Differentiation of Human ES and iPS Cells into Lens Cells

描述（由申请人提供）：本项目的长期目标是通过胚胎干（ES）细胞产生的透镜细胞开发研究人类老年性白内障的新模型。这些模型采用患者来源的诱导多能干（iPS）细胞分化成透镜细胞，以研究在已知诱导人类白内障发生的条件下晶状体细胞的稳态。该R21申请的直接目标是进行系统的探索性研究，以使用规定的细胞培养条件从人胚胎干（ES）细胞生成透镜细胞。眼相关性白内障是一种透镜疾病，造成全世界近一半的失明，并且由于发达国家和不发达国家的预期寿命增加，预期会增加。由于透镜微结构的进行性破坏，40岁以后发生的白内障。白内障是一种遗传和环境因素共同作用的复杂疾病。透镜混浊被认为是由于UV-B暴露、低抗氧化剂摄入和吸烟对透镜蛋白和透镜的其他结构组分的环境损害的累积损伤。由于缺乏合适的动物模型，以及原代透镜细胞和器官培养物的使用有限，阻碍了研究人类白内障的系统方法。我们现在已经确定了一种实验方案，以产生大量的透镜细胞祖细胞分化的人ES细胞系，H1。该提议将（1）建立优化的实验方案以从人ES细胞产生高度富集的透镜祖细胞群，（2）建立产生分化的晶状体样体的条件，随后对其进行表征，和（3）使用六种iPS细胞系的代表性库来测试这些实验条件。这些数据将为利用不同白内障患者的iPS细胞产生透镜细胞奠定基础。患者来源的透镜细胞将以足够的数量用于特定抗氧化防御机制的生物化学研究，以鉴定在白内障发生中起关键作用的那些透镜保护机制。通过确定药物开发的分子靶点，这种新方法将激发合理的药物搜索，以减少老化对透镜的影响。总体而言，这些探索性研究将用于R 01项目，以使用遗传和表型表征的生物材料模拟人类白内障。 公共卫生相关性：由于透镜白内障是全球范围内致盲的主要原因，因此本申请与人类健康相关。年龄相关性白内障在40岁以后的某个时间由于眼部透镜结构的进行性破坏而发展。目前对老年性白内障的治疗通常包括用人工眼内透镜替换不透明透镜的手术。虽然在美国，每年有180万至200万患者定期进行手术，但它代表了一个主要的医疗保险报销类别。据美国国家眼科研究所（National Eye Institute，NIH）（马里兰州贝塞斯达）估计，白内障发病延迟10年，可以减少近一半的手术数量，从而显著降低视力保健成本。

DESCRIPTION (provided by applicant): The long-term goal of this program is to develop new models to study human age-onset cataract via lens cells generated from embryonic stem (ES) cells. These models employ patient-derived induced pluripotent stem (iPS) cells differentiated into lens cells to study lens-cell homeostasis under conditions known to induce human cataractogenesis. The immediate goal of this R21 application is to perform systematic exploratory studies to generate lens cells form human embryonic stem (ES) cells using defined cell culture conditions. Age-related cataract is a disease of the lens that is responsible for nearly half of blindness worldwide, and is expected to increase as a result of increased life expectancy in both developed and underdeveloped countries. Age-onset cataracts develop after the age of 40 as a result of the progressive breakdown of the lens microarchitecture. Age-onset cataract is a complex disease involving both genetic and environmental factors. Lens opacities are thought to originate from the cumulative damage of environmental insults on lens proteins and other structural components of the lens from UV-B exposure, low antioxidant intake, and cigarette smoking. A systemic approach to study human cataract is hampered both by the lack of appropriate animal models, and limited use of primary lens cell and organ cultures. We have now identified an experimental protocol to generate large quantities of lens cells progenitor cells differentiated from human ES cell line, H1. This proposal will (1) establish an optimized experimental protocol to generate a highly enriched population of lens progenitor cells from human ES cells, (2) will establish conditions to generate differentiated lentoid bodies followed by their characterization, and (3) will test these experimental conditions using a representative pool of six iPS cell lines. These data will lay foundation for generation of lens cells using iPS cells from distinct cataract patients. The patient-derived lens cells will be available at quantities sufficient for biochemical studies of specific antioxidant defensive mechanisms to identify those lens protective mechanisms that play key roles in cataractogenesis. By identifying molecular targets for drugs to exploit, this novel approach will inspire a rational search for drugs to reduce the effects of aging on the lens. Collectively, these exploratory studies will be used for R01 projects to model human cataract using genetically and phenotypically characterized biological materials. PUBLIC HEALTH RELEVANCE: This application is relevant to human health as lens cataract is a major cause of worldwide blindness. Age- related cataract develops at some time after 40 years of age as a result of progressive breakdown of the ocular lens structure. Current treatment for senile cataract generally consists of surgery that replaces the opaque lens with an artificial intraocular lens. Although the surgery is performed routinely in the US at a rate of 1.8-2 million patients per annum, it represents a major Medicare reimbursement category. It has been estimated by the National Eye Institute, NIH (Bethesda, MD) that a 10-year delay in the onset of cataracts, could decrease the number of surgeries needed by almost one half, thus significantly decreasing vision care costs.