We submit this proposal in response to RFA-OD-09-004 "Grand Opportunities (GO)" and NHLBI requests for applications to one of its nine (9) selected high-priority topics, "Characterizing Differentiated Heart, Lung, and Blood Cells Derived by Reprogramming Human Embryonic and Induced Pluripotent Stem Cells". Considerable progress has recently been made in the field of cellular reprogramming, including the induction of pluripotent stem cells from a diversity of adult somatic cell types. To assess the value of these unique cell sources in the development of model experimental systems, and to evaluate their safety and efficacy in potential therapies, several urgent questions remain to be addressed. In this proposal, we will address two of three questions raised by NHLBI: (1) How do the differentiated states generated by reprogramming stem cells in the laboratory compare with the characteristics and fates of their normal tissue and organ counterparts? (2) How do the differentiated states generated by reprogramming embryonic stem (ES) and induced pluripotent stem (iPS) cells compare with each other? To these ends, we have established a multidisciplinary team from 5 institutions in 3 states that includes investigators from Johns Hopkins University, University of Maryland, two biotechnology companies (in CA and MA) and NIH. The human hematopoietic system with defined surface markers and functional assays provides us the best choice to characterize the potential as well as safety of human iPS/ES cell progeny massively generated in laboratories, as compared to postnatal stem cells that are always in short supplies. We will first compare hematopoietic stem-progenitor cells (HSPCs) generated from human iPS cells with those of an adult donor from whom the iPS cells are derived (Aim 1). In addition, we will compare iPS cells and their hematopoietic derivatives with NIH-approved human ES cell lines and their hematopoietic derivatives. We will use cutting-edge technologies such as genome-scale analyses of DNA methylation and chromatin-associated histone modifications to establish epigenetic and gene expression signatures of purified HSPCs from different sources. In Aim 2, we will establish genome-wide molecular signatures of red blood cells (erythroblasts) massively generated from human iPS cells, as compared to those generated ex vivo under the same culture condition from the isolated HSPCs of the original adult donor and of human ES cells. In Aim 3, we will develop a DNA methylation-based assay to assess and monitor tumorigenic potential of iPS cell lines and their differentiated hematopoietic progeny. A GO grant will support us to build critical mass and synergy, to accelerate critical breakthroughs using recently established human iPS cells and their hematopoietic derivatives. This focused study will help us to elucidate and modulate epigenetic determinants of gene expression and cell fate determination. The greater understanding at the molecular level gained from this 2-year study can be broadly translated to the study of heart, vascular and lung diseases, in addition to the blood disorders that are our near-term focus. PUBLIC HEALTH RELEVANCE: Human iPS cells that are patient-specific, renewable and pluripotent provide unprecedented opportunities for us to generate unlimited numbers of a specific cell type such as hematopoietic cells from a single defined stem cell clone. This focused study will help us to elucidate and modulate epigenetic determinants of gene expression and cell fate specification applicable to both pluripotent and postnatal stem cells. The human hematopoietic system with defined surface markers and functional assays provides us the best choice to characterize the potential as well as safety of human iPS/ES cells, as compared to postnatal stem cells. The greater understanding at a molecular level gained from this study can be broadly translated to the study of heart, vascular and lung diseases in addition to the blood disorders that are our near-term focuses.