Abstract
The use of mesenchymal stromal cells (MSCs) differentiated toward a smooth muscle cell (SMC) phenotype may provide an alternative for investigators interested in regenerating urinary tract organs such as the bladder where autologous smooth muscle cells cannot be used or are unavailable. In this study we measured the effects of good manufacturing practice (GMP)-compliant expansion followed by myogenic differentiation of human MSCs on the expression of a range of contractile (from early to late) myogenic markers in relation to the electrophysiological parameters to assess the functional role of the differentiated MSCs and found that differentiation of MSCs associated with electrophysiological competence comparable to bladder SMCs. Within 1–2 weeks of myogenic differentiation, differentiating MSCs significantly expressed alpha smooth muscle actin (αSMA; ACTA2), transgelin (TAGLN), calponin (CNN1), and smooth muscle myosin heavy chain (SM-MHC; MYH11) according to qRT-PCR and/or immunofluorescence and Western blot. Voltage-gated Na+ current levels also increased within the same time period following myogenic differentiation. In contrast to undifferentiated MSCs, differentiated MSCs and bladder SMCs exhibited elevated cytosolic Ca2+ transients in response to K+-induced depolarization and contracted in response to K+ indicating functional maturation of differentiated MSCs. Depolarization was suppressed by Cd2+, an inhibitor of voltage-gated Ca2+-channels. The expression of Na+-channels was pharmacologically identified as the Nav1.4 subtype, while the K+ and Ca2+ ion channels were identified by gene expression of KCNMA1, CACNA1C and CACNA1H which encode for the large conductance Ca2+-activated K+ channel BKCa channels, Cav1.2 L-type Ca2+ channels and Cav3.2 T-type Ca2+ channels, respectively. This protocol may be used to differentiate adult MSCs into smooth muscle-like cells with an intermediate-to-late SMC contractile phenotype exhibiting voltage-gated ion channel activity comparable to bladder SMCs which may be important for urological regenerative medicine applications.
Highlights
The smooth muscle of the urinary tract is subjected to a variety of diseases and pathologies
Despite that some protein expression was already evident in mesenchymal stromal cells (MSCs) in control media, differentiation of MSCs resulted in an increase in protein levels of alpha smooth muscle actin, transgelin, calponin and smooth muscle myosin heavy chain (SM-MHC) protein doi:10.1371/journal.pone.0145153.g001
Even though there was a baseline expression of these myogenic markers, we still observed an increase in expression of αSMA, transgelin, and calponin following myogenic differentiation (DM) compared to day 0 and compared to MSCs cultured in control media (CM)
Summary
The smooth muscle of the urinary tract is subjected to a variety of diseases and pathologies. While clinical trials using autologous bladder smooth muscle cells (SMCs) show that such cell-based approaches seem promising for bladder tissue regeneration [1,2,3], one problem with this approach is that bladder cells are unavailable in some patients (e.g. diseased or malignant bladders). In such cases bladder cells cannot be used for regenerative therapies. The use of MSCs differentiated toward a SMC phenotype may provide an alternative for investigators interested in regenerating urinary tract organs
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