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. Author manuscript; available in PMC: 2015 Dec 1.
Published in final edited form as: Am J Hematol. 2014 Oct 25;89(12):E235–E238. doi: 10.1002/ajh.23837

Resveratrol Increases the Bone Marrow Hematopoietic Stem and Progenitor Cell Capacity

Pauline Rimmelé 1, Sébastien Lofek-Czubek 1, Saghi Ghaffari 1,2,3,4,5,&
PMCID: PMC4237620  NIHMSID: NIHMS624996  PMID: 25163926

Abstract

Resveratrol is a plant-derived polyphenol that has shown protective effects against many disorders including, several types of cancers and other age-associated diseases as well as blood disorders in cultured cells and/or animal models. However, whether resveratrol has any impact specifically on normal blood stem cells remains unknown. Here we show that a three-week treatment of resveratrol increases the frequency and total numbers of normal bone marrow hematopoietic stem cells (HSC) without any impact on their competitive repopulation capacity. In addition, we show that resveratrol enhances the bone marrow multipotent progenitor capacity in vivo. These results have therapeutic value for disorders of hematopoietic stem and progenitor cells (HSPC) as well as for bone marrow transplantation settings.

Keywords: Resveratrol, HSC, hematopoietic stem and progenitor cells, SIRT1, aging, anti-oxidant, bone marrow transplantation, leukemia

Introduction

Resveratrol (trans-3,5,4′-trihydroxystilbene) is a polyphenolic compound found in grapes, berries, peanuts and red wine [1]. Resveratrol has been shown to have diverse biological effects although the full spectrum of its bioactivity is still unknown. Indeed, resveratrol extends lifespan in some species in part by mimicking caloric restriction [2, 3] and promotes health and resistance to age-related diseases including cancer, heart disease and type 2 diabetes in rodent models [4–7].

While mechanisms underlying the beneficial effects of resveratrol are still under investigations [8, 9] evidence suggests that several biological effects of resveratrol are mediated by the activation of the NAD-dependent SIRT1 [7, 10–13] of sirtuin family of type III deacetylases, implicated in the regulation of organismal aging. SIRT1 is critical for the maintenance of hematopoietic, leukemic and embryonic stem cell activity [14–20]. In addition to its anti-oxidant, anti-inflammatory and anti-tumoral properties [8] resveratrol treatment has notable effects on leukemic cells [21, 22]. Resveratrol was shown to have a remarkable anti-proliferative and pro-apoptotic activity on cultured leukemic cell lines in vitro [21–23]. However the in vivo effect of resveratrol on leukemic cells does not match its in vitro impact and seems to be significantly less pronounced [21]. Nonetheless, resveratrol treatment improved the hematopoietic stem and progenitor cell (HSPC) compartment and showed beneficial effects in a mouse model of Fanconi anemia [24].

Resveratrol treatment was also shown to impact differentiation of HSPC in culture but whether resveratrol inhibits or activates differentiation may depend on the culture conditions and possibly on hematopoietic lineage [16, 25]. Similarly, the in vitro effects of resveratrol on the proliferation or apoptosis of normal HSPC may depend on the cell type or rodent versus human species [21–23]. Importantly whether resveratrol treatment has any impact in vivo on normal long-term hematopoietic stem cell (LT-HSC) that represents the most quiescent hematopoietic stem cell population endowed with the ability to restore multilineage hematopoiesis in lethally irradiated mice remains unknown [21, 26].

In this study, we found that resveratrol increased the number of LT-HSC in the bone marrow. Resveratrol also enhanced the hematopoietic multipotential progenitor cell compartment in vivo. However, resveratrol did not promote the ability of LT-HSC to restore multililneage hematopoiesis in lethally irradiated mice. These results show that resveratrol treatment increases the HSPC capacity of the bone marrow.

Methods

Mice

Mice were used according to the protocols approved by the Institutional Animal Care and Use Committee of Mount Sinai School of Medicine.

In vivo treatment

Mice received 5mg/kg of resveratrol (Millipore) by intraperitoneal injection every day for three weeks.

Flow cytometry

Antibody staining and flow cytometry analysis were performed as previously described [27, 28]. For Lin-Sca-1−c-Kit+ (c-Kit+) and Lin-Sca-1+c-Kit+ (LSK) cells, freshly isolated bone marrow cells were pre-incubated with 5% rat serum and biotinylated hematopoietic multi-lineage monoclonal antibody cocktail (StemCell Technologies), containing CD5 (lymphocytes), CD11b (leukocytes), CD19 (B cells), CD45R (lymphocytes), 7-4 (neutrophils), Ly-6G-Gr-1 (granulocytes), TER119 (erythroid cells) antibodies to remove mature cells, stained with PE-Sca-1, APC-c-Kit antibodies (BD Biosciences) prior to two rounds of wash followed by incubation with pacific-blue-streptavidin (eBioscience). In addition to LSK staining, total bone marrow cells were stained with FITC-CD48 (eBioscience) and PECy7-CD150 (BioLegend) antibodies to isolate the long term HSC (LSKCD48−CD150+).

Long-term repopulation assay

Lethally irradiated (12 Gy as a split dose, 6.5 and 5,5 Gy, 4–5 hours apart) congenic C57BL6-CD45.1 mice (NCI) were reconstituted with intravenous injections of 100 donor LSKCD48−CD150+ cells from C57BL6 (all CD45.2) 3 weeks after resveratrol or vehicle control treatment along with 2 × 105 competitor bone marrow (CD45.1) cells. Reconstitution of donor-derived cells was distinguished from host cells by the expression of CD45.2 versus CD45.1 antigens (BD Biosciences) in the peripheral blood.

Colony Forming Unit Spleen Assay (CFU-S)

Bone marrow (1 × 105) cells were injected intravenously into recipient C57BL6 mice (Charles River Laboratory) previously subjected to 11 Gy irradiation. Recipient spleens were excised 12 days later, fixed in Telleyesniczky’s solution and macroscopic spleen colonies were counted as described [29].

In vitro clonogenic progenitor assay

Myeloid clonogenic assay was performed as previously described [27, 28, 30]. 5 × 104 bone marrow cells were cultured in semi solid medium (MethoCult 3234; StemCell Tech) containing 50 ng/ml rat stem cell factor (SCF), 10 ng/ml IL6, 10 ng/ml IL3 and 3 U/ml erythropoietin (Peprotech). Colonies were counted after 8–10 days.

Results and discussion

To investigate the effects of resveratrol on HSPC compartment in the bone marrow (BM), C57BL/6 mice were injected daily with resveratrol (5mg/kg) for three weeks (Figure 1A). This resveratrol treatment did not modulate significantly the bone marrow cellularity as compared to mice treated with vehicle control (Figure 1A). However, this regimen led to a significant increase in the frequency (Figure 1B, 1C left panel) and total number (Figure 1C, right panel) of Lin− Sca1+ c-Kit+ (LSK) cells (n=12 mice, P<0.05), a population enriched for HSC and hematopoietic progenitors, in treated versus control mice. Resveratrol treatment also increased significantly the frequency (Figure 1D and 1E, left panel) of LSK CD48−CD150+ cells which are highly enriched for LT-HSCs [31] as well as their total numbers (n=6 mice, P<0.05) (Figure 1E, right panel) in treated mice. Interestingly, total number of LT-HSCs was increased almost two fold in the bone marrow at the end of a three week resveratrol treatment (Figure 1D, 1E). To assess whether resveratrol impacts the competitive repopulation potential of HSC in treated mice, we performed in vivo competitive repopulation assay in which 100 highly purified LSK CD48−CD150+ HSCs isolated from mice treated with resveratrol or control (CD45.2) for three weeks were injected into lethally irradiated congenic recipient mice (CD45.1) along with 200,000 recipient bone marrow cells. Flow cytometry analysis of the peripheral blood of the transplanted recipients, 4, 8, 12 and 16 weeks after transplantation revealed that the competitive repopulation ability of resveratrol-treated HSC was comparable to that of mice reconstituted with control HSC, suggesting that resveratrol did not promote the ability of LT-HSC to reconstitute miltilineage hematopoiesis in lethally irradiated mice (Figure 2, n=8 mice in each group). Since resveratrol treatment did not impact HSC activity (Figure 2) specifically but increased significantly the size of the LSK and LT-HSC compartments (Figure 1), we thought that resveratrol might similarly impact multipotent hematopoietic progenitors. In agreement with this prediction, we found that resveratrol treatment led to significant elevation of the total number of bone marrow derived multipotential colony-forming unit-spleen (CFU-S) capable of producing colonies on the spleen of lethally irradiated hosts in vivo after 12 days (Figure 3A) (n=4 mice, P<0.05). The in vivo resveratrol treatment also increased the total number of myeloid progenitors specifically colony-forming cells (CFC) in vitro (Figure 3B).

Figure 1. Resveratrol treatment increases HSPC numbers in the BM.

Figure 1

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A. HSC phenotype and function are analyzed in wild type mice treated with vehicle control (Ct) or 5mg/kg/day resveratrol (Res) for three weeks (left panel). Total number of BM cells per femur (n=12 mice) (right panel). B. Lineage negative (Lin−) BM cells were analyzed for c-Kit versus Sca-1 expression (LSK). Representative flow cytometry plots of LSK frequency from A are shown. (C) Frequencies (left panel) and numbers (right panel) per femur of LSK cells (n=12 mice). D. Representative flow cytometry plots of LSKCD48−CD150+ (LT-HSC) frequency from A are shown. E. Frequencies (left panel) and numbers (right panel) per femur of LT-HSC (n=6 mice).

Figure 2. Resveratrol treatment does not modulate significantly the competitive repopulation ability of LT-HSC.

Figure 2

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LT-HSC (100 CD45.2 cells) isolated as in Figure 1D were mixed with 2.105 total BM cells (CD45.1) and transplanted into lethally irradiated recipients (CD45.1, n=8), and the contribution to the peripheral blood formation of (CD45.2) cells was evaluated at shown time points. All data are expressed as mean ± SEM (*p<0.05).

Figure 3. Resveratrol increases multipotential myeloid progenitor cell compartment.

Figure 3

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A. Mice were treated for three weeks with vehicle control (Ct) or 5mg/kg/day resveratrol (Res). BM cells were isolated and assayed for CFU-S in lethally irradiated mice. CFU-S-derived colonies were measured 12 days after injection of 105 WT BM into lethally irradiated hosts (n=4 mice in each group). One representative of two independent experiments is shown. All data are expressed as mean±SEM (*p<0.05). B. Isolated BM cells (1,5 ×105) (n=6 mice) were cultured in semi-solid methylcellulose in duplicates and the number of colony-forming cell-derived colonies (BFUE CFU-GM and CFU-GEMM) was measured.

Therefore these results show that numbers of HSC (LT-HSC and LSK) and the multipotential hematopoietic progenitors are increased by resveratrol treatment. This treatment however does not modulate the HSC activity. These results highlight the differences in in vivo versus in vitro effects of resveratrol on hematopoietic stem and progenitor cells [21, 22]. Specifically, the in vivo effect of resveratrol may be tightly correlated with the dose and the duration of the treatment [6, 32] [24].

Using a tamoxifen-inducible conditional deletion approach, it was shown recently that SIRT1 is required for maintenance of HSC under homeostatic and stress condsitions [17, 20]. Our current studies do not address whether the effect of resveratrol on HSPC is mediated by SIRT1. Given that loss of SIRT1 results in an increase in HSPC similar to the impact of resveratrol, it is unlikely that the effect of resveratrol on the bone marrow HSPC compartment is mediated by the activation of SIRT1 [20]. The effect of resveratrol might be mediated by sirtuins other than SIRT1 or alternative mechanisms [12, 33].

These results indicate that resveratrol enhances the bone marrow HSPC capacity for the period of time and under the experimental conditions we used. Consistent with previous findings [34], results shown here suggest that resveratrol may enhance the hematopoietic recovery post bone marrow transplantation. However they also suggest that resveratrol should be used cautiously in hematological malignancies. Overall these findings may have therapeutic value for disorders of stem and progenitor cells and for aging.

Acknowledgments

We thank The Flow Cytometry Shared Research Facility at Icahn School of Medicine at Mount Sinai School. SLC was a visiting student of the Magistère de Génétique, Université Paris Diderot-Paris 7, Paris, France. This work was supported in part by the National Institutes of Health grants RO1 DK077174, RO1 HL116365 (Co-PI), and a Myeloproliferative Neoplasm Foundation (MPN) award to SG.

Footnotes

Authors’ contributions

PR and SG designed experiments, analyzed data and wrote the paper. PR and SLC performed experiments.

The authors do not have any conflict of interest to declare.

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