Choi S H, Jung S Y, Suh W, Baek S H, Kwon S-M
Laboratory of Cardiovascular Regeneration, Division of Cardiovascular Medicine, Seoul St Mary's Hospital, The Catholic University of Korea, School of Medicine, Seoul, South Korea.
Transplant Proc. 2013 Jan-Feb;45(1):420-6. doi: 10.1016/j.transproceed.2012.08.017.
Although cardiac stem cells (CSCs) have emerged in regeneration research, the number of isolated CSCs is low, making a sufficient supply of functional elements an important consideration in cardiovascular research. In this study, we established an efficient method for CSC isolation. We directly compared cultures of single cells to human cardiac-derived c-kit-positive progenitor cells (hCPCs(c-kit+)). The two protocols employed enzymatically digested hCPCs(c-kit+) (ED-hCPCs) with tissue-expanded hCPC(c-kit+) (TE-hCPCs). Using fluorescence-activated cell sorting, we showed the concentration of c-kit in TE-hCPCs to be higher than in ED-hCPCs, although the total number of c-kit positive cells resulting from ED-hCPCs was similar to that resulting from TE-hCPCs. The cardiomyocyte-associated proteins, GATA4 and Nkx2-5, which were expressed during hCPCs expansion, did not differ between the isolation methods. Importantly, the expression of the CSC stem cell marker, c-kit, was more efficiently preserved using the ED-hCPCs versus the TE-hCPCs method. In a cell proliferation assay, the ED-hCPCs method produced a significantly greater number of cells. Finally, hCPCs derived using both protocols differentiated into endothelial, smooth muscle, and cardiomyocyte lineages. In conclusion, the single-cell culture protocol using an enzymatic digestion method may be more useful to isolate human cardiac-derived c-kit-positive elements compared with the tissue expansion method.
尽管心脏干细胞（CSCs）已在再生研究中出现，但分离出的CSCs数量较少，这使得在心血管研究中充分供应功能性元件成为一个重要的考虑因素。在本研究中，我们建立了一种高效的CSC分离方法。我们直接将单细胞培养物与人类心脏来源的c-kit阳性祖细胞（hCPCs(c-kit+)）进行比较。这两种方案均采用酶消化的hCPCs(c-kit+)（ED-hCPCs）和组织扩增的hCPC(c-kit+)（TE-hCPCs）。通过荧光激活细胞分选，我们发现TE-hCPCs中c-kit的浓度高于ED-hCPCs，尽管ED-hCPCs产生的c-kit阳性细胞总数与TE-hCPCs产生的相似。在hCPCs扩增过程中表达的心肌细胞相关蛋白GATA4和Nkx2-5在两种分离方法之间没有差异。重要的是，与TE-hCPCs方法相比，使用ED-hCPCs方法能更有效地保留CSC干细胞标志物c-kit的表达。在细胞增殖试验中，ED-hCPCs方法产生的细胞数量明显更多。最后，两种方案获得的hCPCs均分化为内皮细胞、平滑肌细胞和心肌细胞谱系。总之，与组织扩增方法相比，使用酶消化法的单细胞培养方案在分离人类心脏来源的c-kit阳性元件方面可能更有用。
