In this report, we have demonstrated that several specialized K+ channels and Na+ channels at the mRNA and functional levels were present in undifferentiated human MSCs from the umbilical cord vein. The direct comparison reported here showed that hUC-MSCs and hBM-MSCs express the same gene markers (Fig. 1B) and share common cell surface antigens (Fig. 1C). Also, hUC-MSCs have an ability to undergo multilineage mesenchymal differentiation (supplemental online Fig. 1). Ca2+-activated K+ channel, delayed rectifying K+ current, and transient outward K+ current were present mainly in hUC-MSCs, although inward rectifying K+ current and Na+ current were present in a small portion of the population. Also, we demonstrated characteristics of hUC-MSCs with morphological and immunophenotypical methods. To our knowledge, this is the first time that hUC-MSCs have been extensively characterized from an electrophysiological viewpoint, not just in hBM-MSCs as studied until now [8, 9, 42].
Characteristics and Functional Role of Ion Channels in hUC-MSCs
Electrophysiologically isolated IKDR and Ito from hUC-MSCs were confirmed as Kv1.1, heag1, Kv4.2, and Kv1.4 by RT-PCR. The Kv1.1 subfamily is expressed in the embryonic nervous system [43, 44], and mutations in Kv1.1 are associated with human episodic ataxia type 1 syndrome, which is characterized by movement disorders and epilepsy [45, 46]. Mammalian ether a go-go (EAG) subfamily K+ channels have been studied in several species including rat , mouse , bovine , and human . Human EAG (heag) K+ channels were found to participate in cell proliferation in human breast cancer cells, and inhibition of heag K+ channels arrested cells in the early G1 phase .
Ito was first observed in hMSCs through mRNA expression analysis by Heubach et al. , and then it was detected in a small population of hMSCs (∼8%) by another group . However, in our group, surprisingly, Ito was recorded in over 50% of hUC-MSCs. During development of embryonic stem cell-derived cardiomyocytes, inhibition of Ito by 4-AP changed the duration and frequency of action potentials in the early stage but not in the late stage, suggesting that the Ito of early-stage cardiomyocytes plays an important role in controlling electrical activity . These reports suggest that K+ currents may modulate progression through the cell cycle in proliferating cells and in early embryonic development as well as at later stages of differentiation [37, 38, 40, 52].
IKCa usually coexists with IKDR, and the channel was proven to have a MaxiK channel by pharmacological and molecular biological approaches (Figs. 4, 6). MaxiK channels are usually believed to be sensors of intracellular Ca2+ and are found to regulate membrane potential in an intracellular Ca2+-dependent manner in hMSCs . These results suggest that MaxiK current could, therefore, be an effector of trophic factors within the body fluids or cell culture medium.
Voltage-gated Na+ channels are responsible for action potential initiation and propagation in excitable cells, including nerve, muscle, and neuroendocrine cells. We recorded INa in about 30% of hUC-MSCs and found that the current was highly sensitive to blockage by TTX (Fig. 3A). NaV1.7 (SCN9A or hNE-Na) is highly TTX sensitive and is broadly expressed in neurons, whereas NaV1.5 (SCN5A) is TTX resistant . Electrophysiological properties and mRNA expression pattern of Na+ channel of hUC-MSCs are consistent with the report by Li et al . Unexpectedly, TTX-sensitive Na+ current in hUC-MSCs was also inhibited by verapamil, known as an L-type Ca2+ channel-specific blocker. Furthermore, the current was not eliminated in the absence of Ca2+, and Ca2+ channel transcripts (1αC, 1αD, 1αG, 1αH, and 1αS) were not detected by RT-PCR analysis (data not shown). However, some groups observed the L-type Ca2+ channel in hBM-MSCs using patch clamp and RT-PCR analysis [8, 9]. These results suggest that the variety of ion channel distribution in MSCs may depend on the species or source of MSCs. Na+ channel may play important physiological roles during differentiation of hUC-MSCs into cardiac cells and neuronal cells. Therefore, further studies on the late passages or differentiated cells are needed because these characteristics of the channel may disappear during differentiation of hMSCs. In our view, undifferentiated cells may have undifferentiated channels.
We also demonstrated the presence of an inwardly rectifying K+ current in a small population of hUC-MSCs. Kir2.1 (classic Kir channel or IRK1) transcript was detected by RT-PCR. To our knowledge, this is the first electrophysiological recording of Kir in human MSCs, not in rodent MSCs [42, 54]. Usually Kir likely plays a dominant role in the maintenance of resting membrane potential, in regulating the action potential duration, and thus in controlling the excitability of a variety of cells. It has been reported that Kir is distributed in hemopoietic progenitor cells and neural stem cells [55, 56], raising the possibility that the presence of Kir might be a physiological marker for the pluripotent neural stem cells or neural progenitor cells.
Functional Implications and Conclusions
The present observations focused on the functional expression of ion channels, and we demonstrated that various ion channels are present in hUC-MSCs. An understanding of the regulation of ion channels in undifferentiated hUC-MSCs will be helpful to investigate possible biological solutions, such as gene and/or cell therapy, to medical challenges such as teratogenicity, infection, and immune rejection. Our results provide strong support for the study of the physiological roles of these ionic currents in the proliferation and differentiation of hMSCs in the future, in order to gather more information on human stem cell biology.