Blood-Derived Human iPS Cells Generate Optic Vesicle-Like Structures with the Capacity to Form Retinal Laminae and Develop Synapses
Blood-Derived Human iPS Cells Generate Optic Vesicle-Like Structures with the Capacity to Form Retinal Laminae and Develop Synapses
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DOI：
10.1167/iovs.11-9313
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发表时间：
2012-04-01
影响因子：
4.4
通讯作者：
Gamm, David M.
中科院分区：
文献类型：
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作者：
Phillips, M. Joseph;Wallace, Kyle A.;Gamm, David M.
目的.我们试图确定来自血液的人诱导多能干细胞（iPSC）是否可以产生具有分层和表达细胞间通讯标志物的能力的视泡样结构（OV）。通过逆转录病毒转导将来自常规外周血样品的活化的T淋巴细胞重编程为iPSC。T淋巴细胞衍生的iPSC（TiPSC）被表征为多能性，并使用我们先前公开的方案分化为OV。然后手动分离、合并TiPSC-OV，并连续培养至视网膜发生的更成熟阶段。在整个逐步分化过程中，通过PCR、免疫细胞化学和/或流式细胞术监测前神经、视网膜和突触标志物表达的变化。TiPSC产生了丰富的OV，其含有几乎同质的增殖神经视网膜祖细胞（NRPC）群体。这些NRPC分化成多种神经视网膜细胞类型，类似于来自人胚胎干细胞和成纤维细胞衍生的iPSC的OV培养物。此外，一些TiPSC-OV的部分保持其独特的神经上皮外观并自发形成原始层，使人想起发育中的视网膜。从TiPSC-OV培养的视网膜后代表达了许多对突触发生和间隙连接形成至关重要的基因和蛋白质，伴随着神经胶质细胞的出现和培养物中血小板反应蛋白的上调。我们首次证明了人类血液来源的iPSC可以产生视网膜细胞类型，为基于iPSC的视网膜研究提供了非常方便的供体细胞来源。我们还表明，培养的TiPSC-OV有能力自组装成基本的神经视网膜结构和表达化学和电突触的标志物指示。（Invest Ophthalmol维斯科学。2012;53：2007-2019）DOI：10.1167/iovs.11-9313
PURPOSE. We sought to determine if human induced pluripotent stem cells (iPSCs) derived from blood could produce optic vesicle-like structures (OVs) with the capacity to stratify and express markers of intercellular communication.METHODS. Activated T-lymphocytes from a routine peripheral blood sample were reprogrammed by retroviral transduction to iPSCs. The T-lymphocyte-derived iPSCs (TiPSCs) were characterized for pluripotency and differentiated to OVs using our previously published protocol. TiPSC-OVs were then manually isolated, pooled, and cultured en masse to more mature stages of retinogenesis. Throughout this stepwise differentiation process, changes in anterior neural, retinal, and synaptic marker expression were monitored by PCR, immunocytochemistry, and/or flow cytometry.RESULTS. TiPSCs generated abundant OVs, which contained a near homogeneous population of proliferating neuroretinal progenitor cells (NRPCs). These NRPCs differentiated into multiple neuroretinal cell types, similar to OV cultures from human embryonic stem cells and fibroblast-derived iPSCs. In addition, portions of some TiPSC-OVs maintained their distinctive neuroepithelial appearance and spontaneously formed primitive laminae, reminiscent of the developing retina. Retinal progeny from TiPSC-OV cultures expressed numerous genes and proteins critical for synaptogenesis and gap junction formation, concomitant with the emergence of glia and the upregulation of thrombospondins in culture.CONCLUSIONS. We demonstrate for the first time that human blood-derived iPSCs can generate retinal cell types, providing a highly convenient donor cell source for iPSC-based retinal studies. We also show that cultured TiPSC-OVs have the capacity to self-assemble into rudimentary neuroretinal structures and express markers indicative of chemical and electrical synapses. (Invest Ophthalmol Vis Sci. 2012;53:2007-2019) DOI:10.1167/iovs.11-9313