Department of Histology and Embryology, Medical School of Shinshu University, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan.
Biochem Biophys Res Commun. 2010 Apr 16;394(4):877-83. doi: 10.1016/j.bbrc.2010.03.008. Epub 2010 Mar 4.
Photoreceptors cannot regenerate and recover their functions once disordered. Transplantation of retinal pigment epithelium (RPE) has recently become a possible therapeutic approach for retinal degeneration. In the present study, we investigated the induction of photoreceptors by coculturing primate embryonic stem cells (ESCs) with ESC-derived RPE cells.
RPE cells were derived by coculturing ESCs and Sertoli cells. Photoreceptors were then induced by using ESC-derived RPE cells and retinoic acid (RA) RESULTS: RPE cell generation was confirmed by morphological analysis, which revealed highly pigmented polygonal cells with a compact cell-cell arrangement. After coculturing ESCs and RPE cells, some ESC derivatives became immunopositive for rhodopsin. RT-PCR analysis demonstrated the expression of retina-related gene markers such as Pax6, CRX, IRBP, rhodopsin, rhodopsin kinase, and Muschx10A. When RA was added, a distinct increase in the expression of photoreceptor-specific proteins and genes was found. In addition, the differentiation of bipolar horizontal cells was demonstrated by protein and gene expression. The ESCs that were cocultured with RPE cells and treated with RA were transplanted into the renal capsule or intra-vitreal space of nude mice. Grafted ESC derivatives demonstrated extensive rhodopsin expression, and they survived and organized into recipient tissues, although they formed teratomas.
These results indicate that coculturing ESCs with ESC-derived RPE cells is a useful and efficient method for inducing photoreceptors and providing an insight into the use of ESCs for retina regeneration.
感光细胞一旦发生紊乱，就无法再生和恢复功能。最近，视网膜色素上皮（RPE）移植已成为治疗视网膜变性的一种可能的治疗方法。在本研究中，我们通过将灵长类胚胎干细胞（ESCs）与 ESC 衍生的 RPE 细胞共培养来研究诱导感光细胞的方法。
通过将 ESCs 与支持细胞共培养来获得 RPE 细胞。然后使用 ESC 衍生的 RPE 细胞和视黄酸（RA）来诱导感光细胞。
通过形态分析证实了 RPE 细胞的生成，该分析显示出高度色素化的多边形细胞，细胞间排列紧密。在 ESCs 和 RPE 细胞共培养后，一些 ESC 衍生物对视蛋白呈免疫阳性。RT-PCR 分析表明表达了视网膜相关基因标志物，如 Pax6、CRX、IRBP、视蛋白、视蛋白激酶和 Muschx10A。添加 RA 后，发现感光细胞特异性蛋白和基因的表达明显增加。此外，通过蛋白和基因表达证明了双极水平细胞的分化。与 RPE 细胞共培养并经 RA 处理的 ESCs 被移植到裸鼠的肾囊或玻璃体腔内。移植的 ESC 衍生物表现出广泛的视蛋白表达，尽管它们形成了畸胎瘤，但仍能存活并组织成受者组织。
这些结果表明，将 ESCs 与 ESC 衍生的 RPE 细胞共培养是一种诱导感光细胞的有效方法，为利用 ESCs 进行视网膜再生提供了思路。
