Diversity of ion channels in human bone marrow mesenchymal stem cells from amyotrophic lateral sclerosis patients

Abstract

Human bone marrow mesenchymal stem cells (hBM-MSCs) represent a potentially valuable cell type for clinical therapeutic applications. The present study was designed to evaluate the effect of long-term culturing (up to 10(th) passages) of hBM-MSCs from eight individual amyotrophic lateral sclerosis (ALS) patients, focusing on functional ion channels. All hBM-MSCs contain several MSCs markers with no significant differences, whereas the distribution of functional ion channels was shown to be different between cells. Four types of K(+) currents, including noise-like Ca(+2)-activated K(+) current (IK(Ca)), a transient outward K(+) current (I(to)), a delayed rectifier K(+) current (IK(DR)), and an inward-rectifier K(+) current (K(ir)) were heterogeneously present in these cells, and a TTX-sensitive Na(+) current (I(Na,TTX)) was also recorded. In the RT-PCR analysis, Kv1.1, heag1, Kv4.2, Kir2.1, MaxiK, and hNE-Na were detected. In particular, I(Na,TTX) showed a significant passage-dependent increase. This is the first report showing that functional ion channel profiling depend on the cellular passage of hBM-MSCs.

Keywords: Bone marrow; Functional ion channels; Passage-dependency; Stem cells; Tetrodotoxin-sensitive Na+ current.

Fig. 1

Characterization of hBM-MSCs. (A) Flow cytometry analysis showing that hBM-MSCswere positive for CD29, CD44, CD105, and CD73 and were negative for CD34, CD45, and HLA-DR. The table shows mean values (%). (B) hBM-MSCs expressed markers for OPN, LIFR, ABCG2, CXCR4, CD44, collagen X, collagen1 and alpha1. The hBM-MSCs RNAs were obtained from different donors (I and II, n=4) (C) Differentiation capacity of hBM-MSCs to adipocytes (upper) and osteoblasts (lower). 200× magnification.

Fig. 2

Different patterns of membrane currents recorded in hBM-MSCs. Current traces elicited by the voltage step (inset) in hBM-MSCs. (A) a slowly activating current similar to IK_DR at potentials from +20 to +100 mV that coexisted with IK_Ca. (B) I_to (arrow) with IK_Ca. (C) I_Na,TTX (arrow) with IK_DR and IK_Ca. (D) IK_ir (arrow) with IK_DR and IK_Ca. (E) Current-voltage relationships were plotted from A, B, C and D.

Fig. 3

Pharmacological effects of functional ion channels in hBM-MSCs. (A) Membrane currents were recorded in the presence of 10 mM TEA or co-application of TEA and 300 µM 4-AP. (B) I_Na,TTX was continuously recorded under control conditions and in the presence of TTX. I_Na,TTX was blocked by TTX (left panel) and also blocked by verapamil (right panel). (C) The I-V relationships of K_ir were obtained by ramp voltage induced currents against membrane potentials in bath solutions containing 5, 15, 30, 75, and 150 mM K⁺ as indicated (left panel). Reversal potentials from six patches were observed and plotted as a function of external [K⁺] concentrations (right panel). The dotted line represents the slope from the Nernst equation (slope, 58 mV/decade). Experimental values (O) were fitted by linear regression (slope, 66 mV/decade).

Fig. 4

Comparison of mRNA expression of ion channels between passages. (A) Kv1.1, heag1 (for IK_DR), Kv4.2 (for I_to), Kir2.1 (for K_ir), MaxiK (for IK_Ca) and hNE-Na (for I_Na.TTX) were detected in hBM-MSCs, but not SCN5A (for TTX-resistant I_Na). β-actin was used as the control. (B) The "recording rate (%)" of I_Na,TTX in various passage of hBM-MSCs. (C) Relative passage-dependent mRNA quantities of the hNE-Na gene by quantitative RT-PCR (n=3). The inset in Fig. 4C shows a representative result for the hNE-Na gene from two different samples. (D) Relative passage-dependent MaxiK gene expression levels in hBM-MSCs. Inset in Fig. 4D displays representative passage-dependent MaxiK channel gene expression patterns from two different samples. ^*p<0.05, ^**p<0.005 when compared with each 3^rd passage group.