Human iPSC-based Modeling of RPE Melanogenesis: Reactivation and Protection

 描述(申请人提供)：老年性黄斑变性(AMD)是60岁以上人群失明的主要原因。在AMD中，视网膜色素上皮(RPE)细胞功能失调和退化，导致光感受器丧失，最终导致中央视力丧失。一些研究已经将AMD的易感性与RPE细胞中的低色素水平联系起来。色素的产生，或黑素的产生，是在RPE中出生时完成的，黑色素随着年龄的增长而降解，进一步表明低水平的色素对AMD在以后的生活中的发展做出了贡献。色素沉着如何保护个体免受AMD的发展是一个重要的未解答的问题。由于缺乏合适的人体模型系统，解决色素丢失的机制和功能后果的研究一直受到阻碍，但这些都是需要解决的重要问题，因为重新激活黑素合成的能力不仅可以通过降低对光诱导毒性的敏感性来保护，还可以通过保留RPE细胞的关键功能，如吞噬脱落的光感受器外部片段来保护。在这项提案中，这些问题将通过利用人类诱导多能干细胞(IPSC)技术的发展而成为可能的方法来解决。我们将把老化的人RPE细胞重新编程为人诱导多能干细胞(HiPSCs)。重新编程过程可以将得到的HiPSCs及其衍生物的分子时钟重新设置为未成熟的、年轻的状态。因此，这个系统提供了独特的机会来研究衰老的RPE细胞向年轻的HiPSC来源的RPE(hiPSC-RPE)细胞恢复活力后黑素生成重新激活的机制。初步数据显示，与来自同一供体的老化RPE细胞相比，HiPSC-RPE细胞中的色素水平增加。我们将描述几个等基因老化的RPE和HiPSC-RPE细胞对的黑素生成。基因表达分析将用于检测黑素生成途径，以确定是否存在调节沉默的发育开关。利用重新编程后的年轻状态，HiPSC-RPE细胞将在体外成熟，以观察色素合成何时停止，并确定可以重新激活黑素生成的分子策略。最后，将使用CRISPR/Cas9技术对HiPSC-RPE细胞进行基因工程，以通过丢失GPR143来减少黑色素小体的生物生成，或通过丢失TYR来减少黑色素的合成；这些工程化品系将用于模拟色素随年龄的丧失。基因工程的功能丧失的HiPSC-RPE细胞将与非靶向等基因对照的HiPSC-RPE细胞进行比较，以了解它们对光诱导毒性的易感性和对吞噬细胞光感受器外部片段的能力。这将首次在人体模型系统中确定影响黑素小体数量或黑色素含量是否会改变这些关键的RPE功能。这项建议的目的是了解调节黑素生成的发育开关，并为确定如何刺激黑素生成来预防或治疗AMD奠定基础。

 DESCRIPTION (provided by applicant): Age-related macular degeneration (AMD) is the major cause of blindness in those over 60 years. In AMD, retinal pigment epithelial (RPE) cells become dysfunctional and degenerate, which leads to the loss of photoreceptors and, ultimately, central vision. Several studies have correlated AMD susceptibility with low pigment level in RPE cells. The production of pigment, or melanogenesis, is completed by birth in RPE, and the pigment melanin degrades with aging, further implicating the contribution of low pigment level to the development of AMD later in life. How pigmentation might protect individuals from developing AMD is an important unanswered question. Studies addressing the mechanism and functional consequences of pigment loss have been hampered by lack of appropriate human model systems, yet these are important issues to address because the ability to reactivate melanogenesis could be protective not only by reducing sensitivity to light-induced toxicity but also by preserving key functions of the RPE cells like the phagocytosis of shed photoreceptor outer segments. These questions will be addressed in this proposal by utilizing an approach made possible through the development of human induced pluripotent stem cell (iPSC) technologies. We will reprogram aged human RPE cells to human induced pluripotent stem cells (hiPSCs). The reprogramming process can reset the molecular clock of the resulting hiPSCs and their derivatives to an immature, young-like state. Thus, this system provides the unique opportunity to investigate the mechanisms underlying reactivation of melanogenesis following the rejuvenation of aged RPE cells to young-like hiPSC-derived RPE (hiPSC-RPE) cells. Preliminary data suggests that pigment level increases in hiPSC-RPE cells compared to aged RPE cells from the same donor. We will characterize melanogenesis in several isogenic aged RPE and hiPSC-RPE cell pairs. Gene expression analysis will be used to examine the melanogenesis pathway to determine whether there is a developmental switch that regulates silencing. Taking advantage of their young-like status following reprogramming, hiPSC-RPE cells will be matured in vitro to observe when pigment synthesis ceases and to identify molecular strategies that could reactivate melanogenesis. Finally, hiPSC-RPE cells will be genetically engineered using CRISPR/Cas9 technology to reduce biogenesis of melanosomes, the melanin-containing organelles, through loss of GPR143, or to reduce melanin synthesis through loss of TYR; these engineered lines will serve to mimic the loss of pigment with aging. The genetically engineered loss-of-function hiPSC-RPE cells will be compared to untargeted isogenic control hiPSC-RPE cells for their vulnerability to light-induced toxicity and their abilit to phagocytose photoreceptor outer segments. This will determine for the first time in a human model system whether affecting melanosome number or melanin content alters these key RPE functions. The goal of this proposal is to understand the developmental switch that regulates melanogenesis and to lay the groundwork for determining how melanogenesis might be stimulated to prevent or treat AMD.