Characterization of EGFP-labeled mesenchymal stem cells and redistribution of allogeneic cells after subcutaneous implantation
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- Duan, X., Yang, L., Dong, S. et al. Arch Orthop Trauma Surg (2008) 128: 751. doi:10.1007/s00402-008-0585-y
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Bone marrow mesenchymal stem cells (MSCs) are ideal target cells for cell transplantation and tissue engineering. We investigated their biological characteristics and differentiation mediated by different methods. It is important to study the short-term fate of labeled allogeneic MSCs after subcutaneous implantation in rabbits in order to provide insights into the application of allogeneic MSCs for tissue regeneration.
Materials and methods
Mesenchymal stem cells were labeled by two different methods in vitro and then were incubated with gelatin sponge. Autologous MSCs-Gelatin constructs and allogeneic MSCs-Gelatin constructs were subcutaneously implanted into 32 rabbits. The constructs were analyzed for the survival and migration of labeled MSCs at day 3, week 1, 3, and 5 post-implantation.
EGFP was successfully expressed following transfection of MSCs with the retroviral vector pLEGFP-N1. In addition, EGFP-MSCs can be functionally induced into osteocytes, chondrocytes, and adipocytes in conditioned media. By weeks 3 after implantation, the labeled cells distributed extensively on the surface of gelatin sponge and gradually integrated into host tissues. EGFP-labeled MSCs were observed under fluorescence microscopy and BrdU-expressing cells were detected with immunohistochemical stain in allogeneic or autologous MSCs-Gelatin constructs during the initial five weeks after implantation.
It is a simple and reliable way to trace the changes of MSCs in vivo by EGFP in cell transplantation and gene therapy. Allogeneic rabbit MSCs can survive for at least 5 weeks after subcutaneous implantation and maintain a strong ability of migration, indicating that allogeneic MSCs are of certain value in clinical application for temporary replacement.
KeywordsMesenchymal stem cells (MSCs)Retroviral transfectionEnhanced green fluorescent protein (EGFP)MigrationAllogeneic cell therapy
Stem cells are defined by their ability of self-renew and differentiated into one or more cell types. Because stem cells are the precursors of many tissues, they are good candidates for transplantation aiming to generate replacement tissue for damaged organs as bone and cartilage defects and treat such human conditions as Parkinson’s disease, diabetes, and heart disease [10, 18, 29]. For transplantation purposes, adult stem cells have advantages over embryonic stem cells. The former can be obtained from the patient, expanded by culture, and then re-introduced into the same patient, thus avoiding some of the problems of allotransplantation of embryonic stem cells, especially ethical restrictions .
A well-known type of adult stem cells is bone marrow mesenchymal stem cells (MSCs). Mesenchymal stem cells were first described by Friedenstein, a decade after the characterization of haematopoietic stem cells . MSCs maintain an undifferentiated and stable phenotype after passing many generations in vitro and are progenitors for different types of somatic cells, such as osteocytes, chondrocytes, and adipocytes [18, 29]. MSCs adhere to the tissue culture matrix and, if primary cultures are maintained for 12–14 days, the nonadherent haematopoietic stem cell fraction is depleted. Although MSCs represent a very small fraction of the total population of nucleated cells in the marrow, they can be isolated and expanded with high efficiency.
Although the growing interest in MSCs has led to a number of studies characterizing the biochemical and morphological properties of MSCs [6, 9,12, 23, 27, 33], the survival and migration of transplanted allogeneic MSCs have not been described yet. The purpose of this study is to seek a feasible and stable method of long-term tracking, which will not interfere cell functions, and to investigate the short-term fate of labeled allogeneic MSCs after being subcutaneously implanted into rabbits, so as to provide insights into the application of allogeneic MSCs for tissue regeneration.
Materials and methods
pLEGFP-N1 retroviral vector and packaging cell line PT67 were purchased from Clontech (Grand Island, NY, USA); penicillin–streptomycin, 0.20% trypsin–EDTA, polybrene, dexamethasone, proline, β-glycerol phosphate, papain, indomethacin, insulin, methylxanthine, N-acetyl cysteine and 5-bromo-2′-deoxyuridine from Sigma (St. Louis, MO, USA); Dulbecco’s modified eagles medium (DMEM) and fetal bovine serum (FBS) were from HyClone (Logan, UT); ITS+ supplement from Collaborative Research (Waltham, MA, USA); ascorbic acid-2-phosphate (AA2P) from Wako BioProducts (Richmond, VA, USA); transforming Growth Factor-β1 (TGF-β1) from Novartis Pharmaceuticals; sterile gelatin sponge from Lizu (Guangzhou, China). Adult New Zealand white rabbits weighing 2–2.5 kg were housed indoors. Animals were used in accordance with Novartis Pharmaceuticals Institutional Animal Care and Use Committee guidelines of the nation.
Isolation and culture of rMSCs
The rMSCs were isolated and cultured as previously described . Briefly, heparinized bone marrow was obtained by aspiration from the femur trochanter of healthy rabbits. Bone mononuclear cells (MNCs) were prepared by density gradient centrifugation with Percoll (below 1.073 g/mL, Pharmacia, USA), washed, counted, and plated in T-75 culture flasks at 2 × 105 cells/cm2 in Dulbecco’s modified Eagle’s medium-low glucose (DMEM-LG, Hyclone, USA) supplemented with 10% fetal bovine serum (FBS) (Hyclone, USA), and incubated with 5% CO2 at 37°C. Non-adhered cells were removed by washing in 24–48 h, and adherent cells were cultured with the medium being replaced every 3 days. Upon confluence of 80% cells, the cultures were digested with 0.20% trypsin-0.02% ethylenediamine tetraacetic acid (EDTA, Sigma, USA), and cells were then seeded at 8 × 103 cells/cm2 in a T-75 flask, being used either for gene transfection or for further culture and passing. MSCs were checked for multi-linage differentiation by adipogenic, chondrogenic and osteogenic differentiation assays as described previously [15, 19, 20, 25, 36]. Briefly, osteogenesis was induced by culturing MSC monolayer in medium containing 10−8 mol/L dexamethasone, 10 mmol/L β-glycerol phosphate, and 50 mg/L ascorbic acid 2-phosphate. Alkaline phosphatase staining and Von Kossa staining of calcium deposition were performed histochemically. For chondrogenesis, MSCs were centrifuged (800 g, 5 min) and cultured in the induction medium including high-glucose DMEM, 1% ITS+, 10−6 mol/L dexamethasone, 0.17 mmol/L ascorbic acid 2-phosphate, 35 μg/mL praline, and 5 ng/mL TGF-β1. MSCs were cultured for 21 days, and then frozen sections of them were obtained and immunostained for type II collagen. Adipogenesis was induced by using the induction medium including high-glucose DMEM, 10−6 mol/L dexamethasone, 100μg/mL indomethacin, 10 μg/mL insulin, and 0.5 mmol/L 3-isobutyl-1-methylxanthine. Induction was confirmed by oil red O staining.
Retroviral vector, virus production, and transfection of rMSCs
The pLEGFP-N1 retroviral vector was employed in these experiments. The pLEGFP-N1 plasmids were transfected into DH5[alpha] cells and identified by restriction endonuclease analysis. Viral supernatant containing EGFP virus was generated by packaging cell line PT67 as described below . Briefly, 2 × 105 PT67 cells were plated into a 6-well dish 12–24 h before transfection at 80% confluence. Adding 25 μL DOTAP (Roche, Germany) before transfection may increase the transfection efficiency, then PT67 cells were transfected and PT67 cells stably producing viruses were screened with 800 μg/mL G418 and then the drug resistant clones were amplified. After collecting viral supernatant through a 0.45 μm cellulose acetate filter, viral titer was determined by NIH 3T3 cells as previously described . 1 × 105 primary rMSCs in a 6-well plate were exposed to viral supernatant containing EGFP virus at a multiplicity of infection of one to ensure single-copy integration, in the presence of 8 μg/mL polybrene for 24 h. Subsequently, the viral supernatant was replaced with fresh medium. After having been incubated for another 24 h, infected rMSCs were selected with 300 μg/mL G418 for 2 weeks in order to get MSCs containing EGFP gene, named rEGFP-MSCs . The transfection efficiency and the biological characteristics of rMSCs were tested after harvest of rEGFP-MSCs.
Confirmation of proliferation and multilineage differentiation in EGFP-MSCs
The growth kinetics of rEGFP-MSCs was analyzed at passage 4. The cells were seeded onto 96-well culture plates (1.5 × 103 cells/well). After continuous culture for 9 days, 20 μL of Tetrazolium salt (MTT) solution (5 mg/mL, Fluka Co., Switzerland) was added into each well and then incubated for 4 h at 37°C in a 5%-CO2 atmosphere. After supernatant was discarded, 150 μL of dimethylsulfoxide (Shanghai Bioengineering Co., Shanghai, China) was added into each well and vibrated for 10 min. Absorbance at wavelength of 570-nm was measured for each well by using a Sepctra spectrophotometer (MAX 250, Molecular Devices Corp, Sunnyvale, CA, USA). Experiments were performed three times in duplicate and then cell growth curves were plotted. According to the growth curves and Patterson formula, the doubling time was calculated.
Evaluation of the differentiation potential of rEGFP-MSCs
Osteogenic, chondrogenic, and adipogenic induction were assessed to prove multilineage differentiation in EGFP-MSCs at passage 4. To induce osteogenic differentiation, cells were seeded onto 6-well culture plates (1 × 105 cells/well) and cultured in the osteogenic induction medium as described previously. The cultures were maintained for 2 weeks and the medium was replaced every 3 days. Osteoblasts were confirmed by cytochemical staining for alkaline phosphatase (ALP). To detect bone nodules, calcium deposits were stained by the Von Kossa method. To induce adipogenic differentiation, the cells were seeded onto 6-well plates (3 × 105 cells/well). Adipogenic differentiation was maintained in the aforementioned induction medium for 2 weeks. The cells were rinsed twice with PBS, fixed with 10% neutral buffered formalin for 30 min, and stained with Oil red O to confirm lipid deposition. In chondrogenic induction, MSCs were centrifuged in a 15 mL tube at 1,500 g for 5 min, and cultured in the induction medium, with 50% medium being replaced every 2 days. The cell pellets were cultured for 21 days, and then frozen sections of the cell pellets were obtained, followed by immunohistochemical staining of type II collagen and staining with hematoxylin and eosin.
Implantation of labeled MSCs-Gelatin constructs
Animal group design
Animal experiments were carried out according to a protocol approved by the Animal Experimentation Committee of the University. A total of 32 healthy rabbits were divided into two groups. 16 animals were transplanted with labeled autologous MSCs and the others with labeled allogeneic MSCs. Samples were evaluated at day 3, weeks 1, 3, and 5 after transplantation (n = 4 for each time point).
Preparation labeled MSCs
Two different methods were used to label MSCs in this study: labeling MSCs with EGFP as described previously, or labeling MSCs with 5-bromo-2′-deoxyuridine (BrdU) that can be observed by immunohistochemical staining . Adding 30μmol/L BrdU in DMEM-LG supplemented with 10% FBS 24 h before harvest may efficiently label cultured MSCs. Eventually, all labeled MSCs were trypsinized to prepare MSCs-Gelatin constructs.
Embedding of labeled MSCs into gelatin
Since type I collagen is the main collagen component of skin, gelatin sponge was used as a carrier material to deliver rEGFP-MSCs or rMSCs in vivo. Gelatin sponge was cut using skin biopsy punch to make uniform cubes (5 mm × 5 mm × 5 mm) for implant studies. The cubes were hydrated for 1 h in DMEM, dabbed dry on gauze, and transferred to a 12 × 75 mm tube containing a suspension of 5 × 106 labeled MSCs in 1 mL and incubated for 2 h at 37°C, 5% CO2. Before MSCs-loaded sponge constructs were implanted, non-adherent cells were removed by placing the constructs into fresh DMEM .
Implantation of labeled MSCs-Gelatin constructs
MSCs-Gelatin constructs were transplanted into dorsal subcutaneous tissue from adipose layer to lumbodorsae fascia of rabbits anesthetized with 30 g/L sodium pentobarbital (1 mL/kg). All grafts containing EGFP-labeled MSCs were on the left side, while BrdU-labeled MSCs were on the right side. Following implantation, the rabbits were raised separately and observed everyday.
Tissue harvesting and histological evaluation
After animals were sacrificed, the whole graft was removed from dorsal subcutaneous tissue. After macroscopic evaluation, samples containing EGFP-labeled cells were frozen and cut into 5 μm-thick sections. Sections were observed for EGFP-positive cells under fluorescence microscope. The survival of EGFP-labeled MSCs was evaluated, the mean ± SE number of EGFP-positive cells per field of vision was determined and ten fields of vision per coverslip were counted. The remaining samples should be fixed in 4% paraformaldehyde overnight and embedded in paraffin. The paraffin blocks were cut into 4 μm sections for hematoxylin and eosin staining or immunoperoxidase staining for BrdU observation. Two histologists evaluated these sections to minimize the intra-observer error. To detect BrdU-labeled MSCs in the samples, immunohistochemical staining for BrdU was carried out at each time point. Methanol–H2O2 treatment was applied to eliminate autogenous peroxidase activity and 10% normal goat serum was used to block non-specific immunoreaction. Sections were labeled overnight at 4°C with anti-BrdU monoclonal antibody diluted 1:100 in PBS. The samples were washed three times with PBS and reacted with mouse anti-horseradish peroxidase at a dilution of 1:100 in PBS for 30 min at 4°C. Finally, DAB was added for coloration .
Data were expressed as mean ± SE, and group differences were analyzed by two-sided Student’s t test using the Statistical Analysis System (Version 6.08, SAS Institute Inc., Cary, NC, USA). A P value less than 0.05 was considered statistically significant.
Isolation and culture of rMSCs
Construction of rEGFP-MSCs
Proliferation and differentiation potential of rEGFP-MSCs
Implantation of labeled MSCs-Gelatin constructs
MSCs have been isolated from bone marrow, periosteum, trabecular bone, adipose tissue, synovium, skeletal muscle, and deciduous teeth [3, 18, 29, 39]. These cells can differentiate into cells of connective tissue lineages, including bone, fat, cartilage, and muscle. A great deal research has been learned in recent years about the isolation and characterization of MSCs and the control of their differentiation. Researchers show great interest in these cells because of their potential use in regenerative medicine and tissue engineering [1, 26, 37]. Some impressive examples, derived from both pre-clinical and clinical studies, illustrate their therapeutic value [7, 14, 38]. Although early pre-clinical and clinical data demonstrate the safety and effectiveness of MSCs therapy [16, 31, 34, 37], there are still many questions regarding the mechanism of action need to be answered. Additional information is required concerning the therapeutic efficacy of transplanted cells and the mechanisms of engraftment, homing and in vivo differentiation. There is accumulating evidence of the hypoimmunogenic nature of MSCs, which has broad implications in terms of allogeneic therapy, or the delivery to a recipient of cells derived from an unmatched donor [2, 18, 24]. There are several reports describing the clinical use of allogeneic donor-mismatched cells with little evidence of host immune rejection. First of all, appropriately designed studies are needed to demonstrate that allogeneic MSCs can survive.
How to detect and observe the fate of the donor cells in the recipient is an important question encountered after transplantation. At present, the common solution is to transplant the cells tagged in vitro, and the tagging methods mainly include : (1) transfecting reporter genes to make cells express specific markers such as Green Fluorescent Protein or β-galactosidase [4, 9, 32]; (2) using Y chromosomes as marker for male donors and female recipients ; (3) using dyes such as DiI or BrdU to label cells . Among them, β-galactosidase enzyme can not be directly observed under a microscope without dying, whereas BrdU can not ensure the long-term and effective marking of cells. Hence, EGFP is a commonly used marker and harmless for cells. It gives strongest detecting signal among all cell labeling techniques. Besides these advantages, expressed EGFP is stable in mammal cells and easy to detect under a fluorescence microscope. Since 1994 when Chalfie et al successfully cloned GFP cDNA and expressed the protein in prokaryotic and eukaryotic systems , EGFP has been widely used in tissue engineering and gene therapy [11, 21, 28]. Although plasmid transfection, adenoviral or retroviral gene transfection of EGFP can label rMSCs, whether rEGFP-MSCs maintain the biological features and the potency of multilineage differentiation can not be determined thoroughly. Therefore, the safety of cell transplantation in vivo is still in doubt. Because the method based on retroviral transfection can ensure that the reporter gene is integrated into host cells’ chromosomes and can stably exist for a long time, rEGFP-MSCs are constructed by retroviral transfection. In the present study, we set up a culture of EGFP transfected rMSCs population by pLEGFP-N1 retroviral vector, and investigated whether the biological characteristics and differentiation potential are retained. In our study, it was indicated that rEGFP-MSCs are similar to rMSCs. First of all, their morphological features and immunophenotypes were identical to those of parent rMSCs. rEGFP-MSCs also presented with a long fusiform, fibroblast-like shape and a flocked array. Second, rEGFP-MSCs had similar growth characteristics with those of rMSCs. Third, most importantly, rEGFP-MSCs still functionally retained osteogenic, chondrogenic, and adipogenic potentia1. These results show that EGFP and retroviral transfection are almost harmless to rMSCs, and that rEGFP-MSCs can maintain their biological function. In conclusion, rMSCs transfected with pLEGFP-N1 retroviral vector exhibit morphologic, immunophenotypic, and growth characteristics similar to those of rMSCs, without losing multipotentiality. Therefore, rEGFP-MSCs that maintain their own biological characteristics may be safely used in vivo cell transplantation and gene therapy.
To know the fate of allogeneic stem cells for treatment, we must investigate whether allogeneic stem cells can survive after transplantation. Hence, we carried out a series of experiments. Cells expressing EGFP or containing BrdU were observed in all the samples during the initial five weeks after implantation of allogeneic or autologous MSCs-Gelatin constructs. But the number of labeled MSCs was decreased gradually in local area of MSCs-Gelatin constructs. The labeled MSCs migrated gradually to the interface between gelatin and subcutaneous tissue, and then detained in host tissue. At day 3 and week 1, inflammatory reaction was obvious, and then inflammatory cells were reduced. In addition, no animal developed complication, such as red swelling and effusion. In this study, we demonstrate that allogeneic MSCs labeled with EGFP or BrdU were adopted by host tissue and survived at lest 5 weeks, and that host responses to allogeneic MSCs graft may be depressed. Appropriately designed toxicological studies are still required to demonstrate the long-term safety of these therapies. Once these issues have been solved, new applications of allogeneic MSCs will emerge, leading to novel therapeutic opportunities [5, 30, 34].
This work was financially supported by the National Natural Science Foundation of China (Grant No. 30300079 and No.30400100) and Key Medical Research Projects of PLA (Grant No. 06G079). The authors thank the staff of the State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Combined Injury, The Third Military Medical University for supplying experimental equipment. They also thank Prof. Z. Zou. of our university for critical review of this manuscript.