Basic and Translational Research iPSC-based hematologic and vascular therapies

斯坦福-约翰霍普金斯大学研究中心旨在更深入地了解分子途径，以提高核重编程的效率，确保诱导多能细胞（IPSC）的功能和安全性，为造血和内皮谱系的分化和纯化提供强大的方案，并指导安全性和有效性的临床前研究的开创性工作。斯坦福大学小组提出了三个研究项目。项目 1：增强 IPSC 衍生和分化为 EC 的新型监管机构（Helen Blau、Wing Wong）。使用一种新的细胞融合方法，我们将鉴定在与小鼠ESC细胞融合后人类成纤维细胞核的核重编程过程中发生的早期表观遗传和转录变化。异核转录物中人类 RNA 的物种特异性转录组扩增允许鉴定重编程过程中人类细胞核中最早的转录事件。使用相同的细胞融合策略，我们还将阐明向内皮细胞定向分化的最早事件。在项目 2：IPSC 工程和表征（Renee Reijo Pera、James Swartz）中，我们将开发和完善基于蛋白质的 iPSC 生成策略。我们将合成包含具有转导结构域的山中因子的细胞渗透性融合蛋白，并优化其剂量、持续时间和时间以诱导最佳重编程。项目 1 中确定的新因素将被纳入以加强重新编程。这些细胞的安全性和有效性的综合表征将包括光谱核型分析、线粒体基因表达和功能，以及表观遗传、转录和致瘤分析。在项目 3：用于治疗性血管生成的 iPSC-EC：分化和功能的决定因素 (John Cooke) 中，我们将利用项目 2 中生成的 IPSC 以及项目 1（以及我们的霍普金斯大学同事）的见解，有效地将 IPSC 分化为内皮谱系。将在体外和体内评估 EC 功能，并使用分子成像和激光多普勒灌注在外周动脉疾病的小鼠模型中研究其治疗效果。我们希望这些项目的见解最终会带来新的血管疗法。

The Stanford-Johns Hopkins Research Hub intends to gain a deeper understanding of molecular pathways to enhance the efficiency of nuclear reprogramming, to ensure the function and safety of induced pluripotential cells (IPSCs), to provide robust protocols for differentiation and purification of hematopoietic and endothelial lineages, and to guide pioneering work in pre-clinical studies of safety and efficacy. The Stanford group proposes three research projects. Project 1: Novel Regulators to enhance IPSC Derivation and Differentiation to EC (Helen Blau, Wing Wong). Using a novel cell fusion approach, we will identify the early epigenetic and transcriptional changes occurring during nuclear reprogramming of the human fibroblast nucleus after cell fusion with mouse ESC. Species-specific transcriptome amplification of the human RNA within the heterokaryon transcripts permits identification of the earliest transcriptional events in the human nucleus during reprogramming. Using the same cell fusion strategy, we will also elucidate the earliest events of directed differentiation toward endothelial cells. In Project 2: IPSC Engineering and Characterization (Renee Reijo Pera, James Swartz) we will develop and refine a protein-based strategy for generating iPSCs. We will synthesize cell-permeant fusion proteins comprising the Yamanaka factors with transduction domains, and optimize their dose, duration and timing to induce optimal reprogramming. Novel factors identified in Project 1 will be incorporated to enhance reprogramming. Comprehensive characterization of the safety and efficacy of these cells will include spectral karyotyping, mitochondrial gene expression and function, and epigenetic, transcriptional and tumorigenic profiling. In Project 3: iPSC-ECs for Therapeutic Angiogenesis: Determinants of Differentiation and Function (John Cooke), we will utilize the IPSC generated in Project 2, and the insights from Project 1 (and our Hopkins colleagues), to efficiently direct differentiation of the IPSC to endothelial lineage. EC function will be assessed in vitro and in vivo, and their therapeutic efficacy studied using molecular imaging and laser Doppler perfusion in a murine model of peripheral arterial disease. We intend that the insights from these projects ultimately lead to novel vascular therapies.