Recapitulating developmental pathways to improve iPSC-RPE derivation protocols

概括改进 iPSC-RPE 衍生方案的发育途径

基本信息

批准号：
8289394
负责人：
Peter Dwayne Westenskow
金额：
$ 4.92万
依托单位：
SCRIPPS RESEARCH INSTITUTE, THE
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-12-15 至 2013-12-16
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8289394
关键词：
Acceleration Age related macular degeneration Agonist Animal Model Animals Appearance Autologous Basic Science Benchmarking Biological Assay Biology Blindness Cell Line Cell Maintenance Cell Shape Cells Cessation of life Choroid Clinical Coupled Culture Media Derivation procedure Developed Countries Development Disadvantaged Disease Disease model Doctor of Philosophy Electrical Resistance Electrophysiology (science)Embryonic Development Encapsulated Ensure Epithelial Functional disorder Gene Expression Genes Goals Graft Rejection Human Imaging Techniques In Vitro Ion Transport Karyotype Laboratories Learning Light Maintenance Measurement Mediating Medicine Melanins Methods Modeling Monitor Morphology Neural Retina Oncogenic Ophthalmoscopes Optics Pathway interactions Patients Phagocytosis Photoreceptors Physiology Pigments Platelet Factor 4 Protocols documentation Rattus Reporter Retina Retinal Retinal Degeneration Risk Scanning Signal Pathway Signal Transduction Signaling Molecule Source Stem cell transplant Structure of retinal pigment epithelium Symptoms Techniques Therapeutic Intervention Time Tissues Translational Research Translations Transplantation Visual Acuity Water Work absorption activin A base cell fate specification cost density gene correction human embryonic stem cell improved in vivo induced pluripotent stem cell insight metabolomics monolayer photoreceptor degeneration pluripotency reagent testing research study retina circulation small molecule stem stem cell biology transcription factor translational medicine

项目摘要

DESCRIPTION (provided by applicant): The retinal pigment epithelium (RPE) is a monolayer of cuboidal shaped cells that provide essential support to photoreceptors in the neural retina. RPE functions include the vectorial transport of ions and water between the choroid circulation and retina, light absorption, and phagocytosis of light damaged photoreceptor outer segments. Loss or dysfunction of RPE cells induces devastating secondary effects on photoreceptors and is responsible for inducing some forms of age related macular degeneration (AMD), a progressive blinding disorder that is the leading cause of blindness in industrialized countries. The transplantation of RPE in rat AMD models and in human AMD patients has been shown to stem vision loss and inhibit further degeneration. RPE can be generated from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) although current methods are slow and the yields are low. If derivation times could be accelerated, and if other concerns about iPSCs such as the oncogenic potential of the transcription factors used to induce pluripotency could be alleviated, they would serve as an excellent source of patient-matched RPE cells. Clues for how to enhance iPSC-RPE derivation can be obtained from developmental studies, and critical steps of RPE development can be recapitulated in vitro by activating key signaling pathways that regulate RPE-promoting gene networks. The Wnt/ss-catenin pathway is an excellent iPSC-RPE enhancing candidate. I have shown that Wnt/ss-catenin signaling is required for RPE cell-fate maintenance and that ss-catenin can directly regulate the key transcription factors Mitf and Otx2. I hypothesize that activating the Wnt/ss-catenin pathway in iPSCs will greatly enhance RPE differentiation. We have derived RPE from hESCs and iPSCs induced to pluripotency using all 4 Thomson factors and with only two (Klf4 and Oct4) and biomicking small molecules. I have preliminary evidence that 1-factor iPSC (Oct4 only) is also competent to generate RPE. I will use well-established assays to characterize the 1-factor iPSC-RPE cells and determine if they are correctly polarized and electrically coupled, and if they express correct gene profiles. I will also transplant the derived cell into rat disease models and monitor rescue. This project offers me the unique opportunity of extending my thesis work into a study of translation medicine and to enhance my understanding of retinal physiology.

描述（由申请人提供）：视网膜色素上皮（RPE）是长方体形细胞的单层，可为神经视网膜中的感光体提供基本的支持。 RPE功能包括离子和视网膜循环之间的离子和水的矢量运输，光吸收和光受损的光感受器外部段的吞噬作用。 RPE细胞的损失或功能障碍会引起对光感受器的毁灭性次要作用，并负责诱导某些形式的与年龄相关的黄斑变性（AMD），这是一种进行性盲目疾病，是工业化国家失明的主要原因。 RPE在大鼠AMD模型和人类AMD患者中的移植已被证明会阻止视力丧失并抑制进一步的变性。 RPE可以由人类胚胎干细胞（HESC）产生，尽管当前方法较慢且产率较低，但诱导的多能干细胞（IPSC）可以产生。如果可以加速推导时间，并且可以减轻对IPSC的其他担忧，例如用于诱导多能性的转录因子的致癌潜力，它们将成为患者匹配的RPE细胞的极好来源。如何通过发育研究获得了如何增强IPSC-RPE推导的线索，并且可以通过激活调节促进RPE促进基因网络的关键信号通路来在体外概括RPE发育的关键步骤。 Wnt/SS-Catenin途径是出色的IPSC-RPE增强候选者。我已经表明，RPE细胞命令维护需要Wnt/SS-catenin信号传导，并且SS-catenin可以直接调节关键转录因子MITF和OTX2。我假设激活IPSC中的Wnt/SS-Catenin途径将大大增强RPE分化。我们使用所有4个Thomson因子，仅使用两个（KLF4和OCT4）和生物攻击的小分子衍生出hESC和IPSC的RPE。我有初步的证据表明1因子IPSC（仅OCT4）也有能力生成RPE。我将使用良好的测定法来表征1因子IPSC-RPE细胞，并确定它们是否正确偏振和电耦合，以及它们是否表达正确的基因谱。我还将将衍生细胞移植到大鼠疾病模型中并监测救援。这个项目为我提供了将论文工作扩展到翻译医学研究并增强我对视网膜生理学的理解的独特机会。