A semi-interpenetrating network of polyacrylamide and recombinant basement membrane allows pluripotent cell culture in a soft, ligand-rich microenvironment

Abstract

The physical properties of the extracellular matrix play an essential role in guiding stem cell differentiation and tissue morphogenesis both in vivo and in vitro. Existing work to investigate the role of matrix mechanics in directing stem cell proliferation, self-renewal, and differentiation has been limited by the poor attachment and survival of human pluripotent cells cultured on soft matrices (Young's modulus E ≲ 1000 Pa). To address this limitation we developed a protocol for generating semi-interpenetrating networks of polyacrylamide and recombinant basement membrane. Using these materials, we found that human embryonic stem cells (hESCs) remained proliferative and pluripotent even when grown in small colonies and on surfaces ranging in stiffness from 150 to 12000 Pa, spanning the range of tissue stiffnesses likely to be encountered in the embryo. Considerable recent attention has focused on the role of the transcriptional coactivator and Hippo effector YAP in regulating differentiation and cell proliferation both in the early embryo and in vitro. We found that while YAP localized to the nucleus on substrates of E ≳ 1000 Pa, its localization was heterogeneous on substrates of moduli ≲ 450 Pa, with predominantly nuclear localization at the colony periphery and mixed cytoplasmic and nuclear localization for cells in the colony interior, a pattern reminiscent of YAP subcellular localization in the inner cell mass (ICM) of the early embryo. In addition, hESC colony dynamics were highly responsive to substrate stiffness, with cells assembling into monolayers, multilayer structures, and transient, hollow rosettes in response to decreasing substrate stiffnesses in the range of 12000 to 150 Pa. We suggest that soft, ligand-rich substrates such as are described here provide a promising means of recapitulating aspects of early mammalian development that are otherwise inaccessible, and more broadly may be useful in the derivation of complex tissues from pluripotent cells in an in vitro setting.

Evidence for the Desmosomal Cadherin Desmoglein-3 in Regulating YAP and Phospho-YAP in Keratinocyte Responses to Mechanical Forces

“…YAP has been identified as a mechanosensor independent of the Hippo pathway, with its nuclear relocation being regulated by mechanical strain and substrate stiffness involving Rho GTPase activity and tension of the actomyosin cytoskeleton [2, 4,6,36,37]. Unexpectedly, and apparently, paradoxically, nuclear retention, or retardation of nuclear export, of YAP/pYAP was detected by immunofluorescence in Dsg3 knockdown cells at low cell densities on coverslips but also in confluent cultures in Flexcell wells subjected to cyclic strain.…”

“…YAP has been identified as a mechanosensor [2] with nuclear relocation of YAP being regulated by mechanical strain [36] and substrate stiffness [37]. To elucidate the underlying mechanism of how Dsg3 regulates junction formation induced by mechanical loading, we investigated YAP and phospho-YAP-S127 (pYAP) expression in a time-course experiment in HaCaTs with exposure to the strain for 6 h. In this case, cells were transferred to the stationary state after straining (relaxation) in an incubator, and total lysates were then extracted at various time points at 0, 2, 6, 24, 48, and 72 h, alongside with static control cells ( Figure 3A).…”

The desmosomal cadherin Desmoglein-3 regulates YAP and phospho-YAP in keratinocyte responses to mechanical forces

“…YAP has been identified as a mechanosensor independent of the Hippo pathway, with its nuclear relocation being regulated by mechanical strain and substrate stiffness involving Rho GTPase activity and tension of the actomyosin cytoskeleton [16,24,37,39,41]. Unexpectedly, and apparently paradoxically, nuclear retention, or retardation of nuclear export, of YAP/pYAP was detected by immunofluorescence in Dsg3 knockdown cells at low cell densities on coverslips but also in confluent cultures in Flexcell wells subjected to cyclic strain.…”

“…YAP has been identified as a mechanosensor [39] with nuclear relocation of YAP being regulated by mechanical strain [24] and substrate stiffness [41]. To elucidate the underlying mechanism of how Dsg3 regulates junction formation induced by mechanical loading, we investigated YAP and pYAP-S127 (pYAP) expression in a time-course experiment in HaCaTs with exposure of strain for 6 hours.…”

Emerging Methods for Enhancing Pluripotent Stem Cell Expansion

“…The effects of stiffness observed in mESCs are not translated to hPSCs. There is limited consensus on the effects of stiffness, with some groups finding little to no influence of stiffness on pluripotency (Keung et al, 2012;Maldonado et al, 2015;Przybyla et al, 2016;Price et al, 2017) and others finding the opposite (Musah et al, 2012;Sun et al, 2012;Kim et al, 2018;Sung et al, 2018). Nonetheless, observations made by several groups imply that stiffer substrates are more suitable for hPSC expansion.…”