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  • Thus based on our prior results


    Thus, based on our prior results above, we hypothesized that B cells derived from ESCs are also B-1 cells and HSC independent. To test this hypothesis, we induced mouse ESCs on OP9 stromal cells into B-progenitors and transplanted them into sublethally irradiated NOD/SCID/Il2rγcnull (NSG) neonates. ESC-derived B cells were detected as peritoneal B-1 cells and splenic MZ B cells in the recipient mice, similar to YS-derived B cells in our previous reports. These B-1 and MZ B cells were maintained in NSG mice for more than 6 months and secreted natural immunoglobulin M (IgM) TNF-alpha, recombinant murine protein in vivo. RNA sequencing (RNA-seq) analysis displayed similarity between ESC-derived and YS-derived B-progenitors.
    Discussion We demonstrated that all B-progenitors produced from mouse ESCs differentiate into only B-1 and MZ B cells in recipient mice, similar to YS-derived B-progenitors (Figure 1H). Although ESC-derived B-1 cells are functional in natural antibody secretion and self-replenishing ability, they differentiate to mostly B-1b cells, not to B-1a cells, while almost half of YS-derived B-progenitors differentiate into B-1a cells in vivo (Figure 1E) (Yoshimoto et al., 2011). This may reflect the difference of gene expression between ESC- and YS-derived B-progenitors. Although ESC-derived B-progenitors are molecularly more similar to YS-derived B-progenitors than BM B-2 progenitors, there were still 1,759 genes differentially expressed between them. Potocnik et al. (1997) have reported B cell reconstitution by in vitro differentiated mouse ESCs, including both B-1 and transient B-2 cells. While B-1a cells were dominantly repopulated in their report, our ESC-derived cells repopulated only B-1b cells and not B-2 cells. This difference may be due to dissimilarity in culture conditions such as 3D embryoid body formation versus 2D OP9 co-culture. In both cases, however, HSCs that could sustain B-2 cell production were not detected. We have previously shown that mouse testis-derived PSCs differentiate into erythromyeloid progenitors, and B and T cells in vitro culture, but found very limited PSC-derived HPC engraftment in the recipient BM and spleen, detectable by PCR (Yoshimoto et al., 2009). Pearson et al. (2015) have reported better engraftment of ESC-derived hematopoietic cells after serum-free differentiation culture. However, despite T cell differentiation potential in vitro, T cell engraftment was not shown and B cell engraftment was limited to the spleen. They indicated that the engrafted B cells in the recipient spleen were not B-1 cells because they lacked expression of CD5 and CD11b. However, CD11b is only expressed in peritoneal B-1 cells, not in splenic B-1 cells. Furthermore, CD5 expression is limited to B-1a cells. Since they did not show any B cell engraftment in the peripheral blood (which is considered to represent the B-2 cell population), it is very likely that the ESC-derived B cells engrafted in the recipient spleen were MZ or B-1 cells, consistent with our results. Lu et al. (2016) have reported repopulation of functional adaptive immune T and B cells by engineered mouse ESCs, which was produced by inducing Notch signaling and Hoxb4 overexpression. In their control, conventional ESC differentiation on OP9 (without Notch signaling) produced only B-1b cells, not B-2 cells, supporting our results. Notch signaling seems to have induced functional adaptive B-2 cells via HSCs in their system, although their secondary transplantation of engineered HSCs displayed T cell dominant engraftment. This result suggests that sustained B-2 potential always accompanies HSC potential, different from B-1 cells that can be produced by early HSC-independent progenitors. Although lymphoid potential has been used as an indicator of “definitive hematopoiesis” in the early mouse embryo or ESC differentiation system (Cumano et al., 1996, Kennedy et al., 2012), it is important to reconsider the definition of definitive hematopoiesis, because some T and B-1 lymphoid progenitors can be produced in the absence of HSCs (Chen et al., 2011, Kobayashi et al., 2014, Tian et al., 2017). In this sense, sustained B-2 cell production may be a more accurate indicator of HSC activity. Therefore, understanding how B-2 potential is obtained in ESC-derived HPCs would represent an important area of future investigation.