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

Abstract

The selective in vitro expansion and differentiation of multipotent stem cells are critical steps in cell-based regenerative therapies, while technical challenges have limited cell yield and thus affected the success of these potential treatments. The Rho GTPases and downstream Rho kinases are central regulators of cytoskeletal dynamics during cell cycle and determine the balance between stem cells self-renewal, lineage commitment and apoptosis. Trans-4-[(1R)-aminoethyl]-N-(4-pyridinyl)cylohexanecarboxamidedihydrochloride (Y-27632), Rho-associated kinase (ROCK) inhibitor, involves various cellular functions that include actin cytoskeleton organization, cell adhesion, cell motility and anti-apoptosis. Here, human periodontal ligament stem cells (PDLSCs) were isolated by limiting dilution method. Cell counting kit-8 (CCK8), 5-ethynyl-2'-deoxyuridine (EdU) labelling assay, cell apoptosis assay, cell migration assay, wound-healing assay, alkaline phosphatase (ALP) activity assay, Alizarin Red S staining, Oil Red O staining, quantitative real-time polymerase chain reaction (qRT-PCR) were used to determine the effects of Y-27632 on the proliferation, apoptosis, migration, stemness, osteogenic and adipogenic differentiation of PDLSCs. Afterwards, Western blot analysis was performed to elucidate the mechanism of cell proliferation. The results indicated that Y-27632 significantly promoted cell proliferation, chemotaxis, wound healing, fat droplets formation and pluripotency, while inhibited ALP activity and mineral deposition. Furthermore, Y-27632 induced PDLSCs proliferation through extracellular-signal-regulated kinase (ERK) signalling cascade. Therefore, control of Rho-kinase activity may enhance the efficiency of stem cell-based treatments for periodontal diseases and the strategy may have the potential to promote periodontal tissue regeneration by facilitating the chemotaxis of PDLSCs to the injured site, and then enhancing the proliferation of these cells and maintaining their pluripotency.

Keywords: ROCK inhibitor; Y-27632; chemotaxis; differentiation; periodontal ligament stem cells; proliferation.