Limits...
Haematopoietic stem cell survival and transplantation efficacy is limited by the BH3-only proteins Bim and Bmf.

Labi V, Bertele D, Woess C, Tischner D, Bock FJ, Schwemmers S, Pahl HL, Geley S, Kunze M, Niemeyer CM, Villunger A, Erlacher M - EMBO Mol Med (2012)

Bottom Line: Thereby, we revealed a critical role for Bim and Bmf as regulators of HSPC dynamics both during early engraftment and long-term reconstitution.HSPCs derived from wild-type donors were readily displaced by Bim- or Bmf-deficient or Bcl-2-overexpressing HSPCs as early as 10 days after engraftment.Finally, we provide proof of principle that RNAi-based reduction of BIM or BMF, or overexpression of BCL-2 in human CD34(+) cord blood cells may be an attractive therapeutic option to increase stem cell survival and transplantation efficacy.

View Article: PubMed Central - PubMed

Affiliation: Division of Developmental Immunology, Biocenter, Innsbruck Medical University, Innsbruck, Austria. verena.labi@mdc-berlin.de

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Bim- and Bmf-dependent apoptosis limits the reconstitution potential of transplanted HSPC in vivoTen days after competitive reconstitution, recipient mice were sacrificed and percentages of Ly5.2+ thymocytes were determined by flow cytometry. Thymocyte subsets were defined by surface expression of CD4, CD8, CD25 and CD44. Similarly, percentages of Ly5.2+ Mac1+Gr1− monocytes and Gr1+ granulocytes in the spleen were determined 10 days after transplantation. Symbols represent %Ly5.2+ cells within the indicated cell populations.Two and 4 weeks after competitive reconstitution, blood samples were collected from recipient mice and white blood cells were stained with antibodies against CD4, CD8, B220, Gr1, Mac1, Ly5.1 and Ly5.2. Bars represent absolute numbers of Ly5.2+ lymphocytes (CD4+ and CD8+ T cells plus B220+ B cells) and Ly5.2+ myeloid cells (monocytes plus granulocytes). Symbols represent means of four to five mice/genotype from three independent experiments ± SEM. Significant p values (Mann–Whitney-test): wt versus bmf−/−: p = 0.05 in lymphocytes (2 weeks), p = 0.03 in myeloid cells (2 weeks) and p = 0.01 in myeloid cells (4 weeks). Wt versus bim−/−: p = 0.01 in lymphocytes (4 weeks). Wt versus bcl-2 tg cells: p = 0.01 in lymphocytes and myeloid cells (4 weeks), and p = 0.03 in myeloid cells (2 weeks).Lethally irradiated recipient mice were transplanted with 10,000 total BM cells derived from Ly5.2+ wt or bim−/− mice. Two-hundred thousand Ly5.1+ wt BM cells were used as competitor cells. Fourteen weeks after transplantation, %Ly5.2+ cells were determined in lymphatic and myeloid cell subsets and successful engraftment was defined by >1% Ly5.2+ myeloid and >1% Ly5.2+ lymphatic splenic cells. The right panel shows %Ly5.2+ cells of BM resident LSK cells. Differences in engraftment were determined with the Fisher exact test (p = 0.015).
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fig04: Bim- and Bmf-dependent apoptosis limits the reconstitution potential of transplanted HSPC in vivoTen days after competitive reconstitution, recipient mice were sacrificed and percentages of Ly5.2+ thymocytes were determined by flow cytometry. Thymocyte subsets were defined by surface expression of CD4, CD8, CD25 and CD44. Similarly, percentages of Ly5.2+ Mac1+Gr1− monocytes and Gr1+ granulocytes in the spleen were determined 10 days after transplantation. Symbols represent %Ly5.2+ cells within the indicated cell populations.Two and 4 weeks after competitive reconstitution, blood samples were collected from recipient mice and white blood cells were stained with antibodies against CD4, CD8, B220, Gr1, Mac1, Ly5.1 and Ly5.2. Bars represent absolute numbers of Ly5.2+ lymphocytes (CD4+ and CD8+ T cells plus B220+ B cells) and Ly5.2+ myeloid cells (monocytes plus granulocytes). Symbols represent means of four to five mice/genotype from three independent experiments ± SEM. Significant p values (Mann–Whitney-test): wt versus bmf−/−: p = 0.05 in lymphocytes (2 weeks), p = 0.03 in myeloid cells (2 weeks) and p = 0.01 in myeloid cells (4 weeks). Wt versus bim−/−: p = 0.01 in lymphocytes (4 weeks). Wt versus bcl-2 tg cells: p = 0.01 in lymphocytes and myeloid cells (4 weeks), and p = 0.03 in myeloid cells (2 weeks).Lethally irradiated recipient mice were transplanted with 10,000 total BM cells derived from Ly5.2+ wt or bim−/− mice. Two-hundred thousand Ly5.1+ wt BM cells were used as competitor cells. Fourteen weeks after transplantation, %Ly5.2+ cells were determined in lymphatic and myeloid cell subsets and successful engraftment was defined by >1% Ly5.2+ myeloid and >1% Ly5.2+ lymphatic splenic cells. The right panel shows %Ly5.2+ cells of BM resident LSK cells. Differences in engraftment were determined with the Fisher exact test (p = 0.015).

Mentions: To define the relevance of the early reconstitution advantage of bim−/− or bmf−/− LSK cells for the kinetics of haematopoietic regeneration, we analysed BM, spleen and thymus at 5 and 10 days after reconstitution. At day 5, all haematopoietic organs were reduced in size as well as cellularity and almost no differentiated Ly5.2+ cells were detectable, independent of the donor-genotype (unpublished observation). Similarly, at Day 10 most of the cells were host-derived. At this time point, Ly5.2+ and thus LSK-derived donor cells were detectable up to the DN3-4 stage and a trend to higher percentages of Ly5.2+ cells was already observed in wt:bim−/−, wt:bmf−/− and wt:bcl-2 tg chimeras when compared to wt:wt chimeras. The percentages of splenic Ly5.2+ monocytes and granulocytes were likewise increased in the absence of Bim or Bmf (Fig 4A). In the peripheral blood, significant advantages of bim−/−, bmf−/− and bcl-2 tg cells were first noted 2 and 4 weeks after transplantation and affected both lymphocytes and myeloid cells (Fig 4B). Of note, whereas bmf−/− cells were clearly less potent in replacing wt cells in long-term reconstitution experiments, when compared to bim−/− cells, they appeared equally potent during the initial haematopoietic reconstitution (Fig 4A and B). This observation is in line with our hypothesis that both, increased stem cell survival as well as accumulation of mature lymphatic and myeloid cells, contribute to the phenotypes observed in our long-term assays. Hence, the role of Bmf may be more critical during initial reconstitution events.


Haematopoietic stem cell survival and transplantation efficacy is limited by the BH3-only proteins Bim and Bmf.

Labi V, Bertele D, Woess C, Tischner D, Bock FJ, Schwemmers S, Pahl HL, Geley S, Kunze M, Niemeyer CM, Villunger A, Erlacher M - EMBO Mol Med (2012)

Bim- and Bmf-dependent apoptosis limits the reconstitution potential of transplanted HSPC in vivoTen days after competitive reconstitution, recipient mice were sacrificed and percentages of Ly5.2+ thymocytes were determined by flow cytometry. Thymocyte subsets were defined by surface expression of CD4, CD8, CD25 and CD44. Similarly, percentages of Ly5.2+ Mac1+Gr1− monocytes and Gr1+ granulocytes in the spleen were determined 10 days after transplantation. Symbols represent %Ly5.2+ cells within the indicated cell populations.Two and 4 weeks after competitive reconstitution, blood samples were collected from recipient mice and white blood cells were stained with antibodies against CD4, CD8, B220, Gr1, Mac1, Ly5.1 and Ly5.2. Bars represent absolute numbers of Ly5.2+ lymphocytes (CD4+ and CD8+ T cells plus B220+ B cells) and Ly5.2+ myeloid cells (monocytes plus granulocytes). Symbols represent means of four to five mice/genotype from three independent experiments ± SEM. Significant p values (Mann–Whitney-test): wt versus bmf−/−: p = 0.05 in lymphocytes (2 weeks), p = 0.03 in myeloid cells (2 weeks) and p = 0.01 in myeloid cells (4 weeks). Wt versus bim−/−: p = 0.01 in lymphocytes (4 weeks). Wt versus bcl-2 tg cells: p = 0.01 in lymphocytes and myeloid cells (4 weeks), and p = 0.03 in myeloid cells (2 weeks).Lethally irradiated recipient mice were transplanted with 10,000 total BM cells derived from Ly5.2+ wt or bim−/− mice. Two-hundred thousand Ly5.1+ wt BM cells were used as competitor cells. Fourteen weeks after transplantation, %Ly5.2+ cells were determined in lymphatic and myeloid cell subsets and successful engraftment was defined by >1% Ly5.2+ myeloid and >1% Ly5.2+ lymphatic splenic cells. The right panel shows %Ly5.2+ cells of BM resident LSK cells. Differences in engraftment were determined with the Fisher exact test (p = 0.015).
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fig04: Bim- and Bmf-dependent apoptosis limits the reconstitution potential of transplanted HSPC in vivoTen days after competitive reconstitution, recipient mice were sacrificed and percentages of Ly5.2+ thymocytes were determined by flow cytometry. Thymocyte subsets were defined by surface expression of CD4, CD8, CD25 and CD44. Similarly, percentages of Ly5.2+ Mac1+Gr1− monocytes and Gr1+ granulocytes in the spleen were determined 10 days after transplantation. Symbols represent %Ly5.2+ cells within the indicated cell populations.Two and 4 weeks after competitive reconstitution, blood samples were collected from recipient mice and white blood cells were stained with antibodies against CD4, CD8, B220, Gr1, Mac1, Ly5.1 and Ly5.2. Bars represent absolute numbers of Ly5.2+ lymphocytes (CD4+ and CD8+ T cells plus B220+ B cells) and Ly5.2+ myeloid cells (monocytes plus granulocytes). Symbols represent means of four to five mice/genotype from three independent experiments ± SEM. Significant p values (Mann–Whitney-test): wt versus bmf−/−: p = 0.05 in lymphocytes (2 weeks), p = 0.03 in myeloid cells (2 weeks) and p = 0.01 in myeloid cells (4 weeks). Wt versus bim−/−: p = 0.01 in lymphocytes (4 weeks). Wt versus bcl-2 tg cells: p = 0.01 in lymphocytes and myeloid cells (4 weeks), and p = 0.03 in myeloid cells (2 weeks).Lethally irradiated recipient mice were transplanted with 10,000 total BM cells derived from Ly5.2+ wt or bim−/− mice. Two-hundred thousand Ly5.1+ wt BM cells were used as competitor cells. Fourteen weeks after transplantation, %Ly5.2+ cells were determined in lymphatic and myeloid cell subsets and successful engraftment was defined by >1% Ly5.2+ myeloid and >1% Ly5.2+ lymphatic splenic cells. The right panel shows %Ly5.2+ cells of BM resident LSK cells. Differences in engraftment were determined with the Fisher exact test (p = 0.015).
Mentions: To define the relevance of the early reconstitution advantage of bim−/− or bmf−/− LSK cells for the kinetics of haematopoietic regeneration, we analysed BM, spleen and thymus at 5 and 10 days after reconstitution. At day 5, all haematopoietic organs were reduced in size as well as cellularity and almost no differentiated Ly5.2+ cells were detectable, independent of the donor-genotype (unpublished observation). Similarly, at Day 10 most of the cells were host-derived. At this time point, Ly5.2+ and thus LSK-derived donor cells were detectable up to the DN3-4 stage and a trend to higher percentages of Ly5.2+ cells was already observed in wt:bim−/−, wt:bmf−/− and wt:bcl-2 tg chimeras when compared to wt:wt chimeras. The percentages of splenic Ly5.2+ monocytes and granulocytes were likewise increased in the absence of Bim or Bmf (Fig 4A). In the peripheral blood, significant advantages of bim−/−, bmf−/− and bcl-2 tg cells were first noted 2 and 4 weeks after transplantation and affected both lymphocytes and myeloid cells (Fig 4B). Of note, whereas bmf−/− cells were clearly less potent in replacing wt cells in long-term reconstitution experiments, when compared to bim−/− cells, they appeared equally potent during the initial haematopoietic reconstitution (Fig 4A and B). This observation is in line with our hypothesis that both, increased stem cell survival as well as accumulation of mature lymphatic and myeloid cells, contribute to the phenotypes observed in our long-term assays. Hence, the role of Bmf may be more critical during initial reconstitution events.

Bottom Line: Thereby, we revealed a critical role for Bim and Bmf as regulators of HSPC dynamics both during early engraftment and long-term reconstitution.HSPCs derived from wild-type donors were readily displaced by Bim- or Bmf-deficient or Bcl-2-overexpressing HSPCs as early as 10 days after engraftment.Finally, we provide proof of principle that RNAi-based reduction of BIM or BMF, or overexpression of BCL-2 in human CD34(+) cord blood cells may be an attractive therapeutic option to increase stem cell survival and transplantation efficacy.

View Article: PubMed Central - PubMed

Affiliation: Division of Developmental Immunology, Biocenter, Innsbruck Medical University, Innsbruck, Austria. verena.labi@mdc-berlin.de

Show MeSH
Related in: MedlinePlus