In vitro differentiation of retinal cells from human pluripotent stem cells by small-molecule induction
In vitro differentiation of retinal cells from human pluripotent stem cells by small-molecule induction
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DOI：
10.1242/jcs.050393
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发表时间：
2009-09-01
影响因子：
4
通讯作者：
Takahashi, Masayo
中科院分区：
文献类型：
--
作者：
Osakada, Fumitaka;Jin, Zi-Bing;Takahashi, Masayo
使用干细胞疗法治疗视网膜变性有很大的希望。然而，尚未设计出不依赖于在动物或大肠杆菌细胞中产生的重组蛋白的视网膜分化的确定方法。在这里，我们报告了一种确定的培养方法，使用低分子量的化合物，诱导分化的人胚胎干细胞（ES）细胞和诱导多能干细胞（iPS）细胞成视网膜祖细胞，视网膜色素上皮细胞和感光细胞。酪蛋白激酶I抑制剂CKI-7、ALK 4抑制剂SB-431542和Rho相关激酶抑制剂Y-27632在无血清和无饲养层的漂浮聚集体培养物中诱导RX、MITF、PAX 6和CHX 10阳性的视网膜祖细胞。该处理诱导表达RPE 65和CRALBP的六边形色素细胞，形成ZO 1阳性紧密连接并表现出吞噬功能。随后用视黄酸和牛磺酸处理诱导表达恢复素、视紫红质和参与光转导的基因的光感受器。三因子（OCT 3/4、SOX 2和KLF 4）和四因子（OCT 3/4、SOX 2、KLF 4和MYC）人iPS细胞均可通过小分子诱导成功分化为视网膜细胞。该方法为细胞替代疗法中的跨物种抗原污染问题提供了解决方案，并且还可用于发育、疾病和药物筛选的体外建模。
The use of stem-cell therapy to treat retinal degeneration holds great promise. However, definitive methods of retinal differentiation that do not depend on recombinant proteins produced in animal or Escherichia coli cells have not been devised. Here, we report a defined culture method using low-molecular-mass compounds that induce differentiation of human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells into retinal progenitors, retinal pigment epithelium cells and photoreceptors. The casein kinase I inhibitor CKI-7, the ALK4 inhibitor SB-431542 and the Rho-associated kinase inhibitor Y-27632 in serum-free and feeder-free floating aggregate culture induce retinal progenitors positive for RX, MITF, PAX6 and CHX10. The treatment induces hexagonal pigmented cells that express RPE65 and CRALBP, form ZO1-positive tight junctions and exhibit phagocytic functions. Subsequent treatment with retinoic acid and taurine induces photoreceptors that express recoverin, rhodopsin and genes involved in phototransduction. Both three-factor (OCT3/4, SOX2 and KLF4) and four-factor (OCT3/4, SOX2, KLF4 and MYC) human iPS cells could be successfully differentiated into retinal cells by small-molecule induction. This method provides a solution to the problem of cross-species antigenic contamination in cell-replacement therapy, and is also useful for in vitro modeling of development, disease and drug screening.