Efficient Induction of Myleoid and Lymphoid Hematopoiesis from Nonhuman Primate Pluripotent Stem Cells Using GSK3b Inhibitor

Induced pluripotent stem cells (iPSCs) have created novel opportunities for the scalable manufacture of blood products for clinical use. Recent advances in hematopoietic differentiation from human pluripotent stem cells have brought the clinical translation of iPSC-derived blood products close to reality and evoke an urgent need for preclinical evaluation of their efficacy, safety, and immunogenicity in large animal models.  Due to substantial similarities with humans, outcomes of cellular therapies in nonhuman primate (NHP) models can be readily extrapolated to clinical setting. However, the use of this model is hampered by limited availability of clinically relevant NHP-iPSCs and low efficiency of myeloid and lymphoid differentiation from them. Here, we describe generation of clinically relevant transgene-free iPSCs from different NHP species, including rhesus, Chinese and Mauritian cynomolgus monkeys, and demonstrate that GSK3β inhibition is essential to induce rapid and efficient differentiation of the NHP-iPSCs into multipotential CD34+CD45+CD90+CD38-CD45RA- hematopoietic progenitors in coculture with OP9 bone marrow stromal cells. NHP-iPSC-derived hematopoietic progenitors were capable of differentiating further into mature erythroid cells, megakaryocytes, granulocytes, monocytes/macrophages following exposure to hematopoietic cytokines promoting development corresponding lineages. In addition, iPSC-derived CD34+CD45+ cells generated CD4+CD8+ T lymphoid cells and CD159a+ NK cells when cultured on OP9 cells expressing DLL4. To confirm T cell development, we analyzed the genomic DNA of the hematopoietic cells from OP9-DLL4 cultures for the presence of T cell receptor (TCR) rearrangements. This analysis demonstrated the presence of multiple PCR products of random V-J and D-J rearrangements at the β locus and V-J rearrangements at the γ locus, indicative of a polyclonal T lineage repertoire. To evaluate feasibility of using NHP model for evaluation of safety and clinical efficacy of iPSC-derived blood products, we generated iPSCs from Mauritian cynomolgus macaque and marked them with GFP. GFP-iPSCs were used to generate CD34+CD45+  multipotential hematopoietic progenitors using multiple rounds of differentiation followed by freezing.  Subsequently, iPSC donor animal was subjected to nonmyeloablative conditioning with fludarabine (50 mg/m(2)/day for 3 days) together with low-level total body irradiation (2 Gy) and transfused with 30x10⁶/kg autologous iPSC-derived CD34+CD45+ cells. Transfusion of iPSC-derived CD34+CD45+ cells well tolerated and animal recovered without any adverse events. By flow cytometry, GFP-marked cells (up to 1%) were detected in peripheral blood on days 20-30 post-transfusion, predominately in granulocyte, monocyte and platelet compartments.  Although, GFP-positive cells mostly disappeared in circulation one month after transfusion, GFP-positive cells were detected in circulation for more than 90 days by genomic PCR.  During 6 month follow-up period no signs of myelo- or lympho-proliferative disorder were detected. Overall, these studies lay the foundation for advancing a NHP model for preclinical testing of iPSC-based therapies for blood diseases.