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TISSUE-SPECIFIC STEM CELLS

Promoting Effects of Serotonin on Hematopoiesis: Ex Vivo Expansion of Cord Blood CD34⁺ Stem/Progenitor Cells, Proliferation of Bone Marrow Stromal Cells, and Antiapoptosis

^aLi Ka Shing Institute of Health Sciences, Department of Paediatrics, The Chinese University of Hong Kong, Hong Kong, China;
^bDepartment of Obstetrics and Gynaecology, The Chinese University of Hong Kong, Hong Kong, China;
^cDepartment of Hematology, First Affiliated Hospital, Shantou University Medical College, Shantou, China;
^dLaboratory Animal Services Centre, The Chinese University of Hong Kong, Hong Kong, China

Key Words. Hematopoiesis • CD34⁺ stem cells • Ex vivo expansion • Bone marrow stromal cells • Serotonin • Antiapoptosis

Correspondence: Karen Li, Ph.D., Department of Paediatrics, The Chinese University of Hong Kong, 6^th floor, Clinical Sciences Block, Prince of Wales Hospital, Shatin, New Territories, Hong Kong. Telephone: (852) 2632 2859; Fax: (852) 2636 0020; e-mail: lipang{at}cuhk.edu.hk

Received on January 18, 2007; accepted for publication on March 31, 2007.

First published online in STEM CELLS EXPRESS  April 19, 2007.

	ABSTRACT

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Serotonin is a monoamine neurotransmitter that has multiple extraneuronal functions. We previously reported that serotonin exerted mitogenic stimulation on megakaryocytopoiesis mediated by 5-hydroxytryptamine (5-HT)₂ receptors. In this study, we investigated effects of serotonin on ex vivo expansion of human cord blood CD34⁺ cells, bone marrow (BM) stromal cell colony-forming unit-fibroblast (CFU-F) formation, and antiapoptosis of megakaryoblastic M-07e cells. Our results showed that serotonin at 200 nM significantly enhanced the expansion of CD34⁺ cells to early stem/progenitors (CD34⁺ cells, colony-forming unit-mixed [CFU-GEMM]) and multilineage committed progenitors (burst-forming unit/colony-forming unit-erythroid [BFU/CFU-E], colony-forming unit-granulocyte macrophage, colony-forming unit-megakaryocyte, CD61⁺CD41⁺ cells). Serotonin also increased nonobese diabetic/severe combined immunodeficient repopulating cells in the expansion culture in terms of human CD45⁺, CD33⁺, CD14⁺ cells, BFU/CFU-E, and CFU-GEMM engraftment in BM of animals 6 weeks post-transplantation. Serotonin alone or in addition to fibroblast growth factor, platelet-derived growth factor, or vascular endothelial growth factor stimulated BM CFU-F formation. In M-07e cells, serotonin exerted antiapoptotic effects (annexin V, caspase-3, and propidium iodide staining) and reduced mitochondria membrane potential damage. The addition of ketanserin, a competitive antagonist of 5-HT₂ receptor, nullified the antiapoptotic effects of serotonin. Our data suggest the involvement of serotonin in promoting hematopoietic stem cells and the BM microenvironment. Serotonin could be developed for clinical ex vivo expansion of hematopoietic stem cells for transplantation.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

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Serotonin, or 5-hydroxytryptamine (5-HT), is a monoamine neurotransmitter synthesized mostly in the gastrointestinal tract and central nervous system. Serotonin regulates a wide range of biological activities, mediated by at least 15 receptors, all of which, except R3, are in the superfamily of G-protein-coupled receptors [1–3]. The synthesis and degradation of serotonin is an active process, with the body pool of the compound replaced every 24 hours [4]. Within cells, serotonin is enzymatically metabolized to 5-hydroxyindole acetic acid and finally excreted in the urine [3, 4]. The extracellular level of serotonin is also regulated by a sodium chloride-dependent membrane protein transporter (5-HTT, SERT) [5]. Various drugs, such as serotonin reuptake inhibitors, monamine oxidase inhibitors, and receptor antagonists, have been designed for the treatment of psychotic disorders such as depression and schizophrenia [5, 6].

The physiological and pathological roles of serotonin have been well recognized in the central nervous system [1, 5, 6], gastrointestinal tract [7, 8], and cardiovascular system [9–11]. Serotonin is also indicated in embryonic development and morphogenesis, probably mediated by stage and tissue-specific receptor subtypes. The 5-HT_1D receptor mRNA could be detected in unfertilized mouse oocytes, zygotes, and preimplantation embryos [12]. Serotonin and the 5-HT_2B receptor are actively responsible for normal craniofacial and cardiovascular morphogenesis [13, 14]. There has been increasing evidence that serotonin acts as a mitogen on a variety of normal and malignant cells [3, 15, 16], including fibroblast [17], osteoblast [18, 19], and vascular endothelium [20]. Recent studies demonstrated that platelet-derived serotonin initiated liver regeneration mediated by 2A and 2B receptors [21] and possible roles for serotonin in hepatic stellate cell function and liver fibrosis [22]. The mechanisms of serotonin-induced mitogenesis appear to involve receptor-specific crosstalks between major signaling pathways. In 5-HT_2B-transfected mouse fibroblasts, serotonin induced cell-cycle progression through pRb/cyclin D1/cdk4 and c-Src/cyclin E activation [23]. The signals were mediated by mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) induction, in concert with receptor tyrosine kinase platelet-derived growth factor receptors. Reactive oxygen species-induced phosphorylation of ERK1/ERK2 has been indicated in smooth muscle cell mitogenesis by serotonin, and the process involved SERT, RhoA/ROCK/5-HT_1B/1D, and P13K/Akt/mTOR/S6K1 pathways [24].

In the hematopoietic system, serotonin is closely associated with platelets and megakaryocytes, as most (>90%) of the serotonin in blood is stored in the dense granules of platelets [3, 25]. Our earlier studies showed that serotonin stimulated megakaryocytopoiesis mediated by 5-HT₂ receptors [26, 27]. Serotonin has marked activities on inflammation and immunity, affecting activities of almost all types of mature blood cells [28–32]. It has been suggested that serotonin acts as a mediator of bidirectional interactions between the nervous system and the immune system. A recent study showed that serotonin acted as an immune signal between dendritic cells and T cells [32]. In the present study, we have provided evidence that serotonin enhanced the ex vivo expansion of cord blood CD34⁺ hematopoietic stem and progenitor cells that engrafted nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mice. It also exerted mitogenic and antiapoptotic effects on human bone marrow stromal cells and the megakaryocytic cell line M-07e, respectively.

	MATERIALS AND METHODS

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Enrichment of Human Umbilical Cord Blood CD34⁺ Cells
Cord blood samples were collected from umbilical veins during normal full-term vaginal deliveries and processed within 24 hours. Mononuclear cells were prepared by density gradient centrifugation (Ficoll Hypaque 1.077 g/ml; Amersham, Uppsala, Sweden, http://www.amersham.com). CD34⁺ cells were enriched using the VarioMACS Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com) as described previously [33, 34]. The purity of enriched CD34⁺ cells, evaluated by flow cytometry, was 91.4% ± 0.59% (mean ± SEM, range 86.1%–96.3%, n = 25), and the proportion of CD34⁺CD38^– cells was 4.75% ± 0.37%. Informed consent was obtained from the mothers for all blood collections, and the study was approved by the Ethics Committee for Clinical Research of The Chinese University of Hong Kong.

Ex Vivo Expansion in Serum-Free Medium with Cytokines
Enriched CD34⁺ cells at 2 x 10⁴ per milliliter were cultured in QBSF-60 Serum-Free Medium (Quality Biological, Gaithersburg, MD, http://www.qualitybiological.com) in 24-well culture plates (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). The cultures contained thrombopoietin (TPO, 50 ng/ml), stem cell factor (SCF, 50 ng/ml), and FL-3 ligand (FL, 80 ng/ml) and were incubated at 37°C and 5% CO₂ in a fully humidified atmosphere with or without serotonin (200 nM; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). This concentration of serotonin has been previously shown to stimulate megakaryocytic colony-forming cells (CFU-MK) from mouse bone marrow [26]. All cytokines were purchased from Peprotech (Rocky Hill, NJ, http://www.peprotech.com) and culture reagents from Gibco (Grand Island, NY, http://www.invitrogen.com) unless specified otherwise. At day 4, each culture was split into three portions with fresh medium, and cytokines were added. Total nucleated cell (TNC) counts, flow cytometric analysis of progenitor cells, and colony-forming unit (CFU) assays were performed on day 0 and day 8. Expanded cells at day 8 were infused into sublethally irradiated NOD/SCID mice for the analysis of NOD/SCID repopulating cells.

Flow Cytometric Analysis of Hematopoietic Stem/Progenitor Cells
Enriched CD34⁺ cells or expanded cells were stained with CD34-fluorescein isothiocyanate (FITC), CD38-phycoerythrin (PE), CD61-FITC (Dako, Glostrup, Denmark, http://www.dako.com), CD41-PE (Dako), and respective isotype controls for 20 minutes. All antibodies and cytometric reagents were purchased from BD Pharmingen (San Diego, http://www.bdbiosciences.com/index_us.shtml) unless specified otherwise. The cells were then washed and resuspended in phosphate-buffered saline (PBS) with 0.5% bovine serum albumin (BSA; Sigma). We added 7-amino-actinomycin D to the cells prior to flow cytometric acquisition for the purpose of gating out dead cells. For samples at day 0 and day 8, 10,000 and 60,000 events were acquired, respectively, using a FACSCalibur flow cytometer and the CellQuest software (BD Pharmingen).

Colony-Forming Unit Assay
Colony-forming unit-granulocyte macrophage (CFU-GM), burst-forming unit/colony-forming unit-erythroid (BFU/CFU-E), and colony-forming unit-mixed (CFU-GEMM) were cultured in methylcellulose (1%) supplemented with fetal calf serum (FCS, 30%), 1% BSA, 0.1 mM ß-mercaptoethanol, 3 IU/ml erythropoietin (Cilage AG, Schaffhausen, Switzerland, http://www.cilag.ch), 10 ng/ml granulocyte macrophage-colony stimulating factor (GM-CSF; Sandoz, Basel, Switzerland, http://www.sandoz.com), 10 ng/ml interleukin-3, and 50 ng/ml SCF. Enriched CD34⁺ cells or expanded cells at 3 x 10³ per milliliter were seeded in triplicate and incubated for 14 days. Colonies were scored in a blinded manner. Colony-forming unit-megakaryocytes (CFU-MK) were assayed using the plasma clot system as described previously [33, 34]. CFU-MK was identified as a cluster of three or more strongly stained CD61-FITC (Dako) positive cells examined by fluorescence microscopy.

Engraftment of Expanded Cells in NOD/SCID Mice
NOD/LtSZ-scid/scid mice were obtained from the Walter and Eliza Hall Institute of Medical Research (Melbourne, VIC, Australia) and bred in the Laboratory Animal Services Centre at The Chinese University of Hong Kong. Mice at 8–10 weeks of age (n = 64) were exposed to 280–320 cGy total body irradiation from a ¹³⁷Cs source (Gammacell 1000 Elite Irradiator; MDS Nordion, Kanata, ON, Canada, http://www.mds.nordion.com). In each independent cord blood experiment (n = 16), cells expanded in the presence or absence of serotonin were infused into sex- and age-matched mice (progenies of 3 x 10⁴ CD34⁺ cells at day 0 per mouse). To prevent the loss of data due to animal mortality, two mice were assigned to each treatment group, and the engraftment parameters averaged as a single datum for analysis, as described previously [33, 34]. These animals were sacrificed 6 weeks post-transplantation. All procedures were approved by the Animal Research Ethics Committee, The Chinese University of Hong Kong.

The engraftment of human (hu) CD45⁺ cells and subsets in the bone marrow (BM), spleen, and peripheral blood (PB) were quantified. For flow cytometric analysis, red blood cells were lysed with 0.83% ammonium chloride and washed with PBS/0.1% BSA. The cells were incubated with mouse IgG and 5% human serum (Gibco) before adding monoclonal antibody specific for huCD45 (PC5; Immunotech, Luminy, France, http://www.beckmancoulter.com/products/pr_immunology.asp), CD34-FITC, and propidium iodide (PI, 10 µg/ml; Sigma) for 20 minutes. Seventy thousand events were acquired. For those BM samples that contained more than 1% human cells (n = 7), we performed additional staining using anti-human antibodies CD19-PE, CD14-PE, CD33-PE, CD61-PE (Dako), and their isotypic controls. Nonviable cells (PI positive) were gated out during data analysis. Human CFU were also analyzed in these BM samples, using methylcellulose culture, and scored after 14 days. As described previously [35], this culture duration was selective for human CFU assay and did not support murine CFU formation, which normally took 7 days.

Human Bone Marrow Colony-Forming Unit-Fibroblast
BM cells were obtained from human donors with informed consent. These cells were recovered from the blood filter during infusion of BM into recipients during the procedure of transplantation. After washing twice with Iscove's modified Dulbecco's medium (IMDM), BM cells (2 x 10⁶ cells) were cultured in 2 ml of IMDM and 10% FCS in triplicates, with or without the addition of serotonin. After 9 days, colony-forming unit-fibroblast (CFU-F) colonies appeared morphologic homogenous, and an aggregate of more than 20 fibroblasts was counted as a CFU-F [36]. Effects of serotonin and other cytokines (50 ng/ml each basic fibroblast growth factor [FGF], platelet-derived growth factor [PDGF], or vascular endothelial growth factor [VEGF]) were assessed in the CFU-F cultures.

Annexin V, Caspase-3, and Mitochondrial Membrane Potential Analysis of M-07e Cells by Flow Cytometry
The megakaryoblastic cell line M-07e (American Type Culture Collection, Manassas, VA, http://www.atcc.org) was maintained in IMDM supplemented with GM-CSF (20 ng/ml) and 10% FCS. Apoptotic cell death was induced by cytokine and serum depletion. We added 5-HT (200 nM), ketanserin (KE, 2 µM), or thrombopoietin (50 ng/ml) to the cultures for 72 hours. Apoptotic cell death was examined using the annexin V-FITC/PI, active caspase-3-PE, and JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine) ApoAlert reagent kits (BD Biosciences, San Diego, http://www.bdbiosciences.com) according to the manufacturer's instructions. Ten thousand events were acquired for each sample and analyzed by flow cytometry using the Lysis II software (FACScan; BD Pharmingen).

Statistical Analysis
Treatment groups were compared using analysis of variance and paired t test or Wilcoxon signed rank test, depending on data distribution, using the SigmaStat software (Jandel Scientific Software, San Rafael, CA). We compared survival rates of NOD/SCID mice using Fisher's exact test. A p value of .05 was considered statistically significant. All values were expressed as mean ± SEM.

	RESULTS

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Effects of Serotonin on Ex Vivo Expansion of CD34⁺ Cells
The cell viability remained high after 8 days of ex vivo culture with (96.0% ± 0.64%) or without (93.5% ± 0.99%) the addition of serotonin. In the presence of TPO, SCF, and FL (TSF), efficient expansions were observed in TNC and all subsets of progenitor cells as demonstrated by flow cytometry and CFU assays (Fig. 1A). The addition of serotonin significantly enhanced the expansion of early progenitor cells (CD34⁺, CD34⁺CD38^–, CFU-GEMM) and committed CFU of the myeloid (CFU-GM), erythroid (BFU/CFU-E), and megakaryocytic (CFU-MK) lineage (p < .001, n = 25). There was also increased expansion of CD61⁺CD41⁺ cells in the presence of serotonin (8.22 ± 0.65 x 10⁵ vs. 6.07 ± 0.54 x 10⁵ cells per milliliter, p < .001). The increased expansion was not only contributed by the higher TNC but also by the significant increase in the proportion of CD34⁺CD38^– cells (1.71% ± 0.16% vs. 1.16% ± 0.14%, p < .001) and density of CFU (Fig. 1B) in day 8 cultures.

Six weeks after expanded cells were infused into sublethally irradiated NOD/SCID mice, there was no difference in the mortality rates of the animals in the TSF (9.4%) and TSF + 5-HT (12.5%) groups. HuCD45⁺ cells were detectable in the BM, spleen, and PB of the mice (Fig. 2A). In the BM, there were engraftments of human hematopoietic cells of the early (CD34⁺), myeloid (CD33⁺, CD14⁺), B-lymphoid (CD19⁺), and megakaryocytic (CD61⁺) lineages (Fig. 2B). In animals that received expanded cells in the TSF + 5-HT group, there were significant increases of engraftment when compared with those only exposed to TSF, as demonstrated by the higher level of huCD45⁺ cells in their BM (5.83% vs. 2.86%, p = .013, n = 16). There were trends of increased engraftment in the PB and spleen of these animals (p < .065, n = 7). In the BM of the TSF + 5-HT animals, we also observed increased human myeloid (CD33⁺, CD14⁺), erythroid (BFU/CFU-E), and early progenitors (CFU-GEMM) (p < .05, n = 7) but not CD19⁺ B lymphocytes (Fig. 2B, 2C).

View larger version (21K): [in this window] [in a new window]   Figure 1. Effects of serotonin on ex vivo expansion of CD34+ cells. Enriched cord blood CD34+ cells at 2 x 104 per milliliter were cultured for 8 days in QBSF-60 medium in the presence of thrombopoietin (50 ng/ml), stem cell factor (50 ng/ml), and FL-3 ligand (80 ng/ml) with or without serotonin (5-HT, 200 nM). (A): 5-HT significantly enhanced expansion of various stem and progenitor cell subpopulations. Results are presented as mean and SEM, n = 25, ** p < .01. (B): 5-HT significantly increased the concentrations of CFU of the myeloid (CFU-GM), erythroid (BFU/CFU-E), megakaryocytic (CFU-MK), and mixed lineages (** p < .01). Abbreviations: 5-HT, 5-hydroxytryptamine; BFU/CFU-E, burst-forming unit/colony-forming unit-erythroid; CFU, colony-forming unit; CFU-E, colony-forming unit-erythroid; CFU-GEMM, colony-forming unit-mixed; CFU-GM, colony-forming unit-granulocyte macrophage; CFU-MK, colony-forming unit-megakaryocyte; TNC, total nucleated cells; TSF, thrombopoietin, stem cell factor, FL-3 ligand.

View larger version (15K): [in this window] [in a new window]   Figure 2. Engraftment of expanded human hematopoietic cells in nonobese diabetic (NOD)/severe combined immunodeficient (SCID) mice. Ex vivo expanded cells in the presence of TSF, with or without serotonin, were infused in sublethally irradiated NOD/SCID mice. (A): After 6 weeks, significantly increased human CD45+ cell engraftment was observed in BM of animals that received expanded cells from cultures with serotonin (p = .013, n = 16). (B): A significant increase of myeloid (CD33+ and CD14+) cell engraftment was observed in BM of animals that received expanded cells from cultures with serotonin (p < .05, n = 16 for CD34+ cells and n = 7 for other subsets). (C): Significant increases of BFU/CFU-E and CFU-GEMM were observed in BM of animals that received expanded cells from cultures with serotonin (p < .01, n = 7). Abbreviations: 5-HT, 5-hydroxytryptamine; BFU/CFU-E, burst-forming unit/colony-forming unit-erythroid; BM, bone marrow; CFU, colony-forming unit; CFU-GEMM, colony-forming unit-mixed; CFU-GM, colony-forming unit-granulocyte macrophage; PB, peripheral blood; TSF, thrombopoietin, stem cell factor, FL-3 ligand.

View larger version (18K): [in this window] [in a new window]   Figure 3. Effect of serotonin on CFU-F from human bone marrow cells. Human BM cells at 1 x 106 per milliliter were cultured in Iscove's modified Dulbecco's medium and 10% fetal calf serum with various doses of serotonin and growth factors for 9 days. (A): 5-HT significantly increased CFU-F formation in a dose-dependent manner. Results are presented as mean and SEM, n = 4, * p < .05, ** p .01. (B): BM cells were cultured in the presence of FGF, PDGF, or VEGF (50 ng/ml each) with or without serotonin (200 nM). Individual factors promoted CFU-F formation compared with control cultures (n = 6, p < .006). The addition of 5-HT significantly enhanced CFU-F formation (* p < .02). Abbreviations: 5-HT, 5-hydroxytryptamine; BM, bone marrow; CFU-F, colony-forming unit-fibroblast; Cont, control; FGF, fibroblast growth factor; PDGF, platelet-derived growth factor; VEGF, vascular endothelial growth factor.

The expression of active caspase-3 (M2, Fig. 5), a downstream effector protein of apoptosis, was significantly increased in nutrient depleted cells (n = 6; _* p = .004). The treatment with 5-HT (## p = .005) or TPO (p = .003) significantly reduced caspase-3 expression. The addition of KE compromised the effects of 5-HT (5-HT vs. 5-HT + KE, + p = .034).

Mitochondria with normal mitochondrial membrane potential (m) concentrate JC-1 into aggregates (red/orange fluorescence in FL2), whereas, in depolarized mitochondria, JC-1 forms monomers (green fluorescence in FL1). Compared with normal M-07e cells, serum- and cytokine-depleted control cultures had increased proportions of cells containing JC-1 monomers (R2, green fluorescence), indicating a drop in m (_* p = .006, n = 7) (Fig. 6). This population of apoptotic cells was significantly decreased in cultures containing 5-HT (# p = .013) or TPO (p = .019). Again, the addition of ketanserin increased the proportion of cells containing damaged mitochondria (+ p = .023). Although the total population of apoptotic cells (R1 + R2) showed a similar pattern of results, R1, a transitional cell subset containing both monomers and aggregates, was not significantly affected by these agents.

View larger version (47K): [in this window] [in a new window]   Figure 4. Effects of serotonin on expression of annexin V on M-07e cells. M-07e cells were cultured in Iscove's modified Dulbecco's medium supplemented with granulocyte macrophage-colony stimulating factor and fetal calf serum (normal). Apoptotic cell death was induced by cytokine and serum depletion (control). Serotonin (5-HT, 200 nM), ketanserin (2 µM), or TPO (50 mg/ml) were added to the cultures for 72 hours. The cells were stained with annexin V-fluorescein isothiocyanate (FL-1) and propidium iodide (PI) (FL-2). R2 denotes early apoptotic cells (annexin V positive and PI negative) and R1 + R2 represents total dead cells. These data demonstrated nutrient deprivation increased cell death (n = 5, control vs. normal, * p < .05, ** p < .01); 5-HT significantly reduced apoptosis and total cell death (5-HT vs. control), whereas the addition of KE nullified the effects of 5-HT (5-HT vs. 5-HT + KE). TPO significantly reduced M-07e cell death. Abbreviations: 5-HT, 5-hydroxytryptamine; KE, ketanserin; TPO, thrombopoietin.

View larger version (36K): [in this window] [in a new window]   Figure 5. Effects of serotonin on expression of caspase-3 in M-07e cells. M-07e cells were stained with caspase-3-PE (FL-2). These data demonstrated nutrient deprivation increased caspase-3 expression (M2; n = 6, ** p = .004); 5-HT (## p = .005) or TPO (p = .003) significantly reduced caspase-3 expression. The addition of KE reduced the effects of 5-HT (+ p = .034). Abbreviations: 5-HT, 5-hydroxytryptamine; KE, ketanserin; TPO, thrombopoietin.

View larger version (55K): [in this window] [in a new window]   Figure 6. Effects of serotonin on mitochondria membrane potential of M-07e cells. M-07e cells were stained with JC-1 reagent. Control cultures had increased proportion of cells containing JC-1 monomers (R2, green fluorescence FL1) (n = 7; * p = .006); 5-HT (# p = .013) or TPO (p = .019) decreased mitochondria membrane damage, whereas the addition of KE increased this population of cells (+ p = .023). R1, a transitional cell subset containing both monomers and aggregates, was not affected by these reagents. Abbreviations: 5-HT, 5-hydroxytryptamine; KE, ketanserin; TPO, thrombopoietin.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion Disclosure of Potential... Acknowledgments References

The mechanisms of serotonin on the hematopoietic system appear complicated and remain not fully understood. It was reported that nine 5-HT receptor subtypes have been identified on various mature blood cells [29]. Our results showed that serotonin rescued cytokine-dependent and 5-HT receptors 2A, 2B, and 2C expressing M-07e cells from induced apoptosis, as shown in the expression of annexin V and caspase-3. The signals could be mediated by the intrinsic pathway involving mitochondria membrane integrity. The antiapoptotic effect of serotonin was similar to that of TPO, a known cytokine for hematopoiesis and megakaryocytopoiesis. The addition of ketanserin, a competitive antagonist of serotonin receptor 5-HT₂ subtype, nullified the antiapoptotic effects of serotonin. Our results are in line with those reported on megakaryocytic cell line HEL that pretreatment with a high dose of serotonin (100 µM) protected subsequent nitric oxide-induced apoptosis as demonstrated by the Comet assay [38]. The antiapoptotic effect of serotonin on other cell types has also been suggested to be mediated by mitochondria signaling, caspase activation, and involved Akt/ERK1/2 pathways [11, 39].

The activities of self-renewal, proliferation, differentiation, and mobilization of hematopoietic stem cells are largely in coordination with their microenvironments [40–43]. We demonstrated that serotonin stimulated human BM stromal cells and synergized with other pleiotropic growth factors FGF, PDGF, or VEGF on CFU-F formation. Taken together, serotonin could promote hematopoietic stem and progenitor cells in a direct manner and indirectly mediate by the enhanced microenvironment of the BM. Our data have provided support to the concept that serotonin might act as a mediator to facilitate crosstalks between hematopoiesis, the BM, and the neural system.

The possible involvement of serotonin in promoting hematopoietic stem cells and BM proliferation has added a new dimension to the multiple functions of this neural transmitter and its receptors on regulating tissue proliferation, differentiation, and repair. Serotonin, a relatively inexpensive agent, could be used for clinical expansion of hematopoietic stem cells for transplantation. The mechanisms of serotonin on hematopoiesis and the BM microenvironment at physiological and tissue damage conditions warrant further investigation.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

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This project was supported by the Li Ka Shing Institute of Health Sciences, Strategic Grant SRP2/02 and Direct Grant CUHK, The Chinese University of Hong Kong.

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