Division of Cell Regeneration and Transplantation, Advanced Medical Research Center, Nihon University School of Medicine, Tokyo, Japan.
Stem Cells Dev. 2009 Jan-Feb;18(1):113-26. doi: 10.1089/scd.2008.0045.
Embryonic stem (ES) cells have been proposed as candidates for cell replacement therapy in patients with intestinal failure because these cells can be expanded indefinitely without losing their pluripotent phenotype. We investigated the differentiation capacity of mouse ES cells into gut-like structures, including intestinal stem cells, and defined culture conditions for efficient induction of formation of these structures. ES cell-derived gut-like structures (ES-guts) were reproducibly induced in developing embryoid bodies (EBs) by day 21 of differentiation culture. ES-guts contained an endodermal epithelium, a smooth muscle layer, interstitial cells of Cajal, and enteric neurons and showed spontaneous contraction. Transplantation of ES-guts under the kidney capsules of immunodeficient mice induced formation of highly differentiated epithelium composed of absorptive cells and goblet cells in the grafts. Immunoreactivity for Musashi-1 (Msi-1), a marker of intestinal stem cells, was detected in 1.9% of the columnar epithelial cells in the graft. Culture with 0.1% dimethyl sulfoxide increased the numbers of ES-guts in EBs, and serum-replacement (SR) culture, in comparison to standard ES culture containing 15% serum, increased the area ratio of ES-guts to EBs. SR culture also promoted maturation of epithelium to form a single layer of columnar epithelial cells, including absorptive cells and goblet cells. Expression of Msi-1 mRNA and protein was significantly enhanced when EBs were cultured under SR conditions. In conclusion, SR conditions efficiently induce formation of ES-guts and promote differentiation of epithelium, including intestinal stem cells. These results suggest the feasibility of cell-based therapy for intestinal failure based on ES cell culture systems.
胚胎干细胞（ES 细胞）被提议作为肠衰竭患者细胞替代治疗的候选物，因为这些细胞可以无限期扩增而不会失去其多能表型。我们研究了小鼠 ES 细胞分化为肠样结构的能力，包括肠干细胞，并确定了有效诱导这些结构形成的培养条件。通过分化培养第 21 天，ES 细胞衍生的肠样结构（ES 肠）可在发育中的胚状体（EB）中重现性诱导。ES 肠包含内胚层上皮、平滑肌层、Cajal 间质细胞和肠神经元，并表现出自发收缩。将 ES 肠移植到免疫缺陷小鼠的肾囊下，诱导移植物中高度分化的吸收细胞和杯状细胞组成的上皮形成。在移植物中，检测到肠干细胞标志物 Musashi-1（Msi-1）的免疫反应性，在移植物中 1.9%的柱状上皮细胞中检测到 Msi-1。与含有 15%血清的标准 ES 培养相比，0.1%二甲基亚砜的培养增加了 EB 中的 ES 肠数量，而血清替代（SR）培养增加了 ES 肠与 EB 的面积比。SR 培养还促进了上皮的成熟，形成单层柱状上皮细胞，包括吸收细胞和杯状细胞。当 EB 在 SR 条件下培养时，Msi-1 mRNA 和蛋白的表达显著增强。总之，SR 条件有效地诱导 ES 肠的形成，并促进上皮的分化，包括肠干细胞。这些结果表明基于 ES 细胞培养系统进行肠衰竭的细胞治疗是可行的。
