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Embryonic hematopoietic development has been the hematopoietic stem cells (hematopoietic stem cells, HSC) biology research focus, divided into primitive hematopoietic two stages. The latter is marked by the generation of lymphocytes and HSC. In the past three decades, understanding the origin of the lymphatic system and HSC extraembryonic yolk sac gradually transferred to the aorta - gonad - kidney area (aorta-gonad-mesonephros region within the embryo, the AGM region AGM region). Cumano found: embryonic circulation dirty parietal rather than within the embryo the extraembryonic the yolk sac in vitro by co-culture with stromal cells and thymic tissue blocks production of B and T lymphocytes. However, the conclusions are not other laboratory and other training system can be verified. Compared to the lymphatic system and the in vitro differentiation system, the hematopoietic system of lethally irradiated adult mice by transplantation and continuous transplantation of long-term reconstruction (4-6 months) is the identification of the HSC gold standard. The mainstream view is that the mouse E10.5 AGM region was one of the first independent sites generate HSC. Recently, the researchers found that almost as much time with the AGM region HSC extraembryonic placental tissue, and significantly more than the number of AGM region, suggest that the occurrence of the HSC is a number of points. Previous studies have found that: the typical mouse the E9 embryo's head contains hematopoietic progenitor cells also expressed SCL / Tal, GATA1 hematopoietic flag. However, the site has the potential of hematopoietic still unknown. The first part of the present study clearly can independently produce the anatomical site of the lymphoid lineage, we selected mice E8.5 embryos (1-7 somites) of the yolk sac (embryo) and dirty as experimental subjects-parietal (embryo) , then the blood circulation is not yet established. Standard colony formation assay, both of which can produce typical hematopoietic colony, but different types. OP9 co-culture found: dirty parietal produce the B-lymphocytes, which expressed B220, CD19 and IgM. Using the three-step method induced dirty parietal OP9-DL1 to 16 days, can to produce CD44-/CD25 naive T cell precursors and CD4 / CD8-single-positive T cells. However, the co-culture system were unable to induce the yolk sac produce B and T lymphocytes. Our research showed that: compared with the yolk sac, dirty parietal has strong lymphoid lineage differentiation potential in vitro. The second part of this study, we myeloid progenitor cells, lymphocytes, compared the characteristics of embryonic AGM region and head of hematopoietic HSC three aspects. First, the separation of E9.5-E12.5 mouse embryonic tail (caudal half (CH), the locus development in E10.5 AGM region) / AGM region and head type Ⅰ collagenase digestion into single cell inoculated in accordance with certain of the embryo equivalents (embryo equivalents, ee) hematopoietic colony culture system (containing the SCF, IL-3, IL-6, EPO and other hematopoietic factors). 4 days observed CH / AGM region and head produce CFU-E, and seven days of CFU-GM was followed by CFU-Mix. We found E9.5 mouse head cells the hematopoietic colony erythroid and dense, and CFU-GM less. E10.5, CFU-GM after increased significantly. The AGM region granulocyte colony were highly homogeneous, rounded or oval, the head of the Department of granulocyte colony larger, more dispersed cells, irregular in shape. In addition, we also studied the CH / AGM region and head myeloid hematopoietic progenitor cells (ie of CFU-C) developmental dynamics found four time points (E9.5-E12.5) CH / AGM zone little change in the number of CFU-C. In contrast, compared with E10.5 the E11.5 head of the number of CFU-C significantly increase reached a peak after gradually reduced. Bead sorting, and found that the AGM region and the head of the CFU-C focus on Tie2 cell populations. The above results show that: the mouse embryonic AGM region and head myeloid hematopoietic progenitor cells, but there are differences both in progenitor cell types and developmental dynamics. After that, we examine the CH / AGM zone and the head of the lymphatic system and development potential. We OP9 stromal cell co-culture system investigated B cell differentiation potential. SCF, FL, IL-7 and other cytokines in the role of the first 3-4 days OP9 cell clusters, followed by the semi-adherent cells was significantly amplified, its shape is similar lymphocytes. 7-10 days to collect the cells by flow cytometry analysis found (representative data): CH obtained cells induced expression of B220 (23.0%), CD19 (25.0%); cells of the head of B220, CD19 positive rates were : 36.7% and 25.8%, respectively. The above results show that the CH and the head cell of the E9.5 mice having a B-cell potential. Similarly, the CH and the head of the separation E9.5 of mouse embryos, using magnetic beads separation Tie2 cells, seeded in OP9-DL1 cells, SCF, FL, the role of IL-7 and other cytokines, found OP9-DL1 on a large number of lymphocyte-like cell proliferation. Found in the four consecutive time points: With in vitro induced prolongation of time (7 days, 13 days, 19 days, 26 days), the naive T-cell precursors (CD44-/CD25, DN3), more mature Shuangyang cells (CD4 / CD8) as well as the proportion of CD3 / TCRβ are gradually increased. To 26 days (representative data), head and CH consistent trend, the proportion of the three cell subsets, respectively, 20.1%, 7.5% and 8.2%, respectively. The above results show that: under identical induction conditions, the CH of the mouse E9.5 and head cells are able to differentiate into T lymphocytes, the two sites have the potential of T lymphocytes. Finally, we look at the the HSC the potential of the the AGM area and head. Transplantation AGM region and head separation E12.5GFP transgenic mouse embryos, digested into single cell through the tail vein. 5 months after transplantation, collecting receptor in peripheral blood flow cytometry found: similar proportion of the AGM region (n = 11), and the reconstruction of the head (n = 21) (45% Vs 62%), the chimeric rate is also similar (49. ± 19.0% Vs 50. ± 22.1%). The above data show that: E12.5 AGM region the head containing cells have the ability to long-term reconstruction lethal dose irradiated mouse hematopoietic system. The bead sorting transplantation AGM region and the head of the Tie2 lethally irradiated receptor cells are able to rebuild. These results suggest that in vivo hematopoietic activity of the head cells express Tie2, other hematopoietic tissue similar to the same period. For detecting multi-based chimeric potential after E12.5 mouse embryos AGM region and the head cell transplantation for the receptor's peripheral blood myeloid (Mac-1, GR-1) and the lymphoid lineage (B220, CD3) chimerism testing found: the AGM region head cell transplantation positive receptors, peripheral blood have a significant proportion of donor derived myeloid and lymphoid chimeric. In addition, the receptors on the part of the reconstruction of the bone marrow, spleen, thymus, detection, derived hematopoietic cells of the AGM region and head can be in a high proportion of these adult hematopoietic tissue fitting the description of the head of E12.5 mouse embryos and AGM region hematopoietic cells with multi-lineage differentiation potential in vivo. Verify the hematopoietic cells self-renewal capacity, we apply a second transplant to rebuild the bone marrow cells of the receptor, two months after the detection of to all receptors all reconstruction AGM region and head of hematopoietic cells with self-renewal capacity. Two loci derived hematopoietic cells in the body capable of self-renewal and differentiation, prove E12.5 AGM region and head of the mice contain true HSC.
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