Hematopoietic Potential In The Mouse Embryonic Circulation

There are generally two stages of early mouse hematopoiesis in mammalian embryos. The first stage, primitive hematopoiesis, is characterized by de novo generation of nucleated erythrocytes in the yolk sac at embryonic day 7-7.5. The second stage, definitive hematopoiesis, is marked by generation of adult type hematopoietic stem cells (HSC) in the mid-gestation embryos. HSCs can self-renew and efficiently reconstitute the entire blood system of a recipient.To date, most research showed that primitive hematopoiesis was derived from extra-embryonic yolk sac, while definitive hematopoiesis was generated in the aorta-gonads-mesonephros (AGM) region. Orkin et al demonstrated that HSC occurred in the mouse placenta at E10.5-E11.0, and their number significantly expanded at E12.5. Yoder et al found the yolk sac and P-SP at E9.0 were capable of reconstituting the hematopoietic system of newborn mouse treated by busulfan, and importantly, could transfer to the secondary recipients, demonstrating their self-renew capacity. In 1994, Dzierzak et al tried to probe HSC in the mouse circulation at E11, but failed. In contrast, Medvinsky et al captured HSC in the circulation at E11.5. In 2005, Orkin et al obtained the same result, but the chimerism is less than 1%. In 2010, Dzierzak et al transplanted five embryonic equivalent of circulation cells at E11.5 to the recipient, but yet detected no HSC capacity. There are much difference in CFU-C among the mouse embryos of different genetic background, even with the same somite pairs and under the same culture conditions. Therefore, repopulating ability of HSC was comprehensively determined by several factors, such as mouse body weight, amount and rate of radiation, and status of donor cells.Hence, it remains unknown about whether and how CFU-C and particularly HSC develop in the mouse embryonic circulation. In this study, we investigated the hematopoietic surface markers, CFU-Cs, lymphocyte potential, CFU-S8 and HSC in the mouse circulation at E10.5 and E11.5, the key time points for HSC emergence. Firstly, we isolated circulation blood cells to determine their surface markers.We foundd that most cells expressed ter119, but few expressed CD45 by immunofluorescence. the Ter119+ cells were nucleated as visualized by their DAPI staining, indicating their identity as primitive erythrocytes. The percentage of CD45, Ter119, Tie2, CD31 and c-Kit was 0.9%, 98.9%, 0.7%, 1.1% and 0.8% respectively in the mouse circulation by flow cytometric analysis. These data indicated the presence of hematopoietic progenitor cells. Then, we plated the circulating cells in methylcellulose medium with SCF, IL-3, IL-6 and Epo to quantify the CFU-Cs in the circulation at E10.5 and E11.5. we found that the CFU-mix was predominant in all the types of CFU-Cs, and its morphology was unique. Interestingly, there were three features of the CFU-Cs derived from circulation. First, the CFU-mix was the main type in all CFU-Cs, whereas the number of CFU-E and CFU-GM were low. Second, the number of total CFU-C and of each type at E11.5 was 5 times more than those at E10.5. Finally, between the 2 time points, the numbers of CFU-E and CFU-mix were equal, but the number of CFU-GM was different. Moreover, the circulation and the AGM region cells at E10.5 could not generate CFU-S8, unlike previous literatures. However, 1 day later, a 1ee of the circulation and the AGM region cells generated 0.17 and 0.5 CFU-S8 respectively.Subsequently, we plated the E11.5 circulating cells on OP9 stromal cells to test their B lymphocyte differentiation after 8-10 days. It showed that the expression of B220 and CD19 in the harvested nonadherent cells were higher than 90% and 80% respectively. Next, we plated the E10.5 circulating cells on OP9-DL1 stromal cells to examine their T lymphocyte differentiation. The proportion of CD44+/CD25+ and CD44-/CD25+ cells was 28.1% and 5.1% respectively after 6 days of coculture. Later, there was 7.5% CD4+/CD8+ and 3.6% CD3+/TCRαβ+ cells after 14 days。Therefore, the precursor cells with B and/or T lymphoid potential emerged in the mid-gestation blood-stream.Finally, we tried to investigate the HSC activity in the mouse circulation. We transplanted the circulating cells from GFP-transgenic mouse embryos at E10.5 (36-40sp) and E11.5 (41-50sp) to radiated adult recipients. Then, GFP+ donor cells in peripheral blood of recipients were determined after 6-8 weeks and 6 months post transplantation. As a result, the circulating cells derived from E11.5 (n=20) but not E10.5 embryos could reconstitute three recipients efficiently. The donor chimerisms of the 3 repopulated recipients were 31.3%, 34.5% and 43.7% after 6-8 weeks, and were 28.4%, 48.8% and 89.3% after 6 months. Meanwhile, a significant chimerism was also detected in the bone marrow, spleen, and thymus. Surprisingly, sencondary transplantation showed a poor engraftment in the recipients (1.1%,2.8% and 1.8% respectively), in striking contrast to those of the AGM region in parallel. In summary, the cells in the mouse embryonic blood-stream expressed hematopietic surface markers, possessed myeloid and lymphoid progenitors, and contained a few CFU-S8. Most importantly, in the circulation, the migrating HSC appeared immature, with intact multi-lineage but limited self-renew potential.