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Effect of ROCK Inhibitor Y-27632 on Normal and Variant Human Embryonic Stem Cells (hESCs) In Vitro: Its Benefits in hESC Expansion

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Abstract

The Rho associated coiled coil protein kinase (ROCK) dependent signaling pathway plays an important role in numerous physiological functions such as cell proliferation, adhesion, migration and inflammation. Human embryonic stem cells (hESCs) undergo differentiation and poor survival after single cell dissociation in culture thus limiting their expansion for cell based therapies. We evaluated the role of the selective ROCK inhibitor Y-27632 on hESC colonies and disassociated single hESCs from two different hESC lines. Karyotypically normal hESCs (HES3) and variant hESCs (BG01V) were treated with Y-27632 at 5, 10 and 20 μM concentrations for 72 h and its effects on hESC self renewal, colony morphology, cell cycle and pluripotency were evaluated. Increased cell proliferation of both HES3 and BG01V were observed for all three concentrations compared to untreated controls following passaging of cell clusters or dissociated single cells and some of these increases were statistically significant. Cell cycle assay demonstrated normal cell cycle progression with no peaks evident of apoptosis. No morphological differentiation was evident following treatment with the highest concentration of Y-27632 (20 μM) and the stemness related genes continued to be highly expressed in both HES3 and BG01V cells compared to untreated controls. The results confirmed that Y-27632 is a useful agent that aids in the expansion of undifferentiated hESC numbers for downstream applications in regenerative medicine.

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  1. Y-27632 is a highly specific pharmacologic agent that inhibits the Rho kinase (ROCK) family. Two members of the ROCK family, ROCK1 and ROCK2 have been identified, and ROCK functions as a target protein for Rho, which acts as membrane switches and alternates between the GTP bound active form and GDP bound inactive form to regulate other downstream kinases [3133] leading to many cellular changes including migration and cell proliferation [14].

  2. In the present study, the increased cell proliferation observed in both normal hESCs (HES3) and variant hESCs (BG01V) when low concentrations of Y-27632 (5–10 µM) were exposed to hESCs in singles or clusters makes it an attractive pharmacological agent for use in hESC culture to scale up cell numbers thus helping to overcome the limitation of hESC supply and accelerating cell based therapies for clinical application. These results are consistent with the recent report that Y-27632 increased hESC colony numbers and size following exposure for 24 hours after 5 days of freeze-thawing [21]. Increased retinal angiogenesis following vascular endothelial growth factor stimulation was observed when Rho signaling was inhibited either with specific ROCK inhibitor H1152 or by using ROCKI/ROCKII Si RNA specific targets [34] further indicating that cell proliferation was also observed with other cell types. Focal adhesion points are the binding of integrins providing a physical link of the cytoskeleton to the extracellular matrix. Cell attachment and flattening for other cellular responses to occur are mediated through Rho signaling [25] and inhibition of these focal adhesions in turn inhibit cell proliferation [35]. Our earlier studies showing decreases in cell proliferation when BG01V and various cancer cell types were exposed to statins [29] was due to the cholesterol independent effect of statins mediated by inhibition of Rho kinase. In contrast, the pharmacological effects of Y-27632 were the reverse where cell proliferation was observed. The dual role of focal adhesion, where increase adhesion stimulates proliferation and decreased adhesion arrests growth [14] explains the selective activation of Rho signaling.

  3. Colony formation is a characteristic feature of hESC survival and dissociation of hESCs into single cells is associated with apoptosis and cell death. hESCs are very ‘social’ cells and they undergo spontaneous differentiation and or marked cell death upon minor perturbation of their culture environment. This limiting factor in hESC culture was overcome by the use of ROCK inhibitor Y-27632 that offered protection and facilitated survival of hESCs [15, 36]. The results of the present study also confirms this protection effect of Y-27632 when exposed to dissociated single hESCs across different hESC lines differing in their genetic nature as well as their derivation methods, and in addition was helpful in increasing cell numbers. Although no colony formation was observed which might be due to high plating density of the dissociated hESCs, the cells attached and remained in close contact, and continued to have the characteristic stem cell morphology of hESCs of high nuclear to cytoplamic ratios and prominent nucleoli. Recently, the role of the Rho-ROCK-Myosin signaling axis in the formation of cell to cell interactions that are essential for colony formation was demonstrated and the signaling mechanisms were shown to be different between pluripotent and non-pluripotent cell lines [37]. Rho mediates cell-cell adhesion through two important downstream effectors namely, the ROCK and diaphanous-related forms [38]. Y-27632 was also shown to decrease the cell-cell integrity in mouse embryonic stem cells (mESCs) and also their recovery following removal of the inhibitor from the culture medium [37].

  4. In contrast to the anticancer effects of statins which was mainly due to cholesterol independent effects mediated by Rho/ROCK regulation, Y-27632 does not appear to selectively target normal or abnormal hESCs as its influence on karyotypically abnormal BG01V cells was not different from normal hESCs. Y-27632 was shown to inhibit proliferation of CD34+ progenitor cells isolated from the bone marrow of chronic myeloid leukemia patients. This inhibition was further increased in combination with the activated protein tyrosine kinase specific inhibitor imatinib, which also increased its pro-apoptotic effects [39]. However, the concentration of Y-27632 used in this particular study was up to 50 mM in contrast to most studies where the concentrations were around 10 µM. Probably, in the present study too, the effects of Y-27632 are more at the membrane level influencing cell-cell communication, and much higher concentrations may be required than those used in the present study to observe changes at the genomic level. Further exploration of specific ROCK inhibitors such as Y-27632/Fausidil and other related compounds in cancer therapeutics is necessary which may target cancer cells better than statins because they act by Rho/ROCK regulation to inhibit cancer cells [29, 40].

  5. The results of the cell cycle assays in the present study demonstrated that most of the cells were in the replicative phase of the cell cycle and no peaks in the sub-G1 phase indicative of apoptosis were evident. The Rho family GTPases (Rho, Rac and CDC42) share around 30% sequence identity with RAS family GTPases that are involved in malignant transformation of cells, mainly due to perturbations in cell cycle transition either due to up or down regulation of cell cycle promoters or inhibitors. Many studies have identified ROCK to be the downstream effector of Rho mediated tumours [41]. Recent focus has shifted to study the precise involvement of Rho family GTPases and their effectors in cell cycle signalling pathways that can lead to cancer and possible development of new therapeutics aimed at Rho/ROCK inhibition [42].

  6. Apart from increasing the cell numbers and promoting cell survival upon dissociation into single cells, Y-27632 also helped to maintain the pluripotency of hESCs as reflected in the immunohistochemical studies for pluripotent markers (SSEA-4, TRA-1-81, OCT4 and AP activity). This was also reflected in the high gene expression levels for pluripotent stem cell markers (NANOG, OCT4 and GDF3) compared to the controls. Our findings are further supported by the studies of Harb et al. [37], who demonstrated high expression levels of OCT3/4 and NANOG and the repression of differentiation related genes such as SNAIL and N-CADHERIN in mESCs following treatment with the ROCK inhibitor Y-27632. Most transplanted cells undergo apoptosis as a result of cell dissociation, loss of proper internal milieu and oxidative stress and hypoxia [43, 44]. Rho-GTPase has been implicated in the apoptosis of many cell types, including neurons, but the mechanism by which it acts is not fully understood. In the present study, the anti-apoptotic BCL2 was highly expressed following treatment with Y-27632 than in untreated controls and this further supports the anti-apoptotic mechanism of ROCK inhibitors. If ROCK inhibition offers protection against apoptosis in vivo, similar to the effects seen in vitro, it would greatly enhance the therapeutic potential of hESCs in regenerative medicine.

  7. ROCK inhibitors seem to be attractive compounds with several pharmacological targets. Their usefulness in the protection, survival and enhancement of human embryonic stem cells should be exploited for culture and expansion in stem cell based therapies.

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Acknowledgements

The authors acknowledge the grant support provided by the National University of Singapore (R-174-000-089-133) and National Medical Research Council, Singapore (R-174-000-103-213) and the technical assistance provided by Mr Gary Peh, Ms. N. Manasi, and Mr. A. Subramanian.

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Gauthaman, K., Fong, CY. & Bongso, A. Effect of ROCK Inhibitor Y-27632 on Normal and Variant Human Embryonic Stem Cells (hESCs) In Vitro: Its Benefits in hESC Expansion. Stem Cell Rev and Rep 6, 86–95 (2010). https://doi.org/10.1007/s12015-009-9107-8

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