Interleukin-3 promotes hemangioblast development in mouse aorta-gonad-mesonephros region

Abstract

Background: The hemangioblast is a bi-potential precursor cell with the capacity to differentiate into hematopoietic and vascular cells. In mouse E7.0-7.5 embryos, the hemangioblast can be identified by a clonal blast colony-forming cell (BL-CFC) assay or single cell OP9 co-culture. However, the ontogeny of the hemangioblast in mid-gestation embryos is poorly defined.

Design and methods: The BL-CFC assay and the OP9 system were combined to illustrate the hemangioblast with lymphomyeloid and vascular potential in the mouse aorta-gonad-mesonephros region. The colony-forming assay, reverse transcriptase polymerase chain reaction analysis, immunostaining and flow cytometry were used to identify the hematopoietic potential, and Matrigel- or OP9-based methods were employed to evaluate endothelial progenitor activity.

Results: Functionally, the aorta-gonad-mesonephros-derived BL-CFC produced erythroid/myeloid progenitors, CD19(+) B lymphocytes, and CD3(+)TCRbeta(+) T lymphocytes. Meanwhile, the BL-CFC-derived adherent cells generated CD31(+) tube-like structures on OP9 stromal cells, validating the endothelial progenitor potential. The aorta-gonad-mesonephros-derived hemangioblast was greatly enriched in CD31(+), endomucin(+) and CD105(+) subpopulations, which collectively pinpoints the endothelial layer as the main location. Interestingly, the BL-CFC was not detected in yolk sac, placenta, fetal liver or embryonic circulation. Screening of candidate cytokines revealed that interleukin-3 was remarkable in expanding the BL-CFC in a dose-dependent manner through the JAK2/STAT5 and MAPK/ERK pathways. Neutralizing interleukin-3 in the aorta-gonad-mesonephros region resulted in reduced numbers of BL-CFC, indicating the physiological requirement for this cytokine. Both hematopoietic and endothelial differentiation potential were significantly increased in interleukin-3-treated BL-CFC, suggesting a persistent positive influence. Intriguingly, interleukin-3 markedly amplified primitive erythroid and macrophage precursors in E7.5 embryos. Quantitative polymerase chain reaction analysis demonstrated declined Flk-1 and elevated Scl and von Willebrand factor transcription upon interleukin-3 stimulation, indicating accelerated hemangiopoiesis.

Conclusions: The hemangioblast with lymphomyeloid potential is one of the precursors of definitive hematopoiesis in the mouse aorta-gonad-mesonephros region. Interleukin-3 has a regulatory role with regards to both the number and capacity of the dual-potential hemangioblast.

Figure 1.

Molecular characteristics, hematopoietic and vascular potential of BL-CFC derived from E10.5 mouse AGM region. (A) Morphology of a typical blast colony. (B) Expression of Brachyury, Flk-1 and Runx1 in seven representative blast colonies determined by nested RT-PCR. (C) Immunofluorescence staining of Sca-1, F4/80 and Ter119 in blast colonies. Morphology of individually plucked blast colonies in the liquid expansion system after 48 h (D) and 10 days (E) of incubation. (F–G) DiI-Ac-LDL incorporation (red) combined with CD45 or F4/80 staining (green) of the adherent cells. Arrowheads, CD45 positive in H or F4/80 positive in I. (H) α-SMA+ staining (green) of the adherent cells. (I) Tube-like structures in Matrigel of the adherent cells. (J–K) DiI-Ac-LDL incorporation (red) combined with α-SMA or Calponin staining (green) of the tube-like structures. Inserts show the corresponding bright fields. Original magnification: ×40 (I), ×100 (D, E), and ×200 (A, C, F–H, J, K).

Figure 2.

Identification of hematopoietic and endothelial potential of BL-CFC by the OP9-based system. Individual blast colonies are co-cultured with OP9 for 10 days in a 48-well plate. In one representative well, both CD45⁺ cells (A) and CD31⁺ tube-like structures (B) are detected. (C) Flow cytometry analysis of B lymphocyte markers of blast colonies differentiated on OP9 stroma for 10 days. (D) Flow cytometry analysis of T lymphocyte markers and NK1.1 of blast colonies differentiated on OP9-DL1 stroma for 15 days. Representative data from three independent experiments are shown. Original magnification: ×40 (B), and ×100 (A).

Figure 3.

Development kinetics and surface markers of BL-CFC. (A) BL-CFC and hematopoietic CFC derived from caudal half (E9.5) or AGM region (E10.5–12.5) are analyzed in parallel, showing different development kinetics. (B–D) Immunohistochemical analysis of E10 embryos. Transverse sections of dorsal aortas (DA) are stained with anti-CD31 (B), anti-endomucin (C) and anti-PDGFRβ (D) antibodies. The dorsal aspect is upward. The endothelial layer is positive for CD31 and endomucin, but negative for PDGFRβ (red arrowheads in D). CD31 and endomucin are also expressed in hematopoietic cell clusters (arrowheads in B and C) associated with endothelium. (E–H) The frequency of BL-CFC in CD105 (E), CD31 (F), endomucin (G), and PDGFRβ (H) positive cells sorted from E10.5 AGM region. The results are representative data expressed as means ± s.e.m from three of four independent experiments.

Figure 4.

Influence of BL-CFC development in the E10.5 AGM region by IL-3-mediated signaling. (A) Among a panel of cytokines, IL-3 and bFGF significantly amplified the BL-CFC in the E10.5 AGM region. The IL-3-mediated effect was dose-dependent (B) and temporally regulated (C). Neutralizing tissue-derived IL-3 markedly reduced the number of BL-CFC in the AGM region. Neu denotes IL-3 neutralizing antibody (D). Phosphorylation of STAT5 and ERK1/2 in AGM cells was enhanced by IL-3 (E), and the corresponding pharmacological inhibitors abrogated IL-3-mediated BL-CFC expansion (F). The results represent the means ± s.e.m. Significance was determined using the Student’s t test: *P<0.05; **P<0.01, compared with the control data.

Figure 5.

Increased hematopoietic and vascular potential of IL-3-treated BL-CFC from the E10.5 AGM region. (A) IL-3- and bFGF-stimulated BL-CFC gave rise to increased numbers of hematopoietic CFC. (B–D) A Matrigel-based test revealed that IL-3- and bFGF-stimulated BL-CFC have enhanced tube-forming capacity both with regards to the number and length of the tubes. (E) CD31 staining of blast colonies co-cultured with OP9 for 10 days. Increased tube formation was detected when IL-3 was added during blast colony formation. (F) Real-time PCR analysis of BL-CFC cultures to examine IL-3-mediated molecular changes. Original magnification: ×40 (B, E). The results represent means ± s.e.m. Significance was determined using the Student’s t- test: **, P<0.01, compared with the control data.