In vivo effects of human adipose-derived stem cells reseeding on acellular bovine pericardium in nude mice

Ethics statement

This study was approved by the ethics committee of the Shanghai Sixth People's Hospital (Shanghai Sixth People's Hospital Affiliated to Shanghai Jiao Tong University). All animal experiments were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Ethics Committee on Animal Experiments at Shanghai Jiao Tong University School of Medicine (Permit Number: SYXK [Shanghai] 2013-0050).

Isolation and cultivation of hASCs

hASCs were obtained from the harvested adipose tissue samples of a 35-year-old woman who underwent cosmetic subdermal liposuction, after a written informed consent. The adipose tissue was digested at 37℃ with mild agitation for 60 min using collagenase type I (Sigma, St. Louis, MO). Digestion was terminated with an equal volume of Dulbecco’s Modified Eagle Medium (DMEM) with high glucose (Gibco, NY) containing 10% fetal bovine serum (FBS) (Gibco). Next, the suspension was centrifuged at 400 g (1500 r/min) for 10 min. The cell pellet was seeded onto a six-well plate (Corning, USA) in control medium (DMEM + 10% FBS + 1% penicillin/streptomycin) (Gibco). The culture was maintained at 37℃ in a humidified incubator containing 5% CO₂. The medium was replaced every three days.

Characterization of hASCs in vitro

hASCs were characterized by flow cytometry and multi-directional differentiation according to the minimum criteria set by The International Society for the Cellular Therapy in 2006 for defining multipotent MSCs ¹⁷ : First, MSC must be plastic-adherent when maintained in standard culture conditions. Second, MSC must express CD90, CD105, or CD73, and lack expression of CD45, CD14, CD34, or CD11b, CD79alpha or CD19 and HLA-DR surface molecules. Third, MSC must differentiate to osteoblasts, adipocytes, and chondroblasts in vitro. Specific cell surface antigen expressions (i.e. CD13, CD44, CD90, CD14, CD31, CD45; Sigma) were confirmed by flow cytometry. The verification of adipogenic and osteogenic differentiation were performed by Oil Red O staining, Alizarin Red staining, and ALP staining (Sigma), respectively.

Viral transduction and luciferase assays in vitro

Cell were cultivated until a 70–80% confluence was achieved, and from the third passage onwards, they were transfected with lentiviral vectors (1 × 10⁷ transduction U/mL, multiplicity of infection [MOI] = 50), containing enhanced green fluorescent protein (eGFP) gene and Luciferase (Luc) gene (SunBio Biotech, Shanghai, China). Next, they were incubated in a six-well cell culture cluster containing 10 µg/mL polybrene (Sigma) for 24 h. The transfected hASCs were selected by flow cytometry and purified, to improve the effect of labeling. The selected cells were cultured and used for seeding the scaffolds. The eGFP expression of eGFP·Luc-hASCs was observed in the twelfth passage by fluorescence microscopy, and the transfection efficiency of hASCs was detected by flow cytometry in vitro. GFP transfected cells were stained by monoclonal antibody such as anti-CD13 (Sigma) and anti-CD44 (Sigma) in 100 µL phosphate-buffered saline (PBS) with 3% FBS at room temperature for 30 min. All samples were analyzed on a FACS Calibur flow cytometer (BD Biosciences). Transduction efficiency of eGFP·Luc-hASCs was assessed by GFP positive rate in CD13 and CD44 double positive cells.

The Luc expression was checked by seeding eGFP·Luc-ASCs of different numbers (5 × 10⁴/100 µL, 1 × 10⁴/100 µL, 1 × 10³/100 µL, 5 × 10²/100 µL, 1 × 10²/100 µL, 50/100 µL, 10/100 µL) were seeded onto black, clear bottom 96-well plates in 100 µL medium (DMEM + 10% FBS + 1% P/S), respectively. Before imaging, 15 µL D-luciferin solution (150 µL/mL; Sciencelight, China) was added to every 100 µL culture medium (in a 96-well plate), and after 5 min, the bioluminescent signals were detected using a highly sensitive, cooled charged coupled device (CCD) camera (Xenogen IVIS, USA).

Cell viability

eGFP·Luc-hASCs were seeded on ABP in vitro during the eighth passage. The cell viability was evaluated using the Cell-Counting Kit (CCK)-8 assays. eGFP·Luc-hASCs (5 × 10³) were cultured on ABP, and the medium was changed every three days (on days 1, 4, and 7). At each time point, 20 µL of CCK-8 solution reagent (Dojindo, Kumamoto, Japan) was added to 200 µL medium, and the CCK-8 assay was done. The absorbance of supernatants was measured at 450 nm, using a microplate reader. eGFP·Luc-hASCs on ABP were observed by fluorescence microscopy. The cells without viral transduction and ABP were placed in the same culture condition as control group.

Culture of eGFP·Luc-hASCs on ABP in vitro

ABP, the collagen-based material used to support cell growth, was provided by the Shanghai Cingular Biotech Corporation (Shanghai, China). The collagen was derived from normal bovine pericardial tissue. It was further processed through a number of steps, such as decellularization, cleaning, anti-viral treatment, chemical modification, etc. Finally, it was lyophilized, packaged, and sterilized by electron-beam radiation.

About 10 mm thick ABPs were incubated in 100% FBS under standard culture conditions for 24 h. In the tenth passage, the labeled hASCs were counted and resuspended in DMEM + 10% FBS + 1% P/S. They were reseeded at a density of 1 × 10⁶ cells per ABP scaffold in a sterile six-well plate, and then cultivated in medium for three days. The combinations were observed by scanning electron microscope (SEM; Hitachi TM-1000 tabletop microscope) and fluorescence microscope.

Ectopic implantation of tissue-engineered product

Female athymic nude mice (BALB/C-nu/nu; n = 30), 4–6 weeks old, were obtained from The Animal Institute, School of Medicine, Shanghai Jiao Tong University (Shanghai, China), and were raised in a specific pathogen-free environment throughout the experiment. All experiments were performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

Mice were randomly divided into three groups (n = 10) and anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg/kg). The surgical area was cleaned with povidone–iodine, and a sagittal midline incision was made on the dorsum skin, to create a subcutaneous pocket of 1.5 × 1.5 cm. In the VIVO group (n = 10), ABPs were implanted in the subcutaneous pockets and eGFP·Luc-hASCs (1 × 10⁶ cells/50 µL) were injected on the ABP at the same time. In the VITRO group (n = 10), the mice were implanted with grafts that were co-cultured with eGFP·Luc-hASCs in vitro. The BLANK group (n = 10) mice were implanted with ABP only as controls. The incisions were closed with non-absorbable 6/0 nylon sutures without any additional dressing.

BLI in vivo and analysis

To detect the behavior of cells in vivo, BLI was performed at 1 day, 1 week, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, and 12 weeks, respectively after implantation of grafts in mice. Mice were intraperitoneally injected with luciferin (150 mg/kg) (Sciencelight), and were imaged by IVIS (Xenogen IVIS). Quantification of signals was evaluated using the acquisition and analysis software Living Image (Xenogen).

Histological analysis

To confirm the results of BLI, the expression of eGFP was checked for fluorescence after 4′,6-diamidino-2-phenylindole (DAPI) staining. Twelve weeks after implantation, the animals were sacrificed and the tissue samples from light producers and non-producers were harvested for histological analysis. One histological section (6 µm) from the post-operative frozen sections of implanted tissues was used for DAPI staining. This was followed by detection of eGFP-positive cells by fluorescence microscopy.

Masson trichrome staining was performed to detect the collagen content. In brief, three tissue specimens were fixed in 10% formalin solution, embedded in paraffin, and stained with hematoxylin and eosin (H&E), and Masson trichrome stains. The number of the vascular structures and cell ingrowths were detected by bright field microscopy. Stained images were analyzed using the Leica Qwin V3 system. For quantitative image assessments, we included 10 fields per specimen, two specimens per mouse, and 10 mice per group.

Immunohistochemistry

Immunohistochemical staining was performed on paraffin sections to analyze contractile protein expression levels and detect vascular endothelial cells. The primary antibodies used in this study were mouse anti-human monoclonal antibodies (anti-alpha smooth muscle actin, anti-desmin, anti-FLI-1, anti-CD31; Sigma) in accordance with the manufacturer’s protocol. Paraffin-embedded tissue sections were obtained using semiautomatic microtome approximately of 4 µm thickness. The sections were cleared by passing through two changes of xylene for 10 min each and rehydrated by passing through two changes of absolute alcohol. Then rinsed thoroughly with distilled water and kept in the distilled water until antigen retrieval. Heat-mediated antigen retrieval in pH 6.0 citric acid was performed. After washing off the redundant antibodies, the paraffin sections were treated with secondary antibodies and incubated at room temperature. All the samples were observed under bright field microscopy.

Statistical analyses

SPSS software was used to analyze the data from the images. All the quantitative values were presented as mean ± SD. Regression plots were used to represent the relationship between cell number and bioluminescence. R² values were described to assess the quality of the regression model. One-way analysis of variance (ANOVA) was used for multiple comparisons, followed by post hoc comparisons with the Newman–Keuls test. A P value of < 0.05 was considered statistically significant.

Culture and characterization hASCs

Spindle shaped adherent cells could be observed during culture. After expansion, the cells were collected and immunostained for flow cytometry analysis of immune phenotype. Most cells were positive for CD13, CD44, CD90, and negative for CD14, CD31, and CD45 (Figure 1a). The immune phenotypes of these cells were consistent with the previous reports. ¹⁶ Adipogenic and osteogenic differentiation showed that hASCs possessed the capacity to differentiate into multiple (Figure 1b). OPEN IN VIEWER

In vitro detection of eGFP·Luc-hASCs on ABP

hASCs transfected with lentivirus were spindle shaped. The purified eGFP·Luc-hASCs were expanded, and eGFP was highly expressed in the twelfth passage cells when observed by fluorescence microscopy (Figure 2a). Transduction efficiency of eGFP·Luc-hASCs measured by flow cytometry was up to 91.12% (Figure 2b). The light production of Luc was steady, and mainly depended on the cell number (Figure 2c). The number of eGFP·Luc-hASCs was linearly correlated with light production. The slopes of the linear regression plots showed standard plots of light production versus cell number (R²= 0.99) (Figure 2d). To prepare tissue-engineered products in vitro, the eGFP·Luc-hASCs at passage 8 were seeded on an ABP scaffold in a sterile six-well plate. Figure 3 (a–e) represents the magnification of materials, demonstrating that a great quantity of eGFP·Luc-hASCs survived and proliferated on ABP. CCK-8 assays indicated that viral transduction and ABP did not affect cell viability (P > 0.05) (Figure 3f). OPEN IN VIEWER

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Figure 3

(a) Scanning electron microscope of ABP. (b, c) Scanning electron microscope of the compound of hASCs-ABP, co-culture in vitro (3 days) (scanning electron microscope: original magnification, a, b: 100 ×). (d, e) In vitro eGFP·Luc-hASCs seeded on ABP scaffold for 3 days (fluorescence microscope: original magnification, d: 5 ×, e: 10 ×). eGFP·Luc-hASCs which expressed GFP grew along ABP. (f) CCK-8 assays. Cell proliferation between hASCs and eGFP·Luc-hASCs on ABP. (ABP, acellular bovine pericardium; CCK-8, cell counting kit-8; eGFP, enhanced green fluorescent protein; hASCs, human adipose-derived stem cells; Luc, luciferase gene). (A color version of this figure is available in the online journal)

In vivo non-invasive monitoring for implants by BLI

To analyze the behavior of hASCs seeded on ABP in vivo, BLI was done on eGFP-Luc ASCs that had undergone 10 passages on ABP, one day post-implantation, and thereafter at the indicated times. The results showed that both the eGFP-Luc ASCs in the two groups remained viable in vivo up to 12 weeks. One day after transplantation, the population of cells in VIVO group was 13-times more than that in the VITRO group. The number of cells in both the groups (VIVO and VITRO) decreased in the first three weeks and then remained stable in the following weeks until the end of the experiment (approximately 6.12% and 0.57% of the implanted cells in VIVO and VITRO groups, respectively at 12 weeks; Figure 4a,c). The BLANK group presented no change (Figure 4d). In addition, during the weekly BLI, majority of the implanted eGFP-Luc ASCs existed at the site of inoculation. However, significant indicators were absent in other organs such as lung, liver, heart, or brain (Figure 4b). OPEN IN VIEWER

Histological examination

All the 30 implanted mice survived the entire 12 weeks. However, each group of the samples behaved differently. To confirm the bioluminescent data and identify in which histological layer the implanted cells survived, DAPI staining sections and H&E staining were used at 12 weeks after transplantation. In two samples of the reseeded ABP group, eGFP-positive ASCs were detected in subcutaneous layer (Figure 5b, d). The number of positive cells in the VIVO group was more than that in the VITRO group. On the other hand, no eGFP reactivity was detected in the BLANK group (Figure 5f). The results of H&E staining showed that both VIVO and VITRO group displayed numerous small vessels (Figure 5a2, c2), and compared with VITRO group, the vascular density was more abundant in VIVO group. Few vessels were present in the BLANK group (Figure 5e2). In addition, as the ABP was absorbed, cell ingrowths into the ABP were demonstrated in all groups (Figure 5a1, c1, and e1). By the end of the experiment it was found that cell ingrowth was highest in the VIVO group than that in the other groups. However, the cell ingrowth was limited in the non-reseeded samples (Figure 5e1). OPEN IN VIEWER

Masson trichrome staining was performed at the material site to detect the collagen content. Compared to the BLANK group, more collagen was present in the tissue samples from the reseeded group (Figure 6a–c). According to the collagen content assay of tissue-engineered products at 12 weeks after implantation, the highest percent of collagen content per unit area was in the VIVO group (34.21 ± 3.22%) followed by the VITRO group (28.39 ± 1.30%). The collagen content in BLANK group (22.46 ± 1.20%) was lower than that in the reseeded group (Figure 6d). OPEN IN VIEWER

Immunohistochemical staining

At the end of the monitoring period implant sections were assessed by immunohistochemistry to detect the expression of contractile proteins and neovascularization needed for pelvic floor repair. Desmin (a marker for striated muscle) expression was observed around the ABP in the VIVO group (Figure 7). Subsequent immunostaining at the same site was positive for α-smooth muscle actin (α-SMA), a marker for both myofibroblasts and smooth muscle cells, thus indicating that significantly healthy myofibroblasts or smooth muscle cells were present in the VIVO group. α-SMA was densely positive around the ABP within implanted eGFP-Luc ASCs (Figure 7, black arrows in the VIVO group). There were also numerous muscular vessels existing in the connective tissue adjacent to ABP (Figure 7, pentagram in the VIVO group). However, few smooth muscles were observed in the VITRO and BLANK groups (Figure 7). These results were proved by the FLI-1 (a marker for vascular endothelial cells) and CD31 (a marker for endothelial cells) staining, which suggest that the use of ABP reseeded with eGFP-Luc ASCs in mice, could regenerate smooth muscle and blood vessels. OPEN IN VIEWER