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First published online October 27, 2005
Stem Cells Vol. 24 No. 4 April 2006, pp. 1030 -1041
doi:10.1634/stemcells.2005-0319; www.StemCells.com
© 2006 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

Human Bone Marrow Stromal Cells Express a Distinct Set of Biologically Functional Chemokine Receptors

Marek Honczarenkoa, Yi Lea, Marcin Swierkowskia, Ionita Ghiranb, Aleksandra M. Glodeka, Leslie E. Silbersteina

a Joint Program in Transfusion Medicine, Children’s Hospital Boston, Harvard Medical School, Boston, Massachusetts, USA;
b Divisions of Allergy-Inflammation and Infectious Diseases at the Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA

Key Words. Stromal cells • Chemokines • Transplantation • Migration • Bone marrow microenvironment

Correspondence: Leslie E. Silberstein, M.D., Joint Program in Transfusion Medicine, Children’s Hospital Boston, Harvard Medical School, Karp Family Research Bldg, RB10217, 1 Blackfan Circle, Boston, Massachusetts 02115, USA. Telephone: 617-919-2588; Fax: 617-730-0615; e-mail: leslie.silberstein{at}childrens.harvard.edu

Received July 14, 2005; accepted for publication October 20, 2005.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Stromal cells isolated from bone marrow (BMSCs), often referred to as mesenchymal stem cells, are currently under investigation for a variety of therapeutic applications. However, limited data are available regarding receptors that can influence their homing to and positioning within the bone marrow. In the present study, we found that second passage BMSCs express a unique set of chemokine receptors: three CC chemokine receptors (CCR1, CCR7, and CCR9) and three CXC chemokine receptors (CXCR4, CXCR5, and CXCR6). BMSCs cultured in serum-free medium secrete several chemokine ligands (CCL2, CCL4, CCL5, CCL20, CXCL12, CXCL8, and CX3CL1). The surface-expressed chemokine receptors were functional by several criteria. Stimulation of BMSCs with chemokine ligands triggers phosphorylation of the mitogen-activated protein kinase (e.g., extracellular signal–related kinase [ERK]-1 and ERK-2) and focal adhesion kinase signaling pathways. In addition, CXCL12 selectively activates signal transducer and activator of transcription (STAT)-5 whereas CCL5 activates STAT-1. In cell biologic assays, all of the chemokines tested stimulate chemotaxis of BMSCs, and CXCL12 induces cytoskeleton F-actin polymerization. Studies of culture-expanded BMSCs, for example, 12–16 passages, indicate loss of surface expression of all chemokine receptors and lack of chemotactic response to chemokines. The loss in chemokine receptor expression is accompanied by a decrease in expression of adhesion molecules (ICAM-1, ICAM-2, and vascular cell adhesion molecule 1) and CD157, while expression of CD90 and CD105 is maintained. The change in BMSC phenotype is associated with slowing of cell growth and increased spontaneous apoptosis. These findings suggest that several chemokine axes may operate in BMSC biology and may be important parameters in the validation of cultured BMSCs intended for cell therapy.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The growth and differentiation of hematopoietic progenitor cells in the bone marrow rely on instructive signals provided by a specialized microenvironment. Stromal cells exert their effects on hematopoietic cells via direct cell–cell interactions as well as by releasing soluble factors [1–3]. However, stromal cells in turn also might receive signals provided by developing hematopoietic cells in the bone marrow microenvironment [4, 5]. It is believed that in order to regulate hematopoiesis in vivo, stromal cells are organized into specialized microenvironmental compartments or niches [6]. However, very little is known about the structural organization of those microcompartments [6], as well as about factors that govern the growth, maintenance, and localization of stromal cells. Stromal cells isolated from bone marrow (BMSCs) form a heterogeneous and fibroblastic in appearance population of nonhematopoietic cells [7]. They are often referred to as mesenchymal stem cells (MSCs) because of their potential to differentiate into various connective tissue lineages including adipocytes, osteoblasts, chondrocytes, and myoblasts [8, 9].

Chemokines are a family of small polypeptides that navigate hematopoietic cell trafficking and localization in tissue compartments [10]. In addition, chemokines are thought to play an important role in cell activation, differentiation, and survival [11–13]. We hypothesized that chemokines in the bone marrow milieu might play a role in stromal cell positioning and/or differentiation and thus contribute to the formation of specific bone marrow microenvironments. To this end we examined BMSCs for chemokine receptor expression and function. Our studies show that human BMSCs express three CC chemokine receptors (CCR1, CCR7, and CCR9; of nine tested) and three CXC chemokine receptors (CXCR4, CXCR5, and CXCR6; of six tested). Ligand binding of these chemokine receptors induced phosphorylation of mitogen-activated protein kinase (MAPK) and/or focal adhesion kinase (FAK) pathways and selective activation of specific signal transducer and activator of transcription (STAT) transcription factors (STAT-5 and STAT-1). Chemokines corresponding to BMSC surface receptors also induced cellular responses, for example, specific chemotaxis and ß-actin filament reorganization (CXCL12). BMSCs cultured in vitro were themselves a source of chemokines. These observations indicate that multiple chemokines, in addition to being essential for hematopoiesis [14], also may play an important role in the development of nonhematopoietic stromal cells in bone marrow.

Several preclinical and phase I/II studies in patients have argued for potential clinical application of BMSCs in transplantation [15–17]. In some instances, this treatment modality involved the infusion of culture-expanded BMSCs to achieve cell numbers of 5.6–36.6 x 106/kg [18]. To accomplish the expansion, cells are sequentially passaged in culture for 20–50 days [18–20]. Since chemokine receptors on BMSCs are likely important for trafficking of transplanted stromal cells, we assessed the influence of long-term culture on stromal cell chemokine receptor expression. We found that long-term culture of BMSCs caused a marked decrease in chemokine receptor expression and abrogated stromal cell chemotactic responsiveness to chemokines. This loss of chemokine receptor expression and function was accompanied by downregulation of other surface receptors characteristic for BMSCs and/or MSCs. Taken together, the loss of these surface receptors raises the possibility of in vitro transformation of BMSCs into more differentiated cells. In this regard, long-term, (e.g., 45–60 days) culture of BMSCs is associated with a slowing of cell growth and increased spontaneous apoptosis. The relevance of these findings to cell therapy involving BMSCs is discussed.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Cells
Heparinized bone marrow was obtained by iliac crest aspiration from healthy adult volunteers after informed consent and in accordance with guidelines approved by the Institutional Review Committees of Dana-Farber Cancer Institute and the CBR Institute for Biomedical Research. BMSCs were isolated as previously described [8, 17, 18]. Mononuclear cells were isolated by Ficoll-Hypaque (Amersham Pharmacia Biotech, Uppsala, Sweden, http://www.amersham.com) gradient centrifugation and allowed to adhere to cell culture flasks in Dulbecco’s modified Eagle’s medium (DMEM; Gibco Life Technologies, Grand Island, NY, http://www.invitrogen.com) supplemented with glucose, pyridoxine-HCl, and 110 mg/ml sodium pyruvate and containing 10% fetal calf serum (FCS), glutamine, penicillin, and streptomycin. The first medium change was done after 24 hours to remove nonadherent cells, and the remaining adherent cells were defined as passage 0. The adherent layer reached 90% confluence within 3–7 days. Cells were then detached with 0.25% trypsin-EDTA (Gibco) and split 1:2 and replated as passage 1. The subconfluent cell monolayer underwent another cycle of trypsinization and cells were again replated as passage 2. Upon reaching 90% confluence, passage 2 cells were used in experiments [21]. For long-term culture experiments, cells were passaged 12 times (cultured for 45 days) or 16 times (cultured for 60 days).

Reverse Transcription-Polymerase Chain Reaction (RT-PCR) of Chemokine Receptors
mRNA from second-passage cells was denatured in the presence of 5 µM oligo(dT)12–18 primer and then reverse transcribed using Moloney murine leukemia virus reverse transcriptase (Clontech Laboratories, Palo Alto, CA, http://www.clontech.com) at 42°C for 1 hour. One tenth volume of cDNA was PCR amplified in 1x PCR buffer, 0.5 µM sense and antisense primers, and 0.5 U DNA Taq polymerase (New England Bio-Labs, Beverly, MA, http://www.neb.com).

The following primers were synthesized by Invitrogen. CCR1, sense primer 5'-ACT CAA GCA AGA TTT CAG ATT T-3', antisense primer 5'-TGA ATT GTT TGA TTT TAG TGG A-3', product length 452 bp; CCR4 sense primer 5'-TAC TAT GCA GCA GAC CAG TG-3', antisense primer 5'-TGA TGA TCA TGG AGT AGC AA-3', product length 350 bp; CCR7 sense primer 5'-ATC TCC AAG ACC AGA GAT AGT G-3', antisense primer 5'-AAA TGT TGC TCT CTT AAC GAA T-3', product length 461 bp; CCR9 sense primer 5'-CTT GTC ACT CTT CCC TTC TG-3', antisense primer 5'-AAA ATG CAG TTG TAG GGA AA-3', product length 531 bp; CXCR4 sense primer 5'-GAG GAG TTA GCC AAG ATG TG-3', antisense primer 5'-TTC TTC TGG TAA CCCATG AC-3', product length 480 bp; CXCR5 sense primer 5'-CAT CCT AAT CAT CCA ATG CT-3', antisense primer 5'-AGC TCT TTT CTT CCC TCT GT-3', product length 450 bp; and CXCR6 sense primer 5'-CCT TAA CCC TGT GCT CTA TG-3', antisense primer 5'-CTC ACC TCT TCA ACC TTC AG-3', product length 540 bp. For control gene GAPDH, sense primer was 5'-GGT GAA GGT CGG AGT CAA CG-3' and antisense primer was 5'-CAA AGT TGT CAT GGA TGA CC-3', product length 500 bp. The conditions for amplification were: 5 minutes at 94°C, followed by 35 cycles of 30 seconds at 94°C, 30 seconds at 62°C, 40 seconds at 72°C, followed by an extension for 4 minutes at 72°C. PCR products were resoled by electrophoresis on 1.2% agarose gels and visualized by ethidium bromide staining. The appropriate length of the GAPDH product and the lack of a detectable band in the control without reverse transcriptase (no-RT), indicated that there was no contamination of genomic DNA.

Cell Surface Staining of BMSCs
Cell surface expression of chemokine receptors on BMSCs was analyzed by indirect immunofluorescence staining. Second-passage stromal cells were detached from flasks by incubation with 0.05% trypsin-EDTA (Gibco) at 37°C, washed twice in 1x phosphate-buffered solution (PBS) and resuspended in ice-cold staining buffer (1x PBS/2% FBS). Cells were incubated on ice for 30 minutes with saturating amounts of the following antibodies to chemokine receptors: anti-CCR1 (clone 53504), anti-CCR2 (clone 48607), anti-CCR3 (clone 61828), anti-CCR7 (clone 150503), anti-CCR8 (clone 191704), anti-CCR9 (clone 112509), anti-CXCR1 (clone AIS02), anti-CXCR2 (clone AKT02), anti-CXCR4 (clone 12G5), anti-CXCR5 (clone 51505), anti-CXCR6 (clone 56811), and anti-CX3CL1 (clone 51637) (all from R&D Systems, Minneapolis, http://www.rndsystems.com); anti-CCR4 (clone 1G1), anti-CCR5 (clone 2D7), anti-CCR6 (clone 11A9), and anti-CXCR3 (clone 1C6) (allfromBDPharmingen,SanDiego,CA,http://www.bdbiosciences.com/pharmingen); and anti-CX3CR1 (MBL, Nagoya, Japan, http://www.mblintl.com).

Cells were washed twice and stained with the secondary goat anti-mouse or goat anti-rat IgG-phycoerythrin labeled (Jackson Immunoresearch Laboratories, West Groove, PA, http://www.jacksonimmuno.com) antibody. As a negative control, cells were incubated with identical concentrations of irrelevant mouse IgG1, IgG2a, IgG2b, or rat IgG2b antibodies (BD Pharmingen).

Data were acquired using a MoFlo flow cytometer (Dako, Fort Collins, CO, http://www.dakousa.com) and analyzed with Summit software (Dako).

Cell Stimulation, Immunoblotting, and Immunoprecipitation
Second-passage stromal cells were incubated for 2–24 hours at 37°C in serum-free medium (StemSpan H2000; Stem Cell Technologies, Vancouver, BC, Canada, http://www.stemcell.com) to decrease the level of constitutive protein phosphorylation. Cells were stimulated with the following chemokines: CCL5, CCL19, CCL25, CXCL12, CXCL13, or CXCL16 (all from Peprotech, Rocky Hill, NJ, http://www.peprotech.com; final concentration was 1 µg/ml) for different time periods (1–30 min) at 37°C. The reactions were stopped by adding 1 ml of ice-cold PBS and cells were lysed in lysis buffer (1% Triton X-100, 150 mM NaCl, 50 mM Tris-HCl, pH 7.6, containing protease and phosphatase inhibitors). Protein samples were separated by SDS-PAGE (Invitrogen, Carlsbad, CA, http://www.invitrogen.com) under reducing conditions and transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, http://www.bio-rad.com). Membranes were blocked and incubated with antibodies against phosphorylated forms of extracellular signal–related kinase (ERK)-1/2, STAT-1, STAT-3, or STAT-5 (all from Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com), according to manufacturer’s recommendations. Membranes were washed and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit antibody (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), developed by enhanced chemiluminescence (ELC; Amersham Pharmacia Biotech) and visualized by autoradiography. For immunoprecipitation of FAK, cell lysates were incubated with 2 µg of anti-FAK antibody (A-17; Santa Cruz Biotechnology) for 1 hour, followed by overnight immunoprecipitation with 35 µl of protein A Sepharose beads at 4°C. Immunoprecipitates were separated by 6% SDS-PAGE (NOVEX) under reducing conditions, electrophoretically transferred to nitrocellulose membranes (Bio-Rad), blocked, and incubated for 3 hours with anti-phosphotyrosine monoclonal antibody (mAb; 4G10) and then with HRP-conjugated goat anti-mouse IgG2b antibody (Caltag Laboratories, Burlingame, CA, http://www.caltag.com) for 1 hour at room temperature. Immunoreactive bands were developed and visualized as described for immunoblotting.

To verify the amount of proteins on the gels, membranes were stripped with stripping buffer (Chemicon, Temecula, CA, http://www.chemicon.com) and reprobed with antibodies against total ERK-1/2 (Cell Signaling Technology), total FAK (Santa Cruz Biotechnology), or appropriate STAT proteins (Upstate Technology, http://www.upstate.com), according to the manufacturers’ protocols. The intensity of phosphoprotein was determined by densitometry using ImageQuant Version 1.1 (GE Healthcare Bio-Sciences, Piscataway, NJ, http://www.gehealthcare.com), and is expressed for each lane as the multiple of the control (assigned a value of one).

Filamentous Actin (F-actin) Polymerization Labeling and Analysis
BMSCs were seeded on glass coverslips and grown to 90% confluence. Cells were stimulated with 1 µg/ml of CXCL12 for 0, 5, 15, or 30 minutes. Cells were then fixed, permeabilized, and stained for F-actin using Fix&Perm Kit (Caltag) according to the manufacturer’s protocol. Briefly, adherent cells were covered with 1 ml of Fixation Medium and incubated for 20 minutes at 4°C. After washing in 1x PBS, cells were covered with 1 ml of Permeabilization Medium containing phalloidin-fluorescein isothiocyanate (FITC) (10 µg) (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) and incubated for 30 minutes at 4°C. After washing, cells were covered with 1 ml of PBS for analysis. Fluorescence microscopy was performed using the Bio-Rad Radiance 2000 MP system (Bio-Rad). Images were collected as 13 horizontal sections and displayed as single mid-cell volume sections. F-actin fluorescence intensity was determined by Confocal Assistant software (Bio-Rad), through mean projection of 13 horizontal sections. A minimum of 10 cells in two different experiments was analyzed for each data point.

Cell Migration Assay
Second- or sixteenth-passage BMSCs were first loaded with 1 µM of Cell Tracker Green fluorescent probe (Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com). Cells were then resuspended in Hanks’ balanced salt solution (HBSS) at 105 cells/50 µl of HBSS. Cells were placed into the upper well of the Neuro Probe MBA96 chemotaxis chamber (Neuro-Probe, Inc, Gaithersburg, MD, http://www.neuroprobe.com). In the lower well was placed 28 µl of 1 µg/ml of chemokine, diluted in HBSS. To test for chemokine-induced cell chemokinesis, chemokines were added to both the upper and lower wells. Cells were allowed to migrate through a 12-µm pore size filter for 45 minutes at 37°C. The 12-µm membrane pore size was chosen because this size allowed for proportionately high specific stromal cell migration to chemokines. After 45 minutes of incubation, membranes were processed as per the manufacturer’s instructions to remove nonmigrating cells from the upper chamber. To avoid a bias related to microscope counting of the cells, the whole spots corresponding to each chamber were automatically recorded using an AX-70 Provis Olympus fluorescence microscope equipped with 480/535 filters and a Retiga EXi cooled charged-coupled device camera. Images were acquired using a 4x 0.13 PlanFL objective and were further processed using IPLab 3.9 (Scanalytics, Rockville, MD, http://www.scanalytics.com). In all instances, the exposure time, gain, and offset values of the camera were kept constant, and images were normalized in the same way.

Enzyme-Linked Immunosorbent Assay (ELISA) for Detection of Chemokines
Secretion of CCL2, CCL4, CCL5, CCL17, CCL20, CCL21, CCL25, CXCL8, CXCL12, and CXCL13 was detected by Quantikine Human Immunoassay (R&D Systems), according to the manufacturer’s protocol. Second-passage stromal cells were washed three times with DMEM without FCS and then cultured for 48 hours in serum-free medium (Stem Cell Technologies) at the concentration of 2 x 106 cells/15 ml of medium. Conditioned media derived from cells were analyzed by the quantitative sandwich enzyme immunoassay. The detection threshold of the ELISA assay was >5 pg/ml for CCL2, >11 pg/ml for CCL4, >2 pg/ml for CCL5, >7 pg/ml for CCL17, >0.3 pg/ml for CCL20, >9.9 pg/ml for CCL21, >13.9 pg/ml for CCL25, >0.21 pg/ml for CXCL8, >18 pg/ml for CXCL12, and >1.64 pg/ml for CXCL13.

Flow Cytometric Analysis of Stromal Cell Apoptosis
Stromal cell apoptosis was analyzed by staining for Annexin-V-positive (apoptotic) and opidium odide (PI)-positive (necrotic) cells using the Annexin-V FITC apoptosis detection kit (BD Pharmingen), according to the manufacturer’s protocol. Briefly, cells were resuspended in 1x Binding Buffer at a concentration of 106 cells/ml. Five microliters of Annexin-V FITC (BD Pharmingen) and 2 µl of PI (BD Pharmingen) were added and incubated with cells at room temperature for 15 minutes. Next, 500 µl of 1x Binding Buffer was added to cells and data were acquired immediately using a MoFlo flow cytometer (Cytomation).

Statistical Analyses
Statistical significance was assessed by Student’s t test, with significance accepted at the p < .01 level. Statistical calculations were performed using GraphPad Prism 4 software (Graph-Pad Software, San Diego, CA, http://www.graphpad.com).


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Isolation of Human BMSCs
BMSCs were obtained from total bone marrow aspirates by selecting cells that tightly adhered to plastic and then expanding them to a second passage in culture, in the presence of FBS but in the absence of growth factors. These cells were phenotypically fibroblastic with a spindle-shape morphology and grew in a distinct whorl pattern, with a population doubling time of 26–36 hours. They expressed a combination of surface markers characteristic for BMSCs, that is, CD13, CD29, CD90, CD105, and CD157 [3, 15]. The absence of contaminating hematopoietic cells in second-passage BMSCs was confirmed by the lack of expression of conventional antigens defining stem/progenitor cells (CD34, CD133) or the leukocyte (CD45), B (CD19), T (CD3, CD4, CD8), natural killer (NK) (CD56), or myeloid (CD14, CD15) cell lineages (data not shown).

Human BMSCs Express a Distinct Set of Chemokine Receptors
As a first measure to study the role of chemokine receptors in stromal cells, we investigated their surface expression on BM-SCs. Stromal cells were positive for the expression of CCR1, CCR7, and CCR9 chemokine receptors (60% ± 18%, 67% ± 4%, and 57% ± 4% positive cells, respectively; mean ± standard deviation [SD], n = 5), but were negative for surface expression of all other CC chemokine receptors (CCR2–CCR6 and CCR8) (Fig. 1AGo). They demonstrated surface expression of three CXC chemokine receptors CXCR4, CXCR5, and CXCR6 (43% ± 13%, 70% ± 14%, and 43% ± %7 positive cells, respectively; mean ± SD, n = 5) and were negative for CXCR1, CXCR2, and CXCR3 expression (Fig. 1BGo). Stromal cells were also negative for the expression of CX3CR1, but expressed high levels of its ligand, surface bound chemokine CX3CL1 (68% ± 15% positive cells; mean ± SD, n = 5) (Fig. 1CGo).


Figure 1
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  		Figure 1. Expression of chemokine receptors and CX3CL1 on bone marrow stromal cells (BMSCs). Histograms represent surface expression of CC (A), CXC (B) , and CX3C (C) chemokine receptors and CX3CL1 on second-passage BMSCs. The shaded part of the histogram indicates cells stained with antichemokine receptor antibodies, the black line indicates staining with the isotype control. The numbers represent the percentage of cells positive for a given chemokine receptor, where the threshold line for positive cells was based on maximum staining by a matching isotype with irrelevant specificity, used in the same concentration as the antichemokine receptor antibody. Representative staining of one out of five donor-isolated cells is shown. Photographs show the presence of CC (D) and CXC (E) chemokine receptor expression, analyzed by RT-PCR of mRNA extracted from second-passage BMSCs. no-RT indicates amplification of the mRNA not preceded by reverse transcription, and the product for the GAPDH housekeeping gene is shown as an internal control for mRNA. CCR4 gene expression is shown as a negative control. The results are representative of two experiments. Abbreviations: FACS, fluorescence-activated cell sorting; RT-PCR, reverse transcription-polymerase chain reaction.

 
Expression of CCR1, CCR7, CXCR4, and CXCR5 chemokine receptors and CX3CL1 was homogeneous for the whole population of stromal cells as indicated by the histogram shift of the entire cell population after staining with anti-chemokine receptor antibody (in comparison to isotype control). In contrast, CCR9 and CXCR6 were expressed only by a subset of stromal cells, as indicated by the presence of two distinct cell populations (receptor positive, right peak and receptor negative, left peak) on the flow cytometry histograms.

To confirm flow cytometry data, we examined by RT-PCR, mRNA expression of chemokine receptors that were expressed on the surface of BMSCs. Stromal cells expressed mRNA for the chemokine receptors CCR1, CCR7, and CCR9 (Fig. 1DGo) and CXCR4, CXCR5, and CXCR6 (Fig. 1EGo).

Chemokine-Induced Activation of Signaling Pathways in BMSCs

Chemokines Activate the MAPK Pathway in BMSCs.   Many chemokines mediate their effects through activation of the common signal transduction MAP kinase [22]. Thus, we stimulated stromal cells with 1 µg/ml of chemokine ligands corresponding to the receptors detected on the surface of BMSCs: CCL5 (ligand for CCR1), CCL19 (ligand for CCR7), CCL25 (ligand for CCR9), CXCL12 (ligand for CXCR4), CXCL13 (ligand for CXCR5), and CXCL16 (ligand for CXCR6). As a negative control, BMSCs were stimulated with CCL17, ligand for the CCR4 receptor, which is not expressed on the surface of BMSCs. As shown in Figure 2Go, all the chemokines with corresponding BMSC surface receptors caused an increase in phosphorylation of both ERK-1 and ERK-2. CCL17 stimulation did not cause activation of the MAPK pathway (Fig. 2AGo).


Figure 2
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  		Figure 2. Chemokines induce phosphorylation of stromal cell ERK-1/ERK-2 and FAK. Serum-starved second-passage stromal cells were stimulated with medium alone (–) or with the indicated chemokines for 1–30 minutes. Stimulation with CCL17 is shown as a negative control. The lysates were run on the gel, transferred to a membrane, and immunoblotted with anti-phospho-ERK-1/2 antibodies, followed by reprobing with anti-ERK-1/2 antibodies to verify the amount of proteins on the gels (as shown in the bottom panels). Immunoprecipitation with anti-FAK antibody was then performed followed by Western blot with antiphosphotyrosine antibody (4G10). The membranes were then stripped and reblotted with anti-FAK antibody to ensure equal protein loading (as shown in the bottom panels). Numbers under each lane are based on densitometry values and indicate the amount of phosphorylated ERK-1/2 expressed as a multiple of the control (assigned as a value of one). Each experiment was repeated three times using cells isolated from different donors, from which a representative blot is shown. Abbreviations: ERK, extracellular signal-related kinase; FAK, focal adhesion kinase; MAPK, mitogen-activated protein kinase.

 

Chemokines Stimulate FAK Phosphorylation in BMSCs.   FAK is a signaling molecule that plays an important role in cell adhesion, migration, and survival [23, 24] and can be activated in hematopoietic progenitor cells by chemokines (e.g., CXCL12) [25].

As in the previous experiment, cells were stimulated with 1 µg/ml of CCL5, CCL19, CCL25, CXCL12, CXCL13, and CXCL16 chemokines and examined for FAK phosphorylation (Fig. 2Go). All the chemokines except CCL19 induced FAK phosphorylation. Stimulation with CCL17 (negative control) also did not cause FAK phosphorylation (Fig. 2AGo).

Chemokines Activate Selected STAT Signaling Pathways in BMSCs.   We also assessed whether chemokines could activate STAT signaling pathways in BMSCs, as the STAT signaling proteins also are implicated in cell survival and proliferation [26]. In this experiment, we tested chemokines CCL5 and CXCL12, both of which were secreted by stromal cells (Table 1Go) and for which corresponding receptors (CCR1 and CXCR4) were present on the surface of BMSCs. We theorized that these two chemokines might operate by autocrine regulation as was previously suggested for hematopoietic cells [27].


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  		Table 1. Secretion of chemokines by bone marrow stromal cells (BMSCs)

 
CCL5 stimulation induced phosphorylation of STAT-1 but not of STAT-3 and STAT-5 (Fig. 3AGo). In contrast, CXCL12 mediated phosphorylation of STAT-5 but not of STAT-1 and STAT-3 (Fig. 3BGo).


Figure 3
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  		Figure 3. CXCL12 and CCL5 selectively activate STAT signaling pathways in bone marrow–derived stromal cells. Serum-starved BMSCs were stimulated with medium alone (–) or with CCL5 or CXCL12 chemokines for 1–30 minutes. Total cell lysates were analyzed by Western blot with specific antibodies against the tyrosine phosphorylated forms of STAT proteins (phospho-STAT-1, -3, -5). Upper panels represent the time course of STAT-1, STAT-3, and STAT-5 phosphorylation after CXCL12 or CCL5 stimulation. Equal protein loading was evaluated by stripping the blot and reprobing with an anti-STAT-1, -3, or -5 antibodies (as shown in the bottom panels). Numbers under each lane are based on densitometry values and indicate the amount of phosphorylated STATs expressed as a multiple of the control (assigned as a value of one). Each experiment was repeated twice with cells isolated from independent donors, from which a representative blot is shown. Abbreviation: STAT, signal transducer and activator of transcription.

 
Chemokines Induce Biological Responses of BMSCs

Chemokines Induce Stromal Cell Chemotaxis.   BMSC chemotaxis to selected chemokines corresponding to chemokine receptors expressed on the surface of BMSCs was carried out using a modified cell migration assay (see Materials and Methods). To avoid a bias related to cell counting, stromal cells were fluorescently labeled and the whole chemotactic field was analyzed for the number of migrated cells using a fluorescent microscope.

All the chemokines tested, that is, those with corresponding BMSC surface chemokine receptors, induced migration of BM-SCs (Fig. 4AGo). Dose-response studies showed a bell-shaped migration curve, which is typical for chemokines (data not shown). To confirm that movement of the stromal cells was directional and dependent on the chemokine gradient, chemokines were added to both wells of the chemotactic chamber. In these experiments cell chemotaxis was abolished and the numbers of cells migrated were not different from that recorded for the negative control (data not shown).


Figure 4
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  		Figure 4. Chemokines induce biological responses of second-passage bone marrow–derived stromal cells (BMSCs). (A): Chemokines induce migration of BMSCs. Chemotaxis assay was performed as described in Material and Methods using a modified Boyden chamber. Chemokines were added to the bottom compartment of the chemotaxis chamber and cells were present in the upper compartment. After 45 minutes, the number of migrated cells was evaluated by scanning the fluorescence intensity of the whole chemotactic field. Bars represent mean fluorescence intensity (± standard deviation, n = 6) of all fluorescently labeled input cells that specifically migrated to the chemokine. For the mean fluorescence intensity measurements, the exposure time, gain, and offset values of the camera were kept constant and images were normalized in the same way. Pictures show the chemotactic field containing cells that migrated to each chemokine. Representative spots corresponding to the migration chamber, photographed after 45 minutes, using the fluorescence microscope, are shown. Significance is indicated as * p<.01, ** p<.001, where p represents a statistical difference in cell migration to the chemokine compared with cell migration to the medium alone. (B): CXCL12 induces F-actin polymerization and stress fiber formation in BMSCs. Immunofluorescent labeling of F-actin in BMSCs after CXCL12 stimulation. Adherent stromal cells were serum starved for 2 hours and stimulated with medium or with CXCL12 for 5, 15, or 30 minutes. Cells were then fixed, F-actin was visualized by fluorescein isothiocyanate (FITC)-phalloidin labeling and viewed under confocal microscopy. A representative cell for each stimulation time point is shown. Results shown are representative of two independent experiments. (C): Quantification of stromal cell F-actin content after CXCL12 stimulation. Quantification of actin cytoskeleton reorganization was performed by measuring green fluorescence intensity in each stage of cytoskeleton organization. The graph represents the pixel intensity of the F-actin fluorescence per cell, expressed as mean ± standard deviation, n = 2, from at least 10 cells scanned for each time point.

 

CXCL12 Induces Actin Cytoskeleton Reorganization in BMSCs.   Next, we investigated whether a chemokine–chemokine receptor pair could trigger changes in F-actin polymerization. Quiescent stromal cells were stimulated with CXCL12 and the time course of the changes in F-actin polymerization was recorded using a confocal microscope.

F-actin staining in unstimulated cells was of a low intensity and noted mostly in the cell periphery, next to the cell membrane, with a few randomly disoriented stress fibers within the cytoplasm (Fig. 4BGo). Addition of CXCL12 increased the F-actin staining and formation of organized filamentous networks appearing as thick stress fibers. At 5 minutes after stimulation, stress fibers first appeared in the cell periphery. The presence of stress fibers became more apparent 15 minutes after stimulation. They were present in the cell cytoplasm, extending from the cell surface toward the nucleus. The CXCL12-induced cytoskeletal response was prolonged, lasting for at least 30 minutes (Fig. 4BGo). The increase in F-actin content after CXCL12 stimulation (expressed as a multiple of the chemokine-stimulated cells in comparison to unstimulated cells) was 1.6-fold, 2.5-fold, and 3.8-fold after 5, 15, and 30 minutes of CXCL12 stimulation, respectively (Fig. 4CGo).

Secretion of CC and CXC Chemokines by BMSCs
Stromal cells are known to regulate bone marrow hematopoiesis through secretion of soluble factors [3]. It is also well known that BMSCs express CXCL12 in both membrane and soluble forms [28]. We were curious if stromal cells could also secrete chemokines other than CXCL12. To this end, the presence of secreted CC chemokines CCL2, CCL4, CCL5, CCL17, CCL20, CCL21, and CCL25 and CXC chemokines CXCL8, CXCL12, and CXCL13 was measured in supernatant medium by ELISA (see Methods). The presence of CX3CL1, a chemokine that exists in the membrane-bound form, was analyzed by flow cytometry (Fig. 1CGo).

Stromal cells secreted several CC and CXC chemokines (Table 1Go). Among the CC chemokines tested, stromal cells secreted CCL2, CCL4, CCL5, and CCL20 (Table 1Go). We did not detect secretion of CCL17, CCL21, or CCL25 chemokines. Stromal cells also secreted two CXC chemokines, CXCL8 and CXCL12, but did not secrete CXCL13 (Table 1Go). In addition, stromal cells expressed high levels of membrane-bound CX3C chemokine CX3CL1 (Fig. 1CGo).

It is interesting to note that two of the chemokines (i.e., CCL5 and CXCL12) secreted by stromal cells were found to activate MAPK, FAK, and STAT signaling pathways and induce BMSC biological responses, suggesting that certain chemokines might function as autocrine signaling molecules for BMSCs in the bone marrow microenvironment.

Analyses of Culture-Expanded BMSCs

Culture-Expanded Stromal Cells Lose Chemokine Receptor Expression and Responsiveness to Chemokines.   Extended passaging (i.e., culture for 45 and 60 days, representing the twelfth and sixteenth passages of cells) produced cells with a markedly lower chemokine receptor expression compared with second-passage BMSCs (Figs. 1Go, 5AGo). Interestingly, loss of chemokine receptor expression was not uniform throughout the cell population. As seen in Figure 5AGo, at passage 12, stromal cell histograms exhibited a dual peak population for chemokine receptor expression. Some of the cells still retained a high expression level of chemokine receptors whereas a separate, major subpopulation was completely negative for chemokine receptor expression. By the sixteenth passage, stromal cells almost completely lost chemokine receptor expression. We could not detect surface expression of CCR7 and CCR9, and found very low expression of the remaining receptors (Fig. 5AGo).


Figure 5
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  		Figure 5. Culture-expanded stromal cells show loss of chemokine receptor expression and responsiveness to chemokines. (A): Stromal cells were cultured in Dulbecco’s modified Eagle’s medium through subsequent cycles of growth to confluence, trypsynization, and reseeding for 45 days (12 passages) or 60 days (16 passages). Surface expression of chemokine receptors was analyzed by flow cytometry. The numbers on the histograms represent the percentage of cells positive for a given surface antigen. Representative staining of one out of three experiments is shown. (B): Migration of sixteenth-passage stromal cells was analyzed using a modified Boyden chamber. Bar graphs represent mean fluorescence intensity (± standard deviation, n =3) of fluorescently labeled input cells that specifically migrated to the chemokine.

 
Next, we tested chemotactic responses of sixteenth-passage BMSCs. Loss of chemokine receptor expression correlated with the lack of detectable migration of culture-expanded stromal cells to corresponding chemokines (Fig. 5A, 5BGo). Expanded stromal cells lacking expression of CCR1, CCR7, or CCR9 did not migrate to corresponding ligands, that is, CCL5, CCL19, and CCL25. Moreover, the low residual expression levels of CXCR4, CXCR5, and CXCR6 were not sufficient to induce significant migration of culture-expanded BM-SCs to the corresponding ligands CXCL12, CXCL13, and CXCL16, respectively.

Culture-Expanded BMSCs Exhibit Change in Phenotype and Spontaneous Apoptosis.   BMSCs grown in extended culture also lost expression of adhesion molecules (ICAM-1, ICAM-2, and vascular cell adhesion molecule 1 [VCAM]-1) and the stromal cell marker CD157. They retained, however, expression of other stromal cell markers, for example, CD105 and CD90 (Fig. 6AGo).


Figure 6
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  		Figure 6. Culture-expanded BMSCs exhibit change in phenotype and spontaneous apoptosis. Stromal cells were cultured for 12 or 16 passages. Surface expression of adhesion molecules and CD markers was analyzed by flow cytometry. The numbers on the histograms represent the percentage of cells positive for a given surface antigen. Representative staining of one out of three experiments is shown. The same cells were analyzed for the presence of apoptotic and necrotic cells by the simultaneous staining with Annexin-V and PI. The upper right quadrant represents necrotic cells (Annexin-V+/PI+), the lower right quadrant represents apoptotic cells (Annexin-V+/PI–), and the lower left quadrant represents viable, nonapoptotic cells (Annexin-V–/PI–). The numbers on the dot-blot represent the percentage of positive cells in each quadrant relative to the total number of cells scored. A representative dot-blot of one out of three experiments is shown. Abbreviation: BMSCs, bone marrow–derived stromal cells.

 
Stromal cells grown in culture for extended periods of time displayed a considerably lower growth rate. Whereas second-passage stromal cells needed 24–28 hours to double in number, the population doubling time of cells grown for 12 passages was 68–72 hours and for cells grown for 16 passages was 90–108 hours (Fig. 6BGo). The slower BMSC growth rate was accompanied by the onset of cell apoptotic features. Long-term culture of stromal cells was associated with a significant increase in Annexin-V staining indicative of cell apoptosis. After 12 passages, 39% of the cells became apoptotic and 2.9% were necrotic (Fig. 6BGo). Extending stromal cell culture to 16 passages further increased the percentage of apoptotic cells. The majority, that is, 67%, of the cells became apoptotic, while 1.9% of cells were necrotic (Fig. 6BGo).


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
During ontogeny, BMSCs appear in the marrow cavity once the osteocytic collar has formed but before hematopoiesis begins. After vascularization of the bone cavity, stromal cells provide a framework of microenvironmental niches. Specialized niches with distinct locations in the bone marrow are thought to regulate hematopoietic cell proliferation and differentiation [6, 29]. Factors that influence the positioning of stromal cells to form specialized niches are not known. We hypothesized that chemokines and their receptors might play a similar role in stromal cell development as they do in hematopoietic cell development, where they regulate homing to bone marrow and retention in the bone marrow microenvironment and provide signals for cell growth and differentiation [11, 30].

Here we showed that BMSCs express a unique panel of chemokine receptors, mostly (with the exception of CCR1) belonging to the group of homeostatic (CCR7, CCR9, CXCR4, CXCR5, CXCR6) chemokine receptors (Fig. 1Go) [31, 32]. This class of receptors is known to be involved in homeostatic leukocyte trafficking and cell compartmentalization within bone marrow and/or in secondary lymphoid organs [29, 32]. Interestingly, CCR9 and CXCR6 were expressed only by a subpopulation of second-passage BMSCs, indicating that these BMSCs represent a heterogeneous cell population [33]. The finding that human BMSCs express CX3CL1 is in agreement with the published pattern of expression and function of this chemokine [34]. CX3CL1 is highly and constitutively expressed by a variety of nonhematopoietic cells of mesenchymal origin (e.g., synoviocytes, lung- and skin-derived fibroblasts, myocytes) [35–37], whereas its receptor, CX3CR1, is present on hematopoietic cells (e.g., T cells, NK cells, monocytes) [38]. The functionality of BMSC surface-expressed chemokine receptors was first measured by examining signaling pathways involved in cell growth, differentiation, and migration (e.g., ERK, FAK, and STAT signaling proteins; Figs. 2Go, 3Go) [22, 26, 39]. The significance of the MAPK signaling pathways in particular in stromal cells has been suggested by studies using in vitro cell systems and in animal models [40, 41]. Sustained activation of ERK is associated with osteogenic differentiation, whereas inhibition of ERK activation results in adipogenic differentiation. Moreover, mice deficient for nemo-like kinase, which, like ERK, belongs to the superfamily of MAP kinases [42], have morphologic bone marrow features indicative of aberrant differentiation of stromal cells [41].

It was important to also assess if BMSC surface chemokine receptors can mediate cell biological responses. We found that several CXC and CC chemokines could induce directional migration of BMSCs in an in vitro chemotaxis assay and that CXCL12 triggered stromal cell stress fiber formation (Fig. 4Go). These findings support the notion that certain chemokines, CXCL12 in particular [43, 44], play a role in BMSC homing and localization within the bone marrow, as has been established for hematopoietic cells [45–47]. In this regard, systemically administered BMSCs have the capacity to home to and engraft in the bone marrow of the recipient [16, 17, 48–51]. Moreover, transplanted stromal cells take up residence in bone marrow micro-environments and have been associated with improved bone structure and bone marrow function [48, 49]. In this study, we showed that, in addition to CXCL12 [52], BMSCs secrete considerable amounts of other CC and CXC chemokines, which could signal through corresponding receptors on neighboring developing hematopoietic cells [53]. Some of the BMSC-derived chemokines (Table 1Go), for example, CCL5 and CXCL12, also have corresponding receptors on BMSCs, suggesting the possibility of an autocrine feedback loop [54].

BMSCs are being considered for various clinical applications, including treatment of osteogenesis imperfecta, enhancement of hematopoietic engraftment, and limitation of graft-versus-host disease in stem/progenitor cell transplantation [15]. With regard to the latter application, BMSCs and/or their secreted chemokines, for example, CCL2 [55], might have an immune regulatory role. In this regard, allogeneic human BMSCs do not trigger T-cell activation in vitro in mixed lymphocyte reactions and suppress T-cell activation to allogeneic lymphocytes as well as to tuberculin [56]. This immunosuppressive effect has also been appreciated in a baboon skin graft rejection model, in which the systemic infusion of BMSCs resulted in prolonged skin graft survival [57].

Because, in some protocols, BMSCs are culture expanded before clinical use, we evaluated their growth rate and phenotype under culture conditions as previously reported in the literature [18, 19]. We noted that culture-expanded BMSCs over time showed a slower cell growth rate and greater rate of spontaneous apoptosis (Fig. 6Go). The causative factors of these in vitro changes are not known. However, long-term culture (e.g., more than two passages) was associated with markedly lower expression levels and function of chemokine receptors, and loss of surface markers characteristic for stromal/mesenchymal cells, for example, VCAM-1, ICAM-1, ICAM-2, and CD157, suggesting that these culture-expanded BMSCs might have become a more differentiated cell type, for example, fibroblasts. We considered if the decrease in chemokine receptor expression could be attributed to long-term exposure of BMSCs to secreted chemokines. However, downregulation was noted for all chemokine receptors, including receptors (e.g., CCR9, CXCR5) corresponding to chemokine ligands (i.e., CCL25 and CXCL13; Table 1Go) that were not secreted by BMSCs.

In recent years, several approaches to the isolation and propagation of human BMSCs have been described [58–60]. However, many fundamental biological questions related to cell heterogeneity, survival, growth, and homing capacity remain. The present data suggest that various chemokines and chemokine receptors may play an important role in stromal cell biology. Moreover, our findings indicate that further studies are needed to validate in vitro expansion methods of BMSCs intended for therapy.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
We thank Drs. Li Chai, Diane Krause, John Manis, and Anne Nicholson-Weller for critical reading of the manuscript and helpful suggestions. We thank Dr. David Miklos for help with human bone marrow aspirates.

This work was supported by grants from the National Institute of Health (HL56949 and U24 HL074355).


   DISCLOSURES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors indicate no potential conflicts of interest.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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