Actin-Myosin Contractility Is Responsible for the Reduced Viability of Dissociated Human Embryonic Stem Cells

Abstract

SUMMARY Human ES cells are the pluripotent precursor of the three embryonic germ layers. Human ES cells exhibit basal-apical polarity, junctional complexes, integrin-dependent matrix adhesion, and E-cadherin-dependent cell-cell adhesion, all characteristics shared by the epiblast epithelium of the intact mammalian embryo. After disruption of epithelial structures, programmed cell death is commonly observed. If individualized human ES cells are prevented from reattaching and forming colonies, their viability is significantly reduced. Here we show that actin-myosin contraction is a critical effector of the cell death response to human ES cell dissociation. Inhibition of myosin heavy chain ATPase, downregulation of myosin heavy chain, and downregulation of myosin light chain all increase survival and cloning efficiency of individualized human ES cells. ROCK inhibition decreases phosphorylation of myosin light chain, suggesting that inhibition of actin-myosin contraction is also the mechanism through which ROCK inhibitors increase cloning efficiency of human ES cells.

The non-muscle-myosin-II heavy chain Myh9 mediates colitis-induced epithelium injury by restricting Lgr5+ stem cells

“…Previous studies showed that actomyosin hyperactivation led to dissociation-induced apoptosis of embryonic and induced-pluripotent stem cells [9][10][11] . Although it is still controversial whether this type of cell death belongs to anoikis, the consensus was that the excessive actin-myosin contraction caused by loss of E-cadherindependent intercellular contact is critical for dissociation-induced death of embryonic or induced-pluripotent stem cells.…”

“…These results together suggest that Akt activity is essential for Myh9 inhibition to promote the survival and pluripotency of Lgr5 þ stem cells. Myh9-mediated actin-myosin contraction has been suggested to be involved in dissociation-induced apoptosis of embryonic and induced-pluripotent stem cells [9][10][11] . We then tested whether disruption of actin filament polymerization could also improve the survival of isolated crypts.…”

“…Its heavy chain has three isoforms in mice, including Myh9, Myh10 and Myh14, among which Myh9 accounts 480% of the total 7,8 . Recent studies revealed that dissociation-induced actin-myosin contraction caused the apoptosis of embryonic and induced-pluripotent stem cells [9][10][11] . However, the detailed mechanism of how NMII regulates stem cell survival remains unclear, and the potential roles of NMII in adult stem cells have not yet been investigated.…”

Rho/ROCK pathway is essential to the expansion, differentiation, and morphological rearrangements of human neural stem/progenitor cells induced by lysophosphatidic acid

“…Knockdown of ROCKI and/or ROCKII was performed using Dharmacon SMART pool ON-TARGETplus ROCK1 siRNA (L-003536-00-0005) and ON-TARGETplus ROCK2 siRNA (L-004610-00-0005), which were already demonstrated to be specifi c in hESC ( 47 ). Control for transfection was done using ON-TARGETplus NonTargeting Pool (D-001810-10-05).…”

Chemical approaches to studying stem cell biology

“…Two concurrent studies revealed that hESC dissociation (breakdown of E-cadherinmediated interaction) led to long-lasting apoptotic cellular contraction due to myosin hyperactivation in a Rho/ ROCK-dependent manner. Inhibition of ROCK or myosin heavy chain ATPase (by blebbistatin) diminishes actomyosin contraction and rescues the dissociated hESCs from apoptosis [17,18].…”