Abstract 347Both human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) are pluripotent stem cells (hPSCs) with potential to differentiate into all types of somatic cells. Patients suffering from blood disorders can be cured with hematopoietic cell transplantations (HCT). Technical advancements in hPSC production and handling have revolutionized their potential applications in regenerative medicine and provided enormous hope for patients who may need HCT. hiPSCs derived from autologous cells could provide unlimited leukocyte antigen matched blood cells on a patient-specific basis. A remaining hurdle in this process remains the need for efficient and effective generation of specific blood cells from hPSCs for therapeutic use. Transcription factors play key roles in regulating maintenance, expansion, and differentiation of blood cells from hPSCs. Studies have shown that transcription factor RUNX1 is required for the formation of definitive blood cells. There are several alternatively spliced isoforms of the RUNX1 protein, including the shortest form RUNX1a and two longer forms RUNX1b and RUNX1c. Based on known properties of RUNX1 proteins, we hypothesized that RUNX1a promotes the production of therapeutic hematopoietic stem cells from hPSCs. By employing ectopic expression of RUNX1a on different human ESC and iPSC lines (H9, BC1, iCB5) under a defined hematopoietic differentiation system, we aimed to identify function of RUNX1a on lineage commitment and molecular mechanisms of RUNX1 activity in differentiation of PSCs to hematopoietic cells. We demonstrated that expression of endogenous RUNX1a parallels lineage commitment and hematopoietic emergence from hPSCs. During differentiation process RUNX1a enhanced the expression of several mesoderm and hematopoietic differentiation related factors, including KDR, SCL, GATA2, and PU.1. In addition, over-expression of RUNX1a in embryoid bodies (EBs) showed more efficient and earlier emergence of typical sac structures, which predicts cell lineage commitment and germ layer development at the early stage of EB differentiation. At day 7, EBs derived from hPSCs was dissociated into single cells for flow cytometry analysis. The mean frequency of CD31+CD34+CD45− and total CD34+ cells with hemato-endothelial cell features are 35.1% and 67.1% from RUNX1a-overexpressing EBs, and 8.7% and 24.1% from vector control EBs. Immunohistochemistry analysis of EBs at day 9 of differentiation confirmed that expression of RUNX1a accelerated mesoderm commitment and emergence of hemato-endothelial precursors. Flow cytometry analysis on EBs collected at days 9, 11, 13 showed that ectopic RUNX1a induced a robust increase in the frequency of hematopoietic progenitor cells in all hPSC lines examined. At Day 9, RUNX1a-overexpressing EBs generated 48.5% CD43+CD45+ cells, 45.1% CD34+CD45+ cells, and 8.5 folds higher CD43+ cells than vector EBs. Later at Day 13, 80% CD45+ and 75% CD43+/CD34+CD45+ hematopoietic stem/progenitor cells (HSPCs) achieved from dissociated EBs. In liquid culture, RUNX1a HSPC showed strong expansion and high percentage of CD235a+CD45− (20%) and CD71+CD235a+ (16%), markers for erythroid populations. Flow cytometry and western blots on RUNX1a-EB formed colonies showed significantly higher β-globin production than that of the vector, suggesting expression of RUNX1a in HSPC enhanced definitive hematopoiesis. RUNX1a-hPSCs derived HSPCs possess self-renewal capability and are capable of differentiating into multi-lineages ex vivo. Furthermore HSPCs generated from RUNX1a-EBs possessed the capacity of interacting with surrogate niche and showed long-term repopulation ability under LTC-IC (Long-Term Culture-Initiating Cell Assay) condition. Colonies generated from HSPC of RUNX1a-EBs after 3 week bulk LTC-IC culture showed 300 folds higher than vector control. RUNX1a-hPSCs derived CD34+CD45+ cells could maintain a non-adherent population in ouldCD45+ sEBsND THIS SENTENCE5 week culture on stromal cell M210. In summary we identified that RUNX1a enhances derivation of definitive hematopoietic cells from human PSCs. Our study provides an important and useful system to enhance specificity and efficiency of generating functional blood cells and further differentiated cells from human PSCs, which may provide valuable source for future clinical applications in patients with hematologic disorders. Disclosures:No relevant conflicts of interest to declare.