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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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Displacement of wt haematopoiesis by bim−/− or bcl-2 tg cellsA,B. 16 weeks after competitive transplantation recipient mice were sacrificed, and (A) spleens and (B) thymi were analysed in detail by flow cytometric analysis. Surface markers identifying immature thymocyte subsets, T, B and myeloid cells were combined with antibodies against Ly5.1 and Ly5.2 (DN: CD4−8−; DN1: CD44+25−; DN2: CD44+25+; DN3: CD44−25+; DN4: CD44−CD25−). Bars represent means of n = 4–6 animals per genotype from four independent experiments ± SEM. Significant p values (Mann–Whitney-test): wt versus bmf−/−: p = 0.03 in monocytes and DN3 thymocytes; p = 0.05 in granulocytes, DN1 and DN2 thymocytes. Wt versus bim−/−: p = 0.01 in B cells, myeloid cells and thymocytes; p = 0.03 in CD4+ and CD8+ cells. Wt versus bcl-2 tg cells: p = 0.01 in all cell types. No significant differences between bim−/− and bcl-2 tg cells were obtained.
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fig02: Displacement of wt haematopoiesis by bim−/− or bcl-2 tg cellsA,B. 16 weeks after competitive transplantation recipient mice were sacrificed, and (A) spleens and (B) thymi were analysed in detail by flow cytometric analysis. Surface markers identifying immature thymocyte subsets, T, B and myeloid cells were combined with antibodies against Ly5.1 and Ly5.2 (DN: CD4−8−; DN1: CD44+25−; DN2: CD44+25+; DN3: CD44−25+; DN4: CD44−CD25−). Bars represent means of n = 4–6 animals per genotype from four independent experiments ± SEM. Significant p values (Mann–Whitney-test): wt versus bmf−/−: p = 0.03 in monocytes and DN3 thymocytes; p = 0.05 in granulocytes, DN1 and DN2 thymocytes. Wt versus bim−/−: p = 0.01 in B cells, myeloid cells and thymocytes; p = 0.03 in CD4+ and CD8+ cells. Wt versus bcl-2 tg cells: p = 0.01 in all cell types. No significant differences between bim−/− and bcl-2 tg cells were obtained.

Mentions: We next asked whether the prolonged survival of bim−/− LSK cells under suboptimal conditions in vitro would translate into a better reconstitution of lethally irradiated mice. We also included bmf−/− LSK cells in our in vivo studies because Bmf mRNA was induced strongest after cytokine deprivation. To directly compare the reconstitution ability of bim−/− or bmf−/− to that of wt LSK cells, we performed competitive reconstitution experiments. Lethally irradiated Ly5.1+ recipients were reconstituted with a 50:50 mixture of LSK cells derived from a Ly5.1+ wt and a Ly5.2+ wt, bim−/−, bmf−/− or vav-bcl-2 tg mouse. After 16 weeks, recipient mice were sacrificed and haematological organs analysed. Mature splenic T and B cells of wt:wt BM chimeras displayed nearly the expected 50:50 ratio (Ly5.1+:Ly5.2+ = 44:56%) between the two donors (Fig 2A and Supporting Information Fig 2). In contrast, in wt:bim−/− chimeras almost all splenic T and B cells were derived from the Ly5.2+bim−/− donor. Of note, chimeras that received BM from vav-bcl-2 tg mice were not significantly different from those that received bim−/− cells, implicating that Bim is the major rate limiting BH3-only protein during reconstitution and accounts for all apoptotic cell death blocked by Bcl-2 overexpression (Fig 2A).


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)

Displacement of wt haematopoiesis by bim−/− or bcl-2 tg cellsA,B. 16 weeks after competitive transplantation recipient mice were sacrificed, and (A) spleens and (B) thymi were analysed in detail by flow cytometric analysis. Surface markers identifying immature thymocyte subsets, T, B and myeloid cells were combined with antibodies against Ly5.1 and Ly5.2 (DN: CD4−8−; DN1: CD44+25−; DN2: CD44+25+; DN3: CD44−25+; DN4: CD44−CD25−). Bars represent means of n = 4–6 animals per genotype from four independent experiments ± SEM. Significant p values (Mann–Whitney-test): wt versus bmf−/−: p = 0.03 in monocytes and DN3 thymocytes; p = 0.05 in granulocytes, DN1 and DN2 thymocytes. Wt versus bim−/−: p = 0.01 in B cells, myeloid cells and thymocytes; p = 0.03 in CD4+ and CD8+ cells. Wt versus bcl-2 tg cells: p = 0.01 in all cell types. No significant differences between bim−/− and bcl-2 tg cells were obtained.
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Related In: Results  -  Collection

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fig02: Displacement of wt haematopoiesis by bim−/− or bcl-2 tg cellsA,B. 16 weeks after competitive transplantation recipient mice were sacrificed, and (A) spleens and (B) thymi were analysed in detail by flow cytometric analysis. Surface markers identifying immature thymocyte subsets, T, B and myeloid cells were combined with antibodies against Ly5.1 and Ly5.2 (DN: CD4−8−; DN1: CD44+25−; DN2: CD44+25+; DN3: CD44−25+; DN4: CD44−CD25−). Bars represent means of n = 4–6 animals per genotype from four independent experiments ± SEM. Significant p values (Mann–Whitney-test): wt versus bmf−/−: p = 0.03 in monocytes and DN3 thymocytes; p = 0.05 in granulocytes, DN1 and DN2 thymocytes. Wt versus bim−/−: p = 0.01 in B cells, myeloid cells and thymocytes; p = 0.03 in CD4+ and CD8+ cells. Wt versus bcl-2 tg cells: p = 0.01 in all cell types. No significant differences between bim−/− and bcl-2 tg cells were obtained.
Mentions: We next asked whether the prolonged survival of bim−/− LSK cells under suboptimal conditions in vitro would translate into a better reconstitution of lethally irradiated mice. We also included bmf−/− LSK cells in our in vivo studies because Bmf mRNA was induced strongest after cytokine deprivation. To directly compare the reconstitution ability of bim−/− or bmf−/− to that of wt LSK cells, we performed competitive reconstitution experiments. Lethally irradiated Ly5.1+ recipients were reconstituted with a 50:50 mixture of LSK cells derived from a Ly5.1+ wt and a Ly5.2+ wt, bim−/−, bmf−/− or vav-bcl-2 tg mouse. After 16 weeks, recipient mice were sacrificed and haematological organs analysed. Mature splenic T and B cells of wt:wt BM chimeras displayed nearly the expected 50:50 ratio (Ly5.1+:Ly5.2+ = 44:56%) between the two donors (Fig 2A and Supporting Information Fig 2). In contrast, in wt:bim−/− chimeras almost all splenic T and B cells were derived from the Ly5.2+bim−/− donor. Of note, chimeras that received BM from vav-bcl-2 tg mice were not significantly different from those that received bim−/− cells, implicating that Bim is the major rate limiting BH3-only protein during reconstitution and accounts for all apoptotic cell death blocked by Bcl-2 overexpression (Fig 2A).

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