Actin-myosin contractility is responsible for the reduced viability of dissociated human embryonic stem cells

Abstract

Human ESCs are the pluripotent precursor of the three embryonic germ layers. Human ESCs 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 ESCs 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 ESC 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 ESCs. 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 ESCs.

Figure 1. Inhibition of membrane blebbing improves cell viability after dissociation in human ES cells

(A) A time course of density-dependent survival after dissociation. Different numbers of human ES cells were plated onto each well (3,000/cm² (3K-blue diamond), 10,000/cm² (10K-red square) and 30,000/cm² (30K-green triangle)). The “Survival Index” represents the number of surviving cells divided by the number of input cells. (Note that as cells divide, this index can eventually exceed 100%.) (*) signifies that the 10K density Survival Index differed significantly (p< 0.05) from the 3K density survival at 24 hrs. (**) signifies that the 30K density Survival Index differed significantly (p<0.05) from the 10K survival at 24 hr. For more details, see Experimental Procedures, Statistics section. (B) Phase contrast images of human ES cells dissociated by TrypLE with or without 10 μM blebbistatin. Blebbistatin changed actin organization immediately after dissociation, as visualized by Alexa-633 conjugated phalloidin (Red). (C) Blebbistatin inhibited blebbing and improved cell spreading 1 hr after plating as visualized by Alexa- staining. (D) Colony morphology of human ES cells 24 hours post splitting and treatment with blebbistatin. (E) The 24-hour survival of individualized human ES cells was significantly improved when 10 μM blebbistatin was added to cells plated at 10,000/cm² (*p < 0.05). Comparable experiments were repeated more than 10 times. (F) Treatment of human ES cells with blebbistatin for one hour or longer was sufficient to improve the cloning efficiency (*p <0.05). Individualized cells were plated in the presence and absence of 10 μM Blebbistatin at a density of 150 cells/cm². Blebbistatin was removed after indicated time points. Cloning efficiency was then calculated after 7 days (*p <0.05). Comparable time series experiments were repeated 2 times. In more than 40 single-time point experiments, blebbistatin consistently increased the cloning efficiency of two human ES cell lines (H1 and H9) and two iPS cell lines (iPS-foreskin and iPS-IMR90) (see Table S1).

Figure 2. Knockdown of Non-Muscle Myosin Heavy Chains (MYH) Reduces Blebbing and Improves Survival and Cloning Efficiency of Human ES Cells

Knockdown (by siRNA) of MYH9 alone or in combination with MYH10: (A) changed ES cell morphology in colonies 96 hours after transfection, (B) reduced blebbing immediately after dissociation, (C) increased attachment/spreading 5 hr after plating, (D) increased (*p<0.05) survival 24 hr after plating, and (E) increased (*p<0.05) cloning efficiency. Comparable results were obtained in multiple experimental repetitions using both H1 (n=15) and H9 human ES cell lines (n=2).

Figure 3. Knockdown of Myosin Light Chain (MLC) and Disruption of Actin Filaments Reduce Blebbing and Improve Survival and Cloning Efficiency of Human ES Cells

Knockdown (by siRNA) of the three MLC gene products (MRLC1/2/3): (A) changed human ES cell morphology in colonies 96 hours after transfection, (B) decreased blebbing immediately after dissociation, (C) increased (*p<0.05) cell survival 24 hr after plating, and (D) increased (*p<0.05) cloning efficiency. Disruption of actin filaments by small molecules: (E) reduced membrane blebbing of human ES cells 2 hr after plating in the presence of Cytochalasin D (100 nM), Swinholide A (10 nM) or Mycalolide B (20 μM). (F) increased human ES cell Survival Index at 24 hr, and (G) increased cloning efficiency after an initial 2 hr treatment with Swinholide A (10 nM).

Figure 4. ROCKs Regulate Cell Survival Through MLC Phosphorylation

Knockdown (by siRNA) of ROCK1 and ROCK2 (ROCK1/ROCK2) together: (A) changed human ES cell morphology in colonies 96 hours after transfection, (B) decreased blebbing immediately after dissociation, (C) increased (*p<0.05) cell survival 24 hr after plating, and (D) increased (*p<0.05) cloning efficiency. Increased Phosphorylation of MLC (p-MLC) in dissociated human ES cells over time post-plating: (E) was reduced by the ROCK inhibitor Y27632 (10 μM), and (F) was reduced by siRNA knockdown of ROCK1/ROCK2. The results were confirmed by repeat experiments (n=2). (G) Phosphorylation of MLC (p-MLC) was inversely related to the concentration of Y27632. Dissociated human ES cells were treated with the ROCK inhibitor Y27632 at increasing concentrations at time equal to zero. The treated cells were harvested 5 hours after plating and phosphorylation of MLC examined by western blot. The result was confirmed by repeat experiment (n=2). (H) A decrease in MLC phosphorylation levels correlated with improvements in cloning efficiency (*p<0.05 compared to untreated control). Blue columns represent cloning efficiency, and red columns represent quantified MLC phosphorylation percentage from (G). The result was confirmed by repeat experiment (n=2).