1. Construction of the fgf21-glp1-IgG4fc lentiviral expression plasmid
pGSI-fgf21-glp1-IgG4fc (synthesized by Taihe Biotechnology) was used to subclone the fgf21 and glp1-IgG4fc genes into the lentivector pCDH-EF1 (Addgene) with the EF1α promotor. The amino acid sequence and the nucleotide sequence of the fgf21+ glp1-IgG4fc gene are listed in the supplementary materials. The primers used to amplify the cDNA of the fgf21+ glp1-IgG4fc gene (forward 5′- CGCGGATCCGCCACCATGGACTCGGACGAGACC -3′, reverse 5′- ACGCGTCGACTCATTTACCCGGAGACAG -3′) were synthesized by TsingKe (Beijing). The glp1-igg4fc gene is abbreviated as “glp1 gene”.
2. Lentivirus production
Lentiviral vector plasmids and packaging plasmids (psPAX and pMD.2G) were purchased from Addgene. Lentiviral particles carrying pCDH-EF1-FGF21, pCDH-EF1-FGF21+GLP1 and pCDH-EF1-GLP1 were produced through the transfection of HEK293T packaging cells with a 3rd generation plasmid system. HEK293T cells were transfected with 24 µg of plasmids, 48 µl of Lipofectamine LTX and 24 µl of PLUS reagents, and the proportions of the pMD.2G, psPAX, and pCDH-EF1 plasmids were 1:2:3. The supernatants were collected at 24 and 48 h after transfection, filtered through 0.45-μm filters, and harvested by ultrafiltration with a 100-kDa spin column (Millipore) at 4 °C and 4,000 g for 30 min. The lentiviral particles were aliquoted and stored at -80 °C until use. The transfection efficiency was determined based on EGFP expression using flow cytometry (Beckman), and the viral titers were determined according to the following equation: virus titer (pfu/mL) = cell number in each well × virus dilution factor × 10/ volume of added virus fluid (mL).
3. Mesenchymal stem cell culture, flow cytometry analysis, and characterization
Adipose tissue-derived mesenchymal stem cells were donated by Xijing Hospital and cultured in the same way as traditional cells. Briefly, to obtain the upper adipose tissue, healthy adult adipose tissue extracted by liposuction was transferred to a 50-mL centrifuge tube, completely washed with PBS, and centrifuged at 1500 rpm for 5 minutes. Mixed collagenase (0.2%; Type I, II and IV collagenases=1:1:1) was prepared, and a 1:1 mixture of adipose tissue: collagenase was added to the mixed collagenase digestion solution. The adipose tissue was digested in a 37 °C shaker for 30 minutes. The digested adipose tissue was immediately added to α-MEM cell culture medium containing 10% FBS (Gibco), that is, complete medium. To precipitate the cells and tissue clumps, the mixture was centrifuged at 1500 rpm for 10 min. The cells were resuspended using complete medium, and the undigested tissue was removed by nylon mesh. The cells were inoculated in a culture flask and incubated at 37 °C in a 5% CO2 incubator. Two days later, the nonadherent cells were discarded, and the adherent cells were washed gently with PBS. The cells continued to be cultured in complete medium.
MSCs were harvested from passage 5 and washed three times with PBS. A total of 1 × 106 cells were incubated with 5 μl ECD-conjugated antibodies, 20 μl FITC/PE-conjugated antibodies, or the relevant isotype control antibodies (Beckman Coulter, CD73-PE B68176, CD90-FITC IM1839U, CD105-PE B92442, CD34-PE A07776, CD45-ECD A07784, IgG1 Mouse-FITC IM0639U, IgG1 Mouse-PE IM0670U, IgG1 Mouse-ECD A07797) for 20 min in the dark at room temperature. Then, the cells were washed three times with PBS and examined by flow cytometric analysis (flow cytometer model: Beckman Coulter EPICS XL). In total, more than 95% of the cells expressed CD73, CD90, and CD105, while 2% or less of the cells expressed CD45 and CD34. The released cells were negative for pathogenic microorganisms, HBV, HCV, HIV, cytomegalovirus, syphilis, and ALT, and the endotoxin levels were found to be within 40 IU/L and 0.5 EU/mL. The total cells were counted, and cell viability (≥ 85%) was determined by Trypan blue staining.
4. Transduction of MSCs with lentiviral particles and detection of target gene expression
MSCs (<3 passages) were transduced with concentrated lentivirus at a multiplicity of infection (MOI) of 40 for 6 h in α-MEM containing 8 μg/ml polybrene. To detect the expression patterns of FGF21 and GLP1 in the MSCs, Western blot analyses of the cellular supernatants were performed using anti-FGF21 and human IgG4-Fc monoclonal antibodies. To further measure the secretion of FGF21 and GLP1, the culture medium (CM) of the MSCs and MSCs transduced with pCDH-EF1-FGF21, pCDH-EF1-FGF21+GLP1, pCDH-EF1-GLP1, or pCDH-EF1-vector lentiviral particles was collected after incubation for 48 h. The FGF21 and GLP1 levels secreted into the MSC culture medium were measured by ELISA (Abcam) according to the manufacturer’s protocol. When collecting the culture supernatant for testing, to ensure that the same sample quantity was collected, we inoculated different kinds of cells at a uniform density and then added the same amount of medium. After 48 h of culture, the same amount of centrifuged culture supernatant was analyzed by ELISA and WB. To test the proliferation of the MSCs, each MSC type was seeded in 96-well plates at 5 × 104 cells/well and preconditioned in culture medium. After 48 h of incubation, 20 μl of CCK-8 was added to each well and incubated for 4 h at 37 °C, and the absorbance was measured at 570 nm with a Quant microplate reader. All the samples were analyzed in duplicate, and the samples with coefficient of variation (CV) values > 15% were excluded.
5. Adipogenic and osteogenic differentiation
MSCs were cultured in a 24-well plate in complete α-MEM supplemented with adipogenic- and osteogenic-inducing agents (Sigma Aldrich) at an initial cell density of 1×104 cells/well. The adipogenic medium was α-MEM containing 10% FBS, 1 mmol/L dexamethasone, 5 mg/mL insulin and 100 mmol/L indomethacin. The osteogenic medium was α-MEM containing 10% FBS, 0.1 mmol/L dexamethasone, 50 mmol/L ascorbic acid and 10 mmol/L β-glycerophosphate. The medium was changed every 3 days. After 2-3 weeks, the cells were washed twice with PBS and fixed with 4% paraformaldehyde at room temperature for 30 min. The intracellular lipid droplets were visualized by oil red staining, and calcium deposits were stained with alizarin red S.
6. Western blotting
The cells were washed with PBS buffer and subsequently lysed using cell lysis buffer (Tiangen) with a complete protease inhibitor mix (Biotool). Liver tissue was ground and subsequently lysed using lysis buffer (Tiangen) with a complete protease inhibitor mix (Biotool). The lysates and protein markers were run in SDS-PAGE gels (12% or 15%) and transferred onto nitrocellulose membranes (Millipore). The membranes were blocked with 5% milk in Tris-buffered saline plus Tween 20 (TBST) and exposed to rabbit or mouse primary antibodies (1:3000, Abcam or Cell Signaling). The blots were probed with horseradish peroxidase (HRP)-conjugated goat anti-rabbit (or mouse) IgG (H+L) secondary antibodies and visualized using a Pierce ECL Western Blotting Substrate kit (Thermo Scientific) for signal detection.
7. Relative quantitative real-time polymerase chain reaction (RT-PCR)
Total RNA was isolated with TRIzol (Sigma) in a manner that was counterbalanced across the experimental groups. cDNA was synthesized from 1 μg of total RNA with the cDNA Synthesis Supermix (BioScript All-in-One cDNA Synthesis; Biotool). Quantitative real-time PCRs were performed using SYBR Premix Ex Taq (Tli RNaseH Plus) (Takara) in a 7500 Real-Time PCR System (Applied Biosystems). For normalization, the threshold cycles (Ct-values) were normalized to β-actin/GAPDH within each sample to obtain the sample-specific ΔCt values (ΔCt ¼ Ct gene of interest Ct β-actin/GAPDH). The 2-ΔΔCt values were calculated to obtain the fold expression levels. The primers for the quantitative analyses of the FGF21 gene (forward 5′- ATCGCTCCACTTTGACCCTG -3′, reverse 5′- GGGCTTCGGACTGGTAAACA -3′), GLP1-IgG4Fc gene (forward 5′- CCCCAAAACCCAAGGACACT -3′, reverse 5′- GCCATCCACGTACCAGTTGA -3′), srebp1c gene (forward 5′- CACTGTGACCTCGCAGATCC -3′, reverse 5′- ATAGGCAGCTTCTCCGCATC -3′), insulin gene (forward 5′- TCTCTACCTAGTGTGCGGGG -3′, reverse 5′-GCTGGTAGAGGGAGCAGATG -3′), β-actin gene (forward 5′- CCTGGCACCCAGCACAAT -3′, reverse 5′-GGGCCGGACTCGTCATAC -3′) and GAPDH gene (forward 5′- GGAGCGAGATCCCTCCAAAAT -3′, reverse 5′- GGCTGTTGTCATACTTCTCATGG -3′) were synthesized by TsingKe Company (Beijing).
8. Animal experiments
In our study, BKS.Cg-Dock7m+/+Leprdb/Nju mice (T2DM mouse model) were used, and the mice were purchased from the Model Animal Research Center of Nanjing University. Thirty-six male BKS mice aged 6-8 weeks (>20 g body weight) were randomly divided into six groups. Each group contained six mice housed in two cages. The experiment was divided into six groups. The control group was intraperitoneally injected with 100 μl saline. The liraglutide group was injected with 100 μl of liraglutide drug (0.5 mg/kg) twice a week until the end of the experiment. The MSC group (containing pCDH-EF1-vector lentiviral particles), MSC-FGF21 group, MSC-FGF21+GLP1 group and MSC-GLP1 group were injected with 1×106 MSCs suspended in 0.1 mL of physiological saline once a week for three weeks. Before each injection, the cells were passed through a 70-μm cellular sieve, and then, the cells were injected into the mice at 3-5 time points on each injection day, at intervals of approximately 10 minutes. The drugs were administered by intravenous injection. The glucose levels in the blood obtained from the tails was measured every week during the experiments. On day 28, peripheral blood was collected from the retro-orbital sinus of each mouse.
9. Glucose-stimulated insulin secretion (GSIS)
The rat INS-1 pancreatic β cell line was purchased from CCTCC (China Center for Type Culture Collection). The cells were cultured at 37 °C in a humidified atmosphere containing 5% CO2. The culture medium was RPMI 1640 medium containing 11 mM glucose and supplemented with 10% FBS, 10 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate and 50 μM mercaptoethanol. The culture medium was replaced every second day, and the cells were passaged once a week following trypsinization.
To determine the effect of genetically modified MSCs on GSIS, INS-1 cells were seeded onto 12-well plates and cultured for 24 h. Then, the cells were washed two times with Krebs-Ringer bicarbonate buffer (KRBB, 129 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO4, 1.2 mM KH2PO4, 2.5 mM CaCl2, 5 mM NaHCO3, 0.1% BSA, 10 mM HEPES, (pH 7.4) and 2.8 mM glucose) and starved for 2 h in KRBB. The cells were incubated in fresh KRBB containing different MSC-conditioned media for 1 h in the presence of glucose. The supernatants were collected to measure the insulin concentration.
10. Fasting glucose and glucose tolerance tests
For the weekly fasting glucose test, the mice were starved overnight to assess glycemia. At the end of the experiment, after overnight fasting, the mice were administered glucose (1 g/kg) by oral gavage, and blood samples were collected from the tail vein to determine the glucose levels. Glycemia was assessed using an Accu-Chek glucometer (Roche, Basel, Switzerland, http://www.roche.com), and the area under the curve was calculated.
11. Statistical analysis
All the statistical analyses were conducted using SPSS software. The data were analyzed using one-way ANOVA followed by Tukey’s post hoc test or two-way ANOVA followed by Bonferroni’s post hoc test to determine the differences among the means of the treatment groups. P < 0.05 was considered significant.