Characterization of hematopoietic cells generated from normal and leukemic human induced pluripotent stem cells (hiPSCs) in teratomas

The elucidation of mechanisms that determine the properties of normal and leukemic human hematopoietic stem cells (hHSCs) has been revolutionized by advances in cell separation, clonal tracking, and the generation of new strains of highly immunodeficient mice. However, methods able to generate significant numbers of transplantable hHSCs in vitro either directly from patients or from hiPSCs have not yet been devised. Here we report the types of cells we have found can now be routinely formed in teratomas generated from 5x10ˆ5 hiPSCs transplanted subcutaneously ± mouse fibroblasts engineered to produce hFLT3-L, hSCF, hIL3 into immunodeficient NOD-Rag1-null- IL2Rgc-null mice with a c-kit deficiency (NRG-W41 mice) with or without an endogenous source of transgenically produced hIL3, hGM-CSF and hSCF (NRG-W41-3GS mice). Six to eight weeks post-transplant, the teratomas generated were dissociated into single cell suspensions and analyzed by FACS with various human-specific antibodies. Initial experiments demonstrated the presence of CD34+ cells in all teratomas with >0.1% CD45+ cells. Teratomas generated in NRG-W41-3GS mice from different iPSC lines produced ∼40-fold more CD34+CD45+ cells than those generated in NRG-W41 mice, with yields of up to 7x10ˆ6 CD45+ cells (2% of total live cells in the teratoma). Co-injection of human growth factor (GF) producing mouse fibroblasts further increased the overall production of human CD45+CD34+ cells ∼6-fold in the NRG-W41-3GS mice. Within this subset, we also identified GPI80+ and CD49f+ CD38-CD45RA-CD90+ cells. Teratomas generated from both normal and CML derived hiPSCs were able to produce granulopoietic, erythroid and mixed colonies in standard GF-supplemented methylcellulose cultures providing evidence of functionally competent human hematopoietic progenitors. These experiments lay the foundation for a more detailed investigation of the conditions required for the generation of normal and diseased HSCs and their properties.