Characterization Of Surface Markers Of Hematopoietic Precursor Cells During Mouse Embryogenesis

Development relationship between hematopoietic and endothelial lineages during murine embryogenesis is close. Two models have been proposed, one is that hematopoietic and endothelial cells are more likely derived from a common precursor-the hemangioblast. Another is the hemogenic endothelium, which is mature endothelium that can transdifferentiate into hematopoietic stem cells. The origin of hematopoietic cells is undefined in embryonic development. Since hematopoietic stem cell is difficult to be accurately located, we can not reveal its development dynamics easily. As a core component of hematopoietic tissue, HSC possesses the ability for self-renewal and can differentiate into any blood cell lineages. On the one hand, it can maintain the dynamic balance of the stem cells pool and normal hematopoiesis. On the other hand, it can give rise to a mixture of multiple blood cell types. Hematopoietic stem cells is not homogenous, composed of different subsets of stem cells .Theses cells express different surface antigen, adhesion molecules and have different immune phenotype. Therefore, it is imperative to either find or identify new HSC specific markers to study the origin as well as developmental kinetics of hematopoietic stem cells.The OP9 stromal cells play an important role in the study of the relationship between hematopoietic and endothelial cells and also in the identification of hemangioblast. So we studied the biological characteristics of OP9 from cell morphology, immune phenotype, differentiation capacity, migration features, immunogenicity and immune regulation. The OP9 cells were bipolar fibroblast-like in morphology. They were morphologically homogeneous population. Under the appropriate conditions, the OP9 cells could be induced into osteocytes, chondrocytes and adipocytes. They were negative for CD45, CD11b, Flk-1, CD31, and CD34, indicating their non-hematopoietic and non-endothelial identity. And they were uniformly positive for CD44, CD29 and Sca-1. The OP9 also expressed markers of perivascular cells, including CD140a, CD140b,α-SMA, and Calponin. bFGF, IGF-1, IL-3, PDGF-BB, TGF-β1 and TGF-β3 had relatively stronger effects on migration of the OP9, while SDF-1α, BMP-2, BMP-4 and VEGF had weak chemotactic ability. The OP9 could suppress T lymphocyte proliferation induced by cellular and nonspecific mitogenic stimuli. In conclusion, the OP9 cells have the characteristics of MSCs, and such homogenous cell line will be helpful to delineate biological features of MSCs at the stem cell level. After clear illustration of the cellular identity of the OP9, we introduced it into the study of the relationship between hematopoietic and endothelial cells. The mouse AGM region cells were sorted by immunomagnetic beads and the Tie2+ cells were seeded in the 24-well plates pre-coated with OP9. We verified that OP9 can support hematopoiesis and endothelial growth. Based on OP9, we established a simple identification system of hemangioblast (BL-CFC). Under certain conditions, BL-CFC co-cultured with OP9 cells can form B lymphocytes. This proved that BL-CFC had the potential of definitive hematopoiesis.In order to explore the specific markers of the hematopoietic stem cell in the developmental hematopoiesis, we focused on the expression of Flk-1 and CD43 in multiple types of hematopoietic precursor cells (including myeloid progenitor cells, hemogenic endothelium, B lymphoid precursors, BL-CFC, and HSC). First, E10.5, E11.5, E12.5 AGM were separated and digested into single cell suspension by typeⅠcollagenase, sorted by immunomagnetic beads, and plated in the hematopoietic colony forming assay (including the cytokines SCF, IL-6, IL-3, EPO ). We found Flk-1+ cells were responsible for a proportion of CFU-Cs, whcih reach the climax at E10.5. As compared, all CFU-Cs expressed CD43.The CFU-Cs only reflect the definitive myeloid hematopoietic progenitor cells, while hemogenic endothelium can be quantified by the formation of hematopoietic clusters on OP9. Our results showed that in the presence of SCF,IL-3,FL,VEGF, hematopoietic clusters were more easily generated from Flk-1+ cells than Flk-1- cells. After growth on OP9 for 7 days, the non-adherent cells in both Flk-1+ and Flk-1- groups expressed markers of hematopoietic cells (CD45, Mac-1/Gr-1, B220, CD19 and so on.). The levels of CD45, c-Kit in Flk-1+ cells were higher than in Flk-1- cells, which expressed less Ter119. This suggested that Flk-1- cells could more easily different into mature red cells than form hematopoietic clusters, but the capability of generating hematopoietic stem and progenitor cells from Flk-1+ cells might be stronger. Then, via immunohistochemistry, we found the adherent cells from the two subsets expressed CD31 and Endomucin, but the ability to form tubes in Flk-1+ cells were better than that in Flk-1- cells. As compared, only CD43+ cells could form hematopoietic clusters. Non-adherent cells also expressed surface markers of myeloid lineages and lymphoid lineages. Nevertheless, the adherent cells of CD43+ and CD43- groups both form tube-like structures expressing CD31 and Endomucin, implying that the CD43 expression can distinguish hemogenic from non-hemogenic endothelium.Based on OP9, we studied the relationship of Flk-1 or CD43 with the development of B lymphocyte, respectively. We found Flk-1+ cells and Flk-1-cells of AGM region had the potential to form B lymphocyte in the presence of FL and IL-7. However, only CD43+ cells can different into B lymphocyte.In the methylcellulose BL-CFC system (containing SCF, IL-6, IGF-1, VEGF, LIF, bFGF), we found the BL-CFC was easily formed by Flk-1+ cells than Flk-1-cells, but exclusively concentrated in the CD43+ cells.Finally, we examined the expression of Flk-1 in HSC. The GFP+ AGM region and fetal liver cells were routinely isolated at different time points, and digested into single cells suspension. After sorted, they were transplanted via the tail vein. Four mouth post transplantation, flow cytometry analysis of peripheral blood were implemented. According the standard (chimerism≥10% as repopulated), we found the Flk-1+ cells and Flk-1-cells in E11.5, E12.5 AGM region and E12.5 FL had the ability to long-term reconstitute the hematopoietic system of lethally irradiated mouse. To test the multilineage repopulation post transplantation, the myeloid (Mac-1/Gr1) and lymphoid (B220, CD3) chimerism in the peripheral blood were measured. There was considerable chimerism of donor cells. This was also the case in the thymus, spleen and bone marrow. To verify the self-renewal capacity, the bone marrows cells from the primary recipients were transplanted into the secondary recipients. Two month later, we found the hematopoietic systems of recipients were reconstructed. It indicated the bone marrows cells were able to short-term reconstruct the hematopoietic system of lethally irradiated mice. These date suggested that both Flk-1+ and Flk-1-cells contained bona fide HSC.