Introduction
Mesenchymal or stromal stem cells are present in mature human and animal tissues and are readily harvested from the BM,1–3 periosteum,3 AT,4,5 and synovial membranes.6 Mesenchymal stem cells are pluripotent, capable of differentiating into osteoblasts, chondrocytes, adipocytes, and myocytes; thus, they may be a source of cells to replace those cells affected by damaged musculoskeletal tissues, including intraarticular joint structures, such as the meniscus or intervertebral disks.7–9 Mesenchymal stem cells can be cultured, and they may be subject to as many as 20- to 50-population doubling cycles over approximately 2 to 3 months.10 A focus has been on MSCs derived from AT11 and BM2 because MSCs are readily available from these sites.5,12,13 Studies14,15 reveal that AT-MSCs and BM-MSCs may be used as regenerative treatments in horses with musculoskeletal conditions, and MSCs have been suggested for treatment of various joint diseases in people16 and horses.17
The principal surface markers used to identify MSCs are CD73, CD90, and CD105.18 However, labeling MSCs with GFP is now common in MSC transplantation studies19 to ensure that these MSCs were placed into the receiving tissue. Interest in the structural and ultrastructural characterization of MSC phenotypes for transfected and nontransfected cells has been increasing20; however, the ultrastructure of MSCs has not been thoroughly investigated.
The objective of the study reported here was to characterize AT-MSCs and BM-MSCs that were harvested from healthy horses, including analysis of their ultrastructure, identification of their surface markers, and description of the morphological changes that occur in MSCs after transfection with GFP. Green fluorescent protein stable transfection was speculated to induce morphological changes in the ultrastructure of MSCs.
Materials and Methods
Study protocols complied with the Guidelines of the Council of the European Union (Directive 2010/63/EU [revision of Directive 86/609/EEC] on the protection of animals used for scientific purposes adopted on September 22, 2010) and with Spanish regulations (BOE 67/8509-12, 1998) for the use of laboratory animals. Study protocols were approved by the Scientific Committee of the University of León, Spain.
Isolation of MSCs
Cells were harvested from 6 female Hispano-Bretón horses that were between 4 and 7 years of age. Horses were sedated with romifidinea (0.04 mg/kg, IV) and butorphanolb (0.015 mg/kg, IV). The dorsal area of the gluteal muscles was prepared aseptically, and skin and subcutaneous tissues were desensitized by local infiltration of 2% lidocainec by use of an inverted L-block. A 10- to 15-cm skin incision was made parallel and 15 cm abaxial to the vertebral column. Approximately 15 g of AT was harvested over the superficial gluteal fascia for immediate isolation of AT-MSCs; the skin incision was closed with nylon suture material.d The AT harvested from each horse was then pooled.
The protocol outlined by Zuk et al5 was followed for isolation of the AT-MSCs. Tissue was washed with PBS and its extracellular matrix was degraded with 0.075% type I collagenasee in DMEMe for 1 hour at 37 °C. A high-density stromal vascular fraction was obtained by centrifugation at 1,200 × g for 10 minutes. To remove cellular debris, the resulting pellet was filtered through a 100 hyphenate μm nylon mesh.
Bone marrow–derived MSCs were harvested according to previously published methods.21,22 While the horses were standing, they were sedated with romifidine and butorphanol as before for harvesting of AT-MSCs, and needle-aspirated BM samples were obtained from the 5th and 6th sternebrae. Aspirated BM cells were collected in tubese that contained 0.5 mL of 5,000 U of heparin/mL solution, to yield a final concentration of 250 U of heparin/mL after the addition of 9.5 mL of aspirated BM cells. Then, an aliquot of the heparin-BM cell solution was diluted with DMEM that was prepared with 10% fetal bovine serum.f The subsequent solution was separated by use of a density gradient mediumg (density, 1.077 g/mL) and centrifugation at 1,100 × g for 30 minutes. Then the cell interface layer was collected and cultured.
Both types of MSCs were incubated at 37 °C in 5% CO2 in DMEM containing 10% fetal bovine serum. Adherent cells were grown until they reached 90% confluence (passage, 0). Cells were then trypsinized (0.05% trypsine and 0.04% EDTAe in PBS) for 5 minutes at 37 °C before reseeding. Both types of MSCs were used after 2 to 6 passages.
Transfection
Cells were plated in 24-well plates in growth medium until they reached 90% confluence. Transfection medium was prepared by diluting the plasmid vector pEF1α-GFPh and a lipid-based transfection reagenti (ratio, 1:2.5) in a reduced-serum (minimal essential) medium.j This transfection medium was then added to the plate wells, and the plates were incubated for at least 5 hours at 37 °C in 5% CO2. The transfection medium was replaced with growth medium before cell isolation. Transfection efficiency was determined by use of flow cytometry.
Stable GFP-transfection
Transfected cells were reseeded into 6-well plates and the following day, complete medium supplemented with 500 and 450 μg/mL of G418, an aminoglyco-side related to gentamicin,k was added to the wells that contained AT-MSCs and BM-MSCs, respectively. These concentrations were determined to be optimal for isolating GFP-positive MSCs (data not shown). Cells were grown until the complete selection of stable clones was achieved. Isolated clones were analyzed with fluorescence microscopy,l and the more fluorescent clones were isolated by trypsinization. Fluorescence microscopy was used at each passage to check for positive fluorescence of GFP-positive MSCs. After 15 days, the growth and metabolism of nontransfected cells were altered because of the effect of the G418 on peptide synthesis, thereby only GFP-positive (unaltered) clones remained. Isolated clones were re-seeded and multiplied to obtain sufficient numbers of cells to continue the experiments. Isolated cells were maintained with 200 μg/mL of G418 as an inducer of GFP expression. Transfected cells were quantified by use of a cyan ADP flow cytometry systemm with an acquisition rate of at least 10,000 events/sample. Softwaren was used for the analyses.
Immunocytochemical characterization
Immunocytochemical characterization of MSCs was performed with use of anti-CD90 and anti-CD105° monoclonal antibodies.° Cells were collected after trypsinization, washed twice with 0.2% bovine serum albumin,e transferred to a tube containing 0.1% sodium azidee in 0.1M PBS solution, and then centrifuged. The supernatant was removed, and the remaining pellet was resuspended in the same solution at a concentration of 105 cells/mL and incubated with anti-horse primary antibodies in 1:1,000 dilutions for 30 minutes at 4 °C. Cells were washed, incubated with secondary biotinylated anti-mouse antibodies (dilution, 1:100), and then stained with 2 types of streptavidin-conjugated antibodies (dilution, 1:100): streptavidin conjugated with alexa fluor 488 (a bright, green fluorescent dye)p and streptavidin conjugated with alexa fluor 647 (a bright, far-red fluorescent dye).p Samples were subsequently analyzed with flow cytometry.
Efficiency of stable GFP-transfection as detected by flow cytometry for AT-MSCs and BM-MSCs harvested from 6 adult female Hispano-Bretón horses (A). Representative photomicrographs that were obtained with confocal microscopy of transfected (GFP-positive) AT-MSCs (B) and BM-MSCs (C). Bar = 100 μm.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.770
Efficiency of stable GFP-transfection as detected by flow cytometry for AT-MSCs and BM-MSCs harvested from 6 adult female Hispano-Bretón horses (A). Representative photomicrographs that were obtained with confocal microscopy of transfected (GFP-positive) AT-MSCs (B) and BM-MSCs (C). Bar = 100 μm.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.770
Efficiency of stable GFP-transfection as detected by flow cytometry for AT-MSCs and BM-MSCs harvested from 6 adult female Hispano-Bretón horses (A). Representative photomicrographs that were obtained with confocal microscopy of transfected (GFP-positive) AT-MSCs (B) and BM-MSCs (C). Bar = 100 μm.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.770
Efficiency of stable GFP-transfection as detected by flow cytometry for AT-MSCs and BM-MSCs harvested from 6 adult female Hispano-Bretón horses (A). Representative photomicrographs that were obtained with confocal microscopy of transfected (GFP-positive) AT-MSCs (B) and BM-MSCs (C). Bar = 100 μm.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.770
Efficiency of stable GFP-transfection as detected by flow cytometry for AT-MSCs and BM-MSCs harvested from 6 adult female Hispano-Bretón horses (A). Representative photomicrographs that were obtained with confocal microscopy of transfected (GFP-positive) AT-MSCs (B) and BM-MSCs (C). Bar = 100 μm.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.770
Efficiency of stable GFP-transfection as detected by flow cytometry for AT-MSCs and BM-MSCs harvested from 6 adult female Hispano-Bretón horses (A). Representative photomicrographs that were obtained with confocal microscopy of transfected (GFP-positive) AT-MSCs (B) and BM-MSCs (C). Bar = 100 μm.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.770
Efficiency of stable GFP-transfection as detected by flow cytometry for AT-MSCs and BM-MSCs harvested from 6 adult female Hispano-Bretón horses (A). Representative photomicrographs that were obtained with confocal microscopy of transfected (GFP-positive) AT-MSCs (B) and BM-MSCs (C). Bar = 100 μm.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.770
Transmission electron microscopy
Mesenchymal stem cells were centrifuged at 1,200 × g for 10 minutes, and the resulting pellets were cultured overnight with complete DMEM at 37 °C in 5% CO2 until nodules were formed. Subsequently, cells were washed with PBS, fixed with 2.5% paraformaldehydee for 2 hours at 4 °C, and incubated in 1% osmium tetroxidee in PBS solution for 3 hours at 20 °C. Cells were embedded in 15% gelatin and dehydrated by use of a graded ethanol series and then passed through propylene oxidee and embedded in an epoxy embedding mediumq for 24 hours. Ultra-thin sections were cut by use of an ultramicrotomer and stained with uranyl acetatef and citrate plumb.f Afterwards, samples were analyzed by transmission electron microscopy.s
Statistical analysis
Each portion of the study was performed 3 times. Data were analyzed with commercially available statistical software.t The Kolmogorov-Smirnov test wasused to verify normal distribution of the data. Transfected and nontransfected groups were compared with 1-way ANOVA and then with the Tukey multiple comparison post hoc test. Paired data were compared by use of a t test. Values of P < 0.05 were considered significant.
Results
The yield of transfected (GFP-positive) cells was 4% to 5% for AT-MSCs and BM-MSCs (Figure 1). Greater than 90% of MSCs expressed both CD90 and CD105, except for transfected BM-MSCs (Figure 2). For transfected AT-MSCs, expression of CD90 was significantly (P < 0.05) higher, compared with nontransfected AT-MSCs (97% vs 92%). For transfected BM-MSCs, CD105 expression was significantly (P < 0.05) lower, compared with nontransfected BM-MSCs (85% vs 94%).
Expression of surface markers CD90 (A) and CD105 (B) as detected by flow cytometry by AT-MSCs (with [GFP+ AT-MSCs] and without GFP) and BM-MSCs (with [GFP+ BM-MSCs] and without GFP) that were harvested from the horses of Figure 1. *P < 0.05, compared with AT-MSCs. †P < 0.05, compared with BM-MSCs.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.770
Expression of surface markers CD90 (A) and CD105 (B) as detected by flow cytometry by AT-MSCs (with [GFP+ AT-MSCs] and without GFP) and BM-MSCs (with [GFP+ BM-MSCs] and without GFP) that were harvested from the horses of Figure 1. *P < 0.05, compared with AT-MSCs. †P < 0.05, compared with BM-MSCs.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.770
Expression of surface markers CD90 (A) and CD105 (B) as detected by flow cytometry by AT-MSCs (with [GFP+ AT-MSCs] and without GFP) and BM-MSCs (with [GFP+ BM-MSCs] and without GFP) that were harvested from the horses of Figure 1. *P < 0.05, compared with AT-MSCs. †P < 0.05, compared with BM-MSCs.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.770
Expression of surface markers CD90 (A) and CD105 (B) as detected by flow cytometry by AT-MSCs (with [GFP+ AT-MSCs] and without GFP) and BM-MSCs (with [GFP+ BM-MSCs] and without GFP) that were harvested from the horses of Figure 1. *P < 0.05, compared with AT-MSCs. †P < 0.05, compared with BM-MSCs.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.770
Expression of surface markers CD90 (A) and CD105 (B) as detected by flow cytometry by AT-MSCs (with [GFP+ AT-MSCs] and without GFP) and BM-MSCs (with [GFP+ BM-MSCs] and without GFP) that were harvested from the horses of Figure 1. *P < 0.05, compared with AT-MSCs. †P < 0.05, compared with BM-MSCs.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.770
Transmission electron microscopy helped to identify any ultrastructural morphological differences among the MSCs (with and without GFP transfection; Figures 3 and 4). Each type of MSC had a notched nucleus with a prominent nucleolus; however, the nucleus of each GFP-transfected MSC more often had a nucleolus. At low magnification, AT-MSCs and BM-MSCs appeared similar, in that each MSC had an irregular nucleus containing chromatin within the perinuclear cisternae. Well-developed organelles, including mitochondria, endoplasmic reticulum, and free ribosomes, were observed in all MSCs, with lipid vacuoles as overall the most common. Transfected AT-MSCs had a similar morphology to that of nontransfected AT-MSCs, with their cytoplasms having had high numbers of dense lipid vacuoles and round mitochondria. Their chromatin was concentrated in the perinuclear zone. However, the morphologies of transfected and nontransfected BM-MSCs differed, with each transfected BM-MSC having had a nucleus with a low concentration of perinuclear chromatin. Each type of BM-MSC had rough endoplasmic reticulum, rather than lipid vacuoles as for AT-MSCs, as their most common organelle. Yet, the endoplasmic reticulum was the most common organelle in transfected BM-MSCs and its morphology was irregular and dilated with many mitochondria. Heterochromatin was detected in both types of nontransfected MSCs and in transfected AT-MSCs. Golgi apparatus and other organelles were present but to a lesser extent. Nontransfected and transfected MSCs also had heterogeneous vacuolar inclusions throughout their cytoplasms.
Representative photomicrographs obtained with transmission electron microscopy of nontransfected AT-MSCs (A) and BM-MSCs (C) and transfected AT-MSCs (B) and BM-MSCs (D). Each cell has a notched nucleus (N), many lipid vacuoles (arrows; A through C), and rough endoplasmic reticulum (asterisk; D). Magnification 6,000X. Bar = 2 μm
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.770
Representative photomicrographs obtained with transmission electron microscopy of nontransfected AT-MSCs (A) and BM-MSCs (C) and transfected AT-MSCs (B) and BM-MSCs (D). Each cell has a notched nucleus (N), many lipid vacuoles (arrows; A through C), and rough endoplasmic reticulum (asterisk; D). Magnification 6,000X. Bar = 2 μm
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.770
Representative photomicrographs obtained with transmission electron microscopy of nontransfected AT-MSCs (A) and BM-MSCs (C) and transfected AT-MSCs (B) and BM-MSCs (D). Each cell has a notched nucleus (N), many lipid vacuoles (arrows; A through C), and rough endoplasmic reticulum (asterisk; D). Magnification 6,000X. Bar = 2 μm
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.770
Representative photomicrographs obtained with transmission electron microscopy of the ultrastructure of nontransfected AT-MSCs (A and C) and BM-MSCs (E and G) and of transfected AT-MSCs (B and D) and BM-MSCs (F and H). In panels C, D, G, and H, the nucleoli (dagger) are noted. In panels C through E, chromatin is noted in the perinuclear zone (thin arrows). In panels A, E, and G pinocytotic vacuoles are noted (thick arrows). In panels F through H, the rough endoplasmic reticulum (asterisk) is noted. G = Golgi apparatus. L = Lipid vacuole. M= Mitochondria. N = Nucleus. Magnification 15,000X. Bar = 1 μm.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.770
Representative photomicrographs obtained with transmission electron microscopy of the ultrastructure of nontransfected AT-MSCs (A and C) and BM-MSCs (E and G) and of transfected AT-MSCs (B and D) and BM-MSCs (F and H). In panels C, D, G, and H, the nucleoli (dagger) are noted. In panels C through E, chromatin is noted in the perinuclear zone (thin arrows). In panels A, E, and G pinocytotic vacuoles are noted (thick arrows). In panels F through H, the rough endoplasmic reticulum (asterisk) is noted. G = Golgi apparatus. L = Lipid vacuole. M= Mitochondria. N = Nucleus. Magnification 15,000X. Bar = 1 μm.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.770
Representative photomicrographs obtained with transmission electron microscopy of the ultrastructure of nontransfected AT-MSCs (A and C) and BM-MSCs (E and G) and of transfected AT-MSCs (B and D) and BM-MSCs (F and H). In panels C, D, G, and H, the nucleoli (dagger) are noted. In panels C through E, chromatin is noted in the perinuclear zone (thin arrows). In panels A, E, and G pinocytotic vacuoles are noted (thick arrows). In panels F through H, the rough endoplasmic reticulum (asterisk) is noted. G = Golgi apparatus. L = Lipid vacuole. M= Mitochondria. N = Nucleus. Magnification 15,000X. Bar = 1 μm.
Citation: American Journal of Veterinary Research 82, 9; 10.2460/ajvr.82.9.770
Discussion
In the study reported here, AT-MSCs and BM-MSCs that were harvested from horses were stably transfected with a plasmid containing the gene for GFP. These stably transfected MSCs were characterized, and the morphologies of nontransfected and transfected MSCs were then compared. Genetic modification of MSCs through the use of various nonviral gene delivery systems have been described.23,24 The most common method uses lipids, such as with the lipid-based transfection reagentf used in the present study.25 In a previous study24 that included the use of the same transfection reagent and GFP, MSC transfection efficiency was approximately 2% to 3%, whereas in the present study, AT-MSCs and BM-MSCs were transfected with up to 4% efficiency after 5 hours of incubation (37 °C in 5% CO2) in a transfection medium. To confirm that the isolated cells were MSCs, cells should express the surface markers CD90 and CD105.18 In this study, cells were positive for CD90 and CD105, and although only 85% of transfected BMMSCs expressed CD105, CD90 expression was > 90%, which suggested that these cells were MSCs.
The effect of G418 on cellular metabolism and growth differs on the basis of whether MSCs are transfected or nontransfected.26 Expression of the gene neo in transfected MSCs increases with increasing G418 concentration, such that the negative effect of G418 on cell metabolism is decreased. The optimal concentrations of G418 necessary to isolate transfected MSCs were 500 μg/mL for AT-MSCs and 450 μg/mL for BM-MSCs, on the basis of a previous experiment, and therefore these concentrations were used in the present study.
Some studies20,22,27–33 include de scriptions of MSC morphology, and only 120 includes a description of the morphology of transfected MSCs harvested from adult rats. The present study, however, included a comprehensive analysis of the ultrastructure of equine AT-MSCs and BM-MSCs by use of transmission electron microscopy and a comparison of the ultrastructure between stably transfected MSCs and nontransfected MSCs. Each cultured cell had an undifferentiated cellular pheno-type and were characterized by an irregularly shaped large nucleus, a dense cytoplasm rich in ribosomes, a rough endoplasmic reticulum with dilated cisternae, elongated and rounded mitochondria, and heterogeneous vacuoles, including many lipid vacuoles, which were the most commonly identified organelles in these cells. These ultrastructural features indicated intense metabolic activity (protein synthesis), consistent with the pluripotential of MSCs, and helped to distinguish MSCs from other cell types such as fibroblasts.30–35,28–33 In contrast to other reports,30–35 the MSCs in the present study only had 1 nucleolus/nucleus. Lipid vacuoles were noted in all MSCs; however, a previous study29 of human AT-MSCs reveals that lipid vacuoles are typical because of the cells’ AT lineage, possibly reflecting a cell's memory about the microenvironment of its origin.34 Although MSCs are a heterogeneous population based on their expression of various immunocytochemical markers,10,34,36 the observations described herein indicated that MSC morphology, especially in ultrastructure, was relatively uniform.
Another study29 reveals that each human adult MSC has a euchromatic nucleus and a large cytoplasm that has a rough endoplasmic reticulum and numerous lysosomes, mitochondria, and lipid vacuoles; for some MSCs, pinocytosis is also present. Ozen et al32 reported the presence of vacuoles in the cytoplasm; vacuoles play an important role in the signaling of MSC differentiation and, their presence was confirmed in the present study.
Other studies37–39 indicate similar results. Human BM-MSCs have vacuoles and large irregular nuclei, but these MSCs are frequently binucleate. However, a presumably binucleate MSC may actually only have 1 nucleus; an irregularly shaped nucleus may be erroneously interpreted as having > 1 nucleus.
Transfected and nontransfected AT-MSCs and nontransfected BM-MSCs did not have any important differences in morphological features, with the same organelles identified in all 3 cell types. Transfected AT-MSCs did not alter cell morphology; however, transfected BM-MSCs differed from the other cell types. Specifically, the most common organelle for transfected BM-MSCs was rough endoplasmic reticulum with dilated cisternae and mitochondria, likely because of the relatively high degree of protein synthesis in these MSCs. Previous studies20,29,38,40 reveal characteristics of the ultrastructure of MSCs by electron microscopy; similar characteristics were noted for the MSCs evaluated in the present study. Raimondo et al20 described the ultrastructure of stably transfected BM-MSCs from rats; each MSC has an irregular nucleus and its cytoplasm has organelles similar to those identified in the MSCs evaluated in the present study, including rough endoplasmic reticulum, mitochondria, and lipid vacuoles.
One limitation of the present study was the narrow range of reagents available for research of equine MSCs, which limited mediator analyses. A second limitation was that any changes in the bioactive properties of transfected MSCs were not evaluated.
The protocols for transfection and subsequent isolation of transfected MSCs with the use of G418 were suitable for identifying and isolating AT-MSCs and BM-MSCs of horses in the present study. These findings contribute to the knowledge base of the characteristics of AT-MSCs and BM-MSCs and of transfected and nontransfected MSCs. The data provide a valuable starting point for researchers wishing to further study the morphological characteristics of these cells.
Acknowledgments
This work was funded by the Fundación Leonesa Proneurociencias (grant No. LE013A11-2).
The authors declare that they have no conflict of interest. The authors thank Dr. Margarita Marqués for providing plasmid pEF1α-GFP.
Footnotes
Sedivet, Boehringer Ingelheim Vetmedica GmBH, Ingelheim, Germany.
Torbugesic, Zoetis, Kalamazoo, Mich.
Laboratorios Ovejero SA, León, Spain.
Prolene, Ethicon LLC, Bridgewater, NJ.
MilliporeSigma, St Louis, Mo.
Thermo Fisher Scientific, Waltham, Mass.
Ficoll-Paque Premium, Thermo Fisher Scientific, Waltham, Mass.
Provided by Dr. Margarita Marqués, University of León, León, Spain.
Lipofectamine 2,000, Thermo Fisher Scientific, Waltham, Mass.
Opti-MEM, Thermo Fisher Scientific, Waltham, Mass
Geneticin G418, Thermo Fisher Scientific, Waltham, Mass.
Eclipse Ni-U, Nikon, Tokyo, Japan.
Cyan ADP Analyzer, Dako, Glostrop, Denmark.
CellQuest Summit software, version 4.3, Becton Dickinson, Franklin Lakes, NJ.
Abcam, Cambridge, England.
Invitrogen, Life Technologies, Van Allen Way, Carlsbad, Calif.
Epon 812, MilliporeSigma, St Louis, Mo.
Leica EM UC7, Wetzlar, Germany.
JEM 1010, JEOL, Tokyo, Japan.
IBM SPSS Statistics, version 21, IBM, Armonk, NY.
Abbreviations
| AT | Adipose tissue |
| AT-MSC | Adipose tissue–derived mesenchymal stem cell |
| BM | Bone marrow |
| BM-MSC | Bone marrow–derived mesenchymal stem cell |
| DMEM | Dulbecco modified Eagle medium |
| GFP | Green fluorescent protein |
| MSC | Mesenchymal stem cell |
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