Functional ion channels in human bone marrow-derived mesenchymal stem cells and human cardiac c-kit+ progenitor cells

Abstract

Ion channels play vital roles in maintaining physiological homeostasis and regulating a variety of physiological processes including cell proliferation and migration. The present PhD project investigated the potential roles of ion channels in regulating cell proliferation, differentiation, and/or migration in human bone marrow-derived mesenchymal stem cells (MSCs) and human cardiac c-kit+ progenitor cells using multiple approaches including electrophysiology, molecular biology, cell proliferation assays, flow cytometry, cell migration assays, etc.

In first part of the study, roles of large-conductance Ca2+-activated potassium (BKCa or KCa1.1) channels and ether-à-go-go potassium (hEag1 or KCNH1) channels in regulating cell proliferation and adipogenesis and osteogenesis were determined in human MSCs. We found that inhibition of BKCa with paxilline or hEag1 with astemizole, or silencing BKCa with shRNAs targeting KCa1.1 or hEag1 channels with shRNAs targeting KCNH1 arrested the cells at G0/G1 phase via inhibiting signaling pathways including cyclin D1, cyclin E, p-ERK1/2, and p-Akt. Interestingly, silencing BKCa or hEag1 channels significantly reduced adipogenic differentiation and deceased osteogenic differentiation. These results demonstrate that BKCa and hEag1 channels not only regulate cell proliferation, but also participate in the adipogenic and osteogenic differentiations in human MSCs.

In second part of the study, we found that several ionic currents were heterogeneously expressed in human cardiac c-kit+ progenitor cells, including BKCa in 86% of cells, an inwardly-rectifying K+ current (IKir) in 84% of cells, a transient outward K+ current (Ito) in 47% of cells, a voltage-gated tetrodotoxin-sensitive Na+ current (INa,TTX) in 61% of cells. Molecular identities of these ionic currents were determined with RT-PCR and Western blot analysis. KCa.1.1 (for BKCa), Kir2.1 (for IKir), Kv4.2 and Kv4.3 (for Ito), NaV1.2, NaV1.3, NaV1.6, NaV1.7 (for INa.TTX) were expressed in human cardiac progenitor cells.

In third part of the study, we demonstrated that inhibition of BKCa with paxilline, Ito with 4-aminopyridine, but not INa.TTX with TTX or IKir with Ba2+, decreased cell proliferation in a concentration-dependent manner. Silencing KCa.1.1, or Kv4.2 and Kv4.3, but not Kir2.1 channels, reduced cell proliferation by accumulating the cells at G0/G1 phase. Blockade or knockdown of KCa.1.1, Kv4.2 or Kv4.3 channels, (but not INa.TTX)reduced cell migration. Interestingly, blockade of IKir or silencing Kir2.1 channels enhanced cell migration in human cardiac c-kit+ cells.

Collectively, the present PhD project demonstrates the novel information that 1) BKCa and hEag1 regulate not only cell cycling, but also adipogenesis and osteogenesis in human MSCs; 2) multiple ion currents, BKCa, IKir, Ito, and INa,TTX, are present in human cardiac c-kit+ progenitor cells; and 3) BKCa and Ito, but not IKir and INa.TTX participate in regulating cell cycling. Interestingly, BKCa and Ito (but not INa.TTX) promote, while IKir inhibits cell proliferation in human cardiac c-kit+ progenitor cells