Reduction of pluripotent gene expression in murine embryonic stem cells exposed to mechanical loading or Cyclo RGD peptide

Abstract

BackgroundSelf-renewal and differentiation of embryonic stem cells (ESCs) is directed by biological and/or physical cues that regulate multiple signaling cascades. We have previously shown that mESCs seeded in a type I collagen matrix demonstrate a loss of pluripotent marker expression and differentiate towards an osteogenic lineage. In this study, we examined if this effect was mediated in part through Arginylglycylaspartic acid (RGD) dependent integrin activity and/or mechano-transduction.ResultsThe results from this study suggest that mESC interaction with the local microenvironment through RGD dependent integrins play a role in the regulation of mESC core transcription factors (TF), Oct-4, Sox 2 and Nanog. Disruption of this interaction with a cyclic RGD peptide (cRGDfC) was sufficient to mimic the effect of a mechanical stimulus in terms of pluripotent gene expression, specifically, we observed that supplementation with cRGDfC, or mechanical stimulus, significantly influenced mESC pluripotency by down-regulating core transcription factors. Moreover, our results indicated that the presence of the cRGDfC peptide inhibited integrin expression and up-regulated early lineage markers (mesoderm and ectoderm) in a Leukemia inhibitory factor (LIF) dependent manner. When cRGDfC treated mESCs were injected in Severe combined immunodeficiency (SCID) mice, no tissue growth and/or teratoma formation was observed, suggesting that the process of mESC tumor formation in vivo is potentially dependent on integrin interaction.ConclusionsOverall, the disruption of cell-integrin interaction via cRGDfC peptide can mimic the effect of mechanical stimulation on mESC pluripotency gene expression and also inhibit the tumorigenic potential of mESCs in vivo.

Long‐Term Perfusion Culture of Monoclonal Embryonic Stem Cells in 3D Hydrogel Beads for Continuous Optical Analysis of Differentiation

“…Cell viability was assessed by live/dead flow cytometry based on calcein/ethidium homodimer staining (see the Supporting Information for details). The on‐chip de‐emulsification did not affect cell viability (Figure D; Figure S3, Supporting Information), based on cell growth and reporter gene expression as readouts . In comparison, the de‐emulsification using the fluorosurfactant PFO (as described above and in the Supportin Information) resulted in dramatically lower cell viability that decreased by an order of magnitude with increasing amount of PFO to survival rate of ≈10% with 100% PFO.…”

“…The on-chip de-emulsification did not affect cell viability ( Figure 2D; Figure S3, Supporting Information), based on cell growth and reporter gene expression as readouts. [46,47] In comparison, the de-emulsification using the fluorosurfactant PFO (as described above and in the Supportin Information) resulted in dramatically lower cell viability that decreased by an order of magnitude with increasing amount of PFO to survival rate of ≈10% with 100% PFO. Consequently, the onchip de-emulsification using the developed extraction chip obviates the need for a chemical de-emulsifier, avoiding its detrimental effects on cell survival while facilitating integration of a de-emulsification step into automated workflows.…”

Unchain My Heart: Integrins at the Basis of iPSC Cardiomyocyte Differentiation

“…Specifically, this molecule or mechanical stimulus significantly influenced ESC pluripotency by downregulating core transcription factors. Moreover, RGD peptide, by inhibiting integrin binding and, in turn, integrin expression [6], upregulated early lineage markers (mesoderm and ectoderm) by leukaemia inhibitory factor (LIF) signalling [138]. Interestingly, human ESC, expressing integrin α 6 β 1, preferentially bind human recombinant laminin-111, laminin-332, and laminin-511, which are good substrates able to maintain undifferentiated pluripotent human ESC cultures [139].…”

A high throughput screening system for studying the effects of applied mechanical forces on reprogramming factor expression

“…In addition, mechanical force can lead to chromatin remodeling and altered binding of transcription factors 17 , 18 . Recent studies have also shown that application of mechanical strain can reduce the expression of pluripotency in mouse embryonic stem cells 19 , 20 . In human pluripotent stem cells (hPSCs), mechanical strain helped to maintain pluripotency through increased expression Nodal, transforming growth factor-β (TGF-β), and Activin 21 , 22 .…”