Culture and Transplantation of Human Retinal Spheres

描述（由申请人提供）：以光感受器和/或视网膜色素上皮（RPE）为靶点的遗传性和获得性眼病是人类视力发病率的重要原因。人类组织（包括大脑）中区域特异性干细胞群的存在，引起了人们对干细胞生物学治疗应用的兴趣。从眼组织中繁殖前体细胞的创新方法可能产生能够预防或修复光感受器或RPE变性的可移植细胞群。本提案概述了一个五年计划：1)使用一种新的组织培养技术分离和扩增从胎儿和成人视网膜和/或色素上皮区域获得的前体细胞；2)表征它们在不同培养处理下的生长和分化潜力；3)在视网膜变性动物模型中检测它们重新填充或拯救光感受器的能力。利用宿主实验室开发的培养方法，我们证明了色素细胞和非色素细胞的自由漂浮球体可以成功地从人类胎儿的眼睛后段繁殖。从这些球体中迁移出来的细胞经历分化，并采用不同的形态和色素沉着模式和细胞标记表达。特别是，来自视网膜的非色素球和来自睫状体的色素球产生神经元，而来自RPE的色素球产生有规则排列的色素上皮。在这些初步实验的基础上，我们将立即集中精力解剖更多来自胎儿和出生后去核人眼的标本，并监测它们随着时间的推移的生长和分化潜力。这些研究将延续到资助期的第二年和第三年，届时我们也将开始研究特定生长因子和条件介质对球体行为的影响。在项目的最后几年，提出了在两种不同的视网膜变性大鼠模型中检查不同球体种群的治疗潜力的策略。总之，这些实验旨在建立在干细胞生物学新兴领域的最新进展，并将其应用于眼科的基础和临床科学。

DESCRIPTION (provided by applicant): Inherited and acquired eye diseases that target photoreceptors and/or retinal pigment epithelium (RPE) are a significant cause of visual morbidity in humans. The existence of regionally specific stem cell populations in human tissue, including brain, has raised interest in the therapeutic applications of stem cell biology. Innovative methods of propagating precursor cells from ocular tissue may produce populations of transplantable cells capable of preventing or repairing photoreceptor or RPE degeneration. This proposal outlines a five-year plan to 1) use a novel tissue culture technique to isolate and expand precursor cells obtained from retina and/or pigmented epithelial regions of fetal and adult human eyes, 2) characterize their growth and differentiation potential in response to different culture treatments and 3) examine their capacity to repopulate or rescue photoreceptors in animal models of retinal degeneration. Using a culture method developed in the host laboratory, we demonstrate that free-floating spheres of pigmented and nonpigmented cells can be successfully propagated from the posterior segment of human fetal eyes. Cells prompted to migrate from these spheres undergo differentiation and adopt distinct morphologies and patterns of pigmentation and cell marker expression. In particular, non-pigmented spheres from retina and pigmented spheres from the ciliary body produced neurons, while pigmented spheres from RPE produced regularly arranged sheets of pigmented epithelium. Building on these preliminary experiments, we will focus immediate efforts on dissecting additional specimens from both fetal and postnatal enucleated human eyes and monitoring their growth and differentiation potential over time. These studies will be carried over to the second and third years of the grant period, when we will also begin to examine the effects of specific growth factors and conditioned media on sphere behavior. During the final years of the project, strategies are proposed to examine the therapeutic potential of the different sphere populations in two distinct rat models of retinal degeneration. Together, these experiments are designed to build on recent advances in the burgeoning field of stem cell biology and apply them to the basic and clinical science of ophthalmology.