Drab M, Haller H, Bychkov R, Erdmann B, Lindschau C, Haase H, Morano I, Luft F C, Wobus A M
Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany.
FASEB J. 1997 Sep;11(11):905-15. doi: 10.1096/fasebj.11.11.9285489.
Vascular smooth muscle cell (VSMC) differentiation is important in understanding vascular disease; however, no in vitro model is available. Totipotent mouse embryonic stem (ES) cells were used to establish such a model. To test whether the ES cell-derived smooth muscle cells expressed VSMC-specific properties, the differentiated cells were characterized by 1) morphological analysis, 2) gene expression, 3) immunostaining for VSMC-specific proteins, 4) expression of characteristic VSMC ion channels, and 5) formation of [Ca2+]i transients in response to VSMC-specific agonists. Treatment of embryonic stem cell-derived embryoid bodies with retinoic acid and dibutyryl-cyclic adenosine monophosphate (db-cAMP) induced differentiation of spontaneously contracting cell clusters in 67% of embryoid bodies compared with 10% of untreated controls. The highest differentiation rate was observed when retinoic acid and db-cAMP were applied to the embryoid bodies between days 7 and 11 in combination with frequent changes of culture medium. Other protocols with retinoic acid and db-cAMP, as well as single or combined treatment with VEGF, ECGF, bFGF, aFGF, fibronectin, matrigel, or hypoxia did not influence the differentiation rate. Single-cell RT-PCR and sequencing of the PCR products identified myosin heavy chain (MHC) splice variants distinguishing between gut and VSMC isoforms. RT-PCR with VSMC-specific MHC primers and immunostaining confirmed the presence of VSMC transcripts and MHC protein. Furthermore, VSMC expressing MHC had typical ion channels and responded to specific agonists with an increased [Ca2+]i. Here we present a retinoic acid + db-cAMP-inducible embryonic stem cell model of in vitro vasculogenesis. ES cell-derived cells expressing VSMC-specific MHC and functional VSMC properties may be a suitable system to study mechanisms of VSMC differentiation.
血管平滑肌细胞（VSMC）分化对于理解血管疾病至关重要；然而，目前尚无体外模型。本研究利用全能性小鼠胚胎干细胞（ES细胞）建立了这样一个模型。为了检测ES细胞来源的平滑肌细胞是否表达VSMC特异性特性，通过以下方法对分化细胞进行了表征：1）形态学分析；2）基因表达；3）对VSMC特异性蛋白进行免疫染色；4）特征性VSMC离子通道的表达；5）对VSMC特异性激动剂作出反应时形成[Ca2+]i瞬变。用视黄酸和二丁酰环磷酸腺苷（db-cAMP）处理胚胎干细胞来源的胚状体，67%的胚状体中诱导出了自发收缩的细胞簇，而未处理的对照组中这一比例为10%。当视黄酸和db-cAMP在第7至11天应用于胚状体并频繁更换培养基时，观察到最高的分化率。视黄酸和db-cAMP的其他方案，以及VEGF、ECGF、bFGF、aFGF、纤连蛋白、基质胶或缺氧的单一或联合处理均不影响分化率。单细胞RT-PCR及PCR产物测序鉴定出了区分肠道和平滑肌细胞亚型的肌球蛋白重链（MHC）剪接变体。用VSMC特异性MHC引物进行RT-PCR和免疫染色证实了VSMC转录本和MHC蛋白的存在。此外，表达MHC的VSMC具有典型的离子通道，并对特异性激动剂作出反应，使[Ca2+]i增加。在此，我们展示了一种视黄酸+db-cAMP诱导的体外血管生成胚胎干细胞模型。表达VSMC特异性MHC和功能性VSMC特性的ES细胞来源的细胞可能是研究VSMC分化机制的合适系统。
