Extended Data Figure 1. Screening strategy for identification of minimal set of transcriptions factors (TFs) for reprogramming ECs into hematopoietic cells.

A. Candidate genes tested for reprogramming of HUVECs into hematopoietic colonies. To identify TFs that drive EHT transition, we performed RNA-seq of HUVECs and Lin-CD34⁺ umbilical cord HSPCs to select TFs differentially expressed by HSPCs, but not by HUVECs. We then screened various combinations of differentially expressed TFs to identify a minimal set capable of reprogramming ECs to hematopoietic cells. Levels of expression [log₂(RNA-seq value)] in HUVECs and freshly purified CD34⁺Lin⁻ cord blood cells.

B. One-by-one elimination of TFs experiment revealed a minimal set of TFs; FOSB, GFI1, RUNX1, and SPI1, (FGRS) capable of generating hematopoietic colonies in the HUVEC culture. A pulled set of 26 TFs minus one TF was evaluated for the ability to evoke formation of hematopoietic clusters (day 21 to 25; n=3). Asterisks show statistically significant (p<0.05) reduction of the number of hematopoietic clusters in the transduced HUVECs compared to the full set of TFs. Control represents non-transduced HUVECs. Transduced cells were cultured on a layer of serum-free E4EC monolayers.

C. One-by-one elimination of the FGRS factors shows that all four FGRS-TFs are necessary and sufficient for generation of hematopoietic colonies (day21 to 25; n=3).

“All” in B and C: all TFs are present. “Control” in B and C: all TFs are absent.

D. Schema for reprogramming of ECs into human multipotent progenitor cells (rEC-hMPPs). Clonal or bulk populations of HUVECs or hDMECs were transduced with the FGRS and after 3 days were replated on subconfluent monolayers of E4EC ECs. The emerging colonies of hematopoietic cells were subjected to 1) multi-variate immunophenotypic analyses, 2) clonal and oligo-clonal CFC assay, and 3) molecular profiling (RNA-seq). Tissues of the engrafted mice were processed for histological examination to rule out any malignant transformation.