Adult Limbal Neurosphere Cells: A Potential Autologous Cell Resource for Retinal Cell Generation

The Corneal limbus is a readily accessible region at the front of the eye, separating the cornea and sclera. Neural colonies (neurospheres) can be generated from adult corneal limbus in vitro. We have previously shown that these neurospheres originate from neural crest stem/progenitor cells and that they can differentiate into functional neurons in vitro. The aim of this study was to investigate whether mouse and human limbal neurosphere cells (LNS) could differentiate towards a retinal lineage both in vivo and in vitro following exposure to a developing retinal microenvironment. In this article we show that LNS can be generated from adult mice and aged humans (up to 97 years) using a serum free culture assay. Following culture with developing mouse retinal cells, we detected retinal progenitor cell markers, mature retinal/neuronal markers and sensory cilia in the majority of mouse LNS experiments. After transplantation into the sub-retinal space of neonatal mice, mouse LNS cells expressed photoreceptor specific markers, but no incorporation into host retinal tissue was seen. Human LNS cells also expressed retinal progenitor markers at the transcription level but mature retinal markers were not observed in vitro or in vivo. This data highlights that mouse corneal limbal stromal progenitor cells can transdifferentiate towards a retinal lineage. Complete differentiation is likely to require more comprehensive regulation; however, the accessibility and plasticity of LNS makes them an attractive cell resource for future study and ultimately therapeutic application.角膜缘（Corneal limbus）是眼球前部易于获取的区域，分隔角膜（cornea）与巩膜（sclera）。可通过成人角膜缘在体外（in vitro）培养获得神经集落（神经球，neurospheres）。本团队此前已证实，此类神经球源自神经嵴干细胞/祖细胞（neural crest stem/progenitor cells），且可在体外分化为功能性神经元。本研究旨在探究，小鼠与人类角膜缘神经球细胞（limbal neurosphere cells, LNS）在暴露于发育中的视网膜微环境后，能否在体内（in vivo）与体外向视网膜谱系分化。本文证实，通过无血清培养检测法，可从成年小鼠与高龄人类（最高可达97岁）体内获取LNS。在与发育中小鼠视网膜细胞共培养后，多数小鼠LNS实验样本中均可检测到视网膜祖细胞标志物、成熟视网膜/神经元标志物以及感觉纤毛。将小鼠LNS细胞移植至新生小鼠视网膜下间隙后，其可表达光感受器特异性标志物，但未观察到其整合入宿主视网膜组织。人类LNS细胞在转录水平亦可表达视网膜祖细胞标志物，但无论在体外还是体内均未检测到成熟视网膜标志物。本研究数据表明，小鼠角膜缘基质祖细胞可向视网膜谱系转分化。完全分化可能需要更全面的调控手段，但LNS的易于获取性与可塑性使其成为未来研究乃至治疗应用中极具潜力的细胞资源。