Development Dynamics Of Definitive Hematopoiesis In The Mouse Somite-stage Embryos

Embryonic hematopoiesis has been a hot topic for long time in the field of hematopoietic stem cell (HSC) research. The process includes 2 stages, the primitive and the definitive hematopoiesis, with the latter featured by the generation of lymphocytes and HSC. In the recent 30 years, the recognition concerning the origin of lymphoid system and HSC has moved from the extra-embryonic yolk sac to the intra-embryonic AGM region. Cumano et al. found that, prior to the establishment of circulation, the intra-embryonic splanchnopleura other than the extra-embryonic yolk sac can generate B and T lymphocytes after cocultured with stromal cells and thymus explant. Nevertheless, the conclusion has not been validated in other independent groups or by alternative experimental systems. Unlike the in vitro lymphopoiesis assay, the long term reconstitution (4-6 months) of blood system in leathally irradiated adult recipients by transplantation and serial transplantation is the gold standard of identifying HSC. It is accepted that the mouse E10.5 AGM region is the earlist site for HSC development de novo. Recently, it is revealed that the canonical HSC forms in the placenta nearly paralleling the AGM region, suggesting the ontogeny of HSC may be of multiple source. As previously documented, the mouse E9 head contains typical hematopoietic progenitors and expresses blood markers including SCL/Tal and GATA1. However, the definitive hematopoietic capacity of the head remains unknown.In the first part, the splanchnopleura and the yolk sac from the mouse embryos between E8.0-E8.25 (1-7 somite pairs, embryonic circulation not built) were selected to explore the site with autonomous lymphopoiesis activity. In standard colony forming assay, the cells from both splanchnopleura and yolk sac formed typical myeloerythroid colonies but the types were distinct. When cocultured with the OP9, the splanchnopleura gave rise to B cells expressing B220, CD19 and surface IgM. Using a three-step culture protocols with the OP9 expressing Delta-like 1 as feeders, the splanchnopleura produced immature T precursor cells (CD44-/CD25+) and more mature single positive T cells (CD4+/CD8-) after 16 days of incubation. However, the yolk sac failed to generate B or T lymphocytes under identical conditions. Therefore, our findings suggested that, prior to linked embryonic circulation, the splanchnopleura other than the yolk sac had robust lymphoid potential in vitro. In the future, more reliable evidence from novel model animals will ultimately delineate the embryonic origin of lymphocytes in vivo.In the second part, three aspects (the myeloid progenitors, lymphocytes, and HSC) were compared between the AGM region and the head. Firstly, the caudal half (CH)/AGM and the head from E9.5-E12.5 mouse embryos were separated and digested into single-cell suspension by type I collagenase, and plated in the hematopoietic colony forming assay culture (containing SCF, IL-3, IL-6 and Epo). Both groups developed CFU-E colonies in the forth day, and CFU-GM the seventh day followed by the CFU-Mix. In the culture of E9.5 head cells, the erythroid colony prevailed with compact morphology. In the cultures with the head cells beyond E10.5, the number of CFU-GM increased. Morphologically, the CFU-GM colonies from the AGM group were homogenous, but those from the head group were irregular with dispersed shape. In addition, the development kinetics of myeloid progenitors of the two sites were investigated. Between E9.5 and E12.5, the numbers didn't change significantly in the CH/AGM cultures. In contrast, as compared with E10.5, the number of CFU-C in the E11.5 head dramatically upregulated and reached the climax, and afterwards stepwise decreased. The myeloid progenitors could be exclusively enriched by Tie2 expression. These data indicated that the myeloid progenitors existed in both the AGM region and the head, but their types and kinetics differed remarkably.Futhermore, the lymphoid potential of CH/AGM region and the head were investigated. The OP9 stromal cells were used to test the B cell activity. In the presence of SCF, FL and IL-7, cell clusters appeared in the third or forth day, followed by significant expansion of semi-adherent lymphocyte-like cells. In the seventh to tenth days, the expanded cells were collected for flow cytometry analysis. As representative data showed, the cells from the CH group expressed B220 (23.0%) and CD19 (25.0%). Likewise, the cells from the head group expressed B220 (36.7) and CD19 (25.8). The result indicated that the two sites possessed precursor cells with B cell potential. In addition, the Tie2+ cells from the E9.5 CH and head were respectively plated on the OP9-DL1 feeders with addition of SCF, FL and IL-7. Four time points were chosen to analysis the T cell commitment. We found that with the culture ongoing, the proportion of immature T cell precursors (CD44-/CD25+, DN3), more mature double positive cells (CD4+/CD8+) and CD3+/TCRβ+ cells gradually increased. In the 26th days of induction, the cells from the head group had the above three subpopulations at 20.1%, 7.5% and 8.2% respectively, similar to those of CH group. These data suggested that under similar conditions the CH and head of mouse E9.5 embryos had the potential to fulfill T cell specification.Finally, the HSC activity of the AGM region and head were examined. The E12.5 AGM tissue and the head of GFP transgenic mouse were dispersed into single cells and then infused via tail vein into 9 Gy irradiated congenic wild type recipients. Five months post transplantation, the peripheral blood of recipients were harvested for flow cytometry. The reconstitution ratio (45% Vs 62%) and the chimerism (49% Vs 50%) between the AGM and the head were similar. Futher enrichment by Tie2 expression revealed that such cells with in vivo hematopoietic activity expressed Tie2, identical to other embryonic blood-forming tissues. To test the multilineage repopulation post transplantation, the myeloid (Mac-1 and Gr-1) and lymphoid (B220 and CD3) chimerism in the periphral blood were quantified. The reconstituted recipients in the AGM and head group demonstrated significant chimerism. It was also the case for the bone marrow, spleen and thymus. To further determine the self-renew capacity, the bone marrow cells from the primary engrafted recipients were transplanted into the secondary recipients. After 2 months, all the recipients were successfully repopulated, indicating the replication capacity of the blood cells from the AGM region and the head. Based on the in vivo self-renew and multilineage properties of the AGM and the head cells, we concluded that the two sites contained bona fide HSC.