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.
This work will provide basic insight into how stem cells can be generated from adult cells and how these cells can be directed to develop into endothelial cells to benefit patients with vascular disorders.
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