Assessment of human embryonic stem cells differentiation into definitive endoderm lineage on the soft substrates.

Pluripotent stem cells (PSCs) hold enormous potential for treating multiple diseases owing to their ability to self-renew and differentiate into any cell type. Albeit possessing such promising potential, controlling their differentiation into a desired cell type continues to be a challenge. Recent studies suggest that PSCs respond to different substrate stiffness and, therefore, can differentiate towards some lineages via Hippo pathway. Human PSCs can also differentiate and self-organize into functional cells, such as organoids. Traditionally, human PSCs are differentiated on stiff plastic or glass plates towards definitive endoderm and then into functional pancreatic progenitor cells in the presence of soluble growth factors. Thus, whether stiffness plays any role in differentiation towards definitive endoderm from human pluripotent stem cells (hPSCs) remains unclear. Our study found that the directed differentiation of human embryonic stem cells towards endodermal lineage on the varying stiffness did not differ from the differentiation on stiff plastic dishes. We also observed no statistical difference between the expression of yes-associated protein (YAP) and phosphorylated YAP. Furthermore, we demonstrate that lysophosphatidic acid, a YAP activator, enhanced definitive endoderm formation, whereas verteporfin, a YAP inhibitor, did not have the significant effect on the differentiation. In summary, our results suggest that human embryonic stem cells may not differentiate in response to changes in stiffness, and that such cues may not have as significant impact on the level of YAP. Our findings indicate that more research is needed to understand the direct relationship between biophysical forces and hPSCs differentiation.

Soft substrate maintains stemness and pluripotent stem cell-like phenotype of human embryonic stem cells under defined culture conditions.

Human embryonic stem cells (hESCs) are derived from the inner cell mass (ICM) of the pre-implantation blastocyst. Prior to embryo implantation, the ICM cells are surrounded by trophoblasts which have mechanical stiffness ranging from Pascal (Pa) to kilopascal (kPa). However, under in vitro conditions these cells are cultured on stiff tissue culture treated plastic plates (TCP) which have stiffness of approximately 1 gigapascal (GPa). This obvious dichotomy motivated us to investigate the fate of hESCs cultured on softer substrate, and to probe if the hESCs undergo differentiation or they retain pluripotency on soft substrates. We investigated the expression of pluripotency markers, and lineage-specific markers; we particularly looked at the expression of transcriptional coactivator YAP (Yes-associated protein), an important mediator of extracellular matrix (ECM) mechanical cues and a known downstream transducer of Hippo pathway. Downregulation of YAP has been correlated to the loss of multipotency of human mesenchymal stem cells (hMSCs) and pluripotency in mouse ESCs (mESCs); but we report that hESCs maintain their stemness on soft substrate of varying stiffness. Our findings revealed that on soft substrate hESCs express pluripotency markers and does not undergo substrate-mediated differentiation. Interestingly we show that hESCs maintained basal level of YAP expression for cell survival and proliferation, but YAP expression does not correlate directly with pluripotency in hESCs. To summarize, our results show that hESCs retain their stemness on soft substrate despite downregulation of YAP. Supplementary Information: The online version contains supplementary material available at 10.1007/s10616-022-00537-z.

Biomaterials Regulate Mechanosensors YAP/TAZ in Stem Cell Growth and Differentiation.

Tissue-resident stem cells are surrounded by a microenvironment known as 'stem cell niche' which is specific for each stem cell type. This niche comprises of cell-intrinsic and -extrinsic factors like biochemical and biophysical signals, which regulate stem cell characteristics and differentiation. Biochemical signals have been thoroughly studied however, the effect of biophysical signals on stem cell regulation is yet to be completely understood. Biomaterials have aided in addressing this issue since they can provide a defined and tuneable microenvironment resembling in vivo conditions. We review various biomaterials used in many studies which have shown a connection between biomaterial-generated mechanical signals and alteration in stem cell behaviour. Researchers probed to understand the mechanism of mechanotransduction and reported that the signals from the extracellular matrix regulate a transcription factor yes-associated protein (YAP)/transcriptional coactivator with PDZ-binding motif (TAZ), which is a downstream-regulator of the Hippo pathway and it transduces the mechanical signals inside the nucleus. We highlight the role of the YAP/TAZ as mechanotransducers in stem cell self-renewal and differentiation in response to substrate stiffness, also the possibility of mechanobiology as the emerging field of regenerative medicines and three-dimensional tissue printing.