Differentiation of Human ES and iPS Cells into Lens Cells

描述(申请人提供)：该计划的长期目标是开发新的模型，通过胚胎干细胞产生的晶状体细胞研究人类老年性白内障。这些模型使用患者来源的诱导多能干细胞(IPS)分化为晶状体细胞来研究晶状体细胞在已知诱导人类白内障形成的条件下的动态平衡。这项R21应用的直接目标是进行系统的探索性研究，利用确定的细胞培养条件从人类胚胎干细胞中产生晶状体细胞。老年性白内障是一种晶状体疾病，全世界近一半的失明是由这种疾病造成的，预计随着发达国家和不发达国家预期寿命的延长，这种疾病的数量将会增加。由于晶状体微结构的进行性崩溃，40岁以后才会出现老年性白内障。老年性白内障是一种复杂的疾病，既涉及遗传因素，也涉及环境因素。晶状体混浊被认为是由于紫外线-B暴露、抗氧化剂摄入量低和吸烟对晶状体蛋白质和其他结构成分的环境侮辱造成的累积损害。由于缺乏合适的动物模型，以及有限的原代晶状体细胞和器官培养，系统地研究人类白内障的方法受到阻碍。我们现在已经确定了一种实验方案，可以从人类ES细胞系H1分化出大量的晶状体细胞前体细胞。这项建议将(1)建立一个优化的实验方案，以从人类ES细胞中产生高度丰富的晶状体前体细胞群，(2)将建立条件来产生分化的晶状体样体，然后对其进行鉴定，以及(3)将使用具有代表性的6个iPS细胞系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细胞培养晶状体细胞奠定基础。患者来源的晶状体细胞的数量将足以用于特定抗氧化防御机制的生化研究，以确定在白内障发生中发挥关键作用的晶状体保护机制。通过确定药物的分子靶点，这种新的方法将激发人们理性地寻找药物，以减少老化对晶状体的影响。总的来说，这些探索性研究将用于R01项目，使用遗传和表型特征的生物材料来模拟人类白内障。 公共卫生相关性：这项应用与人类健康相关，因为晶状体白内障是全球失明的主要原因。年龄相关性白内障发生在40岁以后的某个时间，这是由于晶状体结构逐渐崩溃的结果。目前老年性白内障的治疗方法一般包括用人工晶状体取代不透明晶状体的手术。尽管这种手术在美国是例行公事，每年有180-200万名患者接受手术，但它代表着一个主要的医疗保险报销类别。据美国国立卫生研究院(马里兰州贝塞斯达)国家眼科研究所估计，将白内障发病推迟10年可能会使所需的手术次数减少近一半，从而显著降低视力护理成本。

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.