Institute of Medical Sciences, Tzu Chi University, Hualien 970, Taiwan.
Institute of Eye Research, Hualien Tzu Chi General Hospital, Hualien 970, Taiwan.
Cells. 2019 Jan 9;8(1):36. doi: 10.3390/cells8010036.
Human induced pluripotent stem cell (hiPSC)-derived three-dimensional retinal organoids are a new platform for studying the organoidogenesis. However, recurrent genomic aberration, acquired during generation of hiPSCs, limit its biomedical application and/or aberrant hiPSCs has not been evaluated for generation of differentiated derivatives, such as organoids and retinal pigment epithelium (RPE). In this study, we efficiently differentiated mosaic hiPSCs into retinal organoids containing mature photoreceptors. The feeder-free hiPSCs were generated from the human epidermal keratinocytes that were rapid in process with improved efficiency over several passages and maintained pluripotency. But, hiPSCs were cytogenetically mosaic with normal and abnormal karyotypes, while copy number variation analysis revealed the loss of chromosome 8q. Despite this abnormality, the stepwise differentiation of hiPSCs to form retinal organoids was autonomous and led to neuronal lamination. Furthermore, the use of a Notch inhibitor, DAPT, at an early timepoint from days 29⁻42 of culture improved the specification of the retinal neuron and the use of retinoic acid at days 70⁻120 led to the maturation of photoreceptors. hiPSC-derived retinal organoids acquired all subtypes of photoreceptors, such as , and . Additionally, the advanced maturation of photoreceptors was observed, revealing the development of specific sensory cilia and the formation of the outer-segment disc. This report is the first to show that hiPSCs with abnormal chromosomal content are permissive to the generation of three-dimensional retinal organoids.
人类诱导多能干细胞（hiPSC）衍生的三维视网膜类器官是研究类器官发生的新平台。然而，在 hiPSC 生成过程中反复出现的基因组异常限制了其在生物医学中的应用，并且尚未对异常 hiPSC 进行评估，以生成分化衍生物，如类器官和视网膜色素上皮（RPE）。在这项研究中，我们高效地将嵌合 hiPSC 分化为含有成熟光感受器的视网膜类器官。无饲养层 hiPSC 是从人表皮角质形成细胞中生成的，其过程快速，经过多代传代后效率提高，并保持多能性。但是，hiPSC 具有染色体核型嵌合，正常和异常核型并存，而拷贝数变异分析显示 8q 染色体缺失。尽管存在这种异常，但 hiPSC 向视网膜类器官的逐步分化是自主的，并导致神经元分层。此外，在培养的第 29-42 天早期使用 Notch 抑制剂 DAPT，可改善视网膜神经元的特化，在第 70-120 天使用视黄酸可促进光感受器的成熟。hiPSC 衍生的视网膜类器官获得了所有光感受器亚型，如 、 和 。此外，还观察到光感受器的高级成熟，显示出特定感觉纤毛的发育和外节盘的形成。这是首次报道显示具有异常染色体含量的 hiPSC 允许生成三维视网膜类器官。
