Human Embryonic and Fetal Mesenchymal Stem Cells Differentiate toward Three Different Cardiac Lineages in Contrast to Their Adult Counterparts

Mesenchymal stem cells (MSCs) show unexplained differences in differentiation potential. In this study, differentiation of human (h) MSCs derived from embryonic, fetal and adult sources toward cardiomyocytes, endothelial and smooth muscle cells was investigated. Labeled hMSCs derived from embryonic stem cells (hESC-MSCs), fetal umbilical cord, bone marrow, amniotic membrane and adult bone marrow and adipose tissue were co-cultured with neonatal rat cardiomyocytes (nrCMCs) or cardiac fibroblasts (nrCFBs) for 10 days, and also cultured under angiogenic conditions. Cardiomyogenesis was assessed by human-specific immunocytological analysis, whole-cell current-clamp recordings, human-specific qRT-PCR and optical mapping. After co-culture with nrCMCs, significantly more hESC-MSCs than fetal hMSCs stained positive for α-actinin, whereas adult hMSCs stained negative. Furthermore, functional cardiomyogenic differentiation, based on action potential recordings, was shown to occur, but not in adult hMSCs. Of all sources, hESC-MSCs expressed most cardiac-specific genes. hESC-MSCs and fetal hMSCs contained significantly higher basal levels of connexin43 than adult hMSCs and co-culture with nrCMCs increased expression. After co-culture with nrCFBs, hESC-MSCs and fetal hMSCs did not express α-actinin and connexin43 expression was decreased. Conduction velocity (CV) in co-cultures of nrCMCs and hESC-MSCs was significantly higher than in co-cultures with fetal or adult hMSCs. In angiogenesis bioassays, only hESC-MSCs and fetal hMSCs were able to form capillary-like structures, which stained for smooth muscle and endothelial cell markers.Human embryonic and fetal MSCs differentiate toward three different cardiac lineages, in contrast to adult MSCs. Cardiomyogenesis is determined by stimuli from the cellular microenvironment, where connexin43 may play an important role.

间充质干细胞（Mesenchymal stem cells, MSCs）的分化潜能存在尚未阐明的差异。本研究针对源自胚胎、胎儿及成人组织的人类（h）间充质干细胞向心肌细胞、内皮细胞和平滑肌细胞的分化过程展开探究。研究对象包括经标记的胚胎干细胞来源的间充质干细胞（human embryonic stem cell-derived MSCs, hESC-MSCs）、胎儿脐带来源、骨髓来源、羊膜来源以及成人骨髓与脂肪组织来源的人类间充质干细胞，将其分别与新生大鼠心肌细胞（neonatal rat cardiomyocytes, nrCMCs）或新生大鼠心脏成纤维细胞（neonatal rat cardiac fibroblasts, nrCFBs）共培养10天，同时设置血管生成培养条件组。心肌生成能力通过人类特异性免疫细胞化学分析、全细胞膜片钳电流记录、人类特异性定量实时聚合酶链反应（quantitative real-time polymerase chain reaction, qRT-PCR）以及光学标测技术进行评估。与新生大鼠心肌细胞共培养后，胚胎干细胞来源的间充质干细胞中α-辅肌动蛋白阳性染色的细胞比例显著高于胎儿来源的间充质干细胞，而成人来源的间充质干细胞则呈阴性染色。此外，基于动作电位记录的功能性心肌分化能力仅在胚胎与胎儿来源的间充质干细胞中得以证实，成人来源的间充质干细胞未出现该现象。在所有来源的间充质干细胞中，胚胎干细胞来源的间充质干细胞表达的心脏特异性基因最多。胚胎干细胞来源与胎儿来源的间充质干细胞的连接蛋白43（connexin43）基础表达水平显著高于成人来源的间充质干细胞，且与新生大鼠心肌细胞共培养可提升其连接蛋白43的表达量。与新生大鼠心脏成纤维细胞共培养后，胚胎干细胞来源与胎儿来源的间充质干细胞均不表达α-辅肌动蛋白，且连接蛋白43的表达量也有所下降。新生大鼠心肌细胞与胚胎干细胞来源的间充质干细胞共培养体系中的传导速度（Conduction velocity, CV）显著高于与胎儿或成人来源的间充质干细胞共培养的体系。在血管生成生物实验中，仅胚胎干细胞来源与胎儿来源的间充质干细胞能够形成毛细血管样结构，且该结构可被平滑肌细胞与内皮细胞标志物染色呈阳性。与成人来源的间充质干细胞不同，人类胚胎及胎儿来源的间充质干细胞可向三种不同的心脏谱系分化。心肌生成能力受细胞微环境中的刺激信号调控，其中连接蛋白43可能发挥重要作用。