Goldman B I, Wurzel J
Department of Pathology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140.
In Vitro Cell Dev Biol. 1992 Feb;28A(2):109-19. doi: 10.1007/BF02631014.
Previous work has suggested that subcultivated human fetal heart muscle cell cultures contain immature cardiac muscle cells capable only of limited differentiation after mitogen withdrawal. We studied several human fetal heart cultures (14-15 wk gestation) at several passage levels using immunocytochemistry, autoradiography, and Northern blot analysis. Characteristics in high-mitogen (growth) medium were compared with those after serum withdrawal. Cultured cells from one heart, expanded through 2 passages in growth medium, did not beat; however, 75% of cells did beat after subsequent culture for 24 days in low-serum (differentiation) medium containing insulin. In confluent cultures after 1 passage, there was no detectable difference in the number of cardiac myocytes present in growth medium compared with that 7 days after serum withdrawal. After 4 passages, however, serum withdrawal increased the number of cells expressing immunoreactive sarcomeric myosin heavy chain by 100-fold; expression of immunoreactive sarcomeric actin and alpha-cardiac actin mRNA also increased in the same cultures. Similar results were obtained in cultures kept in differentiation medium for 20 days before passage and expansion in growth medium. Using isopycnic centrifugation, a high-density cell fraction was isolated which contained no immunostained myocytes in growth medium but numerous myocytes after serum withdrawal. Combined immunocytochemistry/autoradiography showed that myocytes synthesize DNA in growth medium and in serum-free medium containing fibroblast growth factor, but not in serum-free medium alone. The results indicate that a) human fetal cardiac muscle cells proliferate in vitro and can maintain a phenotype characteristic of fetal myocytes after multiple subcultivations followed by serum withdrawal; b) after subcultivation in growth medium, some myocytes modulate their phenotype into one in which detectable levels of cardiac contractile proteins are expressed only after mitogen withdrawal, and c) the phenotype attained after serum withdrawal is in part dependent on passage level. Cultured human fetal myocardial cells may provide a useful experimental system for the study of human cardiac muscle cell biology.
先前的研究表明，传代培养的人胎儿心肌细胞培养物中含有未成熟的心肌细胞，这些细胞在去除促有丝分裂原后仅能进行有限的分化。我们使用免疫细胞化学、放射自显影和Northern印迹分析，研究了处于几个传代水平的几种人胎儿心脏培养物（妊娠14 - 15周）。将高促有丝分裂原（生长）培养基中的细胞特征与血清去除后的细胞特征进行了比较。来自一颗心脏的培养细胞在生长培养基中传代2次后不会跳动；然而，在随后含有胰岛素的低血清（分化）培养基中培养24天后，75%的细胞会跳动。在传代1次后的汇合培养物中，生长培养基中的心肌细胞数量与血清去除7天后相比没有可检测到的差异。然而，传代4次后，血清去除使表达免疫反应性肌节肌球蛋白重链的细胞数量增加了100倍；在相同培养物中，免疫反应性肌节肌动蛋白和α - 心肌肌动蛋白mRNA的表达也增加了。在传代和在生长培养基中扩增之前，先在分化培养基中培养20天的培养物也得到了类似的结果。使用等密度离心法，分离出了一个高密度细胞组分，该组分在生长培养基中没有免疫染色的心肌细胞，但在血清去除后有大量心肌细胞。免疫细胞化学/放射自显影联合显示，心肌细胞在生长培养基和含有成纤维细胞生长因子的无血清培养基中合成DNA，但在单独的无血清培养基中不合成。结果表明：a）人胎儿心肌细胞在体外增殖，并且在多次传代后去除血清仍可维持胎儿心肌细胞的表型特征；b）在生长培养基中传代后，一些心肌细胞将其表型调节为仅在去除促有丝分裂原后才表达可检测水平的心脏收缩蛋白的表型；c）血清去除后获得的表型部分取决于传代水平。培养的人胎儿心肌细胞可能为研究人心肌细胞生物学提供一个有用的实验系统。
