Pluripotent stem cells have the potential to treat diseases affecting almost every organ system. However, their clinical use is limited by host rejection due to differences in the diverse Major Histocompatibility Complex (MHC) genes. In the past, this problem has been addressed by using MHC-matched donors and/or dangerous immunosuppressive drug regimens. Here, we propose to demonstrate that MHC-negative pluripotent stem cells can avoid recognition as allogeneic in clinical transplantation applications and therefore be used as universal donor stem cells. Prior experiments using MHC-class I-negative human embryonic stem cells (ESCs) derived by targeted disruption of the Beta Microglobulin gene (B2M) showed that these cells are not recognized by immune cells in vitro. We will now perform in vivo mouse experiments in order to show that MHC class I-negative cells are not rejected and can engraft in a normal mouse after allogeneic transplantation. We will first derive induced Pluripotent stem cells (iPSCs) from the well-characterized B2m-/- model, which are MHC class I-deficient. Then, we will induce teratoma formation using these cells in order to analyze the allogeneic immune response against B2m-/- iPSC-derived teratomas. We will derive cardiac progenitor cells from B2m-/- iPSCs and transplant them into allogeneic recipients. We hypothesize that these cells will engraft and survive after transplantation, contrary to B2m+/+ allogeneic iPSCs, bringing in vivo proof of concept that the MHC class I- negative pluripotent cells can act as universal donors. The long-term goal of this proposal is to use MHC- engineering to create stem cell lines that can be used for regenerative medicine applications in multiple recipients without rejection, thereby facilitating the translation of stem cell-based therapies into the clinic.