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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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Factor deprivation triggers induction of BIM and BMF in human CD34+ cellsFresh cord blood derived CD34+ cells were cultured in the presence or absence of SCF, Flt3L, IL6 (100 ng/ml each) and TPO (10 ng/ml). After 14 h of cytokine deprivation, mRNA levels of the indicated BH3-only proteins, BAX and BAK as well as the anti-apoptotic BCL-2 proteins were determined by RT-MLPA®. Blots show mRNA changes in the absence of cytokines when compared to mRNA levels in the presence of cytokines (n = 7, 5 independent experiments). Significant p values (Mann–Whitney-test): BAD p = 0.05; BID p = 0.02; BIM p = 0.009; NOXA p = 0.002; PUMA p = 0.009; BMF p = 0.002; BAK p = 0.05; BCL-XL p = 0.002; MCL-1 p = 0.05; A1 p = 0.03.Addition of single cytokines or combinations thereof led to differential downregulation of BIM and BMF when compared to mRNA expression in the absence of cytokines (1 = mRNA expression in the presence of SCF, Flt3L, TPO and IL-6). Bars represent means of 3–7 from three independent experiments ± SD. Significant p values (Mann–Whitney-test) are indicated.Lentiviruses were constructed by using the pLeGOhU6-G vector, as published by Roelz et al. Transduction efficacy in CD34+ cells was monitored by eGFP expression and generally was 70–80%. qRT-PCR showed that shRNA led to an efficient downregulation of either BIM or BMF in GFP+CD34+ cells. Bars show mean mRNA expression of n = 3–5 per shRNA from three independent experiments ± SD. Significant p values (Mann–Whitney-test) are indicated.
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fig05: Factor deprivation triggers induction of BIM and BMF in human CD34+ cellsFresh cord blood derived CD34+ cells were cultured in the presence or absence of SCF, Flt3L, IL6 (100 ng/ml each) and TPO (10 ng/ml). After 14 h of cytokine deprivation, mRNA levels of the indicated BH3-only proteins, BAX and BAK as well as the anti-apoptotic BCL-2 proteins were determined by RT-MLPA®. Blots show mRNA changes in the absence of cytokines when compared to mRNA levels in the presence of cytokines (n = 7, 5 independent experiments). Significant p values (Mann–Whitney-test): BAD p = 0.05; BID p = 0.02; BIM p = 0.009; NOXA p = 0.002; PUMA p = 0.009; BMF p = 0.002; BAK p = 0.05; BCL-XL p = 0.002; MCL-1 p = 0.05; A1 p = 0.03.Addition of single cytokines or combinations thereof led to differential downregulation of BIM and BMF when compared to mRNA expression in the absence of cytokines (1 = mRNA expression in the presence of SCF, Flt3L, TPO and IL-6). Bars represent means of 3–7 from three independent experiments ± SD. Significant p values (Mann–Whitney-test) are indicated.Lentiviruses were constructed by using the pLeGOhU6-G vector, as published by Roelz et al. Transduction efficacy in CD34+ cells was monitored by eGFP expression and generally was 70–80%. qRT-PCR showed that shRNA led to an efficient downregulation of either BIM or BMF in GFP+CD34+ cells. Bars show mean mRNA expression of n = 3–5 per shRNA from three independent experiments ± SD. Significant p values (Mann–Whitney-test) are indicated.

Mentions: To explore the possibility of a beneficial effect of apoptosis modulation for HSCT in humans, we isolated fresh cord blood-derived CD34+ cells, a cell population enriched for HSPCs and used as a source of HSCT grafts in certain transplantation regimens such as haplo-identical transplantations. Subjecting these cells to cytokine withdrawal in vitro revealed that human CD34+ cells were generally less susceptible to cytokine deprivation-induced apoptosis than murine LSK cells (comp. Fig 1B and Supporting Information Fig 5A). RT-MLPA analysis performed 14 h after cytokine withdrawal indicated a more restricted upregulation of BH3-only proteins with significant induction noted only for BIM and BMF mRNA (2.1- and 9.5-fold, respectively) and only minor induction of PUMA mRNA (1.6-fold), an observation again confirmed by qRT-PCR (Fig 5A and Supporting Information Fig 5B).


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)

Factor deprivation triggers induction of BIM and BMF in human CD34+ cellsFresh cord blood derived CD34+ cells were cultured in the presence or absence of SCF, Flt3L, IL6 (100 ng/ml each) and TPO (10 ng/ml). After 14 h of cytokine deprivation, mRNA levels of the indicated BH3-only proteins, BAX and BAK as well as the anti-apoptotic BCL-2 proteins were determined by RT-MLPA®. Blots show mRNA changes in the absence of cytokines when compared to mRNA levels in the presence of cytokines (n = 7, 5 independent experiments). Significant p values (Mann–Whitney-test): BAD p = 0.05; BID p = 0.02; BIM p = 0.009; NOXA p = 0.002; PUMA p = 0.009; BMF p = 0.002; BAK p = 0.05; BCL-XL p = 0.002; MCL-1 p = 0.05; A1 p = 0.03.Addition of single cytokines or combinations thereof led to differential downregulation of BIM and BMF when compared to mRNA expression in the absence of cytokines (1 = mRNA expression in the presence of SCF, Flt3L, TPO and IL-6). Bars represent means of 3–7 from three independent experiments ± SD. Significant p values (Mann–Whitney-test) are indicated.Lentiviruses were constructed by using the pLeGOhU6-G vector, as published by Roelz et al. Transduction efficacy in CD34+ cells was monitored by eGFP expression and generally was 70–80%. qRT-PCR showed that shRNA led to an efficient downregulation of either BIM or BMF in GFP+CD34+ cells. Bars show mean mRNA expression of n = 3–5 per shRNA from three independent experiments ± SD. Significant p values (Mann–Whitney-test) are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig05: Factor deprivation triggers induction of BIM and BMF in human CD34+ cellsFresh cord blood derived CD34+ cells were cultured in the presence or absence of SCF, Flt3L, IL6 (100 ng/ml each) and TPO (10 ng/ml). After 14 h of cytokine deprivation, mRNA levels of the indicated BH3-only proteins, BAX and BAK as well as the anti-apoptotic BCL-2 proteins were determined by RT-MLPA®. Blots show mRNA changes in the absence of cytokines when compared to mRNA levels in the presence of cytokines (n = 7, 5 independent experiments). Significant p values (Mann–Whitney-test): BAD p = 0.05; BID p = 0.02; BIM p = 0.009; NOXA p = 0.002; PUMA p = 0.009; BMF p = 0.002; BAK p = 0.05; BCL-XL p = 0.002; MCL-1 p = 0.05; A1 p = 0.03.Addition of single cytokines or combinations thereof led to differential downregulation of BIM and BMF when compared to mRNA expression in the absence of cytokines (1 = mRNA expression in the presence of SCF, Flt3L, TPO and IL-6). Bars represent means of 3–7 from three independent experiments ± SD. Significant p values (Mann–Whitney-test) are indicated.Lentiviruses were constructed by using the pLeGOhU6-G vector, as published by Roelz et al. Transduction efficacy in CD34+ cells was monitored by eGFP expression and generally was 70–80%. qRT-PCR showed that shRNA led to an efficient downregulation of either BIM or BMF in GFP+CD34+ cells. Bars show mean mRNA expression of n = 3–5 per shRNA from three independent experiments ± SD. Significant p values (Mann–Whitney-test) are indicated.
Mentions: To explore the possibility of a beneficial effect of apoptosis modulation for HSCT in humans, we isolated fresh cord blood-derived CD34+ cells, a cell population enriched for HSPCs and used as a source of HSCT grafts in certain transplantation regimens such as haplo-identical transplantations. Subjecting these cells to cytokine withdrawal in vitro revealed that human CD34+ cells were generally less susceptible to cytokine deprivation-induced apoptosis than murine LSK cells (comp. Fig 1B and Supporting Information Fig 5A). RT-MLPA analysis performed 14 h after cytokine withdrawal indicated a more restricted upregulation of BH3-only proteins with significant induction noted only for BIM and BMF mRNA (2.1- and 9.5-fold, respectively) and only minor induction of PUMA mRNA (1.6-fold), an observation again confirmed by qRT-PCR (Fig 5A and Supporting Information Fig 5B).

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