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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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CD34+/c-Kithigh aorta-gonad-mesonephros (AGM) cells cultured with bone morphogenetic protein 4 (BMP4) generate adherent colonies resembling CD34+/c-Kitlow colonies. (A) CD34+/c-Kithigh cells isolated by FACS from AGM tissue (10·5 dpc) and cultured for 10 d in serum-free medium alone (−BMP4) generate haematopoietic colonies composed of granulocytic/myeloid cells (CFU-GM). BMP4-treated CD34+/c-Kithigh cells also produce (B) haematopoietic colonies which also contain erythroid cells (CFU-GEMM) and (C) at a similar frequency, adherent colonies which resemble colonies generated from freshly isolated CD34+/c-Kitlow cells of the same embryonic age. Adherent colonies contain three morphologically distinct cell types: phase-dim spindle-shaped cells (arrowhead), large round cells (arrow) and phase-bright small, round cells (hatched arrow).
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fig03: CD34+/c-Kithigh aorta-gonad-mesonephros (AGM) cells cultured with bone morphogenetic protein 4 (BMP4) generate adherent colonies resembling CD34+/c-Kitlow colonies. (A) CD34+/c-Kithigh cells isolated by FACS from AGM tissue (10·5 dpc) and cultured for 10 d in serum-free medium alone (−BMP4) generate haematopoietic colonies composed of granulocytic/myeloid cells (CFU-GM). BMP4-treated CD34+/c-Kithigh cells also produce (B) haematopoietic colonies which also contain erythroid cells (CFU-GEMM) and (C) at a similar frequency, adherent colonies which resemble colonies generated from freshly isolated CD34+/c-Kitlow cells of the same embryonic age. Adherent colonies contain three morphologically distinct cell types: phase-dim spindle-shaped cells (arrowhead), large round cells (arrow) and phase-bright small, round cells (hatched arrow).

Mentions: CD34+/c-Kithigh AGM cells cultured for 10 d or more in serum-free medium alone or with BMP4 retained the capacity to generate granulocyte/monocyte colonies across all embryonic ages tested (Table I, Fig 3A), suggesting that lineage-restricted haematopoietic progenitors (CFU-GM) were maintained or generated within the cultures. BMP4-treated CD34+/c-Kithigh cells also generated colonies that contained erythroid cells, indicating the presence of more primitive, less restricted progenitors (CFU-GEMM) (Fig 3B).


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)

CD34+/c-Kithigh aorta-gonad-mesonephros (AGM) cells cultured with bone morphogenetic protein 4 (BMP4) generate adherent colonies resembling CD34+/c-Kitlow colonies. (A) CD34+/c-Kithigh cells isolated by FACS from AGM tissue (10·5 dpc) and cultured for 10 d in serum-free medium alone (−BMP4) generate haematopoietic colonies composed of granulocytic/myeloid cells (CFU-GM). BMP4-treated CD34+/c-Kithigh cells also produce (B) haematopoietic colonies which also contain erythroid cells (CFU-GEMM) and (C) at a similar frequency, adherent colonies which resemble colonies generated from freshly isolated CD34+/c-Kitlow cells of the same embryonic age. Adherent colonies contain three morphologically distinct cell types: phase-dim spindle-shaped cells (arrowhead), large round cells (arrow) and phase-bright small, round cells (hatched arrow).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2366020&req=5

fig03: CD34+/c-Kithigh aorta-gonad-mesonephros (AGM) cells cultured with bone morphogenetic protein 4 (BMP4) generate adherent colonies resembling CD34+/c-Kitlow colonies. (A) CD34+/c-Kithigh cells isolated by FACS from AGM tissue (10·5 dpc) and cultured for 10 d in serum-free medium alone (−BMP4) generate haematopoietic colonies composed of granulocytic/myeloid cells (CFU-GM). BMP4-treated CD34+/c-Kithigh cells also produce (B) haematopoietic colonies which also contain erythroid cells (CFU-GEMM) and (C) at a similar frequency, adherent colonies which resemble colonies generated from freshly isolated CD34+/c-Kitlow cells of the same embryonic age. Adherent colonies contain three morphologically distinct cell types: phase-dim spindle-shaped cells (arrowhead), large round cells (arrow) and phase-bright small, round cells (hatched arrow).
Mentions: CD34+/c-Kithigh AGM cells cultured for 10 d or more in serum-free medium alone or with BMP4 retained the capacity to generate granulocyte/monocyte colonies across all embryonic ages tested (Table I, Fig 3A), suggesting that lineage-restricted haematopoietic progenitors (CFU-GM) were maintained or generated within the cultures. BMP4-treated CD34+/c-Kithigh cells also generated colonies that contained erythroid cells, indicating the presence of more primitive, less restricted progenitors (CFU-GEMM) (Fig 3B).

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