Metzger J M, Lin W I, Samuelson L C
Department of Physiology, School of Medicine, University of Michigan, Ann Arbor 48109-0622, USA.
Circ Res. 1996 Apr;78(4):547-52. doi: 10.1161/01.res.78.4.547.
Mouse embryonic stem (ES) cells differentiate in vitro into a variety of cell types, including spontaneously contracting cardiac myocytes. The primary aim of this work was to use vital stain techniques for real-time detection of developing cardiac myocytes in ES cell differentiation cultures. The -440 to +6 human cardiac alpha-actin promoter was used to direct expression of the Escherichia coli reporter gene lacZ (pHCActlacZ) into ES cell-derived cardiac myocytes during cardiogenesis in vitro. Undifferentiated ES cells were electroporated with HCActlacZ together with a plasmid containing the neomycin gene under the direction of the phosphoglycerate kinase promoter, and stable transformants were selected in G418. Individual clones were screened for activation of lacZ gene expression in cardiac myocytes developing in vitro. Results showed that expression of the HCActlacZ reporter construct was activated very early during the ES cell differentiation program, at a time point before the appearance of spontaneous contractile activity. The earliest detection was at day 6 of differentiation, when approximately 25% of the differentiation cultures expressed the reporter construct, with expression increasing to approximately 70% at day 9 and continuing throughout the duration of spontaneous contractile activity exhibited by the ES cell-derived cardiac myocytes. Indirect immunofluorescence assays provide evidence that expression was restricted to the cardiac myocytes in culture. In the present study, we show vital staining of transgene expression in living cardiac myocytes using lipophilic fluorogenic beta-galactopyranoside substrates for real-time detection of the reporter gene during continuous contraction of the ES cell myocytes in vitro. The vital stain approach used in the present study will permit the identification of differentiating ES cells that are committed to the cardiac lineage for analysis of gene expression at early time points of ES cell cardiogenesis and, in addition, will aid in selecting genetically modified ES cell cardiac myocytes for use in functional studies.
小鼠胚胎干细胞（ES细胞）在体外可分化为多种细胞类型，包括自发收缩的心肌细胞。本研究的主要目的是运用活体染色技术实时检测ES细胞分化培养物中正在发育的心肌细胞。在体外心脏发生过程中，利用-440至+6的人心脏α-肌动蛋白启动子将大肠杆菌报告基因lacZ（pHCActlacZ）定向表达至ES细胞来源的心肌细胞中。将未分化的ES细胞与携带在磷酸甘油酸激酶启动子调控下的新霉素基因的质粒一起用HCActlacZ进行电穿孔，并在G418中筛选稳定转化体。对各个克隆进行筛选，以检测体外发育的心肌细胞中lacZ基因表达的激活情况。结果显示，HCActlacZ报告构建体的表达在ES细胞分化程序的早期就被激活，即在自发收缩活动出现之前的时间点。最早检测到是在分化的第6天，此时约25%的分化培养物表达报告构建体，在第9天表达增加至约70%，并在ES细胞来源的心肌细胞表现出自发收缩活动的整个期间持续存在。间接免疫荧光分析提供了证据，表明表达仅限于培养中的心肌细胞。在本研究中，我们展示了使用亲脂性荧光β-半乳糖吡喃糖苷底物对活心肌细胞中的转基因表达进行活体染色，以在体外ES细胞来源的心肌细胞持续收缩期间实时检测报告基因。本研究中使用的活体染色方法将有助于鉴定已定向分化为心脏谱系的ES细胞，以便在ES细胞心脏发生的早期时间点分析基因表达，此外，还将有助于选择用于功能研究的基因修饰的ES细胞来源的心肌细胞。
