The effect of Rho-associated kinase inhibition on the proteome pattern of dissociated human embryonic stem cells.

Rho-associated kinase (ROCK) is an immediate downstream target of the Rho GTPase signaling pathway which participates in transducing the Rho GTPase signal to the actin cytoskeleton, leading to the assembly of focal adhesions and stress fibers. Competitive inhibition of ROCK enhances post-thaw viability, improves cloning efficiency and decreases anoikis in human embryonic stem cells (hESCs). The molecular mechanisms by which ROCK inhibition mediates such responses are largely unknown. We have investigated the effect of Y-27632, a competitive ROCK inhibitor (ROCKi), on the proteome of hESCs. HESCs were exposed to ROCK inhibition directly by the addition of Y-27632 to the culture medium or to the Matrigel substratum. ROCK inhibition significantly increased cell survival and plating efficiency without any changes to the morphology, karyotype, or expression of pluripotency markers. We used a two-dimensional gel electrophoresis (2-DE) coupled with tandem mass spectrometry based protein identification and identified 29 ROCKi responsive proteins. As expected, cytoskeleton-related proteins comprised the major ROCKi responsive proteins. Differential proteomic analysis showed that ROCKi induced upregulation of some actin binding proteins such as tropomyosin, F-actin capping protein (CapZ) and transgelin and downregulation of tubulin. In addition, ROCK inhibition was accompanied by changes in expressions of some chromatin modifying proteins such as SMARCB1, ILF3, and Dpy-30-protein, further suggesting a link between ROCK inhibition and the epigenetic mechanism of gene regulation.

Proteome analysis of post-transplantation recovery mechanisms of an EAE model of multiple sclerosis treated with embryonic stem cell-derived neural precursors.

Multiple sclerosis (MS) is a chronic inflammatory and progressive disorder of the central nervous system (CNS), which ultimately causes demyelination and subsequent axonal injury. Experimental autoimmune encephalomyelitis (EAE) is a well-characterized animal model to study the etiology and pathogenesis of MS. This model can also be used to investigate various therapeutic approaches for MS. Herein; we have treated a score 3 EAE mouse model with an embryonic stem cell-derived neural precursor. Clinical analysis showed recovery of the EAE model of MS following transplantation. We analyzed the proteome of spinal cords of healthy and EAE samples before and after transplantation. Proteome analysis revealed that expressions of 86 spinal cord protein spots changed in the EAE or transplanted mouse compared to controls. Mass spectrometry resulted in identification of 72 proteins. Of these, the amounts of 27 differentially expressed proteins in EAE samples returned to sham levels after transplantation, suggesting a possible correlation between changes at the proteome level and clinical signs of EAE in transplanted mice. The recovered proteins belonged to various functional groups that included disturbances in ionic and neurotransmitter release, apoptosis, iron hemostasis, and signal transduction. Our results provided a proteomic view of the molecular mechanisms of EAE recovery after stem cell transplantation. BIOLOGICAL SIGNIFICANCE: In this study, we applied proteomics to analyze the changes in proteome pattern of EAE mouse model after embryonic stem cell-derived neural precursor transplantation. Our proteome results clearly showed that the expression levels of several differentially expressed proteins in EAE samples returned to sham levels after transplantation, which suggested a possible correlation between changes at the proteome level and decreased clinical signs of EAE in transplanted mice. These results will serve as a basis to address new questions and design new experiments to elucidate the biology of EAE and recovery after transplantation. A thorough understanding of stem cell-mediated therapeutic mechanisms might result in the development of more efficacious therapies for MS than are currently available.