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Nitric oxide has contrasting age-dependent effects on the functionality of murine hematopoietic stem cells

View Article: PubMed Central - PubMed

ABSTRACT

Background: The success of hematopoietic stem cell (HSC) transplantation is dependent on the quality of the donor HSCs. Some sources of HSCs display reduced engraftment efficiency either because of inadequate number (e.g., fetal liver and cord blood), or age-related dysfunction (e.g. in older individuals). Therefore, use of pharmacological compounds to improve functionality of HSCs is a forefront research area in hematology.

Methods: Lineage negative (Lin−) cells isolated from murine bone marrow or sort-purified Lin−Sca-1+c-Kit+CD34− (LSK-CD34−) were treated with a nitric oxide donor, sodium nitroprusside (SNP). The cells were subjected to various phenotypic and functional assays.

Results: We found that SNP treatment of Lin− cells leads to an increase in the numbers of LSK-CD34+ cells in them. Using sort-purified LSK CD34− HSCs, we show that this is related to acquisition of CD34 expression by LSK-CD34− cells, rather than proliferation of LSK-CD34+ cells. Most importantly, this upregulated expression of CD34 had age-dependent contrasting effects on HSC functionality. Increased CD34 expression significantly improved the engraftment of juvenile HSCs (6–8 weeks); in sharp contrast, it reduced the engraftment of adult HSCs (10–12 weeks). The molecular mechanism behind this phenomenon involved nitric oxide (NO)-mediated differential induction of various transcription factors involved in commitment with regard to self-renewal in adult and juvenile HSCs, respectively. Preliminary experiments performed on cord blood-derived and mobilized peripheral blood-derived cells revealed that NO exerts age-dependent contrasting effects on human HSCs as well.

Conclusions: This study demonstrates novel age-dependent contrasting effects of NO on HSC functionality and suggests that HSC age may be an important parameter in screening of various compounds for their use in manipulation of HSCs.

Electronic supplementary material: The online version of this article (doi:10.1186/s13287-016-0433-x) contains supplementary material, which is available to authorized users.

No MeSH data available.


NO-mediated upregulation of CD34 involves c-Myb and c-Jun. Sort-purified LSK-CD34− cells were transfected with siRNA to c-Jun and c-Myb. Mock transfected cells were used as controls. After 18 h post-transfection, the cells were exposed to 200 μM sodium nitroprusside (SNP) for 12 h and then subjected to qRT-PCR analyses. Relative expression of c-Jun-specific mRNA (a) and Cd34-specific mRNA (b) in the cells transfected with c-Jun-specific siRNA, with or without the treatment with SNP, is depicted. Relative expression of c-Myb-specific mRNA (c) and Cd34-specific mRNA (d) in the cells transfected with c-Myb-specific siRNA, with or without the treatment with SNP, is depicted. ***p ≤ 0.001. NS not significant
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Fig6: NO-mediated upregulation of CD34 involves c-Myb and c-Jun. Sort-purified LSK-CD34− cells were transfected with siRNA to c-Jun and c-Myb. Mock transfected cells were used as controls. After 18 h post-transfection, the cells were exposed to 200 μM sodium nitroprusside (SNP) for 12 h and then subjected to qRT-PCR analyses. Relative expression of c-Jun-specific mRNA (a) and Cd34-specific mRNA (b) in the cells transfected with c-Jun-specific siRNA, with or without the treatment with SNP, is depicted. Relative expression of c-Myb-specific mRNA (c) and Cd34-specific mRNA (d) in the cells transfected with c-Myb-specific siRNA, with or without the treatment with SNP, is depicted. ***p ≤ 0.001. NS not significant

Mentions: Transcription factors (TFs) such as c-Myb and c-Jun are known to play an important role in stem cell biology and are also known to be regulated by NO in various cell systems [21–23]. First, we determined whether SNP has any effect on their expression in the HSCs. The LSK-CD34− cells showed a basal level expression of both these TFs, and SNP treatment upregulated the expression of c-Jun in LSK-CD34− HSCs, but did not affect c-Myb expression (Fig. 6a and c). Aliquots of LSK-CD34− HSCs were transfected separately with c-Jun- and c-Myb-specific siRNA (Additional file 1: Table S3) 18 h before the treatment with SNP, and were subjected to qRT-PCR analyses. We found that the NO-mediated upregulation of Cd34 mRNA was significantly affected in the cells transfected with siRNA to c-Jun and c-Myb (Fig. 6b and d), showing that the process involved both TFs. The effect of silencing of c-Jun was more striking, as compared to that seen with c-Myb silencing, indicating that c-Jun plays a more crucial role in the process.Fig. 6


Nitric oxide has contrasting age-dependent effects on the functionality of murine hematopoietic stem cells
NO-mediated upregulation of CD34 involves c-Myb and c-Jun. Sort-purified LSK-CD34− cells were transfected with siRNA to c-Jun and c-Myb. Mock transfected cells were used as controls. After 18 h post-transfection, the cells were exposed to 200 μM sodium nitroprusside (SNP) for 12 h and then subjected to qRT-PCR analyses. Relative expression of c-Jun-specific mRNA (a) and Cd34-specific mRNA (b) in the cells transfected with c-Jun-specific siRNA, with or without the treatment with SNP, is depicted. Relative expression of c-Myb-specific mRNA (c) and Cd34-specific mRNA (d) in the cells transfected with c-Myb-specific siRNA, with or without the treatment with SNP, is depicted. ***p ≤ 0.001. NS not significant
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Related In: Results  -  Collection

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Fig6: NO-mediated upregulation of CD34 involves c-Myb and c-Jun. Sort-purified LSK-CD34− cells were transfected with siRNA to c-Jun and c-Myb. Mock transfected cells were used as controls. After 18 h post-transfection, the cells were exposed to 200 μM sodium nitroprusside (SNP) for 12 h and then subjected to qRT-PCR analyses. Relative expression of c-Jun-specific mRNA (a) and Cd34-specific mRNA (b) in the cells transfected with c-Jun-specific siRNA, with or without the treatment with SNP, is depicted. Relative expression of c-Myb-specific mRNA (c) and Cd34-specific mRNA (d) in the cells transfected with c-Myb-specific siRNA, with or without the treatment with SNP, is depicted. ***p ≤ 0.001. NS not significant
Mentions: Transcription factors (TFs) such as c-Myb and c-Jun are known to play an important role in stem cell biology and are also known to be regulated by NO in various cell systems [21–23]. First, we determined whether SNP has any effect on their expression in the HSCs. The LSK-CD34− cells showed a basal level expression of both these TFs, and SNP treatment upregulated the expression of c-Jun in LSK-CD34− HSCs, but did not affect c-Myb expression (Fig. 6a and c). Aliquots of LSK-CD34− HSCs were transfected separately with c-Jun- and c-Myb-specific siRNA (Additional file 1: Table S3) 18 h before the treatment with SNP, and were subjected to qRT-PCR analyses. We found that the NO-mediated upregulation of Cd34 mRNA was significantly affected in the cells transfected with siRNA to c-Jun and c-Myb (Fig. 6b and d), showing that the process involved both TFs. The effect of silencing of c-Jun was more striking, as compared to that seen with c-Myb silencing, indicating that c-Jun plays a more crucial role in the process.Fig. 6

View Article: PubMed Central - PubMed

ABSTRACT

Background: The success of hematopoietic stem cell (HSC) transplantation is dependent on the quality of the donor HSCs. Some sources of HSCs display reduced engraftment efficiency either because of inadequate number (e.g., fetal liver and cord blood), or age-related dysfunction (e.g. in older individuals). Therefore, use of pharmacological compounds to improve functionality of HSCs is a forefront research area in hematology.

Methods: Lineage negative (Lin−) cells isolated from murine bone marrow or sort-purified Lin−Sca-1+c-Kit+CD34− (LSK-CD34−) were treated with a nitric oxide donor, sodium nitroprusside (SNP). The cells were subjected to various phenotypic and functional assays.

Results: We found that SNP treatment of Lin− cells leads to an increase in the numbers of LSK-CD34+ cells in them. Using sort-purified LSK CD34− HSCs, we show that this is related to acquisition of CD34 expression by LSK-CD34− cells, rather than proliferation of LSK-CD34+ cells. Most importantly, this upregulated expression of CD34 had age-dependent contrasting effects on HSC functionality. Increased CD34 expression significantly improved the engraftment of juvenile HSCs (6–8 weeks); in sharp contrast, it reduced the engraftment of adult HSCs (10–12 weeks). The molecular mechanism behind this phenomenon involved nitric oxide (NO)-mediated differential induction of various transcription factors involved in commitment with regard to self-renewal in adult and juvenile HSCs, respectively. Preliminary experiments performed on cord blood-derived and mobilized peripheral blood-derived cells revealed that NO exerts age-dependent contrasting effects on human HSCs as well.

Conclusions: This study demonstrates novel age-dependent contrasting effects of NO on HSC functionality and suggests that HSC age may be an important parameter in screening of various compounds for their use in manipulation of HSCs.

Electronic supplementary material: The online version of this article (doi:10.1186/s13287-016-0433-x) contains supplementary material, which is available to authorized users.

No MeSH data available.