Runx1+23 Enhancer Reporter Marks Erythro-Megakaryocyte Lineages in Human ESC Differentiation Culture

Runx1 is an essential transcriptional factor for hemogenic endothelial cells (HECs) to transit to hematopoietic stem/progenitors in mammalian embryos. In mice, it has been shown that Runx1 +23kb enhancer region (Runx1+23) marks HECs in the embryo and HSCs in the adult bone marrow (BM) specifically.

In order to identify HECs in human embryonic stem cell (ESC) differentiation culture, we utilized Runx1+23 enhancer-reporter hESC lines; H9 Runx1+23 Ds-Red and H1 Runx1+23 venus. We differentiated these Runx1+23 ESCs into hematopoietic lineage on laminin coated plate. We examined CD34+CD43- EC and CD34+CD43+ hematopoietic progenitor (HPC) productions as well as CD235a+ RBC and CD43+CD41 megakaryocyte production on day 6, day 8, and day 10 culture. We found robust Runx1+23+ Ds-Red (denoted Runx1+23) expression on CD235a+ RBCs, CD43+CD41+ cells, and CD34+CD43+ HPCs. However, Runx1+23 expression was not detected in CD34+CD43- EC population. When we gated Run1+23 population, they were relatively large, did not express CD144 EC markers, but expressed CD43 and CD41. Cytospin preparation of sorted Runx1+23+ cells showed that Runx1+23+ cells contained megakaryocytes and erythroblast. We also sorted Runx1+23 positive or Runx1+23 negative CD34+CD43+ HPCs or CD34+CD43-ECs and plated them onto Methylcellulose culture to test colony forming ability and onto MS5 stromal cells for testing B cell potential. Runx1+23+ cells displayed a greater number of megakaryocytes colony-forming cells compared to Runx1+23 negative cells and expressed erythro-megakaryocyte genes including GATA1, GATA2, TAL1, and hemoglobin genes. On MS5 cell co-culture, only Runx1+23 negative ECs produced CD19+B-cells and CD56+ NK cells. Finally, scRNA-sequencing of day 10 differentiation culture revealed that Runx1+23+ cells were exclusively expressed in the erythro-megakaryocytes populations.

These results indicated that Runx1+23 enhancer region is exclusively active in erythro-megakaryocyte lineage specification in human ESC differentiation culture, different from mouse system, suggesting different usage of Runx1+23 enhancer region between mice and humans.

No relevant conflicts of interest to declare.