Bruce Stephen J, Rea Robert W, Steptoe Anita L, Busslinger Meinrad, Bertram John F, Perkins Andrew C
Department of Anatomy and Cell Biology School of Biomedical Sciences Monash University Melbourne, Vic., Australia.
Differentiation. 2007 Jun;75(5):337-49. doi: 10.1111/j.1432-0436.2006.00149.x. Epub 2007 Feb 5.
Embryonic stem (ES) cells have the capacity to differentiate into all cells of the developing embryo and may provide a renewable resource for future cell replacement therapies. The addition of bone morphogenetic protein 4 (BMP4) to serum-free ES cell culture has previously been shown to induce transcription factors, signaling molecules, and cell adhesion proteins expressed during mesoderm specification of the embryo. Here, we show the dynamics of primitive streak mesoderm differentiation in ES cells is comparable between serum and serum-free embryoid body (EB) cultures, supplemented with BMP4. Furthermore, we show a delayed wave of expression of a cohort of genes (Pax2, WT1, podocalyxin, pod-1, and nephrin), which play important roles during embryonic kidney development. The paired box transcription factor, Pax2, is one of the earliest genes expressed during kidney organogenesis and is required for normal urogenital development. ES cell lines containing either a modified Pax2 promoter-lacZ or bacterial artificial chromosome-green fluorescent protein (GFP) transgene were generated, which enabled the quantitative analysis of kidney rather than neuronal Pax2 expression within EBs. Both beta-galactosidase activity and GFP expression were detected by immunohistochemical and flow cytometric analysis following 16 days of EB culture, which correlated with an increase in Pax2 transcript levels. Together, these results suggest a spontaneous kidney gene expression program develops in mature EBs grown in both serum and serum-free conditions, when supplemented with BMP4. Further, the recombinant growth factors BMP2, BMP4, and BMP7 strongly influence gene expression within mesoderm induced EBs. BMP4 promotes ventral (blood) and intermediate (kidney) mesoderm gene expression, whereas BMP2 and BMP7 promote kidney outcomes at the expense of hematopoietic commitment. This induction assay and these unique ES cell lines will be useful for the generation of mesoderm-derived cell populations with implications for future cell therapeutic/integration assays.
胚胎干细胞（ES细胞）有能力分化为发育中胚胎的所有细胞，并可能为未来的细胞替代疗法提供可再生资源。先前已证明，在无血清ES细胞培养中添加骨形态发生蛋白4（BMP4）可诱导胚胎中胚层特化过程中表达的转录因子、信号分子和细胞粘附蛋白。在此，我们表明，在添加BMP4的血清和无血清胚状体（EB）培养物中，ES细胞中原始条纹中胚层分化的动力学具有可比性。此外，我们发现一组基因（Pax2、WT1、足细胞外蛋白、pod-1和nephrin）的表达出现延迟，这些基因在胚胎肾脏发育过程中发挥重要作用。配对盒转录因子Pax2是肾脏器官发生过程中最早表达的基因之一，是正常泌尿生殖系统发育所必需的。我们生成了含有修饰的Pax2启动子-乳糖酶或细菌人工染色体-绿色荧光蛋白（GFP）转基因的ES细胞系，这使得能够对EB内的肾脏而非神经元Pax2表达进行定量分析。在EB培养16天后，通过免疫组织化学和流式细胞术分析检测到β-半乳糖苷酶活性和GFP表达，这与Pax2转录水平的增加相关。总之，这些结果表明，在添加BMP4的血清和无血清条件下生长的成熟EB中，会出现自发的肾脏基因表达程序。此外，重组生长因子BMP2、BMP4和BMP7强烈影响中胚层诱导的EB内的基因表达。BMP4促进腹侧（血液）和中间（肾脏）中胚层基因表达，而BMP2和BMP7以牺牲造血分化为代价促进肾脏发育。这种诱导试验和这些独特的ES细胞系将有助于生成中胚层来源的细胞群体，对未来的细胞治疗/整合试验具有重要意义。
