| Hematopoietic stem cells (HSCs) develop during embryogenesis in a complex process that involves multiple anatomical sites. The HSCs are firstly generated in aorta-gonad-mesonephros (AGM) region, and later placenta and yolk sac. Thereafter, they migrate into fetal liver through the circulation, after which they colonize the bone marrow at birth. During this process, various embryonic niches where developing HSCs reside may provide appropriate signals for their emergence, maturation, expansion and differentiation. Due to the insufficiency of in vitro culture condition to simulate the complex micro-environment in vivo, HSCs are prone to lose"stemness"in vitro. Promisingly, the in vitro tissue culture method, bywhich the in vivo three dimensional and physiological micro-environment may be maintained, can promote the maturation and expansion of HSCs. Investigating the developmental events that lead to the formation of a functioning hematopoietic system in an adult organism has been a challenging pursuit for several reasons: (1) HSCs of the hematopoietic system are scattered throughout the organism, and multiple sites of hematopoietic activity exit in the embryo, thus obscuring its anatomical boundaries; (2) HSCs in the early embryo (E10.5-E11.5) are very rare (approximately 1-3 per embryo); (3) no markers are known to be exclusively expressed on HSCs in the embryo. Therefore, it is imperative to either find or identify new specific markers to isolate and purify HSCs and study the origin as well as the developmental kinetics of HSCs.In the first part, to analyze hematopoietic kinetics of mouse embryonic AGM region, a specific tissue culture method was developed in this study, partly based on previous reports. After 2 days of tissue culture, a significant number of erythromyeloid progenitors, as quantitated by colony forming assay, were detected in the AGM region. Moreover, the cells from cultured E10.5 AGM region could generate 104±18 colony-forming unit in culture (CFU-C) and 10.8±3.5 colony-forming unit in spleen (CFU-S) per tissue on average. More importantly, transplantation of cultured E10.5-E11.0 AGM cells resulted in efficient (85.7% repopulated) and long-term (> 4 months) reconstitution of lethally irradiated adult recipients with remarkable chimerism (51.12%±21.17%). The multilineage contribution of donor cells was validated by significant engraftment of myeloid and/or lymphoid cells in peripheral blood, bone marrow, spleen and thymus of recipients. The results, as well as the classical literature described, demonstrated that vitro tissue culture could promote the mature and expansion of HSCs in the AGM region, which was an effective method to study the developmental kinetics of various hematopoietic precursor cells, particularly HSC, in mid-gestation mouse embryos.In the second part, using the established tissue culture and transplantation system, we focus on the CD43 expression on HSCs during embryogenesis at temporally and anatomically restricted sites. CD43, also called sialophorin or leukosialin, is an integral transmembrane molecule belonging to the sialomucin family, like CD34 and Endoumucin. The study was mainly based on several reasons: (1) previous studies have demonstrated that CD43 was highly expressed on murine and human HSCs; (2) the hematopoietic progenitor cells derived from human embryonic stem cells and induced pluripotent stem cells expressed CD43; (3) CD43 was expressed on hematopoietic cell clusters of the ventral side of the dorsal aorta in the human embryo; (4) .In addition, CD43 exhibits a protein structure highly related to CD34, an important molecule expressed on HSCs during mouse embryogenesis. We are also interested in the the similarities and differences between the two molecules.To investigate the CD43 expression on HSCs of AGM region, the E10.5, E11.5, and E12.5 AGM region cells were sorted by magnetic beads into CD43+ and CD43- subpopulations. To determine whether CD43 expression identifies cells with in vivo repopulating capacity, we transplanted 1-2 embryo equivalents (ee) from each fraction into 9.0 Gy irradiated adult recipient mice. 4 mouths after transplantation, the peripheral blood of recipient mice were collected and tested. The chimerism was calculated as follows:(GFP+CD45+ cells)/ CD45+ cells. Mice that had≥10% chimerism were considered to be reconstituted. The results showed that the repopulating ratios of recipients transplanted with CD43+ subpopulations from E10.5, E11.5 and E12.5 AGM region were 2/15, 6/12 and 4/4 respectively. On the contrary, all of recipients transplanted with CD43- subpopulations were not repopulated (n=26). The multilineage contribution of donor cells was validated by significant engraftment of myeloid and/or lymphoid cells in peripheral blood, bone marrow, spleen and thymus of recipients, indicating that CD43+ subpopulations from AGM region have multilineage potential in vivo. Moreover, we demonstrated CD43+ subpopulations had hematopoietic self-renew ability by secondary transplantation. Previous studies have demonstrated that the HSCs of AGM region are concerntrated in the c-Kit+CD34+ population. By FACS analysis, we found 16.2-22.3% of c-Kit+CD34+ cells from E11 AGM region expressed CD43, implying the enrichment potential of HSC by CD43 expression. These data clearly showed that the HSCs within AGM region exclusively expressed CD43.Previous studies have found that after tissue culture of E11.5 AGM region in vitro, the immuneophenotype of HSCs changed, particularly from CD34+ into CD34-, resembling typical bone marrow HSC. In order to clarify whether CD43 has similar characteristics, the AGM regions (E10.5-E11.5) cells after 2 days of tissue culture were segregated into CD43+ and CD43- subpopulations, and transplanted into irradiated adult recipient mice. The results showed that the repopulated ratios of recipients transplanted with CD43+ subpopulations from E10.5 and E11.5 AGM region were 4/4 and 6/6, respectively. On the contrary, all of recipients transplanted with CD43- subpopulations were not repopulated (n=7). The multilineage contribution of donor cells was validated by significant engraftment of myeloid and/or lymphoid cells in peripheral blood, bone marrow, spleen and thymus of recipients. The data showed that the tissue culture can promote CD43+ HSC expansion in the AGM region; and different from the CD34, expression of CD43 was retained during the process of HSC maturation and proliferation in vitro.Recently, several studies have indicated that the placenta js also an important hematopoietic site. To further determine the CD43 expression on HSCs of placenta and yolk sac, we transplanted CD43+ and CD43- subpopulations fromE11.5 Placenta and yolk sac into irradiated adult recipient mice. The results showed that the repopulated ratios of recipients transplanted with CD43+ subpopulations from E11.5 placenta and yolk sac were 7/7 and 5/8, respectively. On the contrary, all of recipients transplanted with CD43-subpopulations were not repopulated (n=13). Moreover, CD43+ subpopulations from E11.5 placenta and yolk sac have multilineage potential in vivo. Previous studies have demonstrated that HSCs of placenta and yolk sac were included in the c-Kit+CD34+ population. By FACS analysis, we found 21.2%-33.7% and 14.9%-24% of c-Kit+CD34+ cells respectively from E11.5 placenta and yolk sac expressed CD43. These data clearly showed that HSCs of E11.5 placenta and yolk sac exclusively expressed CD43.During fetal hematopoiesis, the majority of the HSCs colonizing the liver are derived from the AGM region, placenta and yolk sac via the blood circulation. The fetal liver is the main site of HSC expansion. To delineate the CD43 expression on HSCs of fetal liver, we transplanted CD43+ and CD43- subpopulations from E12.5 fetal liver into irradiated adult recipient mice. The results showed that only the recipients transplanted with CD43+ subpopulations from E12.5 fetal liver were repopulated (5/5). To further define the CD43 expression on HSCs of fetal liver, we used the CD43 and CD45 antibody to label the E13.5 and E16.5 fetal liver cells, which were sorted into three populations: CD45+CD43high,CD45+CD43mid and CD45+CD43-. After transplantation, the repopulated ratios of recipients transplanted CD45+CD43high subpopulations from E13.5 and E16.5 placenta were 3/3 and 6/7, respectively. On the contrary, all of recipients transplanted with CD45+CD43mid and CD45+CD43- populations were not repopulated (0/19). Therefore, the HSCs of fetal liver highly expressed CD43 as adult bone marrow. By FACS analysis, >95% Lin-Sca-1+c-Kit+ cells enriched with HSCs of E12.5 fetal liver expressed CD43+.As previously documented, CD43 is considered to be a hematopoietic marker like CD45. As a novel specific marker in embryonic hematopoiesis, CD43 was persistently expressed in various stage of HSC development (including emergence, maturation, and expansion)and at multiple hematopoietic sites (AGM region, placenta, yolk sac, fetal liver). In the future, CD43 will be used to study the hematopoietic activity in mouse embryos as a sensitive marker, and its physiological function has to be further studied. |
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