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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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Bone morphogenetic protein 4 (BMP4) increases the plating efficiency (growth/survival) of mid-gestation aorta-gonad-mesonephros (AGM)-derived CD34+/c-Kithigh cells in 10-d culture. (A) Addition of BMP4 (open symbols) results in an increase in the number of colonies produced at 10·5 dpc compared with serum-free conditions (−BMP4, solid symbols). Statistical analyses were performed using the Kruskal–Wallis test (different embryonic ages) and the non-parametric Wilcoxon signed rank test to compare the collective results for a given embryonic age with and without addition of BMP4. Results were found to be significant at 10·5 dpc (P < 0·001) but not significant at 9·5 dpc or 11·5 dpc (P = 0·068 and P = 0·595 respectively). (B) The effect of BMP4 was consistent at 10·5 dpc, increasing plating efficiency/CFUs by 50% or greater in 12 out of 19 separate experiments. BMP inhibitors. (C) soluble BMP type I-receptor (200 ng/ml) and (D) Noggin (200 ng/ml) neutralize the effect of BMP4 on cultured CD34+/c-Kithigh cells (10·5 dpc).
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fig02: Bone morphogenetic protein 4 (BMP4) increases the plating efficiency (growth/survival) of mid-gestation aorta-gonad-mesonephros (AGM)-derived CD34+/c-Kithigh cells in 10-d culture. (A) Addition of BMP4 (open symbols) results in an increase in the number of colonies produced at 10·5 dpc compared with serum-free conditions (−BMP4, solid symbols). Statistical analyses were performed using the Kruskal–Wallis test (different embryonic ages) and the non-parametric Wilcoxon signed rank test to compare the collective results for a given embryonic age with and without addition of BMP4. Results were found to be significant at 10·5 dpc (P < 0·001) but not significant at 9·5 dpc or 11·5 dpc (P = 0·068 and P = 0·595 respectively). (B) The effect of BMP4 was consistent at 10·5 dpc, increasing plating efficiency/CFUs by 50% or greater in 12 out of 19 separate experiments. BMP inhibitors. (C) soluble BMP type I-receptor (200 ng/ml) and (D) Noggin (200 ng/ml) neutralize the effect of BMP4 on cultured CD34+/c-Kithigh cells (10·5 dpc).

Mentions: The plating efficiency of CD34+/c-Kithigh cells isolated from 9·5 dpc and 11·5 dpc AGM was generally low (<10 CFU/104 cells plated) compared with 10·5 dpc (up to 30 CFU/104 cells), suggesting that ex vivo survival or growth capacity of CD34+/c-Kithigh cells differed with embryonic age. Addition of BMP4 to 9·5 or 11·5 dpc CD34+/c-Kithigh cells resulted in only minimal increases in plating efficiency compared to medium only controls (Fig 2A). In contrast, the number of colonies generated from BMP4-treated 10·5 dpc CD34+/c-Kithigh cells increased significantly (P < 0·001) and reproducibly with a greater than twofold increase in 14 out of 19 independent experiments, some reaching as high as 16 times control levels (Fig 2B). Addition of c-Kit ligand (25 ng/ml), either alone or in combination with BMP4, had no observable effect on colony number, size or morphology.


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)

Bone morphogenetic protein 4 (BMP4) increases the plating efficiency (growth/survival) of mid-gestation aorta-gonad-mesonephros (AGM)-derived CD34+/c-Kithigh cells in 10-d culture. (A) Addition of BMP4 (open symbols) results in an increase in the number of colonies produced at 10·5 dpc compared with serum-free conditions (−BMP4, solid symbols). Statistical analyses were performed using the Kruskal–Wallis test (different embryonic ages) and the non-parametric Wilcoxon signed rank test to compare the collective results for a given embryonic age with and without addition of BMP4. Results were found to be significant at 10·5 dpc (P < 0·001) but not significant at 9·5 dpc or 11·5 dpc (P = 0·068 and P = 0·595 respectively). (B) The effect of BMP4 was consistent at 10·5 dpc, increasing plating efficiency/CFUs by 50% or greater in 12 out of 19 separate experiments. BMP inhibitors. (C) soluble BMP type I-receptor (200 ng/ml) and (D) Noggin (200 ng/ml) neutralize the effect of BMP4 on cultured CD34+/c-Kithigh cells (10·5 dpc).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2366020&req=5

fig02: Bone morphogenetic protein 4 (BMP4) increases the plating efficiency (growth/survival) of mid-gestation aorta-gonad-mesonephros (AGM)-derived CD34+/c-Kithigh cells in 10-d culture. (A) Addition of BMP4 (open symbols) results in an increase in the number of colonies produced at 10·5 dpc compared with serum-free conditions (−BMP4, solid symbols). Statistical analyses were performed using the Kruskal–Wallis test (different embryonic ages) and the non-parametric Wilcoxon signed rank test to compare the collective results for a given embryonic age with and without addition of BMP4. Results were found to be significant at 10·5 dpc (P < 0·001) but not significant at 9·5 dpc or 11·5 dpc (P = 0·068 and P = 0·595 respectively). (B) The effect of BMP4 was consistent at 10·5 dpc, increasing plating efficiency/CFUs by 50% or greater in 12 out of 19 separate experiments. BMP inhibitors. (C) soluble BMP type I-receptor (200 ng/ml) and (D) Noggin (200 ng/ml) neutralize the effect of BMP4 on cultured CD34+/c-Kithigh cells (10·5 dpc).
Mentions: The plating efficiency of CD34+/c-Kithigh cells isolated from 9·5 dpc and 11·5 dpc AGM was generally low (<10 CFU/104 cells plated) compared with 10·5 dpc (up to 30 CFU/104 cells), suggesting that ex vivo survival or growth capacity of CD34+/c-Kithigh cells differed with embryonic age. Addition of BMP4 to 9·5 or 11·5 dpc CD34+/c-Kithigh cells resulted in only minimal increases in plating efficiency compared to medium only controls (Fig 2A). In contrast, the number of colonies generated from BMP4-treated 10·5 dpc CD34+/c-Kithigh cells increased significantly (P < 0·001) and reproducibly with a greater than twofold increase in 14 out of 19 independent experiments, some reaching as high as 16 times control levels (Fig 2B). Addition of c-Kit ligand (25 ng/ml), either alone or in combination with BMP4, had no observable effect on colony number, size or morphology.

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