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I. Cortegano et al.
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Figure 7. Proposed pathways for megakaryopoiesis in the mouse E11.5 embryo liver and in adult bone marrow. Schematic differentiation pathways proposed from data obtained in the experiments performed with cell populations identified ex vivo in the E11.5 fetal liver (FL) and adult bone marrow (BM), and with the short-term liquid culture (STLC) differentiation analysis of purified subpopulations after staining with CD45, CD41 and CD42c antibodies. (A) Purified CD45- embryonic c- Kit++CD41+CD45-CD42c- immature embryo-type CD45- megakaryocytes (iEMK) differentiate into c-Kit+CD41++CD45-CD42c+ embryo-type megakaryocytes (EMK) (STLC data in Figure 5B,D), and at 24 h become elongated mobile cells (EMC) and differentiate into proplatelet-bearing megakaryocytes (P-MK) after 48 h (bottom left). (B) Purified adult-type c-Kit++CD41+CD45+CD42c- embryonic immature adult-type CD45+ megakaryocytes (iAMK) [highlight for the authors] (that are CD11b-CD115-, see Figure 6C) differentiate in STLC (data in Figure 5B and 5D) towards CD45- embryonic-type megakaryocytes (EMK) (middle left plot) and to CD45+ adult-type megakaryocytes (AMK) (middle-right plot). (C) Cultures from CD41+/-CD45++CD42c-CD11b+CD115+ embryonic progenitors (data in Figure 6E) give rise to CD11b+ myelo/monocytic cells and to a few CD115+ iAMK (right plot). The bottom-right data correspond to the results from ex vivo staining of cell suspensions from adult BM (Figure 6B). The c-Kit data were obtained from staining with anti-c-Kit, anti-CD45 and anti-CD41 as shown in Online Supplementary Figures S5 and S6. Black solid connecting lines indicate results from STLC; red dotted lines are differentiation in adult BM.
changes nor did they develop proplatelets, and most CD45++ cells were myelo/monocyte-committed CD11b+ cells ex vivo (Figure 6A and Online Supplementary Figures S3 and S5D). We reasoned that CD45++ cells could be a het- erogeneous population, containing cells able to differenti- ate into megakaryocytes. Indeed, low levels of CD41 were expressed in CD45++CD11b+ cells (Figure 6A). We postu- lated that these CD41loCD45++CD11b+ cells may be able to produce CD41++CD45+CD42c+ megakaryocyte-lineage cells. In fact, more CD41++ cells were obtained in STLC from purified CD41loCD45++CD11b+ cells than in those from CD41-CD45++CD11b+ cells (Figure 6A), and there was a bias towards PF4 expression in cells from CD41loCD45++CD11b+ cultures (Online Supplementary Figure S5D).
To confirm these results we used samples from MaFIA transgenic mice, which allow tracking of cells expressing the macrophage-specific promoter for Csf1r/CD115.29 We analyzed BM preparations from adult mice, in which megakaryocytes were identified as Ter119- CD45+CD9++CD41++CD42c+ (Figure 6B). BM megakary- ocytes from C57BL/6 mice expressed CD115 (26% ± 5.4
%; n=4). Accordingly, around 30% ± 3.5 % (n=4) of BM megakaryocytes from MaFIA mice were CD45+EGFP++ cells expressing NF-E2 and VWF transcripts, although at higher and lower levels, respectively, than those from EGFP- megakaryocytes. When E11.5 FL preparations were analyzed, one fifth of CD41-CD45++ cells actually expressed CD115 brightly at E11.5, and the CD45++CD115+ were EGFP+ (Figure 6C). However, among these EGFP+ cells only around 1% were CD41++CD45- EMK ex vivo (Figure 6D), and when plated in STLC these EGFP+ cells mainly produced CD41- CD45++EGFP+ cells and low numbers of CD41+CD45+ EGFP+ cells (Figure 6E). Surprisingly, after STLC some EGFP- cells became CD41+CD45+/-EGFP+ and CD41++CD45-EGFP+ cells. This observation indicates that the FL EGFP- population contains cells with the potential to become CD115+ in vitro and to generate CD41++ megakaryocytes, which may be reflecting what happens in vivo in the adult BM. Overall, our findings support the notion of a low potential of embryo CD45++ cells to pro- duce CD41+ megakaryocytes in vivo, at difference from the adult BM situation.
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haematologica | 2019; 104(9)