Using iPSC-derived hematopoietic stem cells with long-term engraftment capability to model fetal blood disorders

Abstract Introduction: Human developmental hematopoiesis is a complex process occurring in sequential waves at different embryonic sites. This process yields both differentiated blood cells essential for embryonic development and hematopoietic stem cells (HSCs) necessary for lifelong blood cell production. There are at least two main hematopoietic programs during embryogenesis: a transient primitive wave that primarily generates myelo-erythroid progenitors in the yolk sac, and a definitive wave occurring in the aorta-gonad-mesonephros region which produces progenitors with expanded lineage potential and the first transplantable HSCs. A challenge in generating HSCs from induced pluripotent stem cells (iPSCs) is that many current methods recapitulate early developmental stages only, failing to produce transplantable HSCs and limiting their application for in vivo studies. We have previously shown that we can recapitulate fetal erythropoiesis using a protocol that produces definitive hematopoietic cells. Red cells generated with this method primarily produce fetal globin and are functionally distinct from primitive erythroblasts. Here, we adapted this method to a serum-free 3D culture system to produce definitive hematopoietic stem progenitor cells (HSPCs) from iPSCs to 1) evaluate long-term engraftment via xenotransplantation and 2) model a preleukemic disorder of fetal origin, transient abnormal myelopoiesis (TAM), which affects ~20% of neonates with Trisomy 21 (T21) and is associated with mutations in the key hematopoietic transcription factor GATA1. Methods We designed a serum-free 3D culture system that directs mesodermal commitment through Wnt pathway activation by manipulating developmental cues through retinoic acid signaling and shear stress. We transplanted 24 NBSGW mice with 1.2-2 million iPSC-derived HSPCs using 3 distinct wildtype (WT) lines and experimental batches. The 3D definitive differentiation was assessed using isogenic T21 iPSCs with wild type GATA1 or the truncated isoform lacking the N-terminus, GATA1s. Results Long-term repopulation was achieved up to 20 weeks post-transplant in the bone marrow (BM) of all engrafted mice (mean = 3.9±11.7% human HLA-ABC+) and in the peripheral blood of 16/24 mice. In mice with the highest level of engraftment (>10%), we detected human HSCs as well as myeloid, lymphoid, erythroid and megakaryocyte precursors in the BM, indicating complete hematopoietic reconstitution. Human CD34+ cells recovered from the graft retained multilineage potential in colony forming assays. Importantly, no leukemia or teratomas were observed in any of the transplanted mice. To assess whether this definitive iPSC culture protocol could faithfully model the fetal blood disorder TAM, we generated definitive HSPCs from T21/wtGATA1 and T21/GATA1s iPSCs. Both T21 lines generated budding HSPCs at day 15; however, the percentage of CD34+CD45+ HSPCs was lower compared to euploid controls (60.5% of WT ±12.9 for T21, and 64.5% of WT ± 0.06 for T21/GATA1s). In addition, at day 15 we observed a significant CD41+CD42b+ megakaryocyte population in both trisomy lines, consistent with the disease phenotype and that was not previously observed with our primitive hematopoietic differentiation protocols. Compared to WT controls, we observed a 2.3-fold and a 4.2-fold increased megakaryocyte population from T21/wtGATA1 and T21/GATA1s iPSCs, respectively. T21/GATA1s HPSCs also showed aberrant morphology with megakaryocytic and blast-like features. Conclusions These studies show that our serum-free 3D culture system can generate iPSC-derived HSPCs that can engraft immunodeficient mice, reconstitute different blood lineages, and persist for 20 weeks. Using a T21/TAM iPSC model, we found an enhanced megakaryocyte population in T21/wtGATA1 consistent with a phenotype we observed with primary human fetal liver hematopoiesis, but not with iPSC-derived primitive hematopoietic progenitors. Thus, T21/GATA1s definitive HSPCs recapitulate the enhanced megakaryopoiesis consistent with TAM in vitro. Ongoing xenotransplant experiments will help elucidate the interaction of trisomy 21 and GATA1s and provide a novel in vivo model to study and treat human hematological diseases.