Decoding the Endothelial-to-Hematopoietic Transition in Human iPSC-derived Hematopoietic Organoids: Temporal Dynamics, Endothelial Diversity, and Transcriptional Drivers
Blood transfusions and hematopoietic stem and progenitor cell (HSPC) transplantation can be improved by generating patient-specific, engraftable HSCs and transfusable blood cells in vitro. During fetal development, functional HSCs arise from hemogenic endothelium (HE) via endothelial-to-hematopoietic transition (EHT). HE originates in CXCR4⁺ arterial endothelium in the dorsal aorta of the aorta-gonad mesonephros. We developed an induced pluripotent stem cell-derived hematopoietic organoid (HeO) that produces definitive enucleated erythrocytes. We hypothesize hematopoiesis in this model occurs via EHT. Here we (1) characterize HE cell (HEC) dynamics and EHT, (2) examine endothelial heterogeneity during EHT, and (3) identify intrinsic EHT regulators.
IPSCs were allowed to spontaneously form embryoid bodies that eventually form hematopoietic organoids and are subsequently cultured in SCF, EPO and IL-3 containing medium as reported by us previously. HECs were defined broadly as CD309+CD34+ and restrictively as CD309⁺CD34⁺CD43⁻CD73⁻. Timecourse analysis of dissociated HeOs was performed to investigate the dynamics of HEC population. On day 20, index-sorted single cell RNA sequencing using FLASH-sequencing was performed on sorted CD309+CD34+ cells. Broadly and restrictively defined HECs were also isolated and subcultured on day 20 for four days in culture conditions supporting EHT progression.
Flow cytometry of dissociated HeOs showed tightly regulated HEC emergence (days 20–22; 3%), followed by a surge in CD43⁺ hematopoietic cells. Single-cell RNA-sequencing of CD309⁺CD34⁺ endothelial cells at peak HE identified six clusters. Comparison with in vivo EHT datasets revealed shared marker profiles for arterial endothelium (CXCR4⁺, GJA⁺), pre-HE (CXCR4⁺GJA⁺IL33⁺), HE (KCNK17⁺CD44⁺RUNX1⁺), and HSPCs (SPN⁺PTPRC⁺SPINK2⁺) in our arterial and hematopoietic-endothelial clusters. We found known and novel EHT regulators, including upregulated NOTCH signaling in the arterial cluster and hematopoietic regulators (RUNX1, GFI1/GFI1B) and translation genes in the hematopoietic-endothelial cluster. Culturing purified HECs or CD309⁺CD34⁺ populations confirmed EHT progression by flow cytometry and microscopy.
These findings define EHT timing and HE emergence, show endothelial heterogeneity, display alignment with in vivo darasets and confirm, via transcriptomics, cells undergoing EHT in the HeOs. Together, the data support improved HSPC yield from our HeO and validate its use as a developmental hematopoietic model.