Epithelial disruption drives mesendoderm differentiation in human pluripotent stem cells by enabling TGF-β protein sensing

Abstract

The processes of primitive streak formation and fate specification in the mammalian epiblast rely on complex interactions between morphogens and tissue organization. Little is known about how these instructive cues functionally interact to regulate gastrulation. We interrogated the interplay between tissue organization and morphogens by using human induced pluripotent stem cells (hiPSCs) downregulated for the morphogen regulator GLYPICAN-4, in which defects in tight junctions result in areas of disrupted epithelial integrity. Remarkably, this phenotype does not affect hiPSC stemness, but impacts on cell fate acquisition. Strikingly, cells within disrupted areas become competent to perceive the gastrulation signals BMP4 and ACTIVIN A, an in vitro surrogate for NODAL, and thus differentiate into mesendoderm. Yet, disruption of epithelial integrity sustains activation of BMP4 and ACTIVIN A downstream effectors and correlates with enhanced hiPSC endoderm/mesoderm differentiation. Altogether, our results disclose epithelial integrity as a key determinant of TGF-β activity and highlight an additional mechanism guiding morphogen sensing and spatial cell fate change within an epithelium.

Substrate Stiffness Reshapes Layer Architecture and Biophysical Features of Human Induced Pluripotent Stem Cells to Modulate their Differentiation Potential

“…Through our rescue experiments, we present evidence that the kinetics of TJ formation plays a crucial role in the regulation of hiPSC fate acquisition in response to differentiation factors. As per our previous findings, the loss of morphogen regulator GLYPICAN-4 in hiPSCs causes disruption in epithelial integrity with areas of TJ formation being affected 35 . This phenotype results in the Activin A receptors being exposed to the culture medium, thereby enhancing hiPSCs’ capacity to detect Activin A and maintain activation of the Activin A pathway over a prolonged period.…”

“…Interestingly, this disruption of epithelial integrity for hiPSCs grown on gels does not perturb selfrenewal or the expression of pluripotency genes, nor promotes premature expression of mesendoderm, endoderm or other lineage markers (present study and in 35,47 ). Thus, substrate stiffness-dependent alterations in TJs alone do not appear sufficient to promote differentiation or determine lineage fate choices.…”

“…Previously, we have shown that the strength of the hiPSC response to Activin A and the differentiation efficiency into the mesendoderm lineage can be enhanced by disrupting hiPSC epithelial integrity 35 . To understand whether changes in matrix stiffness alter the hiPSC epithelial layer, we used the TJ protein Zona Occludens 1 (ZO1) to map cell-cell contacts.…”

“…We next assessed whether disruption of the epithelial integrity in hiPSCs grown on gels could underlie their increased differentiation potential into mesendoderm and definitive endoderm. This was addressed by restoring the epithelial integrity in hiPSCs using lyso-phosphatidic acid (LPA), a small molecule known to accelerate epithelial organization and the formation of cell contacts through swelling of the apical membrane 35 . As shown in Figure 3A, a 24 hours treatment of hiPSCs grown on both PDMS gels with 5 µM LPA was sufficient to promote TJ organization and epithelial integrity of cells to levels comparable to those of cells grown on glass (almost fully organized following LPA treatment; Figure 3A).…”

In vitro modelling of anterior primitive streak patterning with human pluripotent stem cells identifies the path to notochord progenitors

“…Previous work using micropatterns and BMP or WNT ligands as the triggers of differentiation have shown that cells located at the colony boundary respond more efficiently to signalling molecules than cells in the centre due to a loss of epithelial integrity on the colony edges (Etoc et al, 2016; Legier et al, 2023; Martyn et al, 2018; Martyn et al, 2019; Warmflash et al, 2014). Our data indicate that a similar boundary-driven mechanism is likely taking place here as well given that the size of the peripheral differentiation domain remained constant with increasing colony sizes (Sup Fig 2).…”