Dynamic blebbing: A bottleneck to human embryonic stem cell culture that can be overcome by Laminin-Integrin signaling

Abstract

This study characterizes dynamic and apoptotic blebbing in human embryonic stem cells (hESC), identifies dynamic blebbing as a bottleneck to successful cell attachment during passaging, and demonstrates that dynamic blebbing can be rapidly stopped by plating cells on recombinant human laminin. In freshly plated hESC, dynamic and apoptotic blebbing differed in time of occurrence, bleb retraction rate, mitochondrial membrane potential, and caspase 3&7 activation. While dynamic blebbing can be controlled with drugs that inhibit myosin II, these methods have off-target effects and are not suitable for clinical applications. Recombinant human laminin-521 or addition of laminin-111 to Matrigel provided a safe method to drastically decrease dynamic blebbing and improve cell attachment with proteins normally found in the inner cell mass. Inhibition of focal adhesion kinase, which is activated by binding of integrins to laminin, prolonged dynamic blebbing and inhibited attachment. These data show that hESC bind rapidly to laminins through an integrin, which activates focal adhesion kinase that in turn downregulates dynamic blebbing. Laminins enabled hESC to rapidly attach during passaging, improved plating efficiency, enabled passaging of single pluripotent stem cells, and avoided use of inhibitors that have non-specific off-target effects. These data provide a strategy for improving hESC culture using biologically safe recombinant human proteins.

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“…Their upregulation may have been involved in the formation of the calcium rich blebs seen in submerged cultures. Blebbing involving these proteins/pathways has been reported in response to calcium influx in human embryonic stem cells upon activation of the P2X7 receptor, which causes rapid influx of calcium (Guan et al, 2016;Weng et al, 2018;Weng and Talbot, 2017).…”

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“…This introduces the need to accurately count cells before plating for infection. hESCs can be seeded accurately as single cells using the ROCK inhibitor, Y27632, which blocks Rho‐associated coiled‐coil kinase and reduces cell blebbing and apoptosis (Ohgushi et al., 2010; Watanabe et al., 2007; Weng et al., 2018). Moreover, H9 hESCs can differentiate into the three germ layers (ectoderm, endoderm, and mesoderm) making it possible to apply pseudotyping to later stages of prenatal development.…”

“…This cell number was chosen as the appropriate plating density for H9 hESCs after testing at different MOIs. This protocol involves seeding H9 hESCs using ROCKi to improve single-cell survival (Ohgushi et al, 2010;Watanabe et al, 2007;Weng et al, 2018). Many downstream applications, such as direct differentiation, require accurate counting and plating of single hESCs.…”

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“…But there are some evidences that inhibition of FAK could stimulate metastasis ( Batista et al, 2014 ). It was shown that the inhibition of FAK in human embryonic stem cells prolonged non-apoptotic blebbing ( Weng et al, 2018 ) and that one of the partners of FAK involved in regulation of focal adhesion dynamics RhoA guanine nucleotide exchange factor (GEF) Net1 controls amoeboid motility during invasion of extracellular matrix (ECM) ( Carr et al, 2013 ). The other kinase Src phosphorylates and thus activates FAK.…”