Depolarization Alters Phenotype, Maintains Plasticity of Predifferentiated Mesenchymal Stem Cells

Abstract

Although adult stem cell transplantation has been implemented as a therapy for tissue repair, it is limited by the availability of functional adult stem cells. A potential approach to generate stem and progenitor cells may be to modulate the differentiated status of somatic cells. Therefore, there is a need for a better understanding of how the differentiated phenotype of mature cells is regulated. We hypothesize that bioelectric signaling plays an important role in the maintenance of the differentiated state, as it is a functional regulator of the differentiation process in various cells and tissues. In this study, we asked whether the mature phenotype of osteoblasts and adipocytes derived from human mesenchymal stem cells (hMSCs) could be altered by modulation of their membrane potential. hMSC-derived osteoblasts and adipocytes were depolarized by treatment with ouabain, a Na(+)/K(+) ATPase inhibitor, or by treatment with high concentrations of extracellular K(+). To characterize the effect of voltage modulation on the differentiated state, the depolarized cells were evaluated for (1) the loss of differentiation markers; (2) the up-regulation of stemness markers and stem properties; and (3) differences in gene expression profiles in response to voltage modulation. hMSC-derived osteoblasts and adipocytes exhibited significant down-regulation of bone and fat tissue markers in response to depolarization, despite the presence of differentiation-inducing soluble factors, suggesting that bioelectric signaling overrides biochemical signaling in the maintenance of cell state. Suppression of the osteoblast or adipocyte phenotype was not accompanied by up-regulation of genes associated with the stem state. Thus, depolarization does not activate the stem cell genetic signature and, therefore, does not induce a full reprogramming event. However, after transdifferentiating the depolarized cells to evaluate for multi-lineage potential, depolarized osteoblasts demonstrated improved ability to achieve correct adipocyte morphology compared with nondepolarized osteoblasts. The present study thus demonstrates that depolarization reduces the differentiated phenotype of hMSC-derived cells and improves their transdifferentiation capacity, but does not restore a stem-like genetic profile. Through global transcript profiling of depolarized osteoblasts, we identified pathways that may mediate the effects of voltage signaling on cell state, which will require a detailed mechanistic inquiry in future studies.

Bioelectric modulation of wound healing in a 3D in vitro model of tissue-engineered bone

“…Finally, another group of scaffolds was depolarized by high extracellular [K + ]. Modulation of V mem by high K + was previously shown to down-regulate the progression of hMSC differentiation [29] and also to alter the mature phenotype of differentiated cells [44]. Loss of mature phenotype could be useful for mobilizing cells in the wound environment, similar to de-differentiation events that have been reported in several systems, such as zebrafish heart and fin regeneration, urodele limb regeneration, and Schwann cell de-differentiation following nerve injury [45–48].…”

Depolarization of Cellular Resting Membrane Potential Promotes Neonatal Cardiomyocyte Proliferation In Vitro

“…The other two groups were subjected to sustained depolarization with the addition of either potassium gluconate or ouabain. To assess dose dependent effects, we studied a range of concentrations for both potassium gluconate (5 mM, 10 mM, 20 mM, 40 mM, 60 mM, and 80 mM) and ouabain (10 nM, 100 nM, 1 μM, 10 μM, 100 μM, and 150 μM) , reagents known to depolarize V mem [16, 17, 21-27]. Sustained depolarization was carried out for 72 hours with no media change.…”

“…Of particular significance, he demonstrated that mature neurons from the central nervous system (typically thought of as terminally differentiated and thus non-mitotic) could be stimulated to proliferate by sustained depolarization of V mem via pharmacological treatments [16]. More recently, it was found that human mesenchymal stem cells (hMSCs) could be driven to differentiate via hyperpolarization while depolarization maintained the stem cell phenotype, and that a degree of de-differentiation can be obtained in differentiated hMSCs by manipulation of their resting potential [17]. It is generally accepted that stem cells are significantly more proliferative than differentiated cell types [18, 19], and thus these studies suggest an intriguing link between V mem and a switch from a proliferative to mature state.…”

Membrane potential and cancer progression

“…Pharmacologically-induced V m depolarization suppresses adipogenic and osteogenic differentiation of hMSCs (Sundelacruz et al, 2008). In addition, depolarization reduces the differentiated phenotype of hMSC-derived cells and improves their ability to transdifferentiate, without fully restoring a stem cell-like genetic profile (Sundelacruz et al, 2013). Taken together, these data suggest that V m depolarization may maintain cells in an undifferentiated stage at the gene expression level.…”