Analysis of hematopoietic cells generated from human induced pluripotent stem cells differentiating in teratomas

Much progress has been made in characterizing human hematopoietic cells that can sustain the long-term output of mature blood cells in vivo. However, a method for their expansion ex vivo has remained elusive. One approach is to derive these cells from induced pluripotent stem cells (iPSCs), although a reproducible in vitro protocol for this has also not yet been achieved. Recently, however, some success has been reported in teratomas produced from human iPSCs transplanted into immunodeficient mice. We are now exploring the possibility that this latter approach may be improved using immunodeficient NOD-Rag1-null- IL2Rgc-null mice with a c-kit (W41/W41) deficiency (NRG-W41 mice) with or without a transgenic source of human IL3, GM-CSF and SCF (NRG-W41-3GS mice). Initial experiments showed nonirradiated NRG-W41 mice support higher levels of multi-lineage human hematopoietic cell reconstitution from transplants of normal cord blood or bone marrow CD34+ cells compared to NRG controls. Thus, in our first teratoma experiments, we compared the output of human hematopoietic (CD45+) cells in NRG-W41 and NRG-W41-3GS mice injected subcutaneously with 103-106 human iPSCs ± mouse fibroblasts engineered to produce human FLT3-L, SCF, IL3 and IL6 in Matrigel. Teratomas were consistently produced from 105 human iPSCs. Following their enzymatic dissociation into single viable cell suspensions, and FACS analysis of the cells with various human-specific antibodies, we found CD34+ cells were detected in all teratomas with >0.1% CD45+ human cells. Initial experiments demonstrated that teratomas produced ∼40-fold more CD34+CD45+ cells when generated in NRG-W41-3GS versus NRG-W41 with yields of up to 7x106 CD45+ cells from a teratoma containing 4x108 cells. The co-injection of human growth factor-producing mouse fibroblasts also increased the overall production of human CD34+and CD34+CD45+ cells by ∼6-fold even in the NRG-W41-3GS mice. These teratoma derived cells also produced granulopoietic (GM), erythroid (E) and mixed (GEM) colonies in standard growth factor-supplemented methylcellulose cultures demonstrating the generation of functionally competent human hematopoietic progenitors in iPSC derived teratomas. These experiments lay the foundation for more detailed investigation of the conditions that will promote the generation of hematopoietic stem cells and their properties.