Tag Archives: LBH589 inhibition

Supplementary MaterialsSupplementary Material 41598_2019_41096_MOESM1_ESM. channels on plasma membrane of eMSCs can

Supplementary MaterialsSupplementary Material 41598_2019_41096_MOESM1_ESM. channels on plasma membrane of eMSCs can be a novel indicator of cellular proliferation. Introduction Ion channels play an important role in numerous cellular reactions in living cells. In stem cells, native ion channels participate in various processes including differentiation, proliferation, cell migration, lineage switching, receptor-induced signaling and other. The expression pattern of ion channels in stem cells significantly varies among different species and sources1. Human adult mesenchymal stem cells derived from desquamated endometrium (eMSCs) are promising candidates for use in cell-based therapies due to their availability and non-invasive isolation protocols2C4. To date, little is known about the functional expression and the role of ion channels in eMSCs. At the same time, identification and revealing of functional interplay of ion channels in eMSCs might LBH589 inhibition be important in development of new strategies aimed at control of the behavior of LBH589 inhibition particular stem cell line in course of regenerative therapies. Previously, using single channel patch-clamp technique, we have identified several types of native ion channels and revealed their interplay in the plasma membrane of eMSCs. Particularly, the Ca2+ -mediated coupling was shown between the activity of Ca2+ -dependent potassium ion channels of big conductance (BK, KCa1.1) and mechanosensitive channels5. Moreover, our experiments have showed that BK channels are functionally expressed at high level in the plasma membrane; however, the particular role of BK channels in eMSCs remains to be elucidated. Importantly, due to high expression level, BK channels could significantly contribute to different signaling processes in eMSCs via setting and controlling the membrane potential. It is widely recognized, that ionic permeability and membrane potential significantly changes during cell cycle6. To date, functional interplay between BK channels, cell cycle progression and proliferation of stem cells or other cell types remain rather controversial7,8. Here, we aimed at verification of the putative impact of BK channels as potassium transporting pathway regulating cell cycle passageway of human eMSCs. Results Patch-clamp and immunofluorescent analysis revealed the expression of BK channels in eMSCs In our study, to confirm the presence of native BK channels in the plasma membrane of eMSCs, patch clamp experiments were performed. The typical activity of BK channels in cell-attached configuration on different holding membrane potentials is shown on Fig.?1A. A number of channel openings and NPo increases in potential-dependent manner (Fig.?1B,C) that is characteristical fingerprint of BK-mediated currents9, as well as current saturation (Fig.?1D) at membrane potentials higher than +100?mV10. The biophysical characteristics (single channel conductance and reversal potential) of the channels were similar to those recorded previously5. Immunofluorescent staining of Ocln BK channels with specific antibodies against pore-forming alpha subunit confirmed the expression of BK channels in the plasma membrane of eMSCs (Fig.?2). Importantly, immunofluorescent analysis allowed to detect, that a fraction LBH589 inhibition of cells in exponentially growing eMSC population are not stained with the antibodies (BK-negative cells, Fig.?2). The presence of BK-negative and BK-positive cells could potentially be explained by several factors, including heterogeneity of eMSCs, their differentiation status or the presence of apoptotic cells in culture. To test these possibilities, we confirmed the stemness of eMSCs by immunophenotyping (see Material and Methods and Fig.?S2). Our analysis did not reveal differentiated cells in the cell culture and demonstrated the homogeneity of cell population. Furthermore, staining for apoptotic marker Caspase 3/7 demonstrated extremely low basal level of apoptosis in eMSCs culture (Fig.?S3), and thus heterogeneity in BK channel expression could not be associated with the cell viability. Instead, we proposed that the difference in BK staining could potentially be explained by cell cycle status of the eMSCs. The changes in membrane permeability as well as the role of different ion channels during cell cycle were reported in numerous studies and reviews forming membrane potential hypothesis of cycle progression6,7. Open in LBH589 inhibition a separate window.