Isolation, Expansion And Ion Electrophysiological Characteristics Of Cardiac Stem Cells From Adult Mouse

Following normal development of the heart, mammalian cardiomyocytes withdraw from the cell cycle during the perinatal period. This fact accounts for the long-held belief that the heart is a terminally differentiated organ with no intrinsic capacity to regenerate after myocardial injury. This concept is now being seriously challenged, and compelling evidence has accumulated that suggests the mammalian heart is in a state of continuous cellular turnover and has intrinsic regenerative potential. The presence of myocytes undergoing mitosis and cytokinesis has been demonstrated in human hearts under both normal and pathologic conditions. In addition, resident cardiac stem cells or progenitor cells have been identified in the adult hearts of humans and other mammalian species. These CSCs, through both cell transplantation and in situ activation, have the capacity to regenerate significant segmental and diffuse myocyte losts, restoring anatomical integrity and ventricular function.Therefore, it is believed that CSCs are an ideal cell source for myocardial regeneration. However, cellular electrophysiology is not understood in CSCs. The present study was therefore designed to find a way of isolation and expansion of CSCs and investigate properties of ionic channel currents in undifferentiated CSCs from adullt mouse heart. PartⅠIsolation , expansion and characterization of cardiac stem cells from adult mouseIsolation of cardiac stem cells from adult mouse Hearts were excised and cannulated for retrograde perfusion through the aorta. Cardiac myocytes were isolated enzymatically. After removal of the heart from the cannula, the myocardium was cut into small pieces and shaken in collagenase solution. Intact small cells and myocytes were separated and enriched by centrifugation. The fraction of small cells was then incubated with microbeads coated with c-kit antibody. Small, round, phase-bright cells were obtained, most CSs were between 4 and 10μm in size.Expansion of cardiac stem cells from adult mouse Freshly isolated c-kitPOS cells were plated in enriched F12 medium for 48 hours, when most of the contaminating c-kitNEG cells attached to the plastic while the c-kitPOS remained unatdistached. The nonattached cells were transferred to modified neural stem cell medium (mNSCM) lacking neurogenic factors. After 7–10 days in mNSCM, most cells attached and were continuously grown.Characterization of cardiac stem cells from adult mouse Freshly isolated c-kitPOS cell and attached cells were fixed and analyzed by immunocytochemistry. Cells were observed by Confocal Laser Microscopy techniques. Both expressed c-kit and the attached cell did not differentiation. PartⅡIon electrophysiological characteristics of cardiac stem cells from adult mouseFor subculture, CSCs were detached with 0.25% trypsin and passaged plates when clones grew . For recording ion channel currents, the detached cells were suspended in the culture medium. The whole-cell patch-clamp technique was used. A gradually activating current like a delayed rectifier K+ current (IKDR) was recorded in a representative mouse CSCs upon the depolarizing voltage steps. IKDR was reversibly suppressed by TEA.In conclusion:1. The c-kitPOS cells were separated by repeated panning, using immunomagnetic microbeads. the method result in purified c-kitPOS cells.2. The cardiac c-kitPOS cells are self-renewing.3. Both freshly isolated c-kitPOS cell and attached cells expressed c-kit and the attached cell did not differentiation.4. Delayed rectifier K+current (IKDR) were present in undifferentiated CSCs.