Molecular pathway and cell state responsible for dissociation-induced apoptosis in human pluripotent stem cells.

Human embryonic stem cells (hESCs), unlike mouse ones (mESCs), are vulnerable to apoptosis upon dissociation. Here, we show that the apoptosis, which is of a nonanoikis type, is caused by ROCK-dependent hyperactivation of actomyosin and efficiently suppressed by the myosin inhibitor Blebbistatin. The actomyosin hyperactivation is triggered by the loss of E-cadherin-dependent intercellular contact and also observed in dissociated mouse epiblast-derived pluripotent cells but not in mESCs. We reveal that Abr, a unique Rho-GEF family factor containing a functional Rac-GAP domain, is an indispensable upstream regulator of the apoptosis and ROCK/myosin hyperactivation. Rho activation coupled with Rac inhibition is induced in hESCs upon dissociation, but not in Abr-depleted hESCs or mESCs. Furthermore, artificial Rho or ROCK activation with Rac inhibition restores the vulnerability of Abr-depleted hESCs to dissociation-induced apoptosis. Thus, the Abr-dependent "Rho-high/Rac-low" state plays a decisive role in initiating the dissociation-induced actomyosin hyperactivation and apoptosis in hESCs.

[1]  E. Sahai,et al.  Rac Activation and Inactivation Control Plasticity of Tumor Cell Movement , 2008, Cell.

[2]  K. E. Johnson,et al.  Circus movements and blebbing locomotion in dissociated embryonic cells of an amphibian, Xenopus laevis. , 1976, Journal of cell science.

[3]  G M Bokoch,et al.  Abr and Bcr are multifunctional regulators of the Rho GTP-binding protein family. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[4]  N. Sato,et al.  The Rho-Rock-Myosin Signaling Axis Determines Cell-Cell Integrity of Self-Renewing Pluripotent Stem Cells , 2008, PloS one.

[5]  R. McKay,et al.  New cell lines from mouse epiblast share defining features with human embryonic stem cells , 2007, Nature.

[6]  S. Yamanaka,et al.  Role of ERas in promoting tumour-like properties in mouse embryonic stem cells , 2003, Nature.

[7]  A. Brivanlou,et al.  Molecular signature of human embryonic stem cells and its comparison with the mouse. , 2003, Developmental biology.

[8]  E. Sahai,et al.  Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I , 2001, Nature Cell Biology.

[9]  Yoshiki Sasai,et al.  Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals. , 2008, Cell stem cell.

[10]  C. Der,et al.  GEF means go: turning on RHO GTPases with guanine nucleotide-exchange factors , 2005, Nature Reviews Molecular Cell Biology.

[11]  Yukio Nakamura,et al.  A Method for the Selection of Human Embryonic Stem Cell Sublines with High Replating Efficiency After Single‐Cell Dissociation , 2006, Stem cells.

[12]  Krister Wennerberg,et al.  Rho and Rac Take Center Stage , 2004, Cell.

[13]  M. Trotter,et al.  Derivation of pluripotent epiblast stem cells from mammalian embryos , 2007, Nature.

[14]  Janet Rossant,et al.  Blastocyst lineage formation, early embryonic asymmetries and axis patterning in the mouse , 2009, Development.

[15]  S. Narumiya,et al.  ROCK‐I and ROCK‐II cooperatively regulate closure of eyelid and ventral body wall in mouse embryo , 2005, Genes to cells : devoted to molecular & cellular mechanisms.

[16]  J. Thomson,et al.  Embryonic stem cell lines derived from human blastocysts. , 1998, Science.

[17]  M. Matsuda,et al.  Activity of Rho-family GTPases during cell division as visualized with FRET-based probes , 2003, The Journal of cell biology.

[18]  V. Kaartinen,et al.  Human ABR encodes a protein with GAPrac activity and homology to the DBL nucleotide exchange factor domain. , 1993, The Journal of biological chemistry.

[19]  Karen H. Vousden,et al.  p53 in health and disease , 2007, Nature Reviews Molecular Cell Biology.

[20]  Timothy J. Mitchison,et al.  Reassembly of contractile actin cortex in cell blebs , 2006, The Journal of cell biology.

[21]  J. Thomson,et al.  Human ERas Gene Has an Upstream Premature Polyadenylation Signal That Results in a Truncated, Noncoding Transcript , 2005, Stem cells.

[22]  Alan Hall,et al.  Rho GTPases: biochemistry and biology. , 2005, Annual review of cell and developmental biology.

[23]  L. Mahadevan,et al.  Non-equilibration of hydrostatic pressure in blebbing cells , 2005, Nature.

[24]  Fuminori Tsuruta,et al.  The Phosphatidylinositol 3-Kinase (PI3K)-Akt Pathway Suppresses Bax Translocation to Mitochondria* , 2002, The Journal of Biological Chemistry.

[25]  D. Rancourt,et al.  Human embryonic stem cells: caught between a ROCK inhibitor and a hard place , 2009, BioEssays : news and reviews in molecular, cellular and developmental biology.

[26]  M. Matsuda,et al.  Activation of Rac and Cdc42 Video Imaged by Fluorescent Resonance Energy Transfer-Based Single-Molecule Probes in the Membrane of Living Cells , 2002, Molecular and Cellular Biology.

[27]  M. Okabe,et al.  E-Cadherin-Coated Plates Maintain Pluripotent ES Cells without Colony Formation , 2006, PloS one.

[28]  J. Bertoglio,et al.  Caspase-3-mediated cleavage of ROCK I induces MLC phosphorylation and apoptotic membrane blebbing , 2001, Nature Cell Biology.

[29]  S. Nishikawa,et al.  A ROCK inhibitor permits survival of dissociated human embryonic stem cells , 2007, Nature Biotechnology.

[30]  竹本 研,et al.  Spatio-temporal activation of caspase revealed by indicator that is insensitive to environmental effects , 2004 .

[31]  A. Strasser,et al.  The BCL-2 protein family: opposing activities that mediate cell death , 2008, Nature Reviews Molecular Cell Biology.

[32]  V. Kaartinen,et al.  Abr and Bcr, Two Homologous Rac GTPase-Activating Proteins, Control Multiple Cellular Functions of Murine Macrophages , 2006, Molecular and Cellular Biology.

[33]  C. Simón,et al.  Disruption of Apical‐Basal Polarity of Human Embryonic Stem Cells Enhances Hematoendothelial Differentiation , 2007, Stem cells.

[34]  M. Takeichi,et al.  Shroom3-mediated recruitment of Rho kinases to the apical cell junctions regulates epithelial and neuroepithelial planar remodeling , 2008, Development.

[35]  Anne J. Ridley,et al.  ROCKs: multifunctional kinases in cell behaviour , 2003, Nature Reviews Molecular Cell Biology.

[36]  M. Karin,et al.  Mammalian MAP kinase signalling cascades , 2001, Nature.

[37]  J. Miyazaki,et al.  Phenotypic Complementation Establishes Requirements for Specific POU Domain and Generic Transactivation Function of Oct-3/4 in Embryonic Stem Cells , 2002, Molecular and Cellular Biology.

[38]  Guillaume Charras,et al.  Blebs lead the way: how to migrate without lamellipodia , 2008, Nature Reviews Molecular Cell Biology.

[39]  S. Frisch,et al.  Anoikis mechanisms. , 2001, Current opinion in cell biology.

[40]  J. Downward PI 3-kinase, Akt and cell survival. , 2004, Seminars in cell & developmental biology.