Ability of human pluripotent stem cell-derived osteoprogenitor cells to repair non-union fracture Li Zou1, Frank Chen3, Cory Kellum1, Zach Quanbeck3, Joan E. Bechtold2,3, Dan S. Kaufman1 1 Department of Medicine, 2 Department of Orthopedics, University of Minnesota 3 Excelen Center for Bone & Joint Research and Education, Minneapolis, MN Introduction: Human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are attractive cell sources for bone regeneration. However, it’s difficult to compare the osteogenic potential of these pluripotent stem cells (PSCs) because of the diverse differentiation protocols and/or in vivo models reported in different studies. The objective of this study is to compare differentiation efficiency of several PSCs to potentially provide a better cell source promote bone regeneration. Methods: To optimize the osteogenic differentiation condition, we monitored the differentiation of a RUNX2-driven YFP reporter system integrated into hESCs (RUNX2-YFP hESCs). We used flow cytometric analysis to compare RUNX2-YFP hESCs cultured on various ECM proteins in either serum-free conditions or or FBS in MSC medium containing osteogenic supplements. Next, we used both these hESCs and two iPSC lines (umbilical cord blood derived iPSCs: UC-iPSCs, and peripheral blood derived iPSCs: PB-iPSCs) derived in our lab and cultured in the optimized differentiation conditions. Phenotype and genotype of the differentiated cells was evaluated by flow cytometric and qRT-PCR analysis. More functional testing was done by alizarin red staining of mineral deposition. A rat femoral non-union fracture model was utilized for in vivo studies, and bone repair by the engrafted osteogenic progenitor cells derived from bone marrow (BM)-MSCs, hESCs and the two hiPSCs were investigated by X-ray examination, microCT scanning, biomechanical testing and immunohistology. Results: Flow cytometric analysis of RUNX2-YFPhESCs cultured in different conditions reveals that 0.1% gelatin coating and 10% characterized FBS in MSC+OS culture medium is the best differentiation condition. When cultured in the optimized condition, hESCs and the two hiPSC lines produce a homogeneous cell population with similar percentage of osteocalcin positive cells as compared to BM-MSC-derived osteoprogenitor cells. In vivo studies demonstrate much better fracture repair at 8-weeks by engrafted BM-MSC-derived or UC-iPSC-derived osteoprogenitor cells compared to the other two cell sources. Immunohistologic analysis of the healing bone tissue is in progress to provide more information regarding local vasculature in the healing tissue and the contribution of engrafted cells vs. host cells to fracture healing. Discussion: Our study demonstrates an efficient differentiation protocol to generate homogenous osteoprogenitor cells from human PSCs, although the osteogenic potential of these cells depends on sources of PSCs. For next step, we aim to identify small molecular compounds with osteogenic activity by high throughput screening test in order to search for more efficient but less costly approaches to generate osteogenic cells for in vivo applications. These studies will also allow us to better define underlying signaling pathways that mediate early human osteogenic development.