stiff 67 kPa substrate, found a decrease in Oct4, Sox2, Klf4 and Tbx3 levels and an increase in brachyury, T, Gata6, and Foxa2 levels suggesting that mechanical factors play a role in the induction of early mesoderm and endoderm.   Teratomas were also larger following transplantation of mESCs grown on the 67 kPa substrate, overall suggesting that mESCs are more inclined to differentiate on the softer 7.5 kPa substrate without LIF.

Src, a proto-oncogene tyrosine-protein kinase, has been suggested to play a role in mechanotransduction via a pathway that engages MAPK (Mitogen-activated protein kinase) (Boutahar et al) and this study demonstrated that MAPK activity was enhanced in cells grown on the 7.5 kPa substrate compared to the other elasticities.   Inhibition of MAPK by a MEK1/2 inhibitor (PD0325901) or Src inhibition (CGP77675) restored the pluripotent morphology of mESCs grown on the 7.5 kPa substrate without LIF, and restored the loss of pluripotency associated alkaline phosphatase (AP)⁺ mESC colonies observed without the inhibitors.   Furthermore, Src can phosphorylate ShcA proteins, phosphotyrosine-dependent docking proteins, to allow for Grb2-Sos complex binding leading to MAPK activations (van der Greer et al).   Subsequently, it was found that tyrosine phosphorylation of p46Shc and p52Shc, the major ShcA isoforms expressed in mESCs, was enhanced in mESCs cultured on the 7.5 kPa substrate. Inhibition of Src with CGP77675 inhibited MAPK activation and phosphorylation of ShcA and the loss of MAPK activity upon shRNA-mediated knockdown of ShcA in mESCs cultured on the 7.5 kPa substrate all suggest that Src-ShcA-MAPK signaling is involved in the mechanical regulation of mESCs.    This was confirmed by the restoration of the pluripotent morphology of the mESCs on the 7.5 kPa substrate without LIF by RNAi mediated knock-down of Src, MAPK or ShcA.

mESCs grown in serum-free N2/B27 supplemented medium with the dual inhibition of MAPK and GSK3 signaling pathways (2i) (Ying et al) maintain their pluripotency and pluripotent morphology on standard culture plastics.   In this study, mESCs cultured in serum-free N2/B27 medium showed a flattened spread morphology on the 7.5 kPa substrates even under 2i conditions and exhibited enhanced activity of Src and FAK (Focal adhesion kinase).   However, mESCs maintained their pluripotent-like morphology on the 67 kPa substrate and expressed higher levels of Nanog and Oct4 and lower levels of Fgf5 and Sox1 than cells grown on the softer substrates.   Interestingly, Src inhibition of mESCs grown on the softer substrates restored the undifferentiated morphology and the expression of Oct4 and Nanog suggesting that Src inhibition could complement GSK3 inhibition in place of MAPK inhibition.    The Src inhibitor (CGP77675) alone could not support the growth and survival of mESCs on gelatin alone, but when combined with a GSK3 inhibitor (CHIR99021) could allow for growth as observed under 2i conditions.   GSK3/SRC inhibition (alternative 2i) allowed for long-term self-renewal in serum-free N2/B27 medium, with a 90% cellular homogeneity as measured by analysis of Oct4-GFP-positive mESCs and similar levels of Oct4, Nanog, Sox2, Klf4 and Tbx3 expression compared to 2i conditions.    Teratoma formation using mESCs grown under alternative 2i conditions also gave similar results to mESCs grown under conventional 2i conditions, displaying comparable tumour weights and germ layer differentiation.   ESC-derivation was also analysed and it was demonstrated that alternative 2i plus LIF conditions was highly efficient in deriving ESCs, while subtracting LIF lead to a decrease in derivation.   The derived mESCs also showed homogeneous expression of Oct4 and Nanog and were demonstrated to contribute to chimeric mice production with transmission to the germline.

These findings suggest that integrated regulation that involves both mechanical and soluble factors is important for mESC self-renewal and differentiation, providing new insights into Src-mediated regulation of mESC differentiation and suggesting that the dual inhibition of GSK3 and Src may provide a versatile tool for both the maintenance and derivation of mESCs.

References

Boutahar N et al. Mechanical strain on osteoblasts activates autophosphorylation of focal adhesion kinase and proline-rich tyrosine kinase 2 tyrosine sites involved in ERK activation. J Biol Chem 2004; 279: 30588–30599.

Chowdhury F et al. Soft substrates promote homogeneous self-renewal of embryonic stem cells via downregulating cell-matrix tractions. PLoS One 2010; 5: e15655.
Shimizu T et al. Dual Inhibition of Src and GSK3 Maintains Mouse Embryonic Stem Cells, Whose Differentiation Is Mechanically Regulated by Src Signaling. Stem Cells. 2012; 30 :1394–404.

Sun Y, et al. Mechanics regulates fate decisions of human embryonic stem cells. PLoS One. 2012; 7: e37178.
van der Geer P et al. The Shc adaptor protein is highly phosphorylated at conserved, twin tyrosine residues (Y239/240) that mediate protein-protein interactions.

Curr Biol 1996; 6: 1435–1444.
Ying QL et al. The ground state of embryonic stem cell self-renewal. Nature 2008; 453: 519–523.

STEM CELLS correspondent Stuart Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.