alexa Enhanced Expression of N-Formyl Peptide Receptor in Mesenchymal Stem Cells Facilitates Homing to Inflammatory Lungs | Open Access Journals
ISSN: 2157-7412
Journal of Genetic Syndromes & Gene Therapy
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Enhanced Expression of N-Formyl Peptide Receptor in Mesenchymal Stem Cells Facilitates Homing to Inflammatory Lungs

Anand Viswanathan1 and Guoshun Wang1-3*

1 Department of Genetics, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA

2 Department of Microbiology and Immunology, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA

3 Department of Medicine, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA

*Corresponding Author:
Guoshun Wang
DVM, PhD, Department of Microbiology and Immunology
Genetics and Medicine
Louisiana State University Health Sciences Center
New Orleans, LA 70112, USA
Tel: 504-568-7908
Fax: 504-568-8500
Email: [email protected]

Received date: February 13, 2014; Accepted date:April 28, 2014; Published date: April 30, 2014

Citation: Viswanathan A, Wang G (2014) Enhanced Expression of N-Formyl Peptide Receptor in Mesenchymal Stem Cells Facilitates Homing to Inflammatory Lungs. J Genet Syndr Gene Ther 5:227. doi: 10.4172/2157-7412.1000227

Copyright: © 2014 Viswanathan A, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

Marrow-derived mesenchymal stem cells (MSCs) exhibit certain intrinsic tropism to inflammatory tissues. However, effective stem cell therapy often requires high levels of engraftment, which may not be sufficed by the MSC natural homing process. Here we investigated if lentiviral vector-enhanced expression of N-formyl peptide receptor (FPR) in MSCs could increase their sensitivity to N-formylated peptides (N-FP) and facilitates MSC homing to inflammatory lungs. HIV-6 1-based lentiviral vectors, expressing the FPR-EGFP fusion protein (HIV-FPR-EGFP) or co-7 expression of the FPR and EGFP proteins (HIV-FPR-IRES2-EGFP), were engineered. Expression of 8 FPR in 293T cells, a cell line without the endogenous receptor, rendered the cells responsive to N-FP by intracellular calcium mobilization. Human MSCs, transduced with the FPR-IRES2-EGFP vector, showed a greater sensitivity to and an enhanced chemotaxis towards a low level of N-FP. The FPR-11 engineered hMSCs, expressing a luciferase reporter, were infused systemically into nu/nu mice which were pre-intubated intratracheally with or without a sublethal dose of Pseudomonas aeruginosa. In both groups, the MSCs were largely located in the lungs initially and cleared rapidly within days as shown by in vivo whole body bioluminescence imaging. However, MSC retention in the bacterium- challenged lungs a week after infusion was ~2-fold higher than the non-challenged controls. Biochemical measurement of luciferase enzymatic activity demonstrated low but definite homing of the MSCs in the bacteriumchallenged lungs. Engraftment of MSCs to the lungs was immunohistochemically confirmed. These data provide a proof of principle that engineering MSCs with FPR can enhance the stem cell homing to inflammatory tissues for potential repair.

Keywords

N-formyl peptide receptor; Mesenchymal stem cells; MSC homing and engraftment

Introduction

Multipotent mesenchymal stem cells (MSCs) derived from bone marrow have demonstrated great promise in regenerative medicine. A wealth of literature has documented the potency of MSCs to differentiate into many types of cells beyond their germinal boundary [1-3]. Moreover, MSCs can also secrete paracrine soluble factors to modulate local cells to promote damage recovery [4-7]. In order to achieve these effects, MSCs are required to be physically present in target tissues or organs [8,9]. MSCs have shown some tropism for sites of damage tissues [10,11]. However, experimental data only show low levels of engraftment [12], which limits greater clinical benefits for stem cell therapies. Therefore, engineering MSCs to increase their homing and engraftment to target tissues is critical for potential application of the cells for therapy to achieve maximal clinical benefits.

Even though the mechanisms underlying MSC migration and homing have not been fully elucidated, many receptors involved in migration of leukocytes and other cell types have been identified on MSCs [13]. Thus, it is believed that many of the same receptors are also involved in governing MSC migration. In vitro comparisons of chemotactic activities demonstrate a panel of growth factors and chemokines as MSC chemoattractants [14]. MSCs genetically modified to overexpress insulin-like growth factor (IGF)-1 or CXCR4 accelerate their mobilization for myocardial repair [15-17]. We and others have previously reported that human marrow-derived MSCs functionally express N-formyl peptide receptor (FPR) [18,19]. This G proteincoupled receptor binds to N-18 formylated peptides (N-FP) released from intruding bacteria or dead cells [20,21], allowing leukocytes to approach and infiltrate inflammatory tissues [20-24]. Fibroblast cells also use this same mechanism to migrate into inflammatory sites to repopulate wounds, deposit extracellular matrices for tissue growth, and secrete growth factors and immune modulating cytokines and chemokines [25]. Furthermore, overexpression of FPR in cells of non-leukocyte origin confers these cells with responsiveness to N-FP and results in directional chemotaxis [26-30]. The data from these publications altogether suggest the possibility of engineering MSCs with FPR to enhance their directional migration and homing. The present report demonstrates that lentiviral vector-mediated overexpression of FPR in human MSCs enhances their sensitivity to N-FP stimulation and increases MSC homing to inflammatory lungs.

Materials and Methods

Cell lines and reagents

Human MSCs were isolated from healthy volunteer donors by the NCRR/NIH-sponsored Tulane center for distribution of MSCs prepared with a standardized protocol [11]. These cells were cultured in α-MEM (Invitrogen-Gibco, Carlsbad, CA), containing 20% lot- specified fetal bovine serum (Atlanta Biologicals, Lawrenceville, GA), 100 units/ml penicillin, 100 μg/ml streptomycin and 2 mM L-glutamine (Invitrogen- Gibco). Bovine serum albumin (BSA), N- FMLP (N-formyl-Met-Leu- Phe), N-formyl hexapeptide (N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys), human placental collagen IV and common chemicals were purchased from Sigma (St. Louis, MO).

Lentiviral vector construction and production

Total RNAs from human MSCs were extracted using the RNeasy extraction kit (Qiagen, Valencia, CA) and reverse-transcribed with the ImProm-IITM reverse transcription system (Promega, Madison, WI). The FPR cDNA was amplified using the sense primer 5’-CAGGAGCAGACAAGATGGAGACAA-3’ and the antisense primer 5’-TCACTTTGCC TGTAACTCCACCTC-3’. The PCR product was cloned into the ZeroBlunt II-Topo vector plasmid (Invitrogen) and sequenced for verification. The FPR cDNA was further subcloned into pIRES2-EGFP or pEGFP-N1 (Clontech, Mountain View, CA), resulting in the following two cassettes: CMV-FPR- IRES2-EGFP or CMV-FPREGFP (fusion protein). Then, both cassettes were, respectively, used to replace the CMV-EGFP cassette in the parental lentiviral transgene plasmid (pNL-CMV-EGFP- WPRE-dU3) [31]. The corresponding HIVbased viral vectors (HIV-CMV-EGFP, HIV-FPR-EGFP, and HIV-FPRIRES2- EGFP) were produced by triple-plasmid calcium-phosphate transfection method [32]. Viral particles in the culture medium were collected three times at 48, 60 and 72 hours after HEK293T cells were transfected with the envelope plasmid pLTR-G, the packaging plasmid pCD/NL- BH*ΔΔΔ, and one of the transgene plasmids [31,32]. Vector concentration was achieved by ultracentrifugation on a 20% sucrose cushion. Viral titers were obtained by serial dilution and transduction of HT1080 or HOS cells followed by real-time PCR for quantitation of vector genome.

Vector transduction of MSCs

MSCs were grown in 150 mm culture dishes (Thermo Fisher Scientific, Rochester, NY) at a low density (5000 cells per 150 mm dish). The cells were transduced with either HIV-CMV-EGFP or HIV-CMVFPR- IRES2-EGFP virus at an MOI of 20 in the presence of polybrene (8 μg/ml). Sixty hours after viral transduction, the cells were harvested by trypsin dissociation for use in subsequent experiments. In the case of in vivo application, MSCs were co- transduced with two lentiviral vectors: HIV-CMV-FPR-IRES2-EGFP and HIV-CMV-Luciferase- IRESs-DsRed at an MOI of 20 for each.

Calcium mobilization assay

Vector-transduced HEK293 cells were dissociated from cultures using 0.25% trypsin/EDTA and resuspended in PBS with 1% bovine serum albumin and 1.25 mM CaCl2. The cells were incubated with 2 μM Indo-1 AM (Invitrogen-Molecular Probes, Carlsbad, CA) for 30 minutes at 37°C. After washes to remove excess Indo-1 AM, the cells were resuspended in PBS, followed by fluorescent measurement by spectrofluorometry using excitation wavelength of 338 nm. The ratios of fluorescence emitted at 400 nm and 475 nm from the excitation wavelength were obtained, which reflects the changes of free calcium levels within the cells. After a base line was 18 established, the cells were stimulated with 100 nM N-formyl hexapeptide.

MSC chemotaxis assay

Trypsin-dissociated MSCs were resuspended in PBS solution with 1% BSA. These cells were fluorescently labeled with 15 μM CellTracker Green CMFDA (Invitrogen-Molecular 22 Probes) for 30-45 minutes and then applied to the apical side of the Fluoroblok 24-well transwells (BD, Franklin Lake, NJ). The cells were allowed to migrate towards the N-formyl hexapeptide (5 nM) applied to the basal side of the transwells for 2, 4 and 8 hours. Data were collected by measuring the fluorescence intensities from the basal side using the microplate fluorescence reader. Because the filter membrane of the transwells was specially designed to block any fluorescence from the apical side, the basal fluorescence reading represents cell migration to the basal side [18]. To convert fluorescence readings to cell numbers, a standard curve was preestablished for each experiment. The actual numbers of cells migrated to the basal side in response to the chemoattractant were obtained after subtracting the baseline cell migration of the control group receiving no N-formyl hexapeptide stimulation.

MSC differentiation assays

To assess pluripotency of the vector-transduced MSCs, standard differentiation assay schemes were followed [33]. Human MSCs, transduced with 50 MOIs of HIV- CMV-FPR-IRES2-EGFP vector, were plated at an initial density of 50 cell/cm2 on a 60 mm dish. For osteogenesis, MSCs were cultured with a complete culture medium with 20 mM glycerol phosphate, 50 ng/ml thyroxine, 1 nM dexamethasone, and 0.5 μM ascorbate-2-phosphates (Sigma-Aldrich). After 3 weeks, the cells were fixed with 10% formalin for 20 minutes at room temperature and stained with Alizarin Red (Sigma-Aldrich). For adipogenesis, MSCs were cultured with a complete culture medium with 5 μg/ml insulin, 50 μM indomethacin, 1 x 10–6 M dexamethasone, and 0.5 μM 3-isobutyl-1- methylxanthine (Sigma-Aldrich). After 3 weeks, the cells were fixed with 10% formalin and stained with 0.5% Oil Red O (Sigma- Aldrich).

Bioluminescence imaging of MSCs in mice and luciferase assay of mouse lungs

The animal protocol for this experiment was approved by the LSUHSC institutional IACUC committee. Athymic (nu/nu) male mice received a midline cervical incision to expose the tracheas, followed by an intratracheal intubation of 50 μl of PBS solution with Pseudomonas aeruginosa (PAO1, 5 x 105 cfu) or control PBS. After a 1-day recovery, MSCs (1 x 106), co-transduced with the HIV-CMV-FPR-IRES2- 7 EGFP and HIV-CMV-Luc-IRES2-DsRed vectors at an MOI of 20 for each, were infused into the mice by tail vein injection. For in vivo imaging, the mice were anesthetized by isoflurane inhalation and injected with 150 mg/kg of luciferin (Xenogen, Hopkinton, MA). Bioluminescent signals were detected at 5 minutes after luciferin injection at an integration time of 1 second to 2 minutes using an in vivo imaging system with a cooled charge-coupled device camera (IVIS100; Xenogen, Hopkinton, MA). Similarly, the animals were measured for bioluminescence emission at the different time points (1, 4, and 7 days). To assess the luciferase enzyme activity in the lungs biochemically, the mouse lungs were harvested at 1,4,7 days post MSC infusion and homogenized into 1 ml of the luciferase assay lysis solution (Promega, Madison, WI). The homogenates were assayed immediately after addition of luciferin (Promega) in a luminometer (Turner, Sunnyvale, CA) and the detected luciferase activities were normalized against the protein concentrations determined by BCA analyses (Pierce, Rockford, IL).

Immunohistochemistry staining

Mouse lungs, 8 days post intratracheal intubation of PBS or PAO1, were dissected out and fixed in 2% paraformaldehyde/PBS solution by intra-tracheal inflation and en bloc immersion overnight at 4°C. Then, the fixative was removed by exchange with PBS repeatedly. Paraffin embedment and tissue section were performed using the routine protocol. The tissue slices were stained immunohistochemically with the chicken polyclonal antibody against GFP as the first antibody (Novus Biologicals, Littleton, CO) and the donkey anti-chicken IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) as the second antibody. The biotin/streptavidin- conjugated HRP-DAB system was used for color development. The slides were weakly counter stained with hematoxylin.

Results

Lentiviral vector construction and functional expression of FPR

In order to genetically modify MSCs with FPR, we constructed the following three self-inactivating lentiviral vectors: 1) HIV-CMV- EGFP, 2) HIV-CMV-FPR-EGFP, and 3) HIV-CMV-FPR-IRES2-EGFP (Figure 1A). The high-titer VSV-G-pseudotyped vectors were produced by triple-plasmid co-transfection. To test if each of the vectors correctly expressed the expected transgene, we transduced 293T cells, a cell line without endogenous FPR expression. As displayed, EGFP was expressed in all three transduced cells (Figures 1B-1D). Noticeably, the EGFP subcellular localization patterns appear different. The FPR-EGFP fusion protein was present largely on cytoplasmic membrane (Figure 1C) due to the transmembrane nature of the FPR receptor, while EGFP from the HIV-CMV-EGFP or HIV-CMV-FPR-IRES2-EGFP vector gave rise to the expected cytosolic distribution pattern. Because FPR is a G-protein coupled membrane-bound receptor, its functional expression can be validated by intracellular calcium response to N-FP stimulation. The three transduced cell lines (HEK293-EGFP, HEK293- FPR-EGFP, and HEK- FPR-IRES2-EGFP) were labeled with 2 μM of Indo1-AM, a ratioable calcium probe, and subjected to intracellular calcium mobilization assays by spectrofluorometry. After a baseline was established, 100 nM of fMLP was used to stimulate the cells. The results demonstrated that cells receiving the HIV- CMV-FPR-IRES2-EGFP vector had a higher sensitivity to N-FP than those transduced with the HIV- CMV-FPR-EGFP vector (Figure 1E). In contrast, the cells receiving HIV-CMV-EGFP, a control vector, did not respond to N-FP stimulation at all. Given the strongest response to N-FP from the cells transduced with the HIV-CMV-FPR-IRES2-EGFP vector, we chose this vector to engineer MSCs hereafter.

Lentivector-enhanced FPR expression in MSCs induces greater cell sensitivity to N-FP

Human marrow-derived MSCs were transduced with 20 MOIs of HIV-CMV-FPR-IRES2-EGFP or HIV- CMV-EGFP. The micrographs (Figures 2A-2D) and flow cytometry histograms (Figures 2F-2H) demonstrated that the MSCs were efficiently transduced. The HIVCMV- EGFP transduction gave rise to 95% EGPF-positive cells and the HIV-CMV-FPR-IRES2-EGFP transduction resulted in 53% EGFPpositive cells. Given that equal MOIs of vectors were applied, the EGFP expression difference may have been caused by the IRES sequence. The transduced MSCs were trypsin-dissociated, fluorescently labeled with 15 μM Cell Tracker Green CMFDA and applied to the apical side of the Fluoroblok transwell inserts for chemotaxis assays. To the basal medium, N-FP (0 or 5 nM) was applied. After 2, 4 and 8 hours of incubation, the fluorescent intensities were measured with a fluorescence plate reader from the basal side. To correlate the fluorescence readings to cell numbers, a standard curve was pre- established for each experiment. The actual numbers of cells that migrated to the basal side in response to N-FP was obtained after subtracting the base level random migration of the control cells receiving 0 mM N-FP treatment. As shown in Figure 2E, MSCs with FPR expression showed an increased sensitivity to N-FP by undergoing chemotaxis towards the low nano-molar concentration of N-FP. This level of sensitivity was not seen in the MSCs transduced with HIV-CMV-EGFP, negating any possibility of a lentiviral transduction affecting the sensitivity of hMSCs to N-FP. We also assayed the cells for intracellular calcium response to N-FP by spectrofluorometry. The N-FP (5 nM) stimulation led to a rapid and pronounced elevation of the cytosolic levels of free Ca2++. In contrast, the MSCs transduced with the EGFP control vector had no response to the lower level N-FP stimulation (data not shown).

genetic-syndromes-gene-therapy-expression-transgenes

Figure 1: Lentiviral vector constructs and expression of transgenes in HEK293 cells. Panel A: HIV-1 based self-inactivated vectors using the CMV promoter to drive the expression of EGFP alone (HIV-CMV-EGFP), FPR-EGFP fusion protein (HIV-CMV-FPR-EGFP) or two independent proteins (FPR and EGFP) through an internal ribosomal entry site (HIV-CMV-FPR-IRES2-EGFP). Panels B â D: HEK293 cells were transduced with HIV-CMV-EGFP (B), HIV-CMV-FPR-EGFP (C) or HIV- CMV-FPR-IRES2-EGFP (D). Panel E: Cytosolic calcium responses of HEK293 cells, expressing EFPG alone (Green line), FPR-EGFP fusion protein (Red line) or the two separate proteins (FPR and EGFP) (Blue line), to N-formyl peptide stimulation.

genetic-syndromes-gene-therapy-derived-mesenchymal

Figure 2: Lentivector-enhanced expression of FPR in human marrow-derived mesenchymal stem cells. Panels A-D: Transduction of human marrow-derived mesenchymal stem cells with either HIV-CMV-EGFP or HIV-CMV-FPR-IRES2-EGFP. DIC Micrographs (A & C) and their corresponding fluorescent micrographs are displayed (B & D). Panel E: Chemotaxis assay was performed to determine the functionality of FPR expressed by the lentivector in MSCs. Open column indicates the level of chemotaxis of the MSCs expressing EGFP in response to 5 nM N-FP, while the closed column represents that of the MSCs expressing EGFP and FPR. Panels F-H: Flow cytometric analyses of the MSCs transduced with either HIV-CMV-EGFP (G) or HIV-CMV-FPR-IRES2-EGFP (H). The non-transduced MSC were also examined as shown (F).

These results indicate that an enhanced expression of FPR in MSCs causes the cells to have a greater sensitivity to N-FP and a greater capacity of migration towards a low concentration of agonists. This finding suggests that the FPR-mediated chemotaxis mechanism can be potentially exploited to enhance MSC homing and engraftment.

MSCs with lentivector-enhancing FPR expression retain their pluripotency

Retention of stem cell pluripotency after ex vivo engineering is important for potential therapeutic applications. To confirm if our vector-enhanced FPR expression affects such a property, we assayed the MSCs for their ability to differentiate into osteocytes and adipocytes. MSCs, transduced with 20 MOIs of HIV-CMV- FPR-IRES2-EGFP, expressed EGFP and FPR in all the cells (data not shown). By following the published protocol [33], the transduced cells were cultured in differentiation media that induces either osteogenesis or adipogenesis. After 3 weeks of continuous culture, differentiation was validated by Alizarin red staining for osteogenesis and oil red O staining for adipogenesis. As shown in Figure 3, MSCs modified by vector-mediated expression of FPR showed a rich extracellular matrix and calcium phosphate deposition, indicative of osteogenesis (Figures 3A and 3B). Furthermore, the cells also demonstrated an abundant intracellular accumulation of lipid droplets with variable sizes, suggesting adipogeneis (Figures 3C and 3D). Hence, the stem cells engineered with FPR via a lentiviral vector retain their authentic pluripotency.

MSCs with lentivector-enhanced expression of FPR display an increased homing to bacterial challenged lungs

genetic-syndromes-gene-therapy-expression-transgenes

Figure 3:In vitro differentiation of MSCs with lentivector-enhanced expression of FPR. Panels AâD: Standard differentiation assays for osteogenesis (A & B) and adipogenesis (C & D). Alizarin Red chelates calcium, forming an alizarin red S-calcium complex. Positive staining indicates deposition of calcium phosphate extracellularly (A) and the corresponding fluorescence image (B). Oil Red O is a fat-dissolving dye. Positive staining reflects lipid accumulation in cells (C) and the corresponding fluorescence image (D).

In order to longitudinally observe homing of the MSCs with enhanced FPR expression in vivo, hMSCs were co-transduced with lentiviral vectors expressing FPR and firefly luciferase, as described in Materials and Methods. The experimental scheme is displayed (Figure 4A). Nude mice were challenged with a sublethal dose of Pseudomonas aeruginosa (PAO1, 5 x 105 CFU) or PBS via intra-tracheal intubation. After recovery for one day, the mice were infused with 1 x 106 of the vector-transduced MSCs via tail vein injection. One, 4 and 7 days after MSC administration, the same animals from each treatment were anesthetized and injected with 150 mg/kg of luciferin for in vivo bioluminescent imaging. As shown (Figure 4B), one day after MSC administration, the stem cells were predominantly located in the lungs. At Days 4 and 7 most of the MSCs disappeared from the lungs regardless of treatments. However, the PAO1-challanged lungs appeared to have a slower rate of clearance than those of the controls (Figures 4B and 4C). By Day 7, bioluminescence fell below the detection threshold of this method for both groups (data not shown).

To confirm the observation from the in vivo imaging, we quantitatively measured the luciferase enzymatic activities of the lungs from the animals similarly treated as those for the whole body imaging. One, 4, and 7 days post MSC infusion, the lungs were harvested, homogenized, and quantified for luciferase activities by bioluminescence assays. Consistent with the imaging data, MSCs, infused through tail veins, were largely lodged into the lungs. Analyses of the Day-1 homogenates showed very high bioluminescence. No significant difference was detected between the control and PAO1- challanged lungs. At Day 4 after MSC administration, there was a dramatic reduction in the level of bioluminescence within the lungs for both groups. At Day 7, the control group showed little bioluminescence. Interestingly, the PAO1-treated group maintained a comparable bioluminescence as Day 4 and is significantly higher than that of the day-7 control animals by Student’s t-test (p<0.05, n= at least 3). Thus, the MSCs with FPR overexpression had an enhanced retention and homing to inflamed lungs.

Engraftment of FPR-engineered MSCs to distal lungs and airways

The next question we asked was whether the i.v. infused MSCs extravasated blood vessels and engrafted into lung epithelia. Immunohistochemical staining with the first antibody specific to EGFP was performed on tissue sections of paraffin-embedded lungs. The biotinylated secondary antibody, coupled with streptavidinhorseradish peroxidase, oxidizes 3,3’-diaminobenzidine (DAB) to produce a brown staining for EGFP- positive cells. The nu/nu mice, 8 days after receiving intra-tracheal intubation of PBS followed by i.v. infusion of the FPR-engineered MSCs the next day, resulted in no detectable MSC engraftment in the lungs (Figures 5A and 5B). However, the parallel group of mice intubated with sub-lethal PAO1 (5 x 105 CFU) intratracheally gave rise to MSC engraftment in the airways and alveoli (Figures 5C-5F). Interestingly, clonal expansion of MSCs was clearly seen (Figures 5D and 5E). The positive cells in the examined samples of distal lungs were counted and the percent of positive cells was estimated to be ~0.5%. In contrast, MSC airway engraftment was much lower (~0.03%). Because the samples were collected at Day 8 after intra-tracheal intubation when lung inflammation had been resolved, few inflammatory cells were observed in the lungs. Noticeably, the EGFP-positive cells integrated into the alveolar and airway epithelial structures, suggesting that the FPR-engineered MSCs have the potential of not only homing, but also engrafting to lung epithelia under the condition of bacterial challenge.

Discussion

Even though MSCs possess the great capacity of tissue regeneration and repair, a significant barrier to effective therapy is the inability of MSCs to target tissues of interest with high efficiency of engraftment [34]. Engineering MSCs to enhance their ability to home to particular tissues represent a novel strategy to overcome the low engraftment hurdle facing the stem cell field. In order for MSCs to migrate, the cells must have the ability to sense chemoattractant gradients prior to engaging in directional movement. Marrow-derived MSCs express FPR and migrate towards an N-FP gradient [18,19]. Taking advantage of such an existing chemotactic signaling pathway in MSCs, we demonstrate in this report that lentivector-enhanced expression of FPR enhances MSC homing and engraftment to inflammatory targets such as bacteriuminfected lungs. Because N-FP and its receptor FPR constitute a unique ligand-receptor pair for chemotaxis towards inflammation, the strategy of engineering MSCs with FPR for therapy may have broad clinical applications in potential intervention of inflammation- related disorders.

genetic-syndromes-gene-therapy-enhanced-expression

Figure 4:Lentivector-enhanced expression of FPR in human MSCs facilitates their homing to inflamed lungs. Panel A: Experimental scheme: at Day -1, athymic (nu/nu) mice were intubated intra- tracheally with 50 µl of Pseudomonas aeruginosa suspension solution (5 x 105 CFU) or the same volume of PBS for control. At Day 0, human MSCs (1 x 106), co-transduced with 20 MOI of HIV- CMV-FPR-IRES2-EGFP and HIV-CMV-Luciferase-IRES2-DsRed vectors, were infused into the mice by tail vein injection. At the specified time points (Day +1, +4 and +7), the animals were either subjected to in vivo bioluminescent imaging or sacrificed for measurement of firefly luciferase enzymatic activity in the lung. Panels B & C: Whole-body bioluminescent imaging of MSC localization. Two animals from each group were anesthetized by isoflurane inhalation and injected with 150 mg/kg of luciferin. Bioluminescent signals were detected at 5 minutes after luciferin injection at an integration time of 1 second to 2 minutes using the Xenogen in vivo imaging system. The intensity of bioluminescent emission was pseudo-colored and displayed (B). Photon emission rates in the thorax area were obtained and demonstrated (C). Panel D: Biochemical measurement of firefly luciferase enzymatic activity in the lungs. The animals were sacrificed at Day +1, +4 and +7 post MSC-infusion. The lungs were harvested and homogenized. Luminescence was measured immediately after addition of luciferin in a luminometer and normalized against the protein concentrations determined by BCA analyses. The data were expressed as Relative Luminescent Units (RLU) per milligram protein (mg) per minute. By day 7, significant retention of the MSCs in the lungs challenged with Pseudomonas aeruginosa was detected.

Lung diseases including asthma, emphysema, pulmonary fibrosis, and cystic fibrosis remain amajor cause of morbidity and mortality globally. Although important advances in supportive treatments have been made, there has been no cure for these diseases. MSCs are emerging as a promising cell-based therapy for a wide range of disorders. Clinical trials are evaluating the therapeutic effects of MSCs in patients with multiple sclerosis, graft-versus-host disease, Crohn disease and severe chronic myocardial ischemia [35]. Compelling preclinical data also demonstrate the promise of using MSCs for inflammatory lung diseases [36-40]. However, MSCs engraft undamaged lungs poorly [41-43], even though intravenously infused MSCs are largely lodged into the lungs initially. To increase MSC homing and engraftment, many reagents including bleomycin [44,45], naphthalene [41,43], LPS [46], irradiation [42] and polidocanol [47], were used to damage the lungs to create inflammation milieus. Using E. coli endotoxin or E. coli live bacteria to induce acute injury in isolated human lungs, Lee and colleagues found therapeutic effects of human mesenchymal stem cells on alveolar fluid clearance, inflammation reduction and microbicidal activity [40,48]. In this report, we used live Pseudomonas to infect mouse lungs to evaluate FPR-engineered MSC homing and engraftment. This model is of clinical relevance because bacterium-induced lung inflammation and injury has a high clinical occurrence. MSCs could have therapeutic effects on lung diseases through two possible ways: 1) direct structural constitution via transdifferentiation into lung cells, and 2) indirect functional modulation via secretion of paracrine factors such as growth factors, cytokines and chemokines. Regardless of their working modes, MSCs have to be present in the target tissues. Our data, showing that FPR-overexpression enhances MSC homing and engraftment into inflammatory lungs, suggest the possibility of achieving N-FP-guided stem cell targeting.

genetic-syndromes-gene-therapy-Paraffin-sections

Figure 5:Morphological examination of MSC homing and engraftment in the lungs. Paraffin sections of the lungs, 8 days post intubation with either PBS or PAO1, were immunohistochemically staining for EGFP expression. Panels A & B: Lung sections from the control animals received PBS intra-tracheal intubation and MSC intra-vascular infusion. Panels C-F: Lung sections from the experimental animals received PAO1 intra-tracheal intubation and MSC intra-vascular infusion. The EGFP-positive cells were stained brown (Red arrows). MSCs extravasated out of the blood vessel (C). MSCs engrafted into lung airway epithelia and underwent clonal expansion (D & E). MSCs engrafted into lung alveolar epithelia (F).

Stem cell therapy for different lung diseases may need different modalities [49]. For some lung diseases such as acute lung injury and acute respiratory distress syndrome, transient presence of MSCs in the lungs may be sufficient for injury repair via paracrine mechanisms [38,39,50,51]. However, for some other lung diseases such as cystic fibrosis and COPD, long-term engraftment may be required. As shown in Figure 5, the engrafted MSCs appeared to assume epithelial morphology and underwent clonal expansion in some cases. With regard to therapy, differentiation of the engrafted MSCs into pulmonary epithelia is critical for certain diseases, such as cystic fibrosis, which can correct and replace the diseased lung epithelia. Our previous publication has shown that CFTR gene-corrected CF- MSCs can differentiate into airway epithelial cells and contribute to chloride transport in airway epithelia in vitro in an air-liquid interface system [33]. The data from this report demonstrate that the MSCs engineered with FPR can engraft into distal lung and airway epithelia in vivo, indicating the potential for targeting of gene-engineered MSCs to CF lungs.

In addition to their stem/progenitor properties, MSCs have shown broad immune modulation abilities. They can be either proinflammatory or anti-inflammatory depending on their local environmental cues which induce them into different phenotypes [5,6,52,53]. Our data in Figure 3 demonstrate that the MSCs with FPR overexpression retain their pluropotency. It awaits further characterization as to whether the engineered stem cells retain their natural ability to sense and respond to their surrounding conditions for function.

In conclusion, this research provides the evidence suggesting that marrow-derived MSCs can be engineered to overexpress FPR for specific tissue targeting. Such a manipulation does not affect pluripotency of the MSCs. Importantly, overexpression of FPR enhances the MSC ability to home and engraft to inflammatory tissues such as bacteriuminfected lungs. Thus, it is possible to exploit the existing FPR signaling mechanism and chemotactic machinery within MSCs to achieve a guided targeting of the stem cells to inflammatory sites for therapy.

Acknowledgements

The authors would like to acknowledge the technical assistance from the Vector and Morphology Core Facilities sponsored by the LSUHSC Gene Therapy Program. Human MSC isolation, production, and distribution were supported through National Institutes of Health National Center for Research Resources grant to Darwin Prockop (5P40-RR017447-03).

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