Department of Pharmacology, Dalhousie University, Sir Charles Tupper Building, 5850 College Street, P.O. Box 15000, Halifax, Nova Scotia, Canada B3H 4R2.
Department of Pharmacology, Dalhousie University, Sir Charles Tupper Building, 5850 College Street, P.O. Box 15000, Halifax, Nova Scotia, Canada B3H 4R2.
Differentiation. 2019 Jan-Feb;105:1-13. doi: 10.1016/j.diff.2018.11.001. Epub 2018 Nov 23.
Mid-gestation mouse ventricles (E11.5) contain a larger number of Nkx2.5 cardiac progenitor cells (CPCs). The proliferation rates are consistently higher in CPCs compared to myocyte population of developing ventricles. Recent studies suggested that CPCs are an ideal donor cell type for replacing damaged tissue in diseased hearts. Thus, the ability to isolate and expand CPCs from embryos or stem cell cultures could be useful for cell fate studies and regenerative therapies. Since embryonic CPCs possess fewer mitochondria compared to cardiomyocytes, we reasoned that CPCs can be fractionated using a fluorescent mitochondrial membrane potential dye (TMRM) and these cells may retain cardiomyogenic potential even in the absence of cardiomyocytes (CMs). FACS sorting of TMRM stained embryonic ventricular cells indicated that over 99% of cells in TMRM high fraction stained positive for sarcomeric myosin (MF20) and all of them expressed Nkx2.5. Although majority of cells present in TMRM low fraction expressed Nkx2.5, very few cells (~1%) stained positive for MF20. Further culturing of TMRM low cells over a period of 48 h showed a progressive increase in MF20 positive cells. Additional analyses revealed that MF20 negative cells in TMRM low fraction do not express markers for endothelial cells (vWF, CD31) or smooth muscle cells (SM myosin). Treatment of TMRM low cells with known cardiogenic factors DMSO and dynorphin B significantly increased the percentage of MF20 cells compared to untreated cultures. Collectively, these studies suggest that embryonic CPCs can be separated as a TMRM low fraction and their differentiation potential can be enhanced by exogenous addition of known cardiomyogenic factors.
妊娠中期的小鼠心室（E11.5）含有更多的 Nkx2.5 心脏祖细胞（CPCs）。与发育心室中的心肌细胞群体相比，CPCs 的增殖率始终更高。最近的研究表明，CPCs 是替代病变心脏中受损组织的理想供体细胞类型。因此，从胚胎或干细胞培养物中分离和扩增 CPCs 的能力可能对细胞命运研究和再生疗法有用。由于胚胎 CPCs 比心肌细胞具有更少的线粒体，我们推断 CPCs 可以使用荧光线粒体膜电位染料（TMRM）进行分离，并且即使没有心肌细胞（CMs），这些细胞也可能保留心肌生成潜力。TMRM 染色的胚胎心室细胞的 FACS 分选表明，TMRM 高分数的细胞中超过 99%对肌球蛋白（MF20）呈阳性染色，并且它们都表达 Nkx2.5。尽管 TMRM 低分数中存在的大多数细胞表达 Nkx2.5，但只有很少的细胞（约 1%）对 MF20 呈阳性染色。进一步培养 TMRM 低细胞 48 小时后，MF20 阳性细胞的比例逐渐增加。进一步的分析表明，TMRM 低分数中的 MF20 阴性细胞不表达内皮细胞（vWF、CD31）或平滑肌细胞（SM 肌球蛋白）的标志物。用已知的心肌生成因子 DMSO 和 dynorphin B 处理 TMRM 低细胞与未处理的培养物相比，MF20 细胞的比例显著增加。总的来说，这些研究表明，胚胎 CPCs 可以作为 TMRM 低分数分离出来，并且通过外源添加已知的心肌生成因子可以增强它们的分化潜力。
