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

Show MeSH

Related in: MedlinePlus

Knockdown of BIM or BMF improves engraftment of human CD34+ cellsA. Cord blood derived human CD34+ cells were transduced with the indicated lentiviruses and injected intrahepatically into newborn rag2−/−γc−/− mice. Eight weeks later, animals were sacrificed. Single cell suspensions were stained with antibodies against human CD45 (left) or CD34 (right) and murine CD45, and the percentages of GFP+ cells within the human populations were determined. Dot blots of representative experiments are shown.B. Eight weeks after transplantation, the indicated haematological organs were analysed. Total human engraftment was determined by staining single cell suspensions with antibodies against human and murine CD45. Lentiviruses expressed shRNA specific for Luciferase, BIM or BMF (left panel) or overexpressed BCL-2 under control of the SFFV promoter (right panel). No significant differences in total human engraftment could be observed between the different groups (n = 7–14 from four to six independent experiments, left panel; and n = 4 from two independent experiments, right panel; Mann–Whitney-test).C,D. The percentage of GFP+ cells within the human CD45+ (C) or the immature CD34+ (D) cell population was determined by flow cytometry. Knockdown of BIM or BMF (left panel) as well as overexpression of BCL-2 (right panel) clearly increased percentages of GFP+ cells. Significant p values (Mann–Whitney-test): %GFP+ of hCD45+ cells: Luci shRNA versus BIM shRNA: liver p < 0.0001, BM p = 0.02, spleen p = 0.0002; Luci shRNA versus BMF shRNA: liver p = 0.002, BM p = 0.009, spleen p = 0.004; pIG versus BCL-2 p = 0.03 in liver, spleen and thymus. %GFP+ of hCD34+ cells: Luci shRNA versus BIM shRNA: liver p = 0.006, BM p = 0.0001, spleen p = 0.001; Luci shRNA versus BMF shRNA: liver p = 0.009, BM p = 0.002, spleen 0.005; pIG versus BCL-2: p = 0.03 in liver and spleen.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3569658&req=5

fig07: Knockdown of BIM or BMF improves engraftment of human CD34+ cellsA. Cord blood derived human CD34+ cells were transduced with the indicated lentiviruses and injected intrahepatically into newborn rag2−/−γc−/− mice. Eight weeks later, animals were sacrificed. Single cell suspensions were stained with antibodies against human CD45 (left) or CD34 (right) and murine CD45, and the percentages of GFP+ cells within the human populations were determined. Dot blots of representative experiments are shown.B. Eight weeks after transplantation, the indicated haematological organs were analysed. Total human engraftment was determined by staining single cell suspensions with antibodies against human and murine CD45. Lentiviruses expressed shRNA specific for Luciferase, BIM or BMF (left panel) or overexpressed BCL-2 under control of the SFFV promoter (right panel). No significant differences in total human engraftment could be observed between the different groups (n = 7–14 from four to six independent experiments, left panel; and n = 4 from two independent experiments, right panel; Mann–Whitney-test).C,D. The percentage of GFP+ cells within the human CD45+ (C) or the immature CD34+ (D) cell population was determined by flow cytometry. Knockdown of BIM or BMF (left panel) as well as overexpression of BCL-2 (right panel) clearly increased percentages of GFP+ cells. Significant p values (Mann–Whitney-test): %GFP+ of hCD45+ cells: Luci shRNA versus BIM shRNA: liver p < 0.0001, BM p = 0.02, spleen p = 0.0002; Luci shRNA versus BMF shRNA: liver p = 0.002, BM p = 0.009, spleen p = 0.004; pIG versus BCL-2 p = 0.03 in liver, spleen and thymus. %GFP+ of hCD34+ cells: Luci shRNA versus BIM shRNA: liver p = 0.006, BM p = 0.0001, spleen p = 0.001; Luci shRNA versus BMF shRNA: liver p = 0.009, BM p = 0.002, spleen 0.005; pIG versus BCL-2: p = 0.03 in liver and spleen.

Mentions: To investigate the potency of human HSPCs with reduced levels of BIM or BMF to engraft and to compete against non-manipulated HSPCs we used newborn rag2−/−γc−/− mice and injected them intrahepatically with the progeny of 105 CD34+ cells transduced with lentiviruses either expressing shRNAs targeting Luciferase, BIM or BMF mRNA or a BCL-2 transgene. Transduction efficiencies were monitored before transplantation and found comparable between the vectors (Supporting Information Fig 6A). Eight weeks after transplantation overall human engraftment, defined by percentages of human CD45+ cells was similar in all groups analysed (Fig 7A and B). However, in all analysed organs a clearly higher proportion of GFP+ cells was detected when CD34+ donor cells expressed BIM or BMF shRNA, corroborating our results obtained in the murine in vivo system (Fig 7A and C). Notably, significant competitive advantages of GFP+ cells expressing BIM or BMF shRNAs could not only be observed in differentiated CD19+ B cells and CD33+ myeloid cells (Supporting Information Fig 6B and C) but was already visible in immature human CD34+ cells isolated from murine livers, BM and spleens (Fig 7A and D). The effects obtained by BCL-2 overexpression were similar (Fig 7B–D, right panels) indicating that BIM and BMF account for the majority of apoptosis induction during transplantation of CD34+ cells. Since intrahepatic transplantation may only partially mimic intraosseous engraftment occurring during clinical HSCT, we additionally injected human CD34+ cells intravenously into adult recipient mice. Results were comparable with those obtained in newborn mice with a clear engraftment advantage of CD34+ cells expressing BIM shRNA in all cell subsets analysed (Supporting Information Fig 7).


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)

Knockdown of BIM or BMF improves engraftment of human CD34+ cellsA. Cord blood derived human CD34+ cells were transduced with the indicated lentiviruses and injected intrahepatically into newborn rag2−/−γc−/− mice. Eight weeks later, animals were sacrificed. Single cell suspensions were stained with antibodies against human CD45 (left) or CD34 (right) and murine CD45, and the percentages of GFP+ cells within the human populations were determined. Dot blots of representative experiments are shown.B. Eight weeks after transplantation, the indicated haematological organs were analysed. Total human engraftment was determined by staining single cell suspensions with antibodies against human and murine CD45. Lentiviruses expressed shRNA specific for Luciferase, BIM or BMF (left panel) or overexpressed BCL-2 under control of the SFFV promoter (right panel). No significant differences in total human engraftment could be observed between the different groups (n = 7–14 from four to six independent experiments, left panel; and n = 4 from two independent experiments, right panel; Mann–Whitney-test).C,D. The percentage of GFP+ cells within the human CD45+ (C) or the immature CD34+ (D) cell population was determined by flow cytometry. Knockdown of BIM or BMF (left panel) as well as overexpression of BCL-2 (right panel) clearly increased percentages of GFP+ cells. Significant p values (Mann–Whitney-test): %GFP+ of hCD45+ cells: Luci shRNA versus BIM shRNA: liver p < 0.0001, BM p = 0.02, spleen p = 0.0002; Luci shRNA versus BMF shRNA: liver p = 0.002, BM p = 0.009, spleen p = 0.004; pIG versus BCL-2 p = 0.03 in liver, spleen and thymus. %GFP+ of hCD34+ cells: Luci shRNA versus BIM shRNA: liver p = 0.006, BM p = 0.0001, spleen p = 0.001; Luci shRNA versus BMF shRNA: liver p = 0.009, BM p = 0.002, spleen 0.005; pIG versus BCL-2: p = 0.03 in liver and spleen.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3569658&req=5

fig07: Knockdown of BIM or BMF improves engraftment of human CD34+ cellsA. Cord blood derived human CD34+ cells were transduced with the indicated lentiviruses and injected intrahepatically into newborn rag2−/−γc−/− mice. Eight weeks later, animals were sacrificed. Single cell suspensions were stained with antibodies against human CD45 (left) or CD34 (right) and murine CD45, and the percentages of GFP+ cells within the human populations were determined. Dot blots of representative experiments are shown.B. Eight weeks after transplantation, the indicated haematological organs were analysed. Total human engraftment was determined by staining single cell suspensions with antibodies against human and murine CD45. Lentiviruses expressed shRNA specific for Luciferase, BIM or BMF (left panel) or overexpressed BCL-2 under control of the SFFV promoter (right panel). No significant differences in total human engraftment could be observed between the different groups (n = 7–14 from four to six independent experiments, left panel; and n = 4 from two independent experiments, right panel; Mann–Whitney-test).C,D. The percentage of GFP+ cells within the human CD45+ (C) or the immature CD34+ (D) cell population was determined by flow cytometry. Knockdown of BIM or BMF (left panel) as well as overexpression of BCL-2 (right panel) clearly increased percentages of GFP+ cells. Significant p values (Mann–Whitney-test): %GFP+ of hCD45+ cells: Luci shRNA versus BIM shRNA: liver p < 0.0001, BM p = 0.02, spleen p = 0.0002; Luci shRNA versus BMF shRNA: liver p = 0.002, BM p = 0.009, spleen p = 0.004; pIG versus BCL-2 p = 0.03 in liver, spleen and thymus. %GFP+ of hCD34+ cells: Luci shRNA versus BIM shRNA: liver p = 0.006, BM p = 0.0001, spleen p = 0.001; Luci shRNA versus BMF shRNA: liver p = 0.009, BM p = 0.002, spleen 0.005; pIG versus BCL-2: p = 0.03 in liver and spleen.
Mentions: To investigate the potency of human HSPCs with reduced levels of BIM or BMF to engraft and to compete against non-manipulated HSPCs we used newborn rag2−/−γc−/− mice and injected them intrahepatically with the progeny of 105 CD34+ cells transduced with lentiviruses either expressing shRNAs targeting Luciferase, BIM or BMF mRNA or a BCL-2 transgene. Transduction efficiencies were monitored before transplantation and found comparable between the vectors (Supporting Information Fig 6A). Eight weeks after transplantation overall human engraftment, defined by percentages of human CD45+ cells was similar in all groups analysed (Fig 7A and B). However, in all analysed organs a clearly higher proportion of GFP+ cells was detected when CD34+ donor cells expressed BIM or BMF shRNA, corroborating our results obtained in the murine in vivo system (Fig 7A and C). Notably, significant competitive advantages of GFP+ cells expressing BIM or BMF shRNAs could not only be observed in differentiated CD19+ B cells and CD33+ myeloid cells (Supporting Information Fig 6B and C) but was already visible in immature human CD34+ cells isolated from murine livers, BM and spleens (Fig 7A and D). The effects obtained by BCL-2 overexpression were similar (Fig 7B–D, right panels) indicating that BIM and BMF account for the majority of apoptosis induction during transplantation of CD34+ cells. Since intrahepatic transplantation may only partially mimic intraosseous engraftment occurring during clinical HSCT, we additionally injected human CD34+ cells intravenously into adult recipient mice. Results were comparable with those obtained in newborn mice with a clear engraftment advantage of CD34+ cells expressing BIM shRNA in all cell subsets analysed (Supporting Information Fig 7).

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