Heterogenic transplantation of bone marrow-derived rhesus macaque mesenchymal stem cells ameliorates liver fibrosis induced by carbon tetrachloride in mouse

Animals

Mature female 4-week-old Kunming mice (Charles River, Beijing, China) with a body weight between 30 and 40 g were used for the carbon tetrachloride-induced liver fibrosis mouse model. Mice were housed in a room with a 12 h light:12 h dark cycle and provided with sterile food and water ad libitum. The temperature was controlled at 22 °C. Three male rhesus macaques (two years old) were used as bone marrow donors. The procedures for the monkey bone marrow retrieval, mouse model generation and cell transplantation were approved by the Institutional Animal Care and Use Committee of Kunming University of Science and Technology and were performed in accordance with the Guide for the Care and Use of Laboratory Animals. The IACUC approval number is LPBR20170201. All of the chemicals used in this study were obtained from Sigma Chemical Co. (St. Louis, MO, USA), unless otherwise indicated.

Preparation of rhesus macaque bone marrow-derived MSCs

Bone marrow-derived MSCs were isolated from the tibias of young rhesus macaques. The muscular tissues on the tibias were carefully removed. The ends of the bones were cut, and bone marrow was aseptically flushed ten times by a sterile syringe with 10 mL of Dulbecco’s modified Eagle’s medium (DMEM) (Gibco BRL, Grand Island, NY, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco BRL, Grand Island, NY, USA) and 1% (v/v) penicillin/streptomycin (Gibco BRL, Grand Island, NY, USA). The cell suspension was then centrifuged at 500 g for 5 min, and the supernatant was discarded. Marrow cells were then mechanically dispersed into single-cell suspension and seeded into a 10-cm plastic dish at a density of 1 × 10⁶ cells/mL. The cells were cultured in DMEM supplemented with 10% FBS at 37 °C in an incubator with a humidified atmosphere of 5% CO₂ in the air. The non-adherent cells were removed, and the medium was refreshed every 48 h. Ten days later, the primary cell culture (Passage 0) was passaged at 80% confluency with 0.25% trypsin (Gibco BRL, Grand Island, NY, USA). The cells were resuspended in culture medium at a dilution ratio of 1:3 and expanded on a new plastic petri dish to passage 1. The morphology, surface markers and differentiation potency of the MSCs were identified at passage 3.

Flow cytometry analysis for surface marker profiles of MSCs

Surface marker profiles of the MSCs were examined using a commercial MSC Analysis Kit (BD Biosciences, San Jose, CA, USA) by flow cytometry analysis (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. Briefly, approximately 5 × 10⁵ MSCs were collected and washed with 500 µL PBS (containing 1% FBS, PBSF). The cells were resuspended in 100 µL PBSF. Each cell sample was incubated with anti-human antibodies (Positive markers including CD44, CD90, CD73 and negative markers cocktail including CD45, CD34, CD11b, CD19, HLA-DR, which were provided by BD Co.) at a final concentration of 50 mg/L for 1 h on ice, and the isotype control antibody cocktail (mIgG1, kAPC/mIgG1, kFITC for CD73 and CD90; mIgG2b, k PE for CD44; mIgG1, k/mIgG2a, kPE for negative markers cocktail) was used as the negative control at a final concentration of 50 mg/L for 1 h on ice. Unbound antibodies were washed off with PBSF, and then the cells were re-suspended with 500 µL PBSF for flow cytometry analysis.

Evaluation of the differentiation potential of MSCs

For adipogenic differentiation, MSCs were seeded into 24-well plates and cultured at a density of 8 × 10⁴ cells per well for 12 h. Then, the cells were cultured in an adipogenic differentiation medium (Gibco BRL, Grand Island, NY, USA) for 7 days (Ninomiya et al., 2010). The medium was refreshed every three days. The cells were stained in filtered Oil Red O (0.2% Oil Red O in 60% isopropanol, v/v) for 15 min and washed 3 times with PBS after fixation in 4% methanol. The adipogenic differentiation was confirmed by the cellular accumulation of neutral lipid vacuoles, which were stained red with Oil Red O (Sigma, St. Louis, MO, USA). For osteogenic differentiation, MSCs were seeded into 24-well plates and cultured at a density of 4 × 10⁴ cells per well for 12 h. Then, the culture medium was replaced with an osteogenic differentiation medium (Gibco BRL, Grand Island, NY, USA) and further cultured for 21 days. The medium was refreshed every three days. The cells were stained with fresh 0.5% Alizarin Red solution and washed three times with PBS after fixation with 4% methanol. The osteogenic differentiation was confirmed by the appearance of Alizarin Red stain. For chondrogenic differentiation, MSCs were collected in 15-mL centrifuge tubes at approximately 2 ×10⁵ cells per tube cultured in a chondrogenic differentiation medium (Gibco BRL, Grand Island, NY, USA). The medium was refreshed every three days. After 21 days of differentiation induction, the chondroid pellets were generated and washed with PBS, fixed in 4% paraformaldehyde, and embedded with OTC embedding material (Leica, Wetzlar, Germany). The pellets were sectioned by a freezing microtome, and then sulphated proteoglycans were visualized by staining with 1% toluidine blue (Merck, Darmstadt, Germany) for 10 min. These slices were washed three times with PBS and photographed under an inverted microscope. Differentiation was confirmed by the appearance of Alcian Blue stain.

Cell labeling with EGFP

To track the transplanted cells in vivo, MSCs were labeled with EGFP by lentivirus infection. Briefly, the pLV-CMV-EGFP-Neo vector, PMD2.G and PSPAX2 packaging plasmids, and the X-tremeGENE HP DNA Transfection Reagent (Roche, Basel, Switzerland) were added to 10% FBS DMEM medium, mixed gently, and incubated at room temperature for 20 min. The mixture was added dropwise into 293T cells in a 10-cm plate. After a 48-h incubation period, the virus supernatant was collected and filtered using a 0.45- µm filter. Rhesus macaque bone marrow-derived MSCs were then infected with the virus. After being cocultured for 48 h, the aminoglycoside antibiotic G418 (Gibco-BRL, Carlsbad, CA, USA) was added to the medium at a final concentration of 600 µg/mL to select MSCs with a stable EGFP expression. The MSCs labeled with EGFP were observed with a fluorescence emission ratio at 530 nm using an epifluorescence microscope and an excitation wavelength of 488 nm, and the labeling efficiency was detected by flow cytometry analysis. The differentiation potency of adipogenic, osteogenic and chondrogenic MSCs was further examined by the methods described above after being labeled with EGFP using lentivirus infection.

Carbon tetrachloride-induced liver fibrosis mouse model and heterogenic MSC infusion

The mice were intragastrically administered CCl₄ (mixed with olive oil at a 1:1 volume ratio) at a dose of 0.2 mL/100 g body weight during the first week. Then, the mice were further administered CCl₄ (mixed with olive oil at a 3:1 volume ratio) at a dose of 0.2 mL/100 g body weight three times a week for eight weeks. The mice administered with the same volume of olive oil only were used as controls. Sixty mice were randomly divided into olive oil administered mice (n = 10) and (2) CCl₄ administered mice (n = 50). After eight weeks, several mice died from CCl₄ toxicity, and the development of CCl₄-induced liver fibrosis in the mice that survived was determined by the evaluation of pathological liver sections by a trained pathologist. Then, seven CCl₄ administered mice from the 16 survivors were divided into two groups randomly, and the others were used for anther experiment, which was not involved in the present study. Three mice administered with CCl₄ but without MSCs transplantation were used as CCl₄ group. In contrast, four mice administered with CCl₄ and transplanted with MSCs were used as CCl₄+MSCs group. The EGFP-labeled rhesus MSCs at passage six were washed three times with saline and resuspended at a concentration of 5 ×10⁶ cells/mL. Then, a single dose of MSCs (1 ×10⁶ cells) was infused into the CCl₄ administered mice via tail vein injection. Meanwhile, the same volume of saline (200 µL) was infused into the control mice. Thirty days later, each group was anesthetized using 0.3 mL/100 g of body weight of 10% chloral hydrate (Sigma, St. Louis, MO, USA) solution which was injected intraperitoneally, and the liver and venous blood were collected.

Blood collection and plasma chemistry tests

Blood serum was separated by centrifugation at 3,000 rpm for 15 min within 1 h after blood collection and stored at −80 °C until analysis. The following plasma chemistry parameters were measured using a Roche Modular P800 automatic biochemical analyzer (Roche Diagnostics Ltd., Basel, Switzerland): aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin (ALB) and total protein (TP).

Gene expression analyzed by qRT-PCR

Total RNA was extracted from the liver tissues of mice using Trizol Reagent (Takara, Dalian, China). The RNA was first separated into an aqueous phase by adding chloroform and then was precipitated with isopropanol, rinsed with 75% ethanol and finally solubilized in sterile DEPC water. Complementary DNA (cDNA) was synthesized using a Prime-Script RT reagent kit (Takara, Dalian, China) according to the manufacturer’s recommendations. Highly purified gene-specific primers are listed in Table 1, including the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), α-smooth muscle actin (α-SMA), albumin (ALB), tumor necrosis factor (TNF-β) and alpha fetal protein (AFP), which were commercially synthesized (Shengong, Shanghai, China). Quantification of the cDNA of the specific genes was performed with a Bio-Rad CXF real-time PCR system. All experiments were performed in triplicate, and the data were analyzed by the 2^−△Ct procedure.

Histological and immunofluorescence analysis

Liver tissue samples were collected from the euthanized mice and fixed in 4% formaldehyde for two days. Tissues were then dehydrated, cleared and infiltrated with a histoprocessor for 16 h. The liver tissues were sectioned into 3-µm pieces and stained with hematoxylin and eosin (H&E) or Masson’s Trichrome solutions. For H&E analysis, sectioned samples were stained with hematoxylin solution (Sigma-Aldrich, Munich, Germany) for 5 min, followed by eosin for 5 min. For Masson’s trichrome stain, sectioned samples were placed in Bouin’s solution at 56 °C for 1 h and stained in succession with Mayer’s hematoxylin solution, Biebrich Scarlet-acid fuchsin solution, phosphomolybdic acid–phosphotungstic acid and aniline blue for 5 min, 15 min, 15 min and 5 min, respectively. All of the staining reagents were from Sigma-Aldrich (St. Louis, MO, USA). Specimens were evaluated with respect to inflammation, ballooning degeneration, and collagen deposition by an experienced veterinary pathologist who was blinded to the treatment that the animals received.

For immunofluorescence, liver tissues were cut into cubes of about 1 cm ×1 cm in size, fixed in 4% paraformaldehyde for 48 h, and dehydrated in 15%, 20% and 30% sucrose solution for 12 h. Then, the liver tissue samples were embedded in OTC embedding material (Leica, Wetzlar, Germany) and sectioned into 8-µm sections, which were incubated with 0.1% Triton in PBS for 30 min. After being washed with PBS, the slices were incubated with the blocking solution (PBS containing 10% goat serum), incubated overnight with primary antibodies (EGFP, Ki-67, and caspase-3 purchased from Abcam, Cambridge, UK), and finally incubated with rabbit secondary antibodies (conjugated with FITC and PE, purchased from Abcam, Cambridge, UK) at 37 °C for 2 h after being washed with PBS. All matched samples were photographed using an immunofluorescence microscope. Immunofluorescence images were analyzed using Image J (NIH, Bethesda, MD, USA) after being stained with DAPI (4′,6-dia-midino-2-phenylindole).

DNA extraction and PCR product detection

Genomic DNA was extracted from the liver tissue using DNA extraction kits (Tiangen, Beijing, China) following the manufacturer’s instructions. Genomic DNA extracted from the EGFP-labeled MSCs was used as a positive control. The concentration of the extracted DNA was measured by a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). For PCR, the reaction mixture contained 20 ng of DNA template, 1 µL of EGFP primers (forward and reverse primers at a concentration of 10 µM, primers are listed in Table 1), 10 µL of 2 × Taq PCR Mix (Dingguo, Beijing, China), and ddH₂O of up to 20 µL. The PCR reaction was conducted for 30 cycles. Each cycle consisted of the following steps: 94 °C for 2 min, 94 °C for 40 s, 66 °C for 30 s, and 72 °C for 150 s. After 30 cycles, the samples were subjected to a final step at 72 °C for 10 min. PCR products were separated by agarose gel electrophoresis.

Inhibition of T lymphocyte proliferation in vitro

Lymphocytes derived from the lymph nodes of 10 week-old Kunming mice were first labeled with 5 µM CFDA SE Cell Proliferation Assay and Tracking Kit (Beyotime, Jiangsu, China) for 10 min in a 37 °C water bath and then cultured in 1640 complete medium (RPMI-1640 plus 10% FBS) in a 96-well plate at a density of 2 ×10⁵ cells/100 µL/well. Rhesus MSCs were expanded in a 6-well plate and cultured for five days. Then, the MSCs were dissociated with 0.05% Trypsin-EDTA and added to the 96-well plate cultured with lymphocytes. The ratio of MSCs to lymphocytes was 1:40 or 1:80 per well. The lymphocytes in the mixture were stimulated with or without pre-coated 10-µg/ml anti-CD3 antibodies (BD Biosciences, San Jose, CA, USA) and 2 µg/mL anti-CD28 antibodies (BD Biosciences, San Jose, CA, USA) or on beads. Lymphocytes were collected 5 days later after stimulation and then subjected to flow cytometry analysis. The data were analyzed with Flowjo (TreeStar, Ashland, OR, USA).

Response of rhesus macaque bone marrow-derived MSCs to IFNγ and reverse transcription-polymerase chain reaction (RT-PCR)

A total of 10⁵ MSCs were loaded in a 6-well plate and treated with 20 ng/mL IFNγ for 24 h. Total RNA was extracted from the cultured MSCs using Trizol reagent (Life Technology, Carlsbad, CA, USA), and cDNA was generated using the PrimeScript RT Reagent kit (Clontech, Mountain View, CA, USA). PCR reactions were carried out using the Quick-Load^® Taq 2 × Master Mix (NEB). GADPH, indoleamine 2,3-dioxygenase 1 (IDO1), C-C motif chemokine ligand 2 (CCL2), chemokine (C-X-C motif) ligand 10 (CXCL10) and C-X-C motif chemokine receptor 4 (CXCR4) primers were commercially synthesized, and the sequences are listed in Table 1. The PCR conditions are as follows: an initial denaturation at 95 °C for 25 s, followed by 28–32 cycles of 25 s, denaturation at 95 °C for 20 s, annealing at 56 °C for 25 s, extension at 68 °C, and a final extension at 68 °C for 5 min. The PCR product was analyzed by electrophoresis in a 1.5% gel.

Data analysis

All of the data are expressed as the means ± SEM. The statistical differences were analyzed using Student’s t-test, and a P value less than 0.05 was considered significantly different. Histograms were drawn with GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA).

Morphology, surface marker profiles and differentiation potency of MSCs

During the primary culture, the MSCs derived from macaque bone marrow adhered to the plastic dishes in a scattered manner. The MSCs appeared like typical homogeneous fibroblast-like, elongated, spindle-shaped, heterogeneous, with single nucleus features following subsequent culture (Fig. 1A). The surface marker profiles of the MSCs were analyzed by flow cytometry. The results indicated that the cells positively expressed high levels of CD44 and CD73 but negatively expressed hematopoietic markers (Figs. 1B–1E). The bone marrow-derived MSCs could differentiate into adipocytes (Fig. 1F), osteocytes (Fig. 1G) and chondrocytes (Fig. 1H). After being submitted to an adipogenic induction, numerous neutral lipid droplets stained by Oil Red were observed in the cytoplasm of cells. After being submitted to an osteogenic induction, the cells presented an aggregation of micronodules or calcium deposits that were stained by Alizarin Red. The chondrogenic differentiation of MSCs could be observed using an Alcian Blue stain.

Efficiency of MSCs labeled with EGFP and potency of labeled MSCs

Rhesus macaque bone marrow-derived MSCs were infected by lentiviruses, which were packaged using 293T cells. After 48 h of infection, the MSCs that expressed EGFP emitted an obviously green fluorescence that was observed under a fluorescence microscope (Figs. 2A and 2B). The efficiency of the MSCs labeled with EGFP was 99 ± 0.6% as detected by flow cytometry analysis (Fig. 2C), which indicated that EGFP labeling was successful with high efficiency through the lentivirus infection method. The MSCs labeled with EGFP had the capacity to differentiate into adipocytes (Figs. 2D–2F), osteocytes (Figs. 2G–2I) and chondrocytes (Figs. 2J–2L), which were identified by Oil Red O, Alizarin Red and Alcian Blue staining, respectively.

Generation of the liver fibrosis mouse model and EGFP-labeled MSC therapy

The toxicity of CCl₄ caused 68% of the mice to die after eight weeks of administration, and the final survival rate was 32% as shown in Fig. 3A. The ratio of the body weight to the liver weight of CCl₄-administered mice was lower than that of the control group (Fig. 3B). The livers of both the control and CCl₄-administered groups were collected after necropsy, and the CCl₄-administered liver exhibited enlargement, an ashen appearance and a messy irregular surface structure compared to the normal liver, which was pink or purplish-red and smooth as shown in Figs. 3C and 3D.

Consequently, EGFP-labeled MSCs were transplanted via the tail vein. Four weeks later, the results of the pathological evaluation, serum biochemical index of the liver function and liver fibrosis-related gene expression were discovered, which are shown in Fig. 4. Compared to the control, the histological examination of H&E and Masson stained liver sections of the CCl₄-administered mice revealed the development of necroinflammatory changes and densely deposited collagen fibers. In contrast, the inflammation and ballooning degeneration in liver tissue were obviously alleviated four weeks after MSC infusion (Figs. 4A and 4B). Blood serum ALT, AST, ALB and TP concentrations of the CCl₄-administered mice with liver fibrosis were elevated compared to the control group, and the level of these indexes decreased to the normal level four weeks after the infusion of MSCs as shown in Figs. 5A–5D. The mNA levels of ALB, α-SMA, AFP and TNF-β in the CCl₄-administered mice with liver fibrosis were significantly increased compared to the control group, and the mRNA level of these genes increased to the normal level four weeks after MSCs infusion, as shown in Figs. 5E–5H.

The status of EGFP-labeled MSCs homed into liver tissue of CCl₄- administered mice with liver fibrosis

The EGFP-labeled MSCs homed into the CCl₄-administered mouse livers and were detected by PCR and immunofluorescence after four weeks of cell transplantation. In comparing the positive groups (EGFP-labeled MSCs), the result of the PCR and electrophoresis detected weak electrophoresis bands of EGFP present in the liver tissues with EGFP-labeled MSC infusions (Fig. 6A). Immunofluorescence of the frozen sections from the CCl₄-administered mouse livers infused with EGFP-labeled MSCs showed that only 1.8 ± 0.4% of the cells in the liver tissues were MSCs (Figs. 6B and 6C). The expression levels of the proliferation and apoptosis-related hepatocyte genes (Ki-67 and caspase-3) from the frozen liver sections were compared between the control, CCl₄-administered mice with liver fibrosis, and CCl₄-administered mice with liver fibrosis infused with MSCs by immunofluorescence (Figs. 6D and 6E). The CCl₄ administration significantly increased expression of the hepatic proliferation-related protein Ki-67 compared to the control, and the transplantation of MSCs did not affect the increased expression of Ki-67. In contrast, CCl₄-administration significantly increased the expression of the hepatic apoptosis-related protein caspase-3 compared to the control, and the transplantation of MSCs alleviated the increased expression of caspase-3 to a normal level.

MSCs have better functional characteristics

The capacity of MSCs to inhibit lymphocyte proliferation and secrete cytokines in vitro was determined by flow cytometry and qRT-PCR. The result showed that the co-culture of MSCs with lymphocytes at a ratio of 1:40 significantly decreased the proliferation rate of lymphocytes from 38.1 ± 4.4% to 18.3 ± 2.2%. However, the inhibition effect of MSCs on lymphocyte proliferation was not significant when the MSC cell ratio decreased to 1:80 (32.5 ± 3.1%) (Fig. 7). After being treated with IFNγ for 24 h, the RT-PCR results showed that the chemokine and migration-related genes, including CCL2, IDO1, CXCL10 and CXCR4, in MSCs were up-regulated, which suggested that the MSCs reacted to the inflammatory factor and had the potential to migrate to inflammatory sites (Fig. 8).