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Objective: To compare the differentiation ability difference of hematopoietic, mesenchymal and endothelial potential between CD41⁺ cells derived from the mouse aorta-gonadmesonephros (AGM) region, yolk sac (YS) and embryonic circulating blood (CB)., Methods: CD41⁺ cells were sorted from AGM, YS and CB. The CD45 and c-kit expression were studied in CD41⁺ cells by flow cytometry. IL-3 and bone morphogenetic protein 4 (BMP-4) treatment together with semi solid culture were used to assess hematopoietic potential difference of CD41⁺ cells. Immunofluorescence staining of α-SMA was used to assess mesenchymal potential difference. The endothelial cell induction system was used to assess endothelial potential difference., Results: The proportions of CD45+ cells in CD41⁺ population were 51.9% (AGM), 45.8% (YS) and 22.2% (CB), respectively, while those of c-kit⁺ cells were 40.0% (AGM), 39.6% (YS) and 36.2% (CB), respectively. After stimulated by IL-3 factor, the number of total colonies increased in all three groups-derived CD41⁺ cells compared to that of unstimulated group[(14.1±1.9) vs (1.2±0.2), (32.4±1.1) vs (18.4±2.2) and (41.8±0.9) vs (10.4±1.8)], (P<0.01). After stimulated by BMP-4 factor, compared to unstimulated group, CFU-Mix colony number in CD41⁺ cells from AGM region and YS were significantly decreased[(0.5±0.6) vs (3.2±0.8), (1.3±0.7) vs (7.4±1.7)](P<0.01), but there was no difference in CB group[(2.5±0.5) vs (3.9±1.5)](P>0.01). The mesenchymal marker α-SMA was highly expressed in CD41⁺ cells from AGM region and YS, but lowly expressed in CD41⁺ cells from CB., Conclusion: There are some differences between CD41⁺ cells in AGM region, YS and CB on hematopoietic cell surface marker expression, hematopoietic colony formation with IL-3 and BMP-4 stimulation.

Mesenchymal stem cells (MSCs) represent powerful tools for regenerative medicine for their differentiation and migration capacity. However, ontogeny and migration of MSCs in mammalian mid-gestation conceptus is poorly understood. We identified canonical MSCs in the mouse embryonic day (E) 11.5 dorsal aorta (DA). They possessed homogenous immunophenotype (CD45(-)CD31(-)Flk-1(-)CD44(+)CD29(+)), expressed perivascular markers (α-SMA(+)NG2(+)PDGFRβ(+)PDGFRα(+)), and had tri-lineage differentiation potential (osteoblasts, adipocytes, and chondrocytes). Of interest, MSCs were also detected in E12.5-E13.5 embryonic circulation, 24 hr later than in DA, suggesting migration like hematopoietic stem cells. Functionally, E12.5 embryonic blood could trigger efficient migration of DA-MSCs through platelet-derived growth factor (PDGF) receptor-, transforming growth factor-beta receptor-, but not basic fibroblast growth factor receptor-mediated signaling. Moreover, downstream JNK and AKT signaling pathway played important roles in embryonic blood- or PDGF-mediated migration of DA-derived MSCs. Taken together, these results revealed that clonal MSCs developed in the mouse DA. More importantly, the embryonic circulation, in addition to its conventional transporting roles, could modulate migration of MSC during early embryogenesis., (© 2010 Wiley-Liss, Inc.)

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.

Aorta-gonad-mesonephros (AGM) is well known as a main structure that de novo generates hematopoietic primary stem cells (HSC) in mid-gestation mammalian embryos. Hemogenic endothelium, and recently, subendothelial mesenchyme as well as hemangioblast are shown as contributing to blood formation in AGM region. AGM-HSC displays dynamic changes in surface markers, including CD41, CD45 and several endothelial-specific molecules. The novel finding of interleukin-3 as a potent regulator of AGM-HSC seems very interesting. Moreover, zebra fish model reveals PGE2 as a novel stimulator of HSC in AGM and kidney marrow, which is also the case in mouse hematopoietic tissues. Identification of mesenchymal stem cells with significant hematopoietic supporting capacity in AGM region suggests an alternative pathway to explore new molecules governing embryonic and adult hematopoiesis. In this paper, the hemogenic model in AGM region, surface markers on HSCs in AGM region and regulation of HSCs in AGM region were reviewed.

To investigate the effects of microenvironment of aorta-gonad-mesonephros (AGM) on embryonic hematopoiesis, mesenchymal stem cell like stromal cells (MSC like stromal cells) derived from dorsal aorta (DA) in AGM region were separated and identified by their growth characteristics, related molecules expression and mesenchymal lineage potentials. Stromal cells from DA region in mouse embryos (E11.5) were isolated and cultured in vitro. After transfected by pSV3neo-SV40, the clones with G418 resistance were selected, and their growth characteristics were studied. The related molecules were analyzed by flow cytometry, and each clone was induced to differentiate into adipocytes, osteocytes, and chondrocytes. The results showed that most clones (20 clones) selected in the mouse DA region held the morphology of fibroblastoid cells. mDAF3 and mDAF18 could be grown in culture for more than 50 passages with G418 resistance, both have the potential to differentiate into adipocytes, osteocytes, and chondrocytes. At the logarithmic growth period, the cell population doubling time is about 24 hours. Surface markers, such as CD29, CD44, CD105 and Sca-1 were positively detected, while low levels of CD34, CD45, and CD31 were detected. It is concluded that immortalized mDAF3 and mDAF18 have the specific phenotype and differential potency of MSC, which suggests that MSC maybe exist in mouse embryonic DA region, where the MSC like stromal cells can be used as a cell model for further research on the modulation activity of DA microenvironment for embryonic hematopoiesis.

Mesenchymal stem cells (MSCs) are multipotent stem cells that can generate various microenvironment components in bone marrow, ensuring a precise control over self-renewal and multilineage differentiation of hematopoietic stem cells. Nevertheless, their spatiotemporal correlation with embryonic hematopoiesis remains rudimentary, particularly in relation to the human being. Here, we reported that human aorta-gonad-mesonephros (AGM) resided with bona fide MSCs. They were highly proliferative as fibroblastoid population bearing uniform surface markers (CD45(-), CD34(-), CD105(+), CD73(+), CD29(+), and CD44(+)), expressed pluripotential molecules Oct-4 and Nanog, and clonally demonstrated trilineage differentiation capacity (osteocytes, chondrocytes, and adipocytes). The frequency and absolute number of MSCs in aorta plus surrounding mesenchyme (E26-E27) were 0.3% and 164, respectively. Moreover, they were functionally equivalent to MSCs from adult bone marrow, that is, supporting long-term hematopoiesis and suppressing T-lymphocyte proliferation in vitro. In comparison, the matching yolk sac contained bipotent mesenchymal precursors that propagated more slowly and failed to generate chondrocytes in vitro. Together with previous knowledge, we propose that a proportion of MSCs initially develop in human AGM prior to their emergence in embryonic circulation and fetal liver.