Characterization of intracellular free Ca2+ movements in neural progenitor cells derived from ES cells transfected with MASH1 transcription factor gene

Abstract

Intracellular free Ca2+ movements in neural progenitor cells obtained by transfection of embryonic stem(ES) cells with MASH1 transcription factor gene were studied. The MASH1 transfected cells, majority of which were Islet1 positive, have been shown to improve motor functions of hemiplegic mice when transplanted into the injured brain. In this study RT-PCR was conducted to detect Ca2+ channel mRNAs. We found that mRNAs of L-type, N-type and T-type Ca2+ channel mRNAs were expressed in the MASH1 transfected cells. Next, a Ca2+-sensitive fluorescence probe, fluo-3, and a scanning confocal laser microscope were used to detect any changes of intracellular Ca2+ concentration. Enhanced fluo-3 fluorescence was observed when the cells were stimulated with increased extracellular potassium, a depolarization signal. Depletion of extracellular Ca2+ abrogated the increase of fluorescence intensity upon depolarization. A Ca2+ channel blocker, lead, inhibited the increase of fluorescence intensity upon depolarization. A L-type channel inhibitor, nifedipine, but not a N-type channel inhibitor, omega-conotoxin GVIA, reduced the increase of fluorescence intensity upon depolarization. Thapsigargin, which depletes intracellular Ca2+ stores, did not attenuate the depolarizationinduced signals. Potassium channel inhibitors, tetraethylammonium and 4-aminopyridine inhibited the fluorescence signals. These results indicate that depolarization-induced intracellular Ca2+ increase is mainly due to inward flow (influx) of Ca2+ via Ca2+ channels and marginally due to the release (mobilization) of Ca2+ from intracellular stores in the MASH1 transfected neural progenitor cells. The calcium signals may regulate various aspects of neural cell function such as authentic cellular activity including neurotransmitter secretion and cell differentiation. Our data add the possibility that calcium signals may play roles even in the ectopically transplanted MASH1-transfected neural progenitor cells reconstituting neural network to improve motor functions of hemiplegic mice. Rec.6/6/2005, Acc.7/25/2005, pp452-460 1)Departments of Immunology and Medicine, 2)Department of Neurosurgery, St. Marianna University School of Medicine 3)Laboratory of Neurobiological Engineering, Department of Biological Science and Technology, School of High-Technology for Human Welfare, Tokai University 4)Department of Regenerative Medicine, Institute of Advanced Medical Science, St. Marianna University Graduate School of Medicine