Department of Ophthalmology, The second Xiangya Hospital, Central South University, Changsha, Hunan, People's Republic of China.
Stem Cells. 2011 Jun;29(6):972-80. doi: 10.1002/stem.637.
Absence of a regenerative pathway for damaged retina following injury or disease has led to experiments using stem cell transplantation for retinal repair, and encouraging results have been obtained in rodents. The swine eye is a closer anatomical and physiological match to the human eye, but embryonic stem cells have not been isolated from pig, and photoreceptor differentiation has not been demonstrated with induced pluripotent stem cells (iPSCs) of swine. Here, we subjected iPSCs of swine to a rod photoreceptor differentiation protocol consisting of floating culture as embryoid bodies followed by differentiation in adherent culture. Real-time PCR and immunostaining of differentiated cells demonstrated loss of expression of the pluripotent genes POU5F1, NANOG, and SOX2 and induction of rod photoreceptor genes RCVRN, NRL, RHO, and ROM1. While these differentiated cells displayed neuronal morphology, culturing on a Matrigel substratum triggered a further morphological change resulting in concentration of rhodopsin (RHO) and rod outer segment-specific membrane protein 1 in outer segment-like projections resembling those on primary cultures of rod photoreceptors. The differentiated cells were transplanted into the subretinal space of pigs treated with iodoacetic acid to eliminate rod photoreceptors. Three weeks after transplantation, engrafted RHO+ cells were evident in the outer nuclear layer where photoreceptors normally reside. A portion of these transplanted cells had generated projections resembling outer segments. These results demonstrate that iPSCs of swine can differentiate into photoreceptors in culture, and these cells can integrate into the damaged swine neural retina, thus, laying a foundation for future studies using the pig as a model for retinal stem cell transplantation.
由于受损视网膜在损伤或疾病后缺乏再生途径，因此进行了使用干细胞移植进行视网膜修复的实验，并且在啮齿动物中已经获得了令人鼓舞的结果。猪眼在解剖学和生理学上更接近人类眼睛，但尚未从猪中分离出胚胎干细胞，并且未用猪的诱导多能干细胞（iPSC）证明光感受器分化。在这里，我们将猪的 iPSC 置于杆状光感受器分化方案中，该方案包括作为胚状体的悬浮培养，然后在贴壁培养中进行分化。实时 PCR 和分化细胞的免疫染色显示多能基因 POU5F1、NANOG 和 SOX2 的表达丧失以及杆状光感受器基因 RCVRN、NRL、RHO 和 ROM1 的诱导。虽然这些分化的细胞显示出神经元形态，但是在 Matrigel 基质上培养会触发进一步的形态变化，导致视紫红质（RHO）和杆状外节特异性膜蛋白 1 在类似于杆状光感受器原代培养物的外节样突起中浓缩。分化的细胞被移植到用碘乙酸处理以消除杆状光感受器的猪的视网膜下空间中。移植后 3 周，在光感受器通常存在的外核层中可以检测到移植的 RHO+细胞。这些移植细胞的一部分已经产生了类似于外节的突起。这些结果表明，猪的 iPSC 可以在培养中分化为光感受器，并且这些细胞可以整合到受损的猪神经视网膜中，从而为将来使用猪作为视网膜干细胞移植模型的研究奠定了基础。
