Surface Marker Of The Definitive Hematopoiesis In The Mouse Embryo

At the beginning of the definitive hematopoiesis, the origin of the hematopoietic stemcells(HSC) has attracted extensive attention in study of the hematopoiesis in themouse embryo so far. The first HSCs were found in the AGM region of embryonic10.5(E10.5) by Medvinsky et al in1996. Meanwhile, HSCs also appeared in the um-blilical artery (UA) and vitelline artery (VA). YS, PL and FL, which was alreadydetected by the functional assay in vivo, contain HSCs by E11.0-11.5. FL became themain pool for HSC proliferation after E12.c5. FL was the main HSCs reservoir atmid-gestation until the HSCs start to colonize the BM at E17.0. In summary, thehematopoiesis site(s) change during the development of HSCs in mouse embryo. Theemergence of HSCs is through several anatomical sites danamically and self-renewingactively.It is still difficult to ascertain the accurate anatomical site(s) for HSCs origin in themouse embryo. That is in primarily because the circulation between extra-andintra-embryonic tissues has already been founded at E8.25-8.5which is earlier the thetime (E10.5) of the first HSCs emergence. Moreover, there is not a specific marker wecan use to enrich and trace the HSCs so far.In the first part of this study, we investigated the expression of Flk-1on distincthematopoietic precursor cells in E10.5mouse AGM region. By flow cytometryanalysis, we found that <10%of Flk-1~+cells of E10.5AGM region co-expressedCD41and CD45/Ter119. Then, E10.5AGM cells were fractionated into two subsets,the CD31~+CD45-Ter119-Flk-1~+CD41~+cells (R1, putative immature hematopoieticcells) and the CD31~+CD45-Ter119-Flk-1~+CD41-cells (R2, putative endothelial cells), followed by methylcellulose-based CFU-C assay and OP9-based stromal co-culture toexamine their myeloid or/and lymphoid potential in vitro. Only R1cells could giverise to typical hematopoietic colonies in CFU-C assay. In contrast, after co-culturedwith OP9for7-9days, both subsets could generate abundant hematopoieticprogenitor cells (CD45~+c-Kit~+), myeloid cells (Gr-1~+/Mac-1~+), erythroid cells(Ter119~+), and B lymphocytes (CD19~+). Therefore, we conclude that both maturingCD41~+CD45-hematopoietic percusor cells and hemogenic endothelial cells expressFlk-1in E10.5AGM region. It requires further functional assay in vivo to clarifywhether the hematopoietic stem cells (HSCs) and their precursors retain Flk-1expression at this developmental stage.In the second part of this study, we focused on the expression of CD47on the distincthematopoietic cells in hematopoietic sites in E10.0-E12.5mouse embryo. By flowcytometry analysis, we found that the percentage of the CD47~+cell in E10.5AGMregion, yolk sac(YS) and placenta(PL) is respectively1.0±0.2%(n=3),13.3±2.8%(n=2) and2.1±0.2%(n=2), compared with1.4±0.2%(n=4),13.4±2.1%(n=2),13.6±2.1%(n=2) and0.9±0.03%(n=2) in E11.5AGM, YS, PL and E12.5circulation.Furthermore, these CD47~+cells co-expressed c-Kit, which was considered as a markerof HSCs. And then the cells from E10.0AGM, YS, PL and circulation were sortedinto2subsets, which were the CD47~+and CD47-cells. Then we used the CFU-Cassay to analyze their myeloid potential in vitro. The CFU-C was only detected inCD47~+subset, which suggested only the CD47~+subset contained the myeloidprogenitors in the CFU-C assay. Thereafter, E10.5AGM cells were sorted into thec-Kit~+CD47~+(R1) cells and c-Kit~+CD47-(R2) cells. After co-cultured with OP9cellsfor7days, we found both subsets could generate hematopoietic cells (CD45~+c-Kit~+),erythroid (Ter119~+), myeloid progenitors (Gr-1/Mac-1~+) and B lymphocytes/theirprecursors (B220~+). The results informed us that both CD47~+and CD47-cells inE10.5AGM region showed hematopoietic potential in OP9co-culture assay. Toclarify the CD47expression of hematopoietic stem cells, we similarly sorted the E11.5AGM/YS/PL cells into c-Kit~+CD47~+(R1) subset and c-Kit~+CD47-(R2) subsets,followed by the functional transplantation assay. The chimerism analysis aftertransplantation for16weeks showed the hematopoietic reconsititution was only foundin the R1subset, which confirmed all the HSCs in E11.5AGM, YS and PL expressedCD47. Thus it could be concluded that CD47could enrich HSCs at thisdevelopmental stage. Interestingly, hematopoietic reconsititution only appeared in thec-Kit~+CD47lowsubset after the E12.5circulation cells was sorted into c-Kit~+CD47highand c-Kit~+CD47lowsubsets and transplantation for16weeks. it can be concluded theHSCs in E12.5circulation could be eriched in CD47lowsubset.