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First published online January 18, 2007
Stem Cells Vol. 25 No. 5 May 2007, pp. 1263 -1269
doi:10.1634/stemcells.2006-0522; www.StemCells.com
© 2007 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

Functional Expression of N-Formyl Peptide Receptors in Human Bone Marrow-Derived Mesenchymal Stem Cells

Anand Viswanathan, Richard G. Painter, Nicholas A. Lanson, Jr., Guoshun Wang

Departments of Medicine and Genetics, Gene Therapy Program, Louisiana State University Health Sciences Center, New Orleans, Louisiana, USA

Key Words. Adult bone marrow stem cells • Receptor • Mesenchymal stem cells • Cell migration

Correspondence: Guoshun Wang, D.V.M., Ph.D., Departments of Medicine and Genetics, Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112, USA. Telephone: 504-568-7908; Fax: 504-568-8500; e-mail: gwang{at}lsuhsc.edu

Received August 22, 2006; accepted for publication January 10, 2007.
First published online in STEM CELLS EXPRESS   January 18, 2007.


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures of Potential...
 Acknowledgments
 References
 
Tissue injury enhances homing and engraftment of mesenchymal stem cells (MSCs). However, the mechanisms by which MSCs sense the signals released by injured tissues and migrate toward injury sites have not been fully defined. In the current report, we investigated whether human MSCs express the N-formyl peptide receptor (FPR) and the formyl peptide receptor-like-1 (FPRL1). These receptors bind to N-formylated peptides by which phagocytes migrate to inflammatory sites and fibroblasts repopulate wounds to remodel the damaged tissues. Reverse-transcription polymerase chain reaction (PCR) demonstrated that MSCs express both FPR and FPRL1 at the transcriptional level. Flow cytometric analyses revealed expression of both receptors at the protein level. Fusion of the enhanced green fluorescence protein (eGFP) to the C terminus of each receptor showed localization to the cell surface. Moreover, MSCs responded to stimulation by N-formyl methionyl leucyl phenylalanine (fMLP), a prototypic N-formyl peptide, demonstrating rapid intracellular calcium mobilization that can be blocked by pertussis toxin or cyclosporin H. It is noteworthy that the fMLP-stimulated MSCs had an enhanced adhesion to extracellular matrix protein-coated surfaces. In addition, MSCs migrated toward gradients of increasing fMLP concentration, indicating that the receptors were functionally involved in positive chemotaxis to formylated peptides. Therefore, the N-formyl peptide receptors present in MSCs may play an important role in signaling stem cell adhesion, migration, and homing to injured and inflamed tissue for repair. Such a mechanism could potentially be exploited to direct the stem cells to target specific tissue sites, such as cystic fibrosis lungs, for therapy.

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


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures of Potential...
 Acknowledgments
 References
 
Adult mesenchymal stem cells (MSCs) derived from bone marrow have the property of self-renewal and the ability to differentiate into mesenchymal tissues, including osteoblasts, chondroblasts, adipocytes, and hematopoietic supporting stroma [1–3]. Further investigations suggested that the stem cells can also differentiate into many types of nonmesenchymal cells, such as pulmonary epithelium [4–8], kidney epithelium [9], myocytes [10], cardiomyocytes [11], and neuronal cells [12], which contributes to tissue repair in vivo [6, 9, 13]. Therefore, MSCs are of great promise in regenerative medicine providing novel therapeutic strategies for a variety of disorders. It is noteworthy that MSCs home and engraft into injured tissues [14–16]. The mechanisms underlying such a process are not fully understood.

The N-formyl peptide receptors belong to the G protein-coupled receptor family [17, 18]. The formyl peptide receptor (FPR) and formyl peptide receptor like-1 (FPRL1) bind to N-formyl peptides (FP), allowing leukocytes to signal inflammation and infiltrate at inflammatory sites [17–21]. It is noteworthy that bacteria are not the sole source of N-formyl peptides [22–24]. Apoptotic and necrotic mammalian cells or tissues release large quantities of such peptides derived from their mitochondrial proteins [25]. FPR and FPRL1 are membrane proteins with seven transmembrane domains. FPR binds N-formyl methionyl leucyl phenylalanine (fMLP) with high affinity (Kd < 1 nM) and is activated by picomolar to low nanomolar concentrations of fMLP. In contrast, FPRL1 is activated by micromolar concentrations of fMLP. Although FPR and FPRL1 were initially detected in phagocytic leukocytes, subsequent studies revealed that other cell types also express these receptors, including hepatocytes, astrocytes, dendritic cells, endothelial cells, epithelial cells, tumor cells, and fibroblasts [21, 26–28]. In response to tissue damage, fibroblasts migrate through ligand interactions with the receptors into inflammatory sites, repopulate wounds, deposit extracellular matrices for tissue growth, and secrete growth factors and immune modulating cytokines and chemokines [21]. This report documents that human MSCs express the formyl peptide receptors, suggesting their potential roles in mediating adhesion and migration of the stem cells to the sites of injury and inflammation for tissue repairing.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures of Potential...
 Acknowledgments
 References
 
Cell Lines and Reagents
Human MSCs were extracted from healthy volunteer donors by the Stem Cell Core facility at Tulane University Health Sciences Center. According to the published protocol [29], these cells were cultured in {alpha}-minimal essential medium ({alpha}MEM; Invitrogen, Carlsbad, CA, http://www.invitrogen.com), containing 20% lot-specified fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA, http://www.atlantabio.com), 100 units/ml penicillin, 100 µg/ml streptomycin (Invitrogen), and 2 mM L-glutamine (Invitrogen). bovine serum albumin (BSA), fMLP, and human placental collagen IV were purchased from Sigma Aldrich (St. Louis, http://www.sigmaaldrich.com). Human fibronectin was from BD Biosciences (San Jose, CA, http://www.bdbiosciences.com). Laminin and pertussis toxin were purchased from Invitrogen and Fura-2 acetoxymethyl ester (AM) from Molecular Probes (Eugene, OR, http://probes.invitrogen.com). Cyclosporin H was obtained from LKT Laboratories (St. Paul, MN, http://www.lktlabs.com/).

Reverse Transcription-Polymerase Chain Reaction
Total RNAs from human MSCs were extracted using the RNeasy extraction kit (QIAGEN, Valencia, CA, http://www.qiagen.com) and reverse-transcribed with the ImProm-II reverse transcription system (Promega, Madison, WI, http://www.promega.com). FPR was amplified using the sense primer 5'-CAGGAGCAGACAAGATGGAGACAA-3' and the antisense primer 5'-TCACTTTGCCTGTAACTCCACCTC-3'. FPRL1 was amplified using the sense primer 5'-TGCTGGCAAGATGGAAACCAACTT-3' and the antisense primer 5'-TCACATTGCCTGTAACTCAGTCTC-3'. The PCR products were cloned into the ZeroBlunt II-Topo vector plasmid (Invitrogen) and sequenced for verification.

Construction and Expression of FPR-Enhanced Green Fluorescent Protein and FPRL1-Enhanced Green Fluorescent Protein Fusion Proteins
The fusion protein constructs were generated from pEGFP-N1 (Clontech, Mountain View, CA, http://www.clontech.com). FPR and FPRL1 cDNA isolated from MSCs were separately subcloned into the multiple cloning site of pEGFP-N1, which linked EGFP in-frame to the C terminus of each gene. For expression, 293T cells were transfected with the fusion constructs by calcium precipitation using the published protocol [30]. Two days later, localization of the fusion proteins were observed by fluorescence microscopy. For transfection of MSCs, the Nucleofector electroporation system was used (Amaxa, Gaithersburg, MD, http://www.amaxa.com/), MSCs (4.5 x 105) were transfected with 5 µg of pEGFP-N1, pFPR-eGFP, or pFPRL1-eGFP according to the procedure recommended by the manufacturer. The cells were resuspended in 100 µl of the Nucleofector solution containing the plasmid. The mixture was then transferred to a 2-mm electroporation cuvette and electroporated on the maximum efficiency setting. After electroporation, the cell suspensions were placed in one well of a six-well dish containing 2 ml of prewarmed media. After 48 hours of incubation, the wells were washed twice with Dulbecco's phosphate-buffered saline and were examined for localization of the fusion proteins by fluorescence microscopy.

Immunostaining and Flow Cytometric Analyses
Human MSCs were dissociated from culture flasks by trypsinization (0.25% trypsin/EDTA; Invitrogen). The MSCs were fixed in 4% paraformaldehyde for 30 minutes and blocked with 1% normal rabbit serum. The primary antibodies reactive to FPR and FPRL-1 were all mouse monoclonal IgG1 and were from BD PharMingen (San Diego, http://www.bdbiosciences.com/index_us.shtml) and Genovac (Freiburg, Germany, http://www.genovac.com/), respectively. Isotype mouse IgG1 (Southern Biotech, Birmingham, AL, http://www.southernbiotech.com/) was used as a negative control antibody. Secondary antibody was AlexaFluor 488-conjugated rabbit F(ab)'2 against mouse IgG1 (Molecular Probes). After thorough washings, the samples were then analyzed by flow cytometry.

Calcium Mobilization Assays
Human MSCs were dissociated from culture flasks using 0.25% trypsin/EDTA and resuspended in phosphate-buffered saline (PBS) with 1% human serum and 1.25 mM CaCl2. The cells were incubated with 2 µM Fura-2 AM for 30 minutes at 37°C. After washes to remove any excess Fura-2 AM, the cells were resuspended in PBS, followed by spectrofluorometry using alternating excitation wavelengths of 340 nm and 380 nm. After a baseline was established, the cells were stimulated with 1 µM fMLP. The ratios of fluorescence emitted at 510 nm from the two excitation wavelengths were obtained, which reflects the change of free calcium levels within the cells. For blocking experiments shown in Figure 2B, pertussis toxin and cyclosporin H were used. In brief, after loading the MSCs with Fura-2 AM, the cells were incubated with either 100 ng/ml pertussis toxin for 1 hour at 37°C or 1 µM cyclosporin H for 20 minutes. Then, the MSCs were subjected to analyses by spectrofluorometry, similarly. After a baseline was established, the cells were stimulated with 100 nM fMLP.


Figure 1
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  		Figure 1. Expression of FPR and FPRL1 in human marrow-derived mesenchymal stem cells. (A, B): Reverse transcription-PCR to detect the expression of FPR (A) and FPRL1 (B) at the mRNA level. (C, D): Flow cytometric analyses of the expression of FPR (C) and FPRL1 (D) at the protein level. (E–J): Subcellular localization of EGFP, FPR-EGFP, and FPRL1-EGFP fusion proteins in 293T cells. (E, G, I): Micrographs of DIC. (F, H, J): Fluorescent micrographs. (K–P): Subcellular localization of EGFP, FPR-EGFP, and FPRL1-EGFP fusion proteins in human MSCs. (K, M, O): DIC images. (L, M, P): Fluorescent micrographs. Abbreviations: bp, base pair(s); DIC, differential interference contrast; EGFP, enhanced green fluorescent protein; FPR, N-formyl peptide; receptor; FPRL, N-formyl peptide receptor like; h, human; RT-PCR, reverse transcriptase-polymerase chain reaction.

 


Figure 2
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  		Figure 2. Intracellular calcium mobilization of human mesenchymal stem cells (MSCs) upon fMLP stimulation. (A): Human MSCs were labeled with 2 µM Fura-2 acetoxymethyl ester and subjected to spectrofluorometric measurements for cytosolic calcium levels before and after fMLP (1 µM) stimulation. The self-ratioable fluorescence probe was excited with two different wavelengths (340 nm and 380 nm). Two emissions at a wavelength of 510 nm were collected, and ratios between the two emissions were obtained. The 340 nm/380 nm ratio increase indicates the elevation of free calcium concentration. The data from three separate experiments of one MSC culture were calculated and smoothed using the FL Winlab Program (PerkinElmer Life and Analytical Sciences, Boston, http://www.perkinelmer.com) for the convenience of presentation. (B): Cytosolic calcium responses of MSCs to fMLP (100 nM) stimulation in the presence or absence of pertussis toxin and cyclosporin H. Three MSC preparations from three different donors (donors 2, 3, and 4) were examined. In this case, the data were presented without smoothing. Abbreviations: Exp., experiment; fMLP, N-formyl methionyl leucyl phenylalanine; Sec., seconds.

 
Cell Adhesion Assay
A 96-well cell culture plate was coated with BSA (2%), fibronectin (20 µg/ml), collagen IV (20 µg/ml), or laminin (1 µg/ml) in a carbonate buffer (500 mM Na2CO2, pH 8.5) overnight at 4°C. After coating, the wells were washed twice with PBS. Human MSCs, fluorescently labeled with 15 µM CellTracker Green 5-chloromethyl-fluorescein diacetate (CMFDA; Molecular Probes), were trypsinized and resuspended in {alpha}MEM. The MSCs were stimulated with 100 nM fMLP for 30 minutes at 37°C, and 2.5 x 104 such cells were placed into each well and allowed to attach. After incubation for 8 minutes, the unbound cells were removed by repeated washes with PBS, and the plate was analyzed with a microplate fluorescence reader (BioTek, Inc., Winooski, VT, http://www.biotek.com/). For antagonist blocking assay, the MSCs were incubated with 1 µM cyclosporin H for 30 minutes at 37°C before fMLP stimulation.

Human MSC Chemotaxis Assay
Trypsin-dissociated human MSCs were resuspended in Ringer's buffered saline solution with 1% BSA. These cells were fluorescently labeled with 15 µM CellTracker Green CMFDA for 30–45 minutes and then applied to the apical side of the FluoroBlok chemotaxis chamber (BD Biosciences). For the fMLP-dose-dependent assay shown in Figure 4A, the labeled cells were allowed to migrate toward various concentrations of fMLP applied to the basal side of the transwells for 4 hours. The fluorescence intensity was obtained by reading from the basal side using the microplate fluorescence reader. To compare the MSC migratory activities of multiple MSC preparations, as shown in Figure 4B, the fluorescently labeled stem cells from various donors were placed on the apical side of the FluoroBlok chemotaxis chamber. A single concentration of fMLP (100 nM) was applied to all the basal media. Control cells received no fMLP stimulation. To convert fluorescence readings to cell numbers, a standard curve was established for each experiment. The actual numbers of cells migrated to the basal side in response to fMLP stimulation were obtained after subtracting the baseline cell migration of the control group receiving no fMLP stimulation.


Figure 3
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  		Figure 3. Stimulation of fMLP enhances mesenchymal stem cell adhesion to extracellular matrix protein-coated surfaces. Human MSCs, labeled with 15 µM Cell Tracker Green (5-chloromethyl-fluorescein diacetate [CMFDA]), were stimulated with 100 nM fMLP. These cells were allowed to adhere on BSA-, collagen IV-, fibronectin-, or laminin-coated surfaces for 8 minutes. The plates were washed to remove the unbound cells and the fluorescence of the attached cells was measured by a microplate fluorescence reader. For the blocking assay, the MSCs were incubated with 1 µM cyclosporin H for 30 minutes at 37°C before fMLP application. Abbreviations: BSA, bovine serum albumin; fMLP, N-formyl methionyl leucyl phenylalanine.

 


Figure 4
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  		Figure 4. Positive chemotaxis of hMSCs toward the fMLP gradient. (A): Human MSCs (donor 1) were labeled with 15 µM Cell Tracker Green (5-chloromethyl-fluorescein diacetate [CMFDA]) and applied to the FluoroBlok chemotaxis chamber. Various concentrations of fMLP (1, 10, 50, 100, 500, and 1,000 nM) were added to the bottom medium for 2 hours at 37°C. Fluorescence from the basal side, which is directly proportional to numbers of cells migrated, was measured using a microplate fluorescence reader. Error bars represent quadruplicate for each condition. (B): Comparison of MSC basal migratory activities of multiple donors. Three more MSC preparations were similarly prelabeled with CMFDA. After the MSCs were applied to the chemotaxis chambers, 100 nM fMLP was added to the basal medium. Standard curve for each donor was established to correlate fluorescent readings to cell numbers. The actual number of cells migrated to the basal side in response to fMLP stimulation was obtained after subtracting the baseline migration of the control group receiving no fMLP stimulation. Error bars represent quadruplicate for each condition. Abbreviation: fMLP, N-formyl methionyl leucyl phenylalanine.

 

   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures of Potential...
 Acknowledgments
 References
 
Human MSCs Express FPR and FPRL1 at Both the mRNA and Protein Levels
To define whether human MSCs (hMSCs) express FPR and FPRL1 at the mRNA level, total RNAs were extracted from hMSCs. Reverse transcription-PCR was performed with primers specific to FPR and FPRL1. The amplified products were resolved in agarose gels (Fig. 1A, 1B) and further cloned into the ZeroBlunt II-TOPO plasmid. DNA sequencing showed that the two amplicons had nucleotide sequences identical to FPR and FPRL1 (data not shown). The samples without reverse transcriptase were negative, indicating that the amplifications were not due to genomic contamination. To examine the native expression of the two receptors at the protein level, we performed immunofluorescence staining of one culture of MSCs followed by flow cytometric analyses. Without permeabilization, the cells were immunostained with the monoclonal antibodies against FPR or FPRL1. The FPR staining gave rise to a mean fluorescence intensity (MFI) of 239, whereas FPRL1 staining gave rise to an MFI of 168. However, the isotype antibody resulted in a background staining with an MFI of 43 (Fig. 1C, 1D). To examine the cellular localization of the two receptors, we used the isolated FPR and FPRL1 genes from MSCs to generate the FPR-EGFP and FPRL1-EGFP fusion constructs where EGFP was linked to the C terminus of each receptor in-frame. After transfection of 293T cells, fluorescence microscopy demonstrated typical cell surface expression localization (Fig. 1H, 1J), whereas the parental EGFP plasmid gave rise to a cytoplasmic localization pattern (Fig. 1F). We further investigated the cellular localization of FPR-EGFP and FPRL1-EGFP in hMSCs. The fusion constructs were transiently expressed in the stem cells by electroporation. It is noteworthy that the membrane localization patterns of both constructs was not as obvious as in 293T cells (Fig. 1N, 1P). This result may reflect the difference of the two cell types.

Intracellular Calcium Mobilization of MSCs upon Stimulation with N-Formyl Peptide
In the FPR and FPRL1 signaling pathway, dissociation of the G-proteins after stimulation with N-formyl peptides leads to a number of downstream cellular events including intracellular Ca2+ mobilization. To evaluate whether the two N-formyl peptide receptors in the stem cells functionally respond to N-formyl peptide stimulation, we assayed the cytosolic calcium levels of hMSCs before and after application of fMLP. First, the cells were loaded with the fluorescent dye Fura-2 AM as described in Materials and Methods. The pre-labeled stem cells (1.5 x 106) were stimulated with 1 µM fMLP. The changes of intracellular calcium concentrations were measured by spectrofluorometry. As shown in Figure 2A, fMLP stimulation led to a rapid elevation of the cytosolic levels of free Ca2+, as indicated by the ratio increase in the emitted fluorescence, which was excited at 340 nm and 380 nm. To explore whether a lower concentration of fMLP had a similar effect, we tested three preparations of MSCs from three different donors with 100 nM fMLP. Figure 2B demonstrates that the MSCs responded to the stimulation instantly by increasing the cytosolic calcium level. Such a response was attenuated by pertussis toxin and cyclosporin H. Pertussis toxin interrupts G-protein interactions with G protein-coupled receptors [17, 31], and cyclosporin H is a potent and selective FPR antagonist [32, 33]. These results suggest that modulation of cytosolic calcium in response to fMLP occurred via activation of FPRs.

FMLP Stimulation Increases MSC Adhesion to Extracellular Matrix Protein-Coated Surfaces
Cellular migration is mediated through interactions between integrins and extracellular matrices. We next sought to determine whether hMSCs stimulated with fMLP had an enhanced attachment to extracellular matrix. Culture plates were coated with BSA, collagen IV, fibronectin, or laminin. Human MSCs, labeled with 15 µM CellTracker Green CMFDA, were trypsinized and resuspended in {alpha}MEM. After stimulation with 100 nM fMLP for 30 minutes at 37°C, the cells were allowed to attach to the protein-coated wells for 8 minutes. The unbound cells were removed by repeated washes with PBS, and the attached cells were quantified on a microplate fluorescence reader.

As shown in Figure 3, fMLP stimulation significantly increased hMSC attachment to fibronectin, collagen IV and laminin-coated surfaces. This effect was impeded by the fMLP antagonist cyclosporin H. FMLP stimulation also slightly increased the adhesion of hMSCs to the BSA-coated surface. However, such an increased attachment could not be blocked by cyclosporin H, presumably via a mechanism other than the FP-FPR pathway. Thus, FPRs in MSCs are functionally involved in MSC adhesion to extracellular matrices.

Positive Chemotaxis of hMSCs Toward the fMLP Gradient
Based on the fact that formyl peptide receptors are responsible for neutrophil chemotaxis toward an fMLP gradient, we predicted that FPRs in hMSCs should render the cells chemotactic toward a gradient of increasing fMLP concentration in a similar manner. To test the prediction, we dissociated the cultured hMSCs by trypsinization and fluorescently labeled them with 15 µM CellTracker Green CMFDA. Then, the cells were applied to the apical side of the FluoroBlok chemotaxis chamber. Various concentrations of fMLP (1, 10, 50, 100, 500, and 1,000 nM) were applied to the basal medium. After 4 hours of incubation, the fluorescence was measured with a fluorescence plate reader from the basal side. Because the filter membrane of the transwell is specifically designed to block any fluorescence from the apical side, the basal fluorescence reading represents cell migration to the basal side. As shown in Figure 4A, the stem cell basal migratory activities peaked at an fMLP concentration between 50 and 100 nM for this particular culture. Higher levels of fMLP seemed to inhibit cell migration, which is consistent with results from studies of other cell types [28, 34]. It is known that donor variations and functional heterogeneity of MSC subpopulations may affect experimental results. We expanded the study to multiple cultures of MSCs from multiple donors. In this case, 100 nM fMLP was applied to the basal medium. The results (Fig. 4B) indicated that MSCs from different donors migrated to the basal side upon fMLP stimulation, even though the actual numbers varied from preparation to preparation.


   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures of Potential...
 Acknowledgments
 References
 
MSCs have been recognized as a natural repair system. Because of their easy isolation from bone marrow and fast expansion in culture, the cells are of great potential for treating a variety of diseases. The current report provides the evidence to suggest that MSCs express the N-formyl peptide receptors at the mRNA and protein levels, and the receptors are functionally involved in MSC adhesion and migration upon stimulation with fMLP. This may represent a mechanism for MSCs to home and engraft to the sites of inflammation and injury.

Tissue injury, such as focal cerebral ischemia, results in inflammation, leading to infiltration and accumulation of leukocytes [35]. The injured tissue produces inflammatory mediators, such as monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein-1{alpha} (MIP-1{alpha}), and interleukin-8 (IL-8) [36]. Further evidence suggests that these inflammation mediators act as chemotactic agents to attract MSCs [37]. This process is mediated by interactions of the chemokines with the CC- and CXC-chemokine receptors. Honczarenko et al. [38] reported that MSCs express a special panel of chemokine receptors, including three CC chemokine receptors (CCR1, CCR7, and CCR9) and three CXC chemokine receptors (CXCR4, CXCR5, and CXCR6). Stromal-derived factor-1 (SDF-1) and its ligand CXCR4 play an important role in homing and engraftment of hematopoietic stem/progenitor cells [39]. MSCs express SDF-1 intracellularly, but a small subset of MSCs present SDF-1 on their cell surfaces, which mediates specific migration of MSCs to bone marrow [40]. Our experimental results indicated that MSCs express the N-formyl peptide receptors, which mediated MSC chemotaxis toward N-formyl peptides. This suggests that MSCs have a complex array of cell surface receptors sensing microenvironmental signals and regulating MSC homing, migration, and engraftment.

Preclinical and clinical data demonstrate that MSCs possess unique immunomodulatory properties and immune privilege [41–44]. This is due in part to the absence of the major histocompatibility complex class II molecules on their surface unless specifically stimulated, and no expression of FAS ligand or such costimulatory molecules as B7–1, B7–2, CD40, or CD40L [44, 45]. Thus, MSCs elicit no T-cell proliferation when cocultured with HLA-mismatched lymphocytes. In addition, T- and B-cell proliferations and immunoglobulin production in B cells were inhibited by MSCs. In this manner, the cells have immunosuppressive functions [46–49]. Even though the mechanism underlying the immunomodulation is not fully characterized, soluble factors released by MSCs and/or direct contact of MSCs with immune cells are thought to be required [38, 44–46]. Therefore, positioning of MSCs to the immune sites is apparently essential for the stem cells to have full effects. MSCs functionally express the N-formyl peptide receptors and show positive chemotaxis toward formyl peptides, which may play an important role in repopulating the stem cells. Future studies are warranted to determine whether FPR and FPRL1 are involved in presenting MSCs to the immune sites.

MSCs have demonstrated potency as cellular vectors for gene therapy. However, specific targeting of the cells to specific tissue sites has not been achieved. Functional expression of the formyl peptide receptors in human MSCs, identified in this report, showed that the cells have the machinery for directional migration. It follows that transient overexpression of the formyl peptide receptors in MSCs could be used therapeutically to enhance the homing and engraftment of the engineered MSCs to inflammatory and injured tissues. One potential application is to recruit MSCs to the lungs of patients with cystic fibrosis (CF). CF is a chloride-channel defect that leads to persistent bacterial infection of the lung, small airway obstruction, and lung inflammation and damage. Repair of damaged airways and restoration of airway epithelial functions by a stem cell approach provides a novel strategy. We found that MSCs from patients with CF in which the CFTR gene defect was corrected can differentiate into airway epithelium and contribute to apical transport of chloride [7]. Thus, MSCs have the potential to repair the defective lung epithelia in CF. However, preclinical data indicate that homing and engraftment efficiency of nonmodified MSCs to airways is limited [8]. We predict that overexpression of the formyl peptide receptors may be potentially exploited to directionally recruit the stem cells to CF lungs, where abundant formyl peptides are present.


   DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures of Potential...
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures of Potential...
 Acknowledgments
References
 
We thank Drs. Darwin Prockop and Bruce Bunnell at Tulane University Center for Gene Therapy for providing human mesenchymal stem cells and technical assistance in stem cell culture. Human MSC isolation, production, and distribution were supported through National Institutes of Health National Center for Research Resources and National Heart, Lung, and Blood Institute Grants 5P40-RR017447-03 and 1P01-HL075161-01A1 to Darwin Prockop (Tulane University, New Orleans, LA). This work was supported by a Cystic Fibrosis Foundation Research Grant (WANG05G0), Louisiana Board of Reagents Research grant, and the Louisiana Gene Therapy Research Consortium.


   REFERENCES
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures of Potential...
 Acknowledgments
 References
 

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