Rho kinase inhibitor Y-27632 promotes the differentiation of human bone marrow mesenchymal stem cells into keratinocyte-like cells in xeno-free conditioned medium

Abstract

IntroductionBone marrow mesenchymal stem cells (BMSCs), which have the ability to self-renew and to differentiate into multiple cell types, have recently become a novel strategy for cell-based therapies. The differentiation of BMSCs into keratinocytes may be beneficial for patients with burns, disease, or trauma. However, the currently available cells are exposed to animal materials during their cultivation and induction. These xeno-contaminations severely limit their clinical outcomes. Previous studies have shown that the Rho kinase (ROCK) inhibitor Y-27632 can promote induction efficiency and regulate the self-renewal and differentiation of stem cells. In the present study, we attempted to establish a xeno-free system for the differentiation of BMSCs into keratinocytes and to investigate whether Y-27632 can facilitate this differentiation.MethodsBMSCs isolated from patients were cultured by using a xeno-free system and characterised by using flow cytometric analysis and adipogenic and osteogenic differentiation assays. Human primary keratinocytes were also isolated from patients. Then, the morphology, population doubling time, and β-galactosidase staining level of these cells were evaluated in the presence or absence of Y-27632 to determine the effects of Y-27632 on the state of the keratinocytes. Keratinocyte-like cells (KLCs) were detected at different time points by immunocytofluorescence analysis. Moreover, the efficiency of BMSC differentiation under different conditions was measured by quantitative real-time-polymerase chain reaction (RT-PCR) and Western blot analyses.ResultsThe ROCK inhibitor Y-27632 promoted the proliferation and lifespan of human primary keratinocytes. In addition, we showed that keratinocyte-specific markers could be detected in BMSCs cultured in a xeno-free system using keratinocyte-conditioned medium (KCM) independent of the presence of Y-27632. However, the efficiency of the differentiation of BMSCs into KLCs was significantly higher in the presence of Y-27632 using immunofluorescence, quantitative RT-PCR, and Western blot analyses.ConclusionsThis study demonstrated that Y-27632 could promote the proliferation and survival of human primary keratinocytes in a xeno-free culture system. In addition, we found that BMSCs have the ability to differentiate into KLCs in KCM and that Y-27632 can facilitate this differentiation. Our results suggest that BMSCs are capable of differentiating into KLCs in vitro and that the ROCK pathway may play a critical role in this process.

Rho‐kinase inhibitor Y‐27632 facilitates the proliferation, migration and pluripotency of human periodontal ligament stem cells

“…Furthermore, supplementary Y‐27632 can regulate the ability of stem cells to self‐renew and differentiate into derivatives of all three germ layers . More importantly, Y‐27632 was shown to greatly increase the long‐term proliferation of primary human keratinocytes, and unexpectedly, to enable these cells to efficiently bypass senescence and become immortal without a detectable cell crisis . Moreover, Y‐27632 could prevent mesenchymal stem cell (MSC) sheets from detaching and maintain the multilayered proliferation of confluent MSCs .…”

Modulation of microenvironment for controlling the fate of periodontal ligament cells: the role of Rho/ROCK signaling and cytoskeletal dynamics

“…The earlier studies have reported that Y-27632 can decrease cytoskeletal tension and contractility and can inhibit osteogenesis of MSC and osteoblasts (McBeath et al 2004;Khatiwala et al 2009;Kilian et al 2010;Shih et al 2011). In contrast, Y-27632 shows differentiation lineage specificity; Y-27632 can induce stem cells to differentiate into adipocytes, neuron cells, endothelial cells, and keratinocytes (McBeath et al 2004;Pacary et al 2006;Joo et al 2012;Li et al 2015). Although Y-27632 has been used as a multifunctional agent, the underlying molecular mechanism that determines inhibition or induction of stem cell differentiation remains unclear.…”

A composite Gelatin/hyaluronic acid hydrogel as an ECM mimic for developing mesenchymal stem cell‐derived epithelial tissue patches

“…In order to enhance the differentiation of MSCs to respiratory epithelium we investigated the impact of ROCK inhibition. ROCK inhibition using Rho kinase inhibitors like Y‐27632 has been shown to promote the induction efficiency, self‐renewal, and differentiation of MSCs towards the keratinocyte like cells with good expression of cytokeratin 14 and cytokeratin 5 in the presence keratinocyte‐conditioned medium (Li et al, ). Thus, it was envisaged that this approach can also further push the epithelial differentiation of MSCs in the presence of biocomposite films.…”