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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.


Related in: MedlinePlus

NO-induced CD34 expression has contrasting age-dependent effects on the HSC functionality. Lin− cells isolated from juvenile mice (6–8 weeks; CD45.1) were treated or not with 200 μM sodium nitroprusside (SNP) for 3 days and infused into irradiated recipients (8–10 weeks; CD45.2); 7–8 mice were used per group. The level of engraftment was analyzed by flow cytometry analysis of peripheral blood (PB) and bone marrow (BM). Percentage chimerism by the donor cells in PB of recipients at 4 weeks (a) and 16 weeks (b) post-transplant is depicted. Percentage engraftment of total donor cells (c) and LSK CD34− LT-HSCs (d) in the BM of recipients is illustrated. The engrafted donor cells were sorted from the bone marrow of primary recipients and infused into irradiated secondary recipients. The percentage chimerism by these primary engrafted donor cells was analyzed by flow cytometry in the PB of secondary recipients. Percentage chimerism produced by the primary engrafted cells in the PB of secondary recipients at 4 weeks (e) and 16 weeks (f) post-transplant is shown; 4–5 secondary recipients were used per group. Sort-purified LSK-CD34− cells from juvenile (6–8 weeks) and adult (10–12 weeks) mice were treated with 200 μM SNP for 3 days (g) or 12 h (h), and the cells were subjected to qRT-PCR analyses. Relative expression of Cd34 and various transcription factors is illustrated (means ± SEM; N = 3). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, between control and SNP-treated sets; ###p ≤ 0.001, SNP-treated juvenile versus SNP-treated adult HSCs. Also see Additional file 6 (Figure S4). NS not significant
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Fig7: NO-induced CD34 expression has contrasting age-dependent effects on the HSC functionality. Lin− cells isolated from juvenile mice (6–8 weeks; CD45.1) were treated or not with 200 μM sodium nitroprusside (SNP) for 3 days and infused into irradiated recipients (8–10 weeks; CD45.2); 7–8 mice were used per group. The level of engraftment was analyzed by flow cytometry analysis of peripheral blood (PB) and bone marrow (BM). Percentage chimerism by the donor cells in PB of recipients at 4 weeks (a) and 16 weeks (b) post-transplant is depicted. Percentage engraftment of total donor cells (c) and LSK CD34− LT-HSCs (d) in the BM of recipients is illustrated. The engrafted donor cells were sorted from the bone marrow of primary recipients and infused into irradiated secondary recipients. The percentage chimerism by these primary engrafted donor cells was analyzed by flow cytometry in the PB of secondary recipients. Percentage chimerism produced by the primary engrafted cells in the PB of secondary recipients at 4 weeks (e) and 16 weeks (f) post-transplant is shown; 4–5 secondary recipients were used per group. Sort-purified LSK-CD34− cells from juvenile (6–8 weeks) and adult (10–12 weeks) mice were treated with 200 μM SNP for 3 days (g) or 12 h (h), and the cells were subjected to qRT-PCR analyses. Relative expression of Cd34 and various transcription factors is illustrated (means ± SEM; N = 3). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, between control and SNP-treated sets; ###p ≤ 0.001, SNP-treated juvenile versus SNP-treated adult HSCs. Also see Additional file 6 (Figure S4). NS not significant

Mentions: CD34+ HSCs from juvenile mice (6 weeks old) have been shown to possess engraftment ability [24, 25]. We therefore determined the consequences of upregulated CD34 expression in the HSCs on their in vivo functionality (Additional file 6: Figure S4a). The Lin− cells (6–8 weeks old) were treated with SNP for 3 days and infused into irradiated recipients (8–10 weeks old). At 4 weeks post-transplant, the level of chimerism in the peripheral blood (PB) of the recipients was comparable in the control and SNP-treated sets (Fig. 7a; Additional file 6: Figure S4b). However, at 16 weeks a significant increase in the level of chimerism was observed with the SNP-treated cells as compared to controls (Fig. 7b). No lineage bias was seen in the regenerated blood cells at 4 weeks post-transplant in either set (Additional file 6: Figure S4c). At 16 weeks post-transplant, the percentage of myeloid cells was significantly lower in the SNP-treated set as compared to the control set, but the percentage of B and T cells was similar in both (Additional file 6: Figure S4d). Surprisingly, analysis of BM performed at 16 weeks post-transplant showed that the percentage of total donor cells and LSK-CD34− HSCs were not significantly different between the two groups (Fig. 7c and d). The engrafted donor cells from the bone marrow of primary recipients were sorted and infused into irradiated secondary recipients to assess their long-term functionality. We found that, in secondary hosts, the cells sorted from primary recipients that had received SNP-treated cells showed significantly higher engraftment at both 4 and 16 weeks post-transplant (Fig. 7e and f). These data suggest that the NO-mediated increase in CD34 expression is perhaps a reflection of self-renewal in juvenile HSCs.Fig. 7


Nitric oxide has contrasting age-dependent effects on the functionality of murine hematopoietic stem cells
NO-induced CD34 expression has contrasting age-dependent effects on the HSC functionality. Lin− cells isolated from juvenile mice (6–8 weeks; CD45.1) were treated or not with 200 μM sodium nitroprusside (SNP) for 3 days and infused into irradiated recipients (8–10 weeks; CD45.2); 7–8 mice were used per group. The level of engraftment was analyzed by flow cytometry analysis of peripheral blood (PB) and bone marrow (BM). Percentage chimerism by the donor cells in PB of recipients at 4 weeks (a) and 16 weeks (b) post-transplant is depicted. Percentage engraftment of total donor cells (c) and LSK CD34− LT-HSCs (d) in the BM of recipients is illustrated. The engrafted donor cells were sorted from the bone marrow of primary recipients and infused into irradiated secondary recipients. The percentage chimerism by these primary engrafted donor cells was analyzed by flow cytometry in the PB of secondary recipients. Percentage chimerism produced by the primary engrafted cells in the PB of secondary recipients at 4 weeks (e) and 16 weeks (f) post-transplant is shown; 4–5 secondary recipients were used per group. Sort-purified LSK-CD34− cells from juvenile (6–8 weeks) and adult (10–12 weeks) mice were treated with 200 μM SNP for 3 days (g) or 12 h (h), and the cells were subjected to qRT-PCR analyses. Relative expression of Cd34 and various transcription factors is illustrated (means ± SEM; N = 3). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, between control and SNP-treated sets; ###p ≤ 0.001, SNP-treated juvenile versus SNP-treated adult HSCs. Also see Additional file 6 (Figure S4). NS not significant
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Fig7: NO-induced CD34 expression has contrasting age-dependent effects on the HSC functionality. Lin− cells isolated from juvenile mice (6–8 weeks; CD45.1) were treated or not with 200 μM sodium nitroprusside (SNP) for 3 days and infused into irradiated recipients (8–10 weeks; CD45.2); 7–8 mice were used per group. The level of engraftment was analyzed by flow cytometry analysis of peripheral blood (PB) and bone marrow (BM). Percentage chimerism by the donor cells in PB of recipients at 4 weeks (a) and 16 weeks (b) post-transplant is depicted. Percentage engraftment of total donor cells (c) and LSK CD34− LT-HSCs (d) in the BM of recipients is illustrated. The engrafted donor cells were sorted from the bone marrow of primary recipients and infused into irradiated secondary recipients. The percentage chimerism by these primary engrafted donor cells was analyzed by flow cytometry in the PB of secondary recipients. Percentage chimerism produced by the primary engrafted cells in the PB of secondary recipients at 4 weeks (e) and 16 weeks (f) post-transplant is shown; 4–5 secondary recipients were used per group. Sort-purified LSK-CD34− cells from juvenile (6–8 weeks) and adult (10–12 weeks) mice were treated with 200 μM SNP for 3 days (g) or 12 h (h), and the cells were subjected to qRT-PCR analyses. Relative expression of Cd34 and various transcription factors is illustrated (means ± SEM; N = 3). *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, between control and SNP-treated sets; ###p ≤ 0.001, SNP-treated juvenile versus SNP-treated adult HSCs. Also see Additional file 6 (Figure S4). NS not significant
Mentions: CD34+ HSCs from juvenile mice (6 weeks old) have been shown to possess engraftment ability [24, 25]. We therefore determined the consequences of upregulated CD34 expression in the HSCs on their in vivo functionality (Additional file 6: Figure S4a). The Lin− cells (6–8 weeks old) were treated with SNP for 3 days and infused into irradiated recipients (8–10 weeks old). At 4 weeks post-transplant, the level of chimerism in the peripheral blood (PB) of the recipients was comparable in the control and SNP-treated sets (Fig. 7a; Additional file 6: Figure S4b). However, at 16 weeks a significant increase in the level of chimerism was observed with the SNP-treated cells as compared to controls (Fig. 7b). No lineage bias was seen in the regenerated blood cells at 4 weeks post-transplant in either set (Additional file 6: Figure S4c). At 16 weeks post-transplant, the percentage of myeloid cells was significantly lower in the SNP-treated set as compared to the control set, but the percentage of B and T cells was similar in both (Additional file 6: Figure S4d). Surprisingly, analysis of BM performed at 16 weeks post-transplant showed that the percentage of total donor cells and LSK-CD34− HSCs were not significantly different between the two groups (Fig. 7c and d). The engrafted donor cells from the bone marrow of primary recipients were sorted and infused into irradiated secondary recipients to assess their long-term functionality. We found that, in secondary hosts, the cells sorted from primary recipients that had received SNP-treated cells showed significantly higher engraftment at both 4 and 16 weeks post-transplant (Fig. 7e and f). These data suggest that the NO-mediated increase in CD34 expression is perhaps a reflection of self-renewal in juvenile HSCs.Fig. 7

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.


Related in: MedlinePlus