Transcription Factor Programming of Human iPSC-Derived Sinusoidal Vascular Endothelial Cells Enhances Co-Cultured Hematopoietic Stem and Progenitor Cell Transplantation

Hematopoietic stem and progenitor cells (HSPCs) reside in specialized niche microenvironments in the marrow made of sinusoidal vascular endothelial cells (ECs) and other perivascular supportive cells. Pathological conditions such as primary myelofibrosis (PMF) in the marrow cause HSPC niche defects and pan-cytopenia. Extramedullary HSPC niche can form in the liver or the spleen sinusoids during PMF, but the process is inefficient. Here, we aim to identify the transcription factor (TF) code that specifies the sinusoidal vascular EC fate in the HSPC niche. We performed differential gene expression analysis on ECs from adult zebrafish kidney marrow and liver and identified TF candidates that were uniquely upregulated in the marrow sinusoidal ECs, namely tfec, mafbb, foxp4, irf8, and hoxb8a. To determine whether these candidate TFs can functionally specify a HSPC niche EC fate, we selectively overexpressed them using a zebrafish liver sinusoidal EC-specific enhancer in vivo. Upon the overexpression of tfec and mafbb together, we found adult zebrafish liver sinusoidal ECs were reprogrammed to upregulate key genes known for HSPC niche supportive functions, including mrc1a (log2FC=4.9, p<0.005), lyve1b (log2FC=3.6, p<0.005) and dab2 (log2FC=5.0, p<0.005). Transplant assay of liver cells into irradiated adult hosts showed that primary HSPCs occupy the newly reprogrammed liver vascular niche (7/26 in the reprogram group vs. 0/20 in the control group) (p=0.0296). Therefore, TFs tfec and mafbb were sufficient in reprogramming adult liver sinusoidal ECs to become HSPC niche in vivo. Furthermore, we aim to translate our findings to program human iPSC-derived ECs to support primary HSPCs in vitro. We engineered human iPSC lines with inducible overexpression of human ETV2, TFEC and MAFB. Upon ETV2-directed differentiation hiPSCs into ECs, the overexpression of TFEC and MAFB significantly upregulated the expression of sinusoidal endothelial and HSPC niche supportive genes, such as MRC1 (log2FC=3.6, p<0.005), STAB2 (log2FC=10.3, p<0.005), JAG1 (log2FC=1.7 p<0.005), and CXCL12 (log2FC=5.1 p< 0.005). Methylcellulose assays showed that CD34+CD45+ HSPCs co-cultured with hiPSC-derived ECs induced with ETV2 plus TFEC and MAFB contained significantly more colony-forming units (CFUs) compared to those co-cultured with ECs induced with ETV2 alone or HSPCs cultured without ECs: CFU-GEMM (23.3±2.4 vs. 10.0±2.1, p=0.004), CFU-G (89.3±13.4 vs. 46.0±4.2, p=0.027), and CFU-E (42.3±3.3 vs. 26.7±0.9, p=0.021). Transplant of HSPCs into immunodeficient mouse hosts showed that HSPCs co-cultured with TFEC and MAFB induced ECs have significantly better engraftment potential than control HSPCs (p=0.0164). In summary, TFs TFEC and MAFB could program human iPSC-derived ECs to adopt HSPC niche fate and support primary human cord blood-derived CD34+ HSPCs in vitro. Our findings provide a method to engineer human HSPC niche-like sinusoidal ECs to enhance engraftment of HPSC, which could help transplantation therapies.

Zon:Triveni Bio: Current holder of stock options in a privately-held company; Scholar Rock: Current holder of stock options in a privately-held company; CAMP4 Therapeutics: Current holder of stock options in a privately-held company; Fate Therapeutics: Current holder of stock options in a privately-held company.