| Hematopoietic stem cells (HSC) lie at the foundation of the adult hematopoietic system and provide an individual throughout its life with all blood cell types. There are a lot of key issues concerning the developmental origin of HSCs, including the sites, the timings and the regulatory mechanisms. The AGM region has been well known to be the first source of HSC. However, after the AGM and fetal liver were removed, the remnants of the E11embryo proper demonstrated robust HSC potential in vivo. The clues prompted us to comprehensively analyze the hematopoietic activity in the rostral half of embryo proper during mouse midgestation.In our study, various research means were used to clarify the HSC potential and fate of mouse embryonic head cells. By long-term transplantation, we found that, with1.7-2.5ee injection,30%(14/40) of recipients were repopulated, with an average long-term chimerism of46.6±22.4%(>16weeks). In parallel, transplantation with E10.5head cells led to successful reconstitution in5out of40recipients, with an average long-term chimerism of57.2±22.9%. However, transplantation with1.7-2.5ee cells from E10.5embryonic circulation did not repopulate the recipients. At E11.5, transplantation of head cells resulted in40%of recipients (8/20) being efficiently repopulated, with an average long-term chimerism of54.6±23.8%. The efficiency was approaching to that of the AGM region (60%,9/15). Therefore, paralleling the AGM region, the E10.5-E11.5mouse head harbors bona fide HSC,as verified by multi-lineage repopulation and self-renewal capacity. Like the AGM region, only the CD34+c-Kit+population in the E12.5head could efficiently repopulate irradiated adult recipients.The frequencies and numbers of CD34+c-Kit+, CD41+c-Kit+, and CD45+c-Kit+cells were comparable between the head and AGM region of E10.5embryos. Next, real-time PCR was used to quantify the mRNA levels of hematopoietic/endothelial-related molecules in the head and AGM region. At E10.5, the transcription levels of Scl, P2-Runxl, and GATA-2in the head were1.5-,1.6-, and2.3-fold respectively relative to that in the AGM region of littermates. The expression levels of endothelial markers were comparable. Furthermore, at E10.5or E11.5, the expression levels of three transcriptional factors (Scl, P2-Runxl, and GATA-2) were similar between head and AGM in specific subpopulations (Tie2+or CD31+c-Kit+) that enriched hematopoietic potential in vitro and in vivo.And then, the hemogenic potential in the head cells was investigated through the staining in vivo, flow cytometry analysis, blood-forming capacity of putative endothelial cells in vitro and lineage tracing. Firstly, We have found that the close association of the CD31+cells (clustered or scattered) with the arterial endothelium was observed in the El0.5head region by immunostaining. The El0.5head had a unique CD31+c-Kithi8h population (0.063-0.113%), resembling the AGM region (0.088-0.157%) by the flow cytometry. Secondly, putative endothelial cells defined as CD3rCD41-CD45Ter119-in the E10.0(31-36sp) head were sorted by FACS and plated on the OP9stromal cells. After7-9days, a considerable number of CD45+hematopoietic cells and CD19+B lymphocytes were detected, similar to those from AGM region. Lineage tracing via an inducible VE-cadherin-Cre transgene was used, and we found that several hematopoietic clusters derived from the head became tdTOMATO+and were positive for CD45, like those from the AGM region. Flow cytometry further confirmed the presence of CD45+hematopoietic cells in tdTOMATO+population of both the head and AGM cultures and the lack of tdTOMATO+population in the circulating blood cultures.SP-A-Cre transgenic mouse line, in which Cre activity in endothelial cells is restricted to the brain vasculature from El1.5, is optimal for tracing the hematopoietic fate of cerebrovascular endothelial cells. First, LacZ staining was performed in E12.5SP-A-Cre;ROSA-LacZ double transgenic embryos. The head sections showed that in some of the morphologically defined arterioles and arteries, blue-stained LacZ+blood cells were detected and some appeared to be "budding" from the endothelial layer. In comparison, no LacZ+cells were detected in the aortic endothelium or in the fetal liver.Next, the hematopoietic and endothelial populations were quantified by flow cytometry and hemogenic activities were analyzed by OP9co-culture system in vitro.10.5%of the CD3+population and4.6%of the CD45+population contained the recombined allele in the head of El3.5SP-A-Cre;ROSA-LacZ double transgenic mice. In SP-A-Cre;ROSA-LacZ double transgenic mice, positive signals for FDG were found in20%of the CD31+population,8.4%of the CD45+population,12.3%of the CD45+c-Kit+population, and27.6%of B220+CD19+B lymphocyte population. These data collectively verified an in situ hematopoietic activity and hemogenic potential in vitro of the cerebrovasculature endothelial cells.Finally, the physiological contribution of SP-A-expressing cells to the postnatal hematopoietic system was investigated through the SP-A-Cre;ROSA-LacZ, SP-A-Cre;ROSA-EYFP mice. We found that in the BM of2-week-old SP-A-Cre;ROSA-LacZ double transgenic mice, approximately1.1-1.3%of the HSC-enriched population (LinSca-l+c-Kit+) was LacZ+. In the BM of4-week-old mice, approximately4.2-4.5%of the HSC-enriched population (CD150+CD48LinSca-l+c-Kit+, CD150+CD48-LSK) was LacZ+. Likewise, in the4-6-week old SP-A-Cre;ROSA-EYFP double transgenic mice, EYFP+cells constituted5.9-7.7%of the BM CD150+CD48-LSK population. Higher levels of contribution were detected in the multi-lineages in BM or peripheral blood. These data validated the continuous physiological contribution of cerebrovascular-specific endothelial cells to postnatal hematopoietic system.Conclusively, by probing the HSC activity, the endothelial-derived hemogenic potential, and the physiological hematopoietic fate, we report here for the first time that the mouse embryonic head is a new site for HSC development. Expectedly, clarification of the key regulatory signals controlling the endothelial-hematopoietic transition will provide new strategies for regeneration medicine of the hematopoietic system. |
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