The animal experiment performed in this study satisfied the requirements of the Institutional Animal Care and Use Committee at the South China Agricultural University (approval ID: 2021-C018).
Animal and cells
1 to 14-day-old chicken used in cell isolation were purchased from Xufeng Farming Co., Ltd. (Kaiping, China).
Chicken macrophage cell line (HD11) used as positive control in the identification of cMSCs was from Guangdong Provincial Key Laboratory of Animal Health Aquaculture and Environmental Control.
Isolation of cMSCs
Primary cMSCs were isolated from femurs and tibia in 1 to 14-day-old chicken. 4-hour differential adhesion method was used in this study. Chickens were killed and sterilized with 75 % alcohol for 5-10 min. The legs of chicken were dissected in super-clean worktable, soaked in 75 % alcohol for 5 min and kept in serum-free Dulbecco's Modified Eagle Medium (DMEM) (ThermoFisher, MA, USA) for dissection. Getting rid of muscles and connective tissues sticked to femurs and tibia bones, and then removing bone epiphysis to expose bone marrow cavity. Marrow cavity was flushed using serum-free DMEM in order to collect total cells existed in marrow cavity. The medium containing bone marrow cells were filtered with 70 μm sterile strainer (Corning, NY, USA) and the filtrate was centrifuged at 1000 rpm for 5 min to get rid of plasma and lipids. Discarding supernatant and using 5 ml growth medium (GM, Dulbecco's Modified Eagle Medium: F-12 (DMEM-F-12) (ThermoFisher, MA, USA) containing 10 % fetal bovine serum (FBS) (ThermoFisher, MA, USA), 100 U/mL penicillin (ThermoFisher, MA, USA) and 100 g/mL streptomycin (ThermoFisher, MA, USA)) to resuspend cell layer. Cells were plated in 60-mm cell culture dishes and were marked as P0 (passage 0). The medium was changed completely after 4 hours in order to remove the non-adherent cells (mainly non-MSCs cells).
Cell culture and subculture
Cultures were incubated at 37 ℃ in a humidified incubator containing 5 % CO2. When cMSCs at P0 reached 90 % confluently, the cells needed to be subcultured. Cells were washed by PBS (ThermoFisher, MA, USA) firstly, and dissociated with 0.25 % Trypsin-EDTA (ThermoFisher, MA, USA) for 2.5 min. After that, adding 5 ml GM to stop dissociation, and then collecting cell suspension to centrifuge at 1000 rpm for 5 min. Finally, using 10 ml GM to resuspend cell layer, and then subculturing at new cell culture dishes. The sub-culture cMSCs were named as P1 (passage 1), and subsequent passaged cells were named as P2, P3, P4.
Growth curve assay
cMSCs were cultured in 24-well plates with 2 x 104 cells/ml per well in advance. In the next 8 days, cells were dissociated by 200 μL 0.25 % trypsin-EDTA for 2.5 min. After that, adding 400 μL GM to stop dissociation and prepare cell suspension. Countstar Automated Cell Counter (ALIT Life Science, Shanghai, China) was used to calculate the cell number of cMSCs on 3 wells daily for 8 days. Every well was counted three times to get the mean value.
RNA extraction and cDNA synthesis
Total RNA of cells was extracted with RNAiso Plus (Takara, Kyoto, Japan) and HiPure Unviersal RNA Mini Kit (Magen, Guangzhou, China), following the manufacturer's protocol. cDNA was synthesized using MonScript™ 5× RTIII All-in-One Mix kits (Monad, Jiangsu, China) for reverse transcription. RNA samples (50 ng-1 μg) were subjected to thermocycling at 37 ℃ for 2 min, followed by 55 ℃ for 15 min, 85 ℃ for 5 min to synthesize cDNA.
Reverse transcription PCR (RT-PCR)
cMSCs were plated in 6-well plates to culture. Cells were harvested to extract total RNA and synthesize cDNA. cDNA sample was subjected to PCR amplification using 2×EasyTaq® PCR SuperMix for PAGE (TransGen Biotech, Beijing, China), and were subjected to thermocycling at 98 ℃ for 3 min, followed by 35 cycles of 98 ℃ for 10 s, 56 ℃ for 10s and 72 ℃ for 10s and followed by 72 ℃ for 2 min.HD11 was employed as positive control, and housekeeper gene GAPDH was employed as an internal control. PCR produced were separated by 1.5% agarose gel electrophoresis to visualize the band was detected using GoldView II Nuclear Staining Dyes (Solarbio, Beijing, China). Primers for each marker gene were designed and checked for target identity using the National Center for Biotechnology Information (NCBI). The information of primers used for RT-PCR assays was presented in Table 1.
cMSCs were plated in 12-well plate for immunofluorescence assay to detect cell surface markers CD44 and CD45. HD11 was employed as positive control in the detection of CD45. Cells were washed with PBS to get rid of medium. Cells were fixed by 20-minute incubation in 4% formaldehyde, washed with PBS three times. The fixed cells were incubated with 0.1% Triton X-100 in PBS for 15 min, washed with PBS three times. After that, cells were blocked with PBS containing 10% goat serum (Beyotime, Shanghai, China) for 30 min and incubated overnight with primary antibody diluted with Immunol staining primary antibody dilution buffer (Beyotime, Shanghai, China). Primary antibodies were directed against cell surface markers CD44 (1:700, Southern Biotech) and CD45 (1:700, Invitrogen). After rinsing with PBS three times, the cells were incubated with the secondary antibody (1:1000, Abcam) for 1 h at room temperature. After rinsing with PBS three times, cells were incubated with PBS containing 10% DAPI staining solution (Beyotime, Shanghai, China) for 5 min and rinsed with PBS three times again. Cells were observed in epifluorescence microscope (Leica DMi8).
The first method is that cMSCs were exposed for 3 days to DMEM-F12 medium containing 10 µM 5-Aza (Macklin, Shanghai. China) and 2 % horse serum (Solarbio, Beijing, China) followed by 2-4 days of further culture in DMEM-F12 medium containing 2 % horse serum (Solarbio, Beijing, China).
The second method is that cMSCs were exposed for 3 days to DMEM-F12 medium containing 0.1 µM DXMS (Sigma-Aldrich, MO, USA), 50 µM HC (Solarbio, Beijing, China) and 2 % horse serum followed by 2-4 days of further culture in DMEM-F12 medium containing 2 % horse serum.
Quantitative real-time PCR (qPCR)
cMSCs was plated in 12-well plates. Total RNA extraction and cDNA synthesis were following the method described above. cDNA samples were subjected to ChamQ Universal SYBR qPCR Master Mix (Vazyme Biotech, Nanjing, China) following the manufacturer's protocol. 2^-ΔΔCt method and internal normalization were used to analyze quantification results. GAPDH was employed as housekeeper gene. Primers of muscle-specific genes were designed in NCBI and the information of primers was presented in Table 1.
Treated cells and control cells were collected to extract total RNA using Trizol reagent kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Three biological replicates were performed for both of groups. RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel electrophoresis. After that, eukaryotic mRNA was enriched by Oligo(dT) beads. Then the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP and buffer. Then the cDNA fragments were purified with QiaQuick PCR extraction kit (Qiagen, Venlo, The Netherlands), end repaired, poly(A) added, and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, PCR amplified, and sequenced using Illumina HiSeq2500 by Gene Denovo Biotechnology Co. (Guangzhou, China)
The RNA-sequence data reported in this study was archived in the NCBI SRA database with the accession number PRJNA756416.
Alignment of RNA-sequence reads onto reference genome and expression analysis
RNAs differential expression analysis was performed by DESeq2 software between two different groups. The genes with the parameter of false discovery rate below 0.05 and absolute fold change ≥2 was considered DEGs.
GO and KEGG enrichment analysis
Gene ontology (GO) enrichment analysis provided all GO terms that significantly enriched in DEGs comparing to the genome background, and filtered the DEGs that correspond to biological functions. Firstly, all DEGs were mapped to GO terms in the GO database (http://www.geneontology.org/). Gene numbers were calculated for every term, and significantly enriched GO terms in DEGs were defined by hypergeometric test. The calculated p-value were gone through false discovery rate correction, taking false discovery rate ≤ 0.05 as a threshold. GO terms meeting this condition were defined as significantly enriched GO terms.
KEGG enrichment analysis identified significantly enriched metabolic pathways or signal transduction pathways in DEGs comparing with the whole genome background. The calculated p-value was gone through false discovery rate correction, taking false discovery rate ≤ 0.05 as a threshold. Pathways meeting this condition were defined as significantly enriched pathways in DEG.
All experiment in this study were repeated 3 times at least to ensure repeatability and all data are expressed as means ± SEM (standard error of the mean). Independent sample t.test was used to compare differences between two group and P＜0.05 was considered as statistically significant between the group. All statistical analyses were performed using SPSS 23.0 for Window (SPSS, Inc., Chicago, IL, USA). Symbol “*” indicates significant difference at P < 0.05 and “ns” indicates significant difference at P > 0.05.