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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Downregulation of BIM but not BMF leads to survival advantages of CD34+ cells in vitroCD34+ cells were transduced with the indicated lentiviral particles. GFP+ cells were cultured for 2 or 4 days in the absence of cytokines, stained with AnnexinV and 7-AAD and analysed by flow cytometry. Bars show a mean of n = 4–5 from three independent experiments ± SEM. Significant p values (Mann–Whitney-test) are indicated.CD34+ cells transduced with the indicated lentiviral particles were cultured in Methocult medium supplemented with G-CSF, GM-CSF, IL-3, IL-6 and EPO. On Day 11 of culture, colonies were analysed by light microscopy and quantified based on typical morphological features (mixed: GEMM, myeloid: GM + M + G, erythroid: E). Bars represent means of n = 6–8 from five independent experiments ± SEM; no significant differences were observed (Mann–Whitney-test).Fourteen days following plating, cells were harvested from Methocult plates and viability was determined by combined staining with AnnexinV and 7-AAD (left panel). Part of the cells was replated in fresh Methocult plates, harvested 17 days later and analysed accordingly to the first plating (right panel). Bars represent means of n = 5–8 from five independent experiments (left) or n = 3–6 from three independent experiments (right) ± SEM, significant p values are indicated (Mann–Whitney-test).
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fig06: Downregulation of BIM but not BMF leads to survival advantages of CD34+ cells in vitroCD34+ cells were transduced with the indicated lentiviral particles. GFP+ cells were cultured for 2 or 4 days in the absence of cytokines, stained with AnnexinV and 7-AAD and analysed by flow cytometry. Bars show a mean of n = 4–5 from three independent experiments ± SEM. Significant p values (Mann–Whitney-test) are indicated.CD34+ cells transduced with the indicated lentiviral particles were cultured in Methocult medium supplemented with G-CSF, GM-CSF, IL-3, IL-6 and EPO. On Day 11 of culture, colonies were analysed by light microscopy and quantified based on typical morphological features (mixed: GEMM, myeloid: GM + M + G, erythroid: E). Bars represent means of n = 6–8 from five independent experiments ± SEM; no significant differences were observed (Mann–Whitney-test).Fourteen days following plating, cells were harvested from Methocult plates and viability was determined by combined staining with AnnexinV and 7-AAD (left panel). Part of the cells was replated in fresh Methocult plates, harvested 17 days later and analysed accordingly to the first plating (right panel). Bars represent means of n = 5–8 from five independent experiments (left) or n = 3–6 from three independent experiments (right) ± SEM, significant p values are indicated (Mann–Whitney-test).

Mentions: Lentivirally transduced shRNA specific for BIM or BMF was used to test the relevance of these two BH3-only proteins for the induction of human HSPC apoptosis. In addition, lentiviruses were generated for expression of their antagonists BCL-2 and BCL-XL. The RNAi-knockdown efficiency was determined by qRT-PCR for BIM and BMF mRNA (Fig 5C) and by immunoblotting for target proteins isolated from sorted GFP+ CD34+ cells (Supporting Information Fig 5C). RNA levels of BIM and BMF were reduced by ∼69 and ∼78%, respectively, at the time of analysis (Fig 5C). Culturing GFP+ CD34+ cells for 2 days in the absence of cytokines revealed that knockdown of BIM but not BMF delayed cytokine deprivation-induced apoptosis ex vivo, although this effect was not as pronounced as that seen in Bim-deficient mouse HSPCs (comp. Figs 6A, 1B). After 4 days BIM knockdown proved inefficient, whereas overexpression of BCL-2 or BCL-XL still effectively increased survival (Fig 6A, right panel).


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)

Downregulation of BIM but not BMF leads to survival advantages of CD34+ cells in vitroCD34+ cells were transduced with the indicated lentiviral particles. GFP+ cells were cultured for 2 or 4 days in the absence of cytokines, stained with AnnexinV and 7-AAD and analysed by flow cytometry. Bars show a mean of n = 4–5 from three independent experiments ± SEM. Significant p values (Mann–Whitney-test) are indicated.CD34+ cells transduced with the indicated lentiviral particles were cultured in Methocult medium supplemented with G-CSF, GM-CSF, IL-3, IL-6 and EPO. On Day 11 of culture, colonies were analysed by light microscopy and quantified based on typical morphological features (mixed: GEMM, myeloid: GM + M + G, erythroid: E). Bars represent means of n = 6–8 from five independent experiments ± SEM; no significant differences were observed (Mann–Whitney-test).Fourteen days following plating, cells were harvested from Methocult plates and viability was determined by combined staining with AnnexinV and 7-AAD (left panel). Part of the cells was replated in fresh Methocult plates, harvested 17 days later and analysed accordingly to the first plating (right panel). Bars represent means of n = 5–8 from five independent experiments (left) or n = 3–6 from three independent experiments (right) ± SEM, significant p values are indicated (Mann–Whitney-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig06: Downregulation of BIM but not BMF leads to survival advantages of CD34+ cells in vitroCD34+ cells were transduced with the indicated lentiviral particles. GFP+ cells were cultured for 2 or 4 days in the absence of cytokines, stained with AnnexinV and 7-AAD and analysed by flow cytometry. Bars show a mean of n = 4–5 from three independent experiments ± SEM. Significant p values (Mann–Whitney-test) are indicated.CD34+ cells transduced with the indicated lentiviral particles were cultured in Methocult medium supplemented with G-CSF, GM-CSF, IL-3, IL-6 and EPO. On Day 11 of culture, colonies were analysed by light microscopy and quantified based on typical morphological features (mixed: GEMM, myeloid: GM + M + G, erythroid: E). Bars represent means of n = 6–8 from five independent experiments ± SEM; no significant differences were observed (Mann–Whitney-test).Fourteen days following plating, cells were harvested from Methocult plates and viability was determined by combined staining with AnnexinV and 7-AAD (left panel). Part of the cells was replated in fresh Methocult plates, harvested 17 days later and analysed accordingly to the first plating (right panel). Bars represent means of n = 5–8 from five independent experiments (left) or n = 3–6 from three independent experiments (right) ± SEM, significant p values are indicated (Mann–Whitney-test).
Mentions: Lentivirally transduced shRNA specific for BIM or BMF was used to test the relevance of these two BH3-only proteins for the induction of human HSPC apoptosis. In addition, lentiviruses were generated for expression of their antagonists BCL-2 and BCL-XL. The RNAi-knockdown efficiency was determined by qRT-PCR for BIM and BMF mRNA (Fig 5C) and by immunoblotting for target proteins isolated from sorted GFP+ CD34+ cells (Supporting Information Fig 5C). RNA levels of BIM and BMF were reduced by ∼69 and ∼78%, respectively, at the time of analysis (Fig 5C). Culturing GFP+ CD34+ cells for 2 days in the absence of cytokines revealed that knockdown of BIM but not BMF delayed cytokine deprivation-induced apoptosis ex vivo, although this effect was not as pronounced as that seen in Bim-deficient mouse HSPCs (comp. Figs 6A, 1B). After 4 days BIM knockdown proved inefficient, whereas overexpression of BCL-2 or BCL-XL still effectively increased survival (Fig 6A, right panel).

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