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Bone morphogenetic protein 4 modulates c-Kit expression and differentiation potential in murine embryonic aorta-gonad-mesonephros haematopoiesis in vitro.

Marshall CJ, Sinclair JC, Thrasher AJ, Kinnon C - Br. J. Haematol. (2007)

Bottom Line: The addition of BMP4 to AGM cells resulted in expansion of the CD34(+)/c-Kit(low) cell pool within 48 h, via a combination of down modulation of the c-Kit receptor in CD34(+)/c-Kit(high) cells and proliferation.CD34(+)/c-Kit(high) progenitors cultured with BMP4 also generated adherent colonies typical of c-Kit(low) cells.These results suggest that BMP4 regulates c-Kit expression and differentiation potential in CD34(+) AGM cells and supports a role for BMP signalling in the maintenance of multipotency during embryonic haematopoiesis, providing an insight into stem cell homeostasis within the mammalian haematopoietic niche.

View Article: PubMed Central - PubMed

Affiliation: Molecular Immunology Unit, UCL Institute of Child Health, London, UK.

ABSTRACT
The transforming growth factor-beta-related factor bone morphogenetic protein 4 (BMP4) is expressed in the human embryonic aorta-gonad-mesonephros (AGM) coincident with the emergence of haematopoietic cells and influences postnatal mammalian haematopoietic stem cells in vitro. To investigate the role of BMP4 in mammalian embryonic haematopoiesis, cells were isolated from murine AGM and two populations of CD34(+) cells with different levels of c-Kit expression and multipotency were identified. CD34(+)/c-Kit(high) cells express CD45 and are haematopoietic-restricted progenitors. In contrast, CD34(+)/c-Kit(low) cells are Flk1+/CD45(neg) and generate adherent colonies in ex vivo culture that resemble haemangioblast colonies identified in other systems. The addition of BMP4 to AGM cells resulted in expansion of the CD34(+)/c-Kit(low) cell pool within 48 h, via a combination of down modulation of the c-Kit receptor in CD34(+)/c-Kit(high) cells and proliferation. In long-term culture, BMP4 increased the growth/survival of CD34(+)/c-Kit(high) haematopoietic progenitors, effects that were blocked by BMP inhibitors. CD34(+)/c-Kit(high) progenitors cultured with BMP4 also generated adherent colonies typical of c-Kit(low) cells. These results suggest that BMP4 regulates c-Kit expression and differentiation potential in CD34(+) AGM cells and supports a role for BMP signalling in the maintenance of multipotency during embryonic haematopoiesis, providing an insight into stem cell homeostasis within the mammalian haematopoietic niche.

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Mid gestation murine CD34+ aorta-gonad-mesonephros (AGM) cells contain subpopulations that differ in levels of c-Kit expression, differentiation potential and response to bone morphogenetic protein 4 (BMP4). (A) AGM cells (10·5 dpc) were analysed by flow cytometry (CyAn ADP Flow Cytometer) for expression of CD34, c-Kit, Flk1 and CD45 to identify subpopulations. Representative results are shown. CD34+/c-Kit+ cells can be subdivided into c-Kithigh and c-Kitlow fractions (areas a & b). Differential expression profiles of (a) CD34+/c-Kithigh and (b) CD34+/c-Kitlow fractions for expression of Flk1 and CD45. Dotted arrows indicate expansion of gated areas a and b as indicated. To maintain accuracy, analysis of the c-Kitlow and c-Kithigh cell fractions could only be performed on small numbers of cells per experiment (pooled littermates) however the distribution of expression was consistent between experiments. (B) CD34+/c-Kithigh cells generate haematopoietic colonies of typical CFU-GM morphology. CD34+/c-Kitlow cells generate exclusively adherent colonies containing multiple cell types: phase-dim spindle-shaped cells (lower arrowhead), large round cells (arrow) and phase-bright small, round cells (upper arrowhead). (C) Unsorted (total) AGM cells were cultured in serum-free conditions −/+ recombinant BMP4 (10 ng/ml) for 2 d and changes in the CD34+/c-Kithigh/low subpopulations analysed by flow cytometry (CyAn ADP cytometer). Without added BMP4 (−BMP4), the percentage of CD34+/c-Kithigh/low cells increases slightly in culture from day 0 but the ratio of CD34+/c-Kithigh to CD34+/c-Kitlow remains relatively constant. With the addition of BMP4, there is a considerable increase in the CD34+/c-Kitlow population compared to day 0 and to cells cultured without added BMP4. Irradiated S17 feeder cells appear to express CD34, accounting for the apparent increase in CD34+/c-Kitneg in cultured cells, but are c-Kitneg.
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fig01: Mid gestation murine CD34+ aorta-gonad-mesonephros (AGM) cells contain subpopulations that differ in levels of c-Kit expression, differentiation potential and response to bone morphogenetic protein 4 (BMP4). (A) AGM cells (10·5 dpc) were analysed by flow cytometry (CyAn ADP Flow Cytometer) for expression of CD34, c-Kit, Flk1 and CD45 to identify subpopulations. Representative results are shown. CD34+/c-Kit+ cells can be subdivided into c-Kithigh and c-Kitlow fractions (areas a & b). Differential expression profiles of (a) CD34+/c-Kithigh and (b) CD34+/c-Kitlow fractions for expression of Flk1 and CD45. Dotted arrows indicate expansion of gated areas a and b as indicated. To maintain accuracy, analysis of the c-Kitlow and c-Kithigh cell fractions could only be performed on small numbers of cells per experiment (pooled littermates) however the distribution of expression was consistent between experiments. (B) CD34+/c-Kithigh cells generate haematopoietic colonies of typical CFU-GM morphology. CD34+/c-Kitlow cells generate exclusively adherent colonies containing multiple cell types: phase-dim spindle-shaped cells (lower arrowhead), large round cells (arrow) and phase-bright small, round cells (upper arrowhead). (C) Unsorted (total) AGM cells were cultured in serum-free conditions −/+ recombinant BMP4 (10 ng/ml) for 2 d and changes in the CD34+/c-Kithigh/low subpopulations analysed by flow cytometry (CyAn ADP cytometer). Without added BMP4 (−BMP4), the percentage of CD34+/c-Kithigh/low cells increases slightly in culture from day 0 but the ratio of CD34+/c-Kithigh to CD34+/c-Kitlow remains relatively constant. With the addition of BMP4, there is a considerable increase in the CD34+/c-Kitlow population compared to day 0 and to cells cultured without added BMP4. Irradiated S17 feeder cells appear to express CD34, accounting for the apparent increase in CD34+/c-Kitneg in cultured cells, but are c-Kitneg.

Mentions: Flow cytometric analysis of cells isolated from mid-gestation murine embryos (pooled 10·5 dpc littermates) suggested that the AGM contained two subpopulations of CD34+ cells that differentially expressed c-Kit (Fig 1A). These two subpopulations were also analysed for expression of the vascular endothelial growth factor receptor Flk1 and the pan-leucocyte marker CD45. The majority of CD34+/c-Kithigh cells were Flk1neg/CD45+ and probably correspond to cells within the intraaortic haematopoietic clusters. In contrast, CD34+/c-Kitlow cells were mainly Flk1+/CD45neg.


Bone morphogenetic protein 4 modulates c-Kit expression and differentiation potential in murine embryonic aorta-gonad-mesonephros haematopoiesis in vitro.

Marshall CJ, Sinclair JC, Thrasher AJ, Kinnon C - Br. J. Haematol. (2007)

Mid gestation murine CD34+ aorta-gonad-mesonephros (AGM) cells contain subpopulations that differ in levels of c-Kit expression, differentiation potential and response to bone morphogenetic protein 4 (BMP4). (A) AGM cells (10·5 dpc) were analysed by flow cytometry (CyAn ADP Flow Cytometer) for expression of CD34, c-Kit, Flk1 and CD45 to identify subpopulations. Representative results are shown. CD34+/c-Kit+ cells can be subdivided into c-Kithigh and c-Kitlow fractions (areas a & b). Differential expression profiles of (a) CD34+/c-Kithigh and (b) CD34+/c-Kitlow fractions for expression of Flk1 and CD45. Dotted arrows indicate expansion of gated areas a and b as indicated. To maintain accuracy, analysis of the c-Kitlow and c-Kithigh cell fractions could only be performed on small numbers of cells per experiment (pooled littermates) however the distribution of expression was consistent between experiments. (B) CD34+/c-Kithigh cells generate haematopoietic colonies of typical CFU-GM morphology. CD34+/c-Kitlow cells generate exclusively adherent colonies containing multiple cell types: phase-dim spindle-shaped cells (lower arrowhead), large round cells (arrow) and phase-bright small, round cells (upper arrowhead). (C) Unsorted (total) AGM cells were cultured in serum-free conditions −/+ recombinant BMP4 (10 ng/ml) for 2 d and changes in the CD34+/c-Kithigh/low subpopulations analysed by flow cytometry (CyAn ADP cytometer). Without added BMP4 (−BMP4), the percentage of CD34+/c-Kithigh/low cells increases slightly in culture from day 0 but the ratio of CD34+/c-Kithigh to CD34+/c-Kitlow remains relatively constant. With the addition of BMP4, there is a considerable increase in the CD34+/c-Kitlow population compared to day 0 and to cells cultured without added BMP4. Irradiated S17 feeder cells appear to express CD34, accounting for the apparent increase in CD34+/c-Kitneg in cultured cells, but are c-Kitneg.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2366020&req=5

fig01: Mid gestation murine CD34+ aorta-gonad-mesonephros (AGM) cells contain subpopulations that differ in levels of c-Kit expression, differentiation potential and response to bone morphogenetic protein 4 (BMP4). (A) AGM cells (10·5 dpc) were analysed by flow cytometry (CyAn ADP Flow Cytometer) for expression of CD34, c-Kit, Flk1 and CD45 to identify subpopulations. Representative results are shown. CD34+/c-Kit+ cells can be subdivided into c-Kithigh and c-Kitlow fractions (areas a & b). Differential expression profiles of (a) CD34+/c-Kithigh and (b) CD34+/c-Kitlow fractions for expression of Flk1 and CD45. Dotted arrows indicate expansion of gated areas a and b as indicated. To maintain accuracy, analysis of the c-Kitlow and c-Kithigh cell fractions could only be performed on small numbers of cells per experiment (pooled littermates) however the distribution of expression was consistent between experiments. (B) CD34+/c-Kithigh cells generate haematopoietic colonies of typical CFU-GM morphology. CD34+/c-Kitlow cells generate exclusively adherent colonies containing multiple cell types: phase-dim spindle-shaped cells (lower arrowhead), large round cells (arrow) and phase-bright small, round cells (upper arrowhead). (C) Unsorted (total) AGM cells were cultured in serum-free conditions −/+ recombinant BMP4 (10 ng/ml) for 2 d and changes in the CD34+/c-Kithigh/low subpopulations analysed by flow cytometry (CyAn ADP cytometer). Without added BMP4 (−BMP4), the percentage of CD34+/c-Kithigh/low cells increases slightly in culture from day 0 but the ratio of CD34+/c-Kithigh to CD34+/c-Kitlow remains relatively constant. With the addition of BMP4, there is a considerable increase in the CD34+/c-Kitlow population compared to day 0 and to cells cultured without added BMP4. Irradiated S17 feeder cells appear to express CD34, accounting for the apparent increase in CD34+/c-Kitneg in cultured cells, but are c-Kitneg.
Mentions: Flow cytometric analysis of cells isolated from mid-gestation murine embryos (pooled 10·5 dpc littermates) suggested that the AGM contained two subpopulations of CD34+ cells that differentially expressed c-Kit (Fig 1A). These two subpopulations were also analysed for expression of the vascular endothelial growth factor receptor Flk1 and the pan-leucocyte marker CD45. The majority of CD34+/c-Kithigh cells were Flk1neg/CD45+ and probably correspond to cells within the intraaortic haematopoietic clusters. In contrast, CD34+/c-Kitlow cells were mainly Flk1+/CD45neg.

Bottom Line: The addition of BMP4 to AGM cells resulted in expansion of the CD34(+)/c-Kit(low) cell pool within 48 h, via a combination of down modulation of the c-Kit receptor in CD34(+)/c-Kit(high) cells and proliferation.CD34(+)/c-Kit(high) progenitors cultured with BMP4 also generated adherent colonies typical of c-Kit(low) cells.These results suggest that BMP4 regulates c-Kit expression and differentiation potential in CD34(+) AGM cells and supports a role for BMP signalling in the maintenance of multipotency during embryonic haematopoiesis, providing an insight into stem cell homeostasis within the mammalian haematopoietic niche.

View Article: PubMed Central - PubMed

Affiliation: Molecular Immunology Unit, UCL Institute of Child Health, London, UK.

ABSTRACT
The transforming growth factor-beta-related factor bone morphogenetic protein 4 (BMP4) is expressed in the human embryonic aorta-gonad-mesonephros (AGM) coincident with the emergence of haematopoietic cells and influences postnatal mammalian haematopoietic stem cells in vitro. To investigate the role of BMP4 in mammalian embryonic haematopoiesis, cells were isolated from murine AGM and two populations of CD34(+) cells with different levels of c-Kit expression and multipotency were identified. CD34(+)/c-Kit(high) cells express CD45 and are haematopoietic-restricted progenitors. In contrast, CD34(+)/c-Kit(low) cells are Flk1+/CD45(neg) and generate adherent colonies in ex vivo culture that resemble haemangioblast colonies identified in other systems. The addition of BMP4 to AGM cells resulted in expansion of the CD34(+)/c-Kit(low) cell pool within 48 h, via a combination of down modulation of the c-Kit receptor in CD34(+)/c-Kit(high) cells and proliferation. In long-term culture, BMP4 increased the growth/survival of CD34(+)/c-Kit(high) haematopoietic progenitors, effects that were blocked by BMP inhibitors. CD34(+)/c-Kit(high) progenitors cultured with BMP4 also generated adherent colonies typical of c-Kit(low) cells. These results suggest that BMP4 regulates c-Kit expression and differentiation potential in CD34(+) AGM cells and supports a role for BMP signalling in the maintenance of multipotency during embryonic haematopoiesis, providing an insight into stem cell homeostasis within the mammalian haematopoietic niche.

Show MeSH
Related in: MedlinePlus