Cells with hematopoietic potential reside within mouse proepicardium

During embryonic development, hematopoietic cells are present in areas of blood-vessel differentiation. These hematopoietic cells emerge from a specific subpopulation of endothelial cells called the hemogenic endothelium. We have previously found that mouse proepicardium contained its own population of endothelial cells forming a network of vascular tubules. We hypothesize that this EC population contains cells of hematopoietic potential. Therefore, we investigated an in vitro hematopoietic potential of proepicardial cell populations. The CD31⁺/CD45^-/CD71^- cell population cultured for 10 days in MethocultTM gave numerous colonies of CFU-GEMM, CFU-GM, and CFU-E type. These colonies consisted of various cell types. Flk-1⁺/CD31^-/CD45^-/CD71^-, and CD45⁺ and/or CD71⁺ cell populations produced CFU-GEMM and CFU-GM, or CFU-GM and CFU-E colonies, respectively. Immunohistochemical evaluations of smears prepared from colonies revealed the presence of cells of different hematopoietic lineages. These cells were characterized by labeling with various combinations of antibodies directed against CD31, CD41, CD71, c-kit, Mpl, Fli1, Gata-2, and Zeb1 markers. Furthermore, we found that proepicardium-specific marker WT1 co-localized with Runx1 and Zeb1 and that single endothelial cells bearing CD31 molecule expressed Runx1 in the proepicardial area of embryonic tissue sections. We have shown that cells of endothelial and/or hematopoietic phenotypes isolated from mouse proepicardium possess hematopoietic potential in vitro and in situ. These results are supported by RT-PCR analyses of proepicardial extract, which revealed the expression of mRNA for crucial regulatory factors for hemogenic endothelium specification, i.e., Runx1, Notch1, Gata2, and Sox17. Our data are in line with previous observation on hemangioblast derivation from the quail PE.