Dopamine Mobilizes Mesenchymal Progenitor Cells Through D2-Class Receptors and Their PI3K/AKT Pathway

Authors


Abstract

As the nervous system exerts direct and indirect effects on stem cells mobilization and catecholamines mobilize hematopoietic stem cells, we hypothesized that dopamine might induce mesenchymal progenitor cells (MPCs) mobilization. We show that dopamine induced in vitro MPCs migration through D2-class receptors, and their alternative phosphoinositide 3-kinase/Akt pathways. Also, administration of catecholamines induced in vivo mobilization of colony-forming unit-fibroblast in mice. In contrast, in vitro and in vivo MPCs migration was suppressed by D2-class receptors antagonists and blocking antibodies, consistent with dopamine signaling pathway implication. In humans, patients treated with L-dopa or catecholaminergic agonists showed a significant increase of a MPC-like population (CD45−CD31−CD34−CD105+) in their peripheral blood. These findings reveal a new link between catecholamines and MPCs mobilization and suggest the potential use of D2-class receptors agonists for mobilization of MPCs in clinical settings. Stem Cells 2014;32:2529–2538

Introduction

Over the last 2 decades, the field of mesenchymal progenitor cells (MPCs) has progressed rapidly from the laboratory to the early clinical trials for a wide range of diseases [1]. Overall, the types of reagents and methods that helped the field of hematopoietic stem cells (HSCs) research can guide, at least in part, what is needed for the MPCs field. It is well known that HSC continuously egress out from the bone marrow (BM) to the circulation under homeostatic conditions and the sympathetic nervous system regulates the hematopoietic niche in BM [2]. At present, the hypothesis of MPCs circulating through the peripheral blood (PB) in steady state is controversial and the molecular mechanisms are very poorly understood [3]. However, there are numerous studies suggesting that under injury conditions, MPCs are stimulated to leave their niche, released into circulation from remote tissues and recruited into damaged tissues [4]. In this regard, hematopoietic and endothelial progenitors also increase transitorily their number in PB after a traumatic event or pharmacological treatment [5].

High amount of catecholamines are secreted into PB in stress situations, mobilizing HSCs [2, 5, 6], with controversial effects on endothelial cells mobilization [6, 7]; conversely, a role on MPCs mobilization by catecholamines is scarcely known. Spiegel et al. proposed the existence of a “brain-bone-blood triad” in which some signals are delivered to the HSCs pool directly, while other effects are exerted indirectly on niche-supporting stromal cells [8]. Recently, it has been demonstrated that MPCs constitute an intrinsic element of the stem cell niche in BM, in close physical association with HSCs, implying a neural and hormonal regulation [9].

The actions of dopamine (DA) are mediated by D1- and D2-classes of dopamine receptors. Usually dopamine receptors activation is mediated by adenylyl cyclase (AC); however, it could also be mediated by transactivation of tyrosine kinase receptors [10]. These receptors are able to active several downstream signal networks, as the phosphoinositide 3-kinase (PI3k)/Akt signaling pathway, which is a survival pathway that regulates cell proliferation, apoptosis, differentiation, and migration [11]. It has been described that several PI3K/Akt stimulators increase MPCs migration, although the downstream target proteins of the PI3K/Akt pathway in MPCs migration are not well understood [12-15].

Here, we show data gathered in vitro, in a in vivo mouse model, and also in primary samples from human patients demonstrating that dopamine receptors are expressed by and have a function in the migration of MPCs, in mice and humans. Dopamine induced D2-class receptors activation in MPCs, which in turn stimulated the noncanonical PI3K/Akt signaling pathway.

Materials and Methods

Culture of hMPCs from PB

Mobilized PB samples were obtained from healthy donors at Hospital Niño Jesús (Madrid, Spain), after informed consent. Samples were centrifuged in a density gradient using Ficoll-Paque (GE Healthcare, Piscataway, NJ, www.gelifesciences.com) to obtain the mononuclear cells (MNC). In experiments where we tested matrix, nontreated culture flasks were cover with several matrix: 7.5 µg/cm2 of matrigel (BD Biosciences, San Jose, CA, www.bdbioscience.com), 6.25 µg/cm2 of fibronectin (R&D Systems, Minneapolis, MN, www.rndsystems.com), 6.25 µg/cm2 of vitronectin, 0.1 mg/ml of Cultrex Basement Membrane Extract (R&D Systems), or 10% of dextrose-gelatin-veronal (Lonza, Allendale, NJ, www.lonza.com). MNC were incubated in commercial MPC-specific medium: BMMPC (Lonza), NH Expansion Medium (Miltenyi Biotec, Bergisch Gladbach, Germany, www.miltenyibiotec.com), Stemline MPC Expansion Medium (Sigma-Aldrich, St. Louis, MO, www.sigmaaldrich.com), or Dulbecco's modified Eagle's medium (DMEM) (Sigma-Aldrich) plus 10%–20% of fetal bovine serum (FBS). In cultures where growth factors were used, their concentration was 10 ng/ml. Individual cell cultures in which we obtained a homogeneous human PB-MPCs at fourth passage were defined as positive.

Human PB-Derived MPCs Characterization

Human PB-MPCs were characterized following The International Society for Cellular Therapy (ISCT) position statement [16]. In coculture experiments, human CD3+ cells were isolated using immunomagnetics methods (Miltenyi Biotec). PB-MPC and T lymphocytes were cocultures for 96 hours and were costimulated with 10 µg/ml of anti-CD3 and 1 µg/ml of anti-CD28 antibodies (BD Bioscience). Mitogen-activated proliferation assays were performed by incubating 1 × 105 MNC with 10 µg/ml phytohaemagglutinin (PHA). The cells were harvested on day 3 and T-cell activation and cell cycle was measured by flow cytometry.

Human PB-MPCs In Vivo Immunomodulation Assays

A model of BM haploidentical transplantation was conducted by transplanting BM cells from C57Bl/6 (H2b/b) mice into B6D2F1 (H2b/d) recipients as previously reported [17]. Briefly, female mice B6D2F1 recipients were irradiated with 9 Gy of x-ray and injected with 5 × 106 BM cells mixed with 2 × 106 T cells from spleen. After 0, 7, and 14 days, the recipients were injected with 1 × 104 hPB- or hBM-MPCs. Procedures were approved by the Animal Experimentation Ethical Committee according to all external and internal biosafety and bioethics guidelines.

In Vitro Cell Migration Assays

Transwells (8-µm pore filters, BD Biosciences) were coated with 0.1% gelatin (Sigma-Aldrich) and 5 × 104 hMPCs were transferred to the upper chambers. Cells were incubated in the presence of 50 µM dopamine, 50 µM bromocriptine, 50 µM SKF-38393, 50 µM eticlopride, 50 µM SCH-23390, or 400 µM AS-604850 in the bottom chamber for 24 hours. A simplified checkerboard assay was also performed by incubating the cells with 50 µM DA in the transwell and DMEM alone in the bottom chamber. Treatment with eticlopride, SCH-23390, and AS-604850 was performed by preincubating the cells with these reagents for 1 hour. Blocking of dopamine receptors was achieved by preincubating the cells for one hour with 10 µg/ml anti-DR2 or anti-DR3 rabbit polyclonal antibodies (Alomone Labs, Jerusalem, Israel, www.alomone.com). An unrelated rabbit polyclonal antibody (Santa Cruz Biotechnology, Dallas, Texas, www.scbt.com) was used as an isotype control. Migrated cells were fixed and stained with crystal violet. For statistical analysis, cells were manually counted in 10 high-power fields and each experiment was repeated three times (n = 6).

Measurement of Intracellular cAMP Levels in Human MPCs

cAMP levels in human PB-MPCs were measured using the Cyclic AMP XP Assay Kit (Cell Signaling Technology, Danvers, MA, www.cellsignal.com) according to manufacturer protocol. Briefly, 50 × 106 cells were incubated in serum-free DMEM in presence of 50 µM of dopamine for 10, 20, and 30 minutes. Each experiment was repeated three times (n = 6).

Western Blot Analysis

Proteins were extracted with SDS sample buffer, separated by 10% SDS-PAGE, blotted on polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA, www.bio-rad.com), and detected using Immobilon Western Chemiluminescent horseradish peroxidase (HRP) Substrate (Merck Millipore, Billerica, MA, www.millipore.com). Primary antibodies were rabbit monoclonal anti-AKT1 (phospho-S473) antibody, 1:1,000 dilution (Epitomics, Burlingame, CA, www.epitomics.com) and mouse monoclonal anti-β-actin, clone AC-15, 1:5,000 dilution (Sigma-Aldrich). Secondary antibodies were polyclonal goat anti-rabbit and anti-mouse immunoglobulins/HRP, 1:3,000 dilution (Dako, Glostrup, Denmarkh, www.dako.com). pAKT-S473 protein signal intensities were quantified using the ImageJ software (http://rsbweb.nih.gov/ij/) after normalization to β-actin signal.

Murine Mobilization Experiments

FVB/NHanTMHsd female mice, between 8 and 12 weeks of age, were treated with different drugs. Epinephrine was diluted in water and administered intraperitoneally (i.p.) at a dose of 2 mg/kg. Dopamine (Grifols, Barcelona, Spain, www.grifols.com) was diluted in 0.9% saline and administered i.p. at a dose of 50 mg/kg. The control group was inoculated i.p. with 0.9% saline. The granulocyte colony-stimulating factor (G-CSF) (200 µg/kg; Amgen, Thousand Oaks, CA, www.amgen.com) was diluted in 5% dextrose solution and administered subcutaneously. Dopamine receptors antagonist, eticlopride (10 mg/kg), U99194 (20 mg/kg), and SCH-23390 (10 mg/kg) were diluted in water and administered subcutaneously 30 minutes before of dopamine inoculation. All drugs were administered for 4 consecutive days, every 24 hours. The protocol was approved by the Animal Experimentation Ethical Committee.

Blood was drawn by cardiac puncture. MNC were resuspended in DMEM plus 20% FBS and plated at 650,000 MNC/cm2 on plates coated with fibronectin. The spleens were dispersed mechanically in 5 ml PBS by passing the cell suspension through 70 µm filter to obtain a suspension of cells. BM, femur, and tibia of mice were also collected. The count of colony-forming unit-fibroblast (CFU-F) was performed at 7 days of culture.

Flow Cytometry Analysis

In vitro human PB-MPCs cultures were analyzed by flow cytometry as previously described [18]. Basically, cultured cells were incubated with appropriate monoclonal antibodies (Supporting Information Table S1) or an appropriate negative control (isotype immunoglobulins). For T-cell activation analysis, cells were staining with anti-human antibodies (Supporting Information Table S1). In addition, for cell cycle studies of T lymphocytes, 2 × 105 cells were fixed and resuspended in 1 ml of PBS containing 50 µg/ml of propidium iodide (PI) and 200 µg/ml RNase. Cells were incubated during 30 minutes at 37°C and analyzed by flow cytometry in a FACSCalibur cytometer (BD Biosciences).

PBs from 26 healthy volunteers and 56 patients with a cerebral stroke were obtained and analyzed. The study protocol was approved by the Centro de Referencia Estatal de Atención al Daño Cerebral Internal Review Board, and patients gave written consent. MNCs were obtained using Ficoll-Hypaque and 2 × 106 cells were incubated with fluorochrome-conjugated monoclonal antibodies (Supporting Information Table S1), washed, and analyzed.

Immunostaining

Tissues were fixed in 10% neutral formaline and cells in 4% formaldehyde. After immunoperoxidase staining, they were visualized using the Vectastain ABC Kit (Vector Laboratories, Peterborough, UK, www.vectorlabs.co.uk). Cells were counterstained with hematoxylin, placed in histoclear (National Diagnostic, Atlanta, Georgia, www.nationaldiagnostics.com), and mounted. Images were taken using a light microscope and digital camera (Nikon, Dusseldorf, Germany, www.nikon.com).

For the immunofluorescence staining, cells were grown on glass chamber slides (Thermo Fisher Scientific, Waltham, MA, www.thermofisher.com), stained with specific primary antibodies, and nuclei cells were counterstained with Dapi. For negative controls, cells were incubated with isotype matched control antibodies. Vectashield mounting media (Vector Laboratories) were added and images were taken using a confocal microscope (Leica, Wetzlar, Germany, www.leica-microsystems.com).

In Vivo Transplantation of Subcutaneous Ossicles Generated with PB-Derived Human Subpopulations

Cell subpopulations were isolated from human mobilized PB using immunomagnetic MACS microbeads, and 1 × 105 CD45−CD31−CD34−CD105+ and 1 × 106 CD45−CD31−CD34−CD105− cells were used to prepare each implant, as previously described [19]. Briefly, 40 mg of hydroxyapatite/tricalcium phosphate (HA/TCP) powder (Biomatlante, Vigneux de Bretagne, France, www.biomatlante.com) was deposited in a 50 ml falcon tube and washed with 1 ml of DMEM culture medium. Fresh cells were mixed with HA/TCP powder and centrifuged (1,000 rpm, 5 minutes). Homogeneous cell seeding was checked and then the ceramic/MPCs compound was cultured overnight. Culture medium was carefully removed and culture medium with 35 µg of BMP2 (Noricum, Madrid, Spain, www.noricumimplants.com) and fibrin was incorporated in order to prepare final ossicles. Finally, the ossicles were implanted s.c. under the dorsal skin of anesthetized NOD/SCID mice and 1 month later, the ossicles were harvested for their analysis.

Statistics

All statistical analyses were performed using Stata/IC 11.0 (College Station, TX, www.stata.com). The nonparametric Wilcoxon rank-sum test was used to compare quantitative variables. Results were considered statistically significant with p < .05. All graphics present the mean ± SE.

Results

Obtaining Human MPCs from PB Mobilized with G-CSF

The frequency of MPCs in peripheral circulation seems to be very rare in steady-state conditions. Even using mobilizing agents, such as G-CSF, the PB-derived-MPCs (PB-MPCs) usually have a limited expanding potential [20]. As it is well known, human PB-MPCs do not form classic CFU-F, so we defined a semiquantitative method: a cell culture was defined as positive when we obtained a homogeneous culture of hPB-MPCs and it could be expanded, at least during four passages. In order to improve the protocol for obtaining human PB-MPC, we performed a combination of multiple cell culture conditions. Our results showed that the initial cell density was critical for obtaining hPB-MPCs. Thus, the best result was obtained with an initial concentration of 1.25 × 106 MNC per square centimeter (Supporting Information Fig. S1A), regardless of other parameters. Also, as shown in Figure 1A, there were differences among substrates, being fibronectin the most effective. In addition, fresh mobilized-MNC resulted in a higher hPB-MPCs production in all experiments. Finally, these hPB-MPCs could be in vitro expanded for a long time, maintaining a normal karyotype (Supporting Information Fig. 1B).

Figure 1.

Isolation and characterization of mesenchymal progenitor cells derived from human G-CSF-mobilized peripheral blood. (A): Cultures of human MPCs from fresh (black bar) or frozen (gray bar) mononuclear cells from complete mobilized apheresis or from their fresh (white bar) or frozen (striped bar) CD34− fraction, and MNCs from nonmobilized buffy coat (dotted bar). Data are shown as mean of eight experiments. (B): Adherent cell growth of primary cultures of hPB-MNCs and their differentiation capacities when cells were cultured in adipogenic (left) or osteogenic (right) specific mediums. (C): Histograms show immunophenotype of hPB-MPCs, using fluorescence intensity with specific antibodies (black area) and isotype as a negative control (white area). (D, E): Isolated CD3+ T cells were cultured in the absence (c) or presence of allogeneic hPB-MNCs, at different ratios (1:1 and 1:10). Data are shown as mean SE of three experiments, where the asterisk (*) indicates statistically significant difference with p < .05. (F): In vivo effect of PBS (black line), human peripheral blood-MPCs (dotted line), and human bone marrow-MPCs (striped line) upon the generation of GvHD produced after haploidentical hematopoietic transplantation in mice. Abbreviations: DGV, dextrose-gelatin-veronal; G-SCF, Granulocyte colony-stimulating factor.

Characterization of Human PB-Derived MPCs

We successfully isolated and expanded homogenous spindle-shaped populations with lineage-differentiation properties (Fig. 1B). The phenotype of hPB-MPCs was determined by flow cytometry, corresponding with a real MPCs population (Fig. 1C). Moreover, we decided to study their immunomodulatory properties according to ISCT guidelines [21]. We isolated CD3+ T cells from PB-derived human MNC using immunomagnetic beads and these cells were mixed in vitro with hPB-MPCs at different ratios. CD3+ cells were activated using anti-CD3 plus anti-CD28 monoclonal antibodies. As shown in Figure 1D, the percentage of activated T cells (CD3+CD69−CD25+) was reduced when hPB-MPCs were presented in the culture. In addition, hPB-MPCs were able to inhibit lymphocytes cell cycle in a dose-dependent manner (Fig. 1E). Similar results were obtained when we analyzed cocultures of hPB-MPCs with T lymphocytes activated with phytohemmaglutinine (Supporting Information Fig. S1). MPCs have been efficiently used for the control of graft-versus-host disease (GvHD) in murine models and also in human clinical trials [22, 23]. Thus, we used a xenogenic GvHD mice model for testing the immunomodulatory properties of hPB-MPCs (Fig. 1F). hPB-MPCs protected the mice from lethal GvHD at the same level as hBM-MPCs did. In summary, our results indicated that the cells we obtained from human mobilized PB fulfilled all ISCT criteria for hMPCs.

Dopamine Induces In Vitro Migration of hBM- and hPB-MPCs Through D2-Class Receptors

In order to test the role of dopamine in mobilization of MPCs into PB we studied the in vitro expression of dopamine receptors in human BM- and PB-derived MPCs by immunocytochemistry. We found that steady state hBM- and hPB-MPCs expressed the six subtypes of dopamine receptor (Fig. 2A), indicating a potential role of dopamine in their cellular homeostasis.

Figure 2.

Expression of dopamine receptors in human mesenchymal progenitor cells (hMPCs) and analysis of their DA-induced in vitro migration. (A): Immunocytochemical analysis of dopamine receptors expression in human MPCs derived from BM or PB. Scale bar = 100 µm. Data shown are representative of three independent experiments. (B): Representative images showing the effect of 50 µM DA on in vitro migration of hMPCs in transwell chambers, growing in DMEM plus 10% serum or in DMEM alone (serum starved; SS) for 24 hours. Migrated cells were fixed and stained with crystal violet. (C): Graph showing the average number of migrated hMPCs per HPF (high-power field) after 50 µM DA treatment. The graph shows the average number of migrated hMPCs in 10 HPFs from three experiments combined (n = 6) and bars represent the SEM. Statistical analysis was performed according to the Wilcoxon test. *, p < .05; **, p < .01. Abbreviations: BM, bone marrow; DA, dopamine; MSC, mesenchymal progenitor cells; PB, peripheral blood; DR, dopamine receptor.

Next, we used an in vitro migration assay to investigate the ability of dopamine to induce migration of hMPCs. As shown in Figure 2B, the basal migration observed in hMPCs in the presence of medium alone was increased by the addition of dopamine. The increase was higher in human MPCs from PB (2.9-fold) than from BM (2-fold). Serum starvation induced a higher increase in hMPCs migration in both types of cells (5.3-fold and 2.8-fold in cells from PB and BM, respectively) (Fig. 2C). In addition, a simplified checkerboard assay was performed with serum-starved PB-MSCs adding DA to the transwell only (the bottom chamber contained medium alone) and no significant cell migration was observed (Supporting Information Fig. S1).

We next studied the type of dopamine receptors involved in the observed migration. First, we used agonists and antagonists for D1-class and D2-class receptors and assessed their effects on hMPCs in vitro migration (Fig. 3A). SKF-38393, a D1R-class agonist, had no effects on hMPCs migration, nor did SCH-23390, a D1R-class antagonist. These results indicate that dopamine hMPCS migration seems not to be directed by D1-class receptors. However, bromocriptine, a D2R-class agonist, was found to clearly increase hMPCs migration. In addition, eticlopride, a D2R-class antagonist, was able to block both dopamine-induced and basal migration in BM- and PB-MPCs (Fig. 3A). Then, we attempted to block D2-class receptors more specifically, using anti-DR2 or anti-DR3 antibodies that recognize their extracellular domain. Figure 3 shows that preincubation of PB-MSCs with anti-DR2 antibody resulted in no significant DA-induced migration whereas the anti-DR3 antibody did not abolish completely the DA effect, as certain level of significant migration was observed (Fig. 3B). Taken together these results indicate that the increased in vitro migration induced by DA occurs mostly through D2-class receptors. Moreover, they suggest that D2R receptors play a predominant role in this mechanism.

Figure 3.

Analysis of the pathway involved in DA signaling in human mesenchymal progenitor cells (hMPCs). (A): Effect of D1R- and D2R-class agonists and antagonists on in vitro migration of hMPCs. DA, SKF-38393 (D1-class agonist), SCH-23390 (D1-class antagonist), bromocriptine (D2-class agonist), and eticlopride (D2-class agonist) were all assayed at 50 µM concentrations in transwell chambers. (B): Effect of anti-DR2 or -DR3-specific antibodies on in vitro migration of hMPCs from PB. All antibodies were preincubated for 1 hour and assayed at 10 µg/ml in transwell chambers. (C): Determination of cAMP levels in hPB-MPCs in response to 50 µM DA. Intracellular levels of cAMP were measured as explained in Materials and Methods at the indicated times (minutes). (D): Effect of PI3K inhibition on in vitro migration of hMPCs in transwell chambers. Experimental conditions and statistical analysis were identical as above with the difference that AS-604850 was assayed at a 400 µM concentration and the control and DA samples contained ethanol (AS-604850 solvent). (E): Western blot analysis of phospho-AKT-S473 in the absence (control) or presence of 50 µM DA from 1 to 40 minutes. (F): Densitometric analysis of relative levels of pAKT-S473. The graph shows fold changes of normalized pAKT-S473 protein signal intensities (compared with 0 minute control). Results are means ± SEM (n = 2, experimental repeats). For significance of data the asterisk (*) indicates comparison of the sample versus control group whereas the hash (#) indicates comparison of the sample versus DA group. *, p < .05; **, p < .01; ##, p < .01. Abbreviations: BM, bone marrow; DA, dopamine; HPF, high-power field; MSC, mesenchymal progenitor cells; PB, peripheral blood; SS, serum starvation.

Dopamine Induces the PI3K/Akt Pathway

The classic pathway of D2R-class activation induces inhibition of AC. However, we did not see changes in AC activity of hMPCs after dopamine treatment (Fig. 3C) indicating the activation of an alternative signaling pathway. D2R-class activation also could be mediated by transactivation of a tyrosine kinase receptor [10]. These receptors are able to activate several downstream signal networks, as the PI3k/Akt signaling pathway [11]. To assess the function of the PI3K pathway in dopamine-induced BM- and PB-MPCs migration, we incubated hMPCs in transwell chambers with dopamine, in the presence of AS-604850, a PI3K inhibitor. As shown in Figure 3D, cell migration was significantly suppressed when hMPCs were incubated with AS-604850, as the number of migrated cells was decreased beyond the basal migration. In addition, hMPCs were incubated with dopamine from 1 to 40 minutes and we studied the phosphorylation of Akt. As shown in Figure 3E, 3F, the amount of phosphorylated Akt increased significantly with dopamine incubation. In summary, our data suggest that dopamine-induced hMPCs migration is mediated via the PI3K/Akt pathway.

In Vivo Mobilization of Murine MPCs Using Dopamine

We injected groups of mice during 4 days with catecholamines (dopamine and epinephrine) and G-CSF, as a positive control. We collected their PB and cultured their PB-MNC in a CFU-F assay. We obtained an increase of CFU-F numbers in the G-CSF-treated group compared to those found with PBS. Interestingly, we obtained a higher number of CFU-F in the dopamine and epinephrine-treated groups (Fig. 4A). The differences were statistically significant only when comparing the mice mobilized with DA versus those mobilized with PBS, indicating the ability of dopamine to mobilize murine MPCs (mMPCs) to PB. In all experiments, we found a low decrease (no statistically significant) of CFU-F numbers in the BM of the G-CSF-treated, but not in the dopamine- and epinephrine-treated groups (Fig. 4B). This decrease in CFU-F in BM of mice injected with G-CSF was also previously reported by other group [24]. These data indicated a more consistent mobilization of mMPCs using dopamine than G-CSF, without depletion of CFU-F in BM, and without aberrant CFU-F accumulation in spleen of mobilized subjects (Fig. 4C).

Figure 4.

Dopamine-induced mobilization in mice. Mice were inoculated during 4 days with PBS (control), G-CSF, dopamine, or epinephrine. Mononuclear cells were obtained from peripheral blood (A), bone marrow (B), and spleen (C), cultured during 7 days and then CFU-Fs were counted. (D): Identical experiments were performed with mice treated with D2-class antagonist (eticlopride and U-99194) and the D1-class antagonist SCH-23390, plus dopamine. Data are shown as mean SE of three experiments, where the asterisk (*) indicates statistically significant difference with p < .01. Abbreviation: CFU-F, colony-forming unit-fibroblast.

In order to test the direct implication of dopamine receptors in mMPCs mobilization, we used D1R-class and D2R-class antagonists. In agreement with in vitro data, both eticlopride and U-99194, D2R-class antagonists, prevented the dopamine mobilization of mMPCs. In contrast, the D1R-class antagonist SCH-23390 did not affect mMPCs mobilization (Fig. 4D). These data indicate that in vivo mMPCs mobilization by dopamine is induced specifically through dopamine D2-class receptors.

Analysis and Characterization of Circulating Human MPCs

MPCs do not have a specific surface marker which enables their in vivo detection. Aiming to phenotypically characterize hMPCs in fresh PB apheresis, we studied diverse combinations of markers. Other groups have published that CD105 can be used to identify MPCs in BM and PB [25-31], including a most primitive human MPCs subpopulation [30]. We defined a cell subpopulation characterized by the profile CD45-CD31-CD34-CD105+ (Fig. 5A). This subpopulation was negative for other hematopoietic and endothelial markers in PB samples processed in fresh (Supporting Information Fig. S2). However, these cells upregulated specific hMPCs markers in culture (Fig. 5B). Unfortunately, all attempts for in vitro expansion of this subpopulation were unsuccessful. In this sense, other authors have previously published similar negative results, using CD105+ cells from human PB as the starting cell population [29]. A possible explanation could be that the hMPC-like population in PB could be in G0, as it has been reported for the hematopoietic precursors in PB after G-CSF mobilization [32, 33]. We next studied the cell cycle status of the hMPC-like subpopulation and found that all CD45−CD31−CD34−CD105+ cells from PB were in G0/G1, with no cells in S or G2/M. Also, a small cell fraction was at the sub-G0-phase, indicating an apoptotic process (Fig. 5C).

Figure 5.

Characterization in vitro and in vivo of human peripheral blood-derived CD45−CD31−CD34−CD105+ subpopulation. (A): Representative flow cytometry analysis of human peripheral blood showing the CD45−CD31−CD34−CD105+ subpopulation. (B): Immunofluorescence of CD45−CD31−CD34−CD105+ cells after their adhesion in cell culture flask. (C): Cell cycle status of immunomagnetically isolated CD45−CD31−CD34−CD105+ subpopulations. (D): Representative ectopic ossicles formed from CD45−CD31−CD34−CD105− or CD45−CD31−CD34−CD105+ cell subpopulations. (E): Immunohistochemistry, using anti-human β2 microglobulin, showing positive cells in ectopic bone marrow (BM) from CD105+ subpopulation. Scale bar = 50 mm. (F): Immunohistochemistries using anti-human vimentin (left panel) and anti-human mitochondria (right panel). Images showing positive cells in ectopic BM from CD105+ subpopulation. Scale bar = 100 mm. (G): Immunohistochemistry, using anti-human adipophilin (left panel) and anti-human osteonectin (right panel), showing positive cells in ectopic BM from CD105+ subpopulation. Scale bar = 50 mm.

Ectopic implantation of hMPCs has been recently reported as a bona fide method to assess in vivo differentiation potential and characterization of these cell populations [19, 30, 34, 35]. To further define the PB-derived CD45−CD31−CD34−CD105+ cells as a real hMPCs population, we attached the cells onto a ceramic compound and implanted it into immunodeficient mice. Figure 5D shows a gross morphology of the harvested ossicles, where powders were clearly visible. Ossicles from the CD105+ subpopulation showed a harder consistency and denser vascularization when comparing with ossicles generated from the CD105− subpopulation. Using anti-human antibodies, we found positive cells only in ectopic ossicles generated from the CD105+ subpopulation (Fig. 5E). Human cells were observed in calcified areas, fibrous, and BM-like tissues, where positive cells either formed adipose tissue or appeared as interstitial cells. These results were confirmed using an anti-human vimentin antibody (Fig. 5F). In addition, we used tissue-specific anti-human antibodies. Figure 5G shows representative images, indicating the capacity of these cells to engraft and differentiate into appropriate tissues. In sum, all data indicated that the PB-derived CD45−CD31−CD34−CD105+ subpopulation was highly enriched in human MPCs.

Catecholamines Mobilize MPCs in Humans

In order to study the mobilization of MPC-like subpopulations by catecholamines in humans, we identified a group of patients treated with catecholamines or their agonists. These patients suffered of a parkinsonian syndrome after a cerebral stroke. As controls we studied either patients with stroke but without parkinsonism symptoms (not treated with catecholaminergic drugs), or healthy donors. We found a statistically significant increase of the CD45−CD31−CD34−CD105+ subpopulation in patients treated with catecholamines, compared with any other group (Fig. 6), indicating a mobilization of MPCs in humans treated with catecholamines.

Figure 6.

Catecholamines induced mobilization in humans. Percentage of CD45−CD31−CD34−CD105+ subpopulation in three groups: normal donors (healthy), patients of a cerebral stroke treated with catecholamines (catecholamines), and patients of a cerebral stroke without catecholamines treatment (brain damage) (mean ± SD, n = 12 samples per group). The higher percentage of “Catecholamines” group is statistically significant respect the other groups (p < .005).

Discussion

The nervous system exerts direct and indirect effects on stem cells, the immune system, the bone, and the supportive stromal microenvironment, in order to maintain homeostasis [8]. Diverse neurotransmitters regulate retention, proliferation, and recruitment of HSCs through noradrenergic sympathetic nerve fibers [36], which are closely associated with MPCs, forming the neuroreticular complex [9]. Recent data support the “brain-bone-blood” regulatory system hypothesis; since a G-CSF activation of peripheral noradrenergic neurons induces mobilization of HSCs [2], and also the neuropeptide substance-P is able to mobilize CD29+ stromal-like cells in mice and rabbits [37]. Stress, injury, and inflammation induce the mobilization of stem cells as a steady-state homeostatic process [38]. Hematopoietic and endothelial progenitors increase transitorily their numbers in PB after a traumatic event. In addition, recent reports suggested that mesenchymal cells would be mobilized in humans after injuries such as wound healing [39, 40], hip fracture [41], or cardiac and/or respiratory failure [42]. In this regard, we previously described a similar result in acute skeletal muscle injury in subjects running a long distance race [43]. We decided to study the effect of catecholamines on MPCs mobilization since it is well known that catecholamines significantly increase after an intensive exercise [44] and, also, catecholamines mobilize HSCs [2], while contradictory data exist about endothelial cells mobilization using catecholamines [6, 7]. A previous study showed that treatment with eticlopride, a D2 receptor antagonist, increased the numbers of circulating MPCs in PB blood of wound bearing mice [45], although treatment with other D1R-class and D2R-class antagonists had no effects on the mobilization of MPCs. Results from this article did not define the precise role of dopamine over MPCs in steady state. However, our results indicate a clear in vitro MPCs migration and in vivo MPCs mobilization using dopamine and their D2-class receptors, mainly the D2 receptor.

Most studies attempting to isolate MPCs from PB have used culture conditions similar to those defined for BM-derived MPCs [46]. The mechanisms underlying the migration of MPCs remain unclear but it would seem likely that MPCs transmigrate into tissues by a similar mechanism to that of leukocytes, using some (but not all) of the same molecules [47]. In addition, it was published that adhesion of MPCs to fibronectin activates the mechanism that controls MPCs adhesion and migration [48]. This data are in agreement with our results where we found a high expression of CD49d (α4) in fresh circulating hMPCs, and it is well known that activated α4β1 and α4β7 integrins are receptors of fibronectin [49].

Some authors have claimed a word of caution about circulating MPCs because they indicate that although there is evidence for the existence of circulating MPCs, an unpersuasive fulfillment of the MPCs criteria is still lacking, as proof of in vivo function [50]. In this regard others authors have shown MPCs functions using in vivo models [37, 51-53]. We here demonstrate a fully characterization of human PB-derived MPCs following the ISCT criteria, including in vitro and in vivo immunological and differentiation studies.

Classic dopamine receptors signal by AC and cAMP production. AC is stimulated by D1R-class, while D2R-class inhibits AC. However, dopamine receptors activation also could be mediated by transactivation of tyrosine kinase receptors [10]. These receptors are able to active the PI3k/Akt signaling pathway [11]. In this regard, Akt is phosphorylated by PI3K and thereby seems to be linked to migration and adhesion in several cell types [54, 55], including MPCs [48, 56, 57]. Tyrosine kinase receptors of basic fibroblast growth factor and platelet-derived growth factor-BB also use this pathway to increase the migratory activity of human MPCs [13].

Conclusion

In conclusion, our results suggest that catecholamines can mobilize MPCs into PB, both in mice and humans. These findings reveal a new link between catecholamines and MPCs mobilization supporting the brain-bone-blood regulatory system hypothesis and open the door to mobilize, in the future, human MPCs for clinical applications.

Acknowledgments

We thank Iván Gutierrez, Ander Abarrategi, Manuel Masip, Mar Arriero, and Daniel Pérez for his assistance in several techniques. This work was supported by grants from the Fondo de Investigaciones Sanitarias (FIS; PI05/2217 and PI08/0029), the Madrid Regional Government (S-BIO-0204–2006, MesenCAM; P2010/BMD-2420, CellCAM) in Spain, and Consejería de Salud de la Junta de Andalucía (0027/2006) to J.G.-C. The experiments were approved by the appropriate committees.

Author Contributions

I.M. and M.Á.R.: collection and/or assembly of data, and data analysis and interpretation; I.C., L.M.-P., and T.d.l.C.: collection and/or assembly of data; A.Z. and M.R.: conception and design and manuscript writing; C.G.: conception and design and provision of study material of patients; J.G.-C.: conception and design, data analysis and interpretation, financial support, manuscript writing, and final approval of manuscript. I.M. and M.Á.R. contributed equally to this work.

Disclosure of Potential Conflicts of Interest

The authors indicate no potential conflicts of interest.

Ancillary