In vitro differentiation of murine embryonic stem cells toward a renal lineage
In vitro differentiation of murine embryonic stem cells toward a renal lineage
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DOI：
10.1111/j.1432-0436.2006.00149.x
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发表时间：
2007-06-01
期刊：
影响因子：
2.9
通讯作者：
Perkins, Andrew C.
中科院分区：
文献类型：
--
作者：
Bruce, Stephen J.;Rea, Robert W.;Perkins, Andrew C.
胚胎干细胞（ES细胞）具有分化为发育中胚胎的所有细胞的能力，并可能为未来的细胞替代疗法提供可再生资源。骨形态发生蛋白4（BMP 4）的添加到无血清ES细胞培养之前已被证明诱导转录因子，信号分子和细胞粘附蛋白在胚胎中胚层特化过程中表达。在这里，我们显示了ES细胞中原条中胚层分化的动力学在补充有BMP 4的血清和无血清胚状体（EB）培养物之间是相当的。此外，我们显示了一组基因（Pax 2，WT 1，podocalyxin，pod-1和nephrin）表达的延迟波，这些基因在胚胎肾脏发育过程中起着重要作用。Pax 2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。产生含有修饰的Pax 2启动子-lacZ或细菌人工染色体-绿色荧光蛋白（GFP）转基因的ES细胞系，这使得能够定量分析EB内肾脏而不是神经元Pax 2表达。EB培养16天后，通过免疫组织化学和流式细胞术分析检测β-半乳糖苷酶活性和GFP表达，这与Pax 2转录水平的增加相关。总之，这些结果表明，当补充有BMP 4时，在血清和无血清条件下生长的成熟EB中，自发的肾脏基因表达程序发展。此外，重组生长因子BMP 2、BMP 4和BMP 7强烈影响中胚层诱导的EB内的基因表达。BMP 4促进腹侧（血液）和中间（肾脏）中胚层基因表达，而BMP 2和BMP 7以造血承诺为代价促进肾脏结果。这种诱导试验和这些独特的ES细胞系将用于产生中胚层衍生的细胞群，并对未来的细胞治疗/整合试验产生影响。
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.