Stem cell emergence and hemopoietic activity are incompatible in mouse intraembryonic sites

Abstract

In the mouse embryo, the generation of candidate progenitors for long-lasting hemopoiesis has been reported in the paraaortic splanchnopleura (P-Sp)/ aorta-gonad-mesonephros (AGM) region. Here, we address the following question: can the P-Sp/AGM environment support hemopoietic differentiation as well as generate stem cells, and, conversely, are other sites where hemopoietic differentiation occurs capable of generating stem cells? Although P-Sp/AGM generates de novo hemopoietic stem cells between 9.5 and 12.5 days post coitus (dpc), we show here that it does not support hemopoietic differentiation. Among mesoderm-derived sites, spleen and omentum were shown to be colonized by exogenous cells in the same fashion as the fetal liver. Cells colonizing the spleen were multipotent and pursued their evolution to committed progenitors in this organ. In contrast, the omentum, which was colonized by lymphoid-committed progenitors that did not expand, cannot be considered as a hemopoietic organ. From these data, stem cell generation appears incompatible with hemopoietic activity. At the peak of hemopoietic progenitor production in the P-Sp/AGM, between 10.5 and 11.5 dpc, multipotent cells were found at the exceptional frequency of 1 out of 12 total cells and 1 out of 4 AA4.1+ cells. Thus, progenitors within this region constitute a pool of undifferentiated hemopoietic cells readily accessible for characterization.

Figure 1

Evolution of absolute numbers of B cell progenitors detected per AGM over the midgestation period. Progenitors endowed with B lymphoid potential were quantified through limiting dilution analysis.

Figure 2

Structure and dissection of the AGM. (A) Scanning electron microscopy of a partially dissected AGM (bar, 0.22 mm). (B) Scanning electron microscopy of an explanted AGM. The anterior part of the explant (left) is considerably larger than the caudal part (bar, 1 mm). (C) Semithin section of an AGM explant. Note the hemopoietic cell cluster (reference 27) associated with the endothelium of the ventral part of the aorta (arrow; bar, 0.26 mm). Ao, aorta; GR, genital ridges; Mn, mesonephros; Mt, mesentery; SV, subcardinal vein; WD, Wolffian duct.

Figure 2

Structure and dissection of the AGM. (A) Scanning electron microscopy of a partially dissected AGM (bar, 0.22 mm). (B) Scanning electron microscopy of an explanted AGM. The anterior part of the explant (left) is considerably larger than the caudal part (bar, 1 mm). (C) Semithin section of an AGM explant. Note the hemopoietic cell cluster (reference 27) associated with the endothelium of the ventral part of the aorta (arrow; bar, 0.26 mm). Ao, aorta; GR, genital ridges; Mn, mesonephros; Mt, mesentery; SV, subcardinal vein; WD, Wolffian duct.

Figure 2

Structure and dissection of the AGM. (A) Scanning electron microscopy of a partially dissected AGM (bar, 0.22 mm). (B) Scanning electron microscopy of an explanted AGM. The anterior part of the explant (left) is considerably larger than the caudal part (bar, 1 mm). (C) Semithin section of an AGM explant. Note the hemopoietic cell cluster (reference 27) associated with the endothelium of the ventral part of the aorta (arrow; bar, 0.26 mm). Ao, aorta; GR, genital ridges; Mn, mesonephros; Mt, mesentery; SV, subcardinal vein; WD, Wolffian duct.

Figure 3

Flow cytometry profile showing the lymphoid progeny of a single micromanipulated AGM cell. (Top) After culture in T lymphoid conditions. Cells of donor origin were gated as Ly5.1⁻. (Bottom) After culture in B lymphoid conditions. Lymphocytes were gated on forward and side scatter.

Figure 3

Flow cytometry profile showing the lymphoid progeny of a single micromanipulated AGM cell. (Top) After culture in T lymphoid conditions. Cells of donor origin were gated as Ly5.1⁻. (Bottom) After culture in B lymphoid conditions. Lymphocytes were gated on forward and side scatter.

Figure 4

Anatomical relationship between omentum and spleen. The omentum and spleen explanted from a 14.5-dpc embryo are shown after being removed from the underlying stomach. The pancreas is then dissected out, and the two organs are separated (bar, 0.5 mm). Omentum, arrowheads; Sp, spleen; P, pancreas.

Figure 6

Flow cytometry profile showing CD19 (FL1) and Ly5 (FL3) staining of explanted omental-splenic rudiments (11 and 12 dpc), omenta, or spleens (12.5 and 13.5 dpc), after 12 d of organotypic culture. Lymphocytes were gated on forward and side scatter. The ratio of explants containing CD19 cells appears above the corresponding profile.

Figure 5

Quantification of erythromyeloid precursors CFU-E, CFC-Mix, and B lymphoid precursors in omentum and spleen over the midgestation period. Ovals, explant comprising both the omental and splenic rudiments (before 13 dpc); circles, splenic explant; squares, omental explant.