Understanding the Activity of Mechanosensors YAP and TAZ in Differentiating Human Pluripotent Stem Cells

Abstract

Background: Human embryonic stem cells (hESCs) are pluripotent cells that have unique ability to self-renew and differentiate into specialized cell types. Due to these qualities, they have greater potential in stem-cell based regenerative medicine therapies, where hESCs are coaxed to differentiate into specific cell type. Multiple pre-clinical and clinical trials using stem-cell based therapies are on-going, however, critical aspect of cell survival and differentiation after transplantation still remains a challenge. Growth factors and chemical cues are well known regulators of stem cells proliferation and differentiation, but recent studies demonstrate a crucial role of biophysical signals in regulating stem cell proliferation and differentiation through mechanotransduction pathways. More importantly, data on mesenchymal stem cells (MSCs) have shown that the stiffness of the substrate on which the stem cells are cultured can regulate stem cell via Hippo pathway transcriptional coactivator, Yes-associated protein (YAP). However, how YAP regulates lineage specification in hESCs in response to mechanical signals still remains a mystery. Our study aims to understand the expression of YAP in pluripotent hESCs and in differentiated cells cultured on substrates of different stiffnesses. newlineMethodology: The hESCs line, KIND1 cells were first cultured on traditional plastic culture plates (TCP) in pluripotency sustaining medium, and were characterized for their pluripotency. Next, we cultured hESCs on soft substrates (0.2kPa, 0.5kPa, 2.0kPa, 8kPa, 16kpa, 32kPa, 64kPa) firstly in serum-free medium that supports pluripotency and later in medium with minimum serum which would encourages differentiation but without adding lineage-specific growth factors; followed by characterization of markers that define pluripotency and lineage specification. Further, we differentiated hESCs on TCP and soft substrates towards definitive endoderm lineage by using high concentrations of ACTIVIN A.