TSPCs isolation and culture

The procedures for the isolation of TSPCs from rat Achilles tendon have been well-established7. Briefly, rat TSPCs were isolated from 4-month-old (abbreviated as Y-TSPC), 8-month-old and 20-month-old (abbreviated as A-TSPC) male Sprague−Dawley rats (n = 10). The Achilles tendons were gently minced, digested with type I collagenase (3 mg/ml; Sigma-Aldrich), and passed through a 70 μm cell strainer (Becton Dickinson) to yield a single-cell suspension. The released cells were washed in phosphate buffered saline (PBS) and resuspended in Dulbecco’s modified essential medium (DMEM) containing 10% fetal bovine serum, 1% penicillin-streptomycin (all from Gibco). The isolated nucleated cells were plated at an optimal low cell density (50 nucleated cells/cm2) for the isolation of stem cells and cultured at 37 °C, 5% CO 2 to form colonies. At day 7, they were trypsinized and mixed together as passage 0 (P0). Cells from P2 to P6 were used for all experiments. Medium was changed every 3 days. The clonogenicity and multilineage differentiation potential of these cells were confirmed before being used for the experiments in this study using standard assays as described previously7. All surgical interventions and postoperative animal care were carried out in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council) and were approved by the Animal Research Ethics Committee of Southeast University. All efforts were made to minimize the number of animals used and their suffering.

Cell transfection

Lentivirus encoding AQP1 which labeled with GFP (LV- AQP1) and negative control lentivirus (LV-GFP) were purchased from GeneChem Corporation (Shanghai, China). 8 × 104 cells were plated on six-well plates; cells at 20–30% confluence were transfected with lentivirus in accordance with the manufacturer’s recommendations. All cells were transfected by HitransG Transfection Reagent P (Genechem). The transfected cells were selected with 2 μg/ml puromycin (Beyotime Biotechnology) for 10 days to establish stably expressing cells and verified by Western blotting.

AQP1-siRNA was designed and synthetized from GenePharma (Shanghai, China); the sequences of AQP1-siRNA were as follows: CCAUGACCCUCUUCGUCUUTT (sense: 5′−3′), AAGACGAAGAGGGUCAUGGTT (antisense: 5′−3′). Transfection was performed when the cells reached 50% confluence. Cells were transfected by Lipofectamine 2000 (Invitrogen). Transfected TSPCs were used for the subsequent experiments 48 h after transfection.

Microarray analysis

Gene expression profiles were examined by Beijing CapitalBio Corporation (Beijing, China). Briefly, poly-A containing mRNA molecules was purified from 3 μg of total RNA by using poly-T oligo-attached magnetic beads. The cleaved RNA fragments were reversely transcribed into first-strand cDNA using random hexamers, followed by second-strand cDNA synthesis using DNA Polymerase I and RNase H. The cDNA fragments were purified, end blunted, “A” tailed, and adaptor ligated. PCR was used to selectively enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. The number of PCR cycles was minimized to avoid skewing the representation of the library. The library was qualified by Agilent 2100 bioanalyzer and quantified by Qubit and qPCR. The produced libraries were sequenced on the HiSeq 2500 platform. After robust multiarray average normalization, fold change threshold ≥2 and P value < 0.05 were recognized to be statistically significant alterations. Clustering analysis and heatmap generation were performed using Cluster3.0 software. The functional assignments were mapped onto Gene Ontology (GO). GSEA (http://software.broadinstitute.org/gsea/index.jsp) was employed to verify the biological processes in the two groups as described above47. NES and false discovery rate were calculated to verify the significant difference for GSEA.

Immunofluorescence staining

For immunofluorescence staining, cultured TSPCs were fixed in 4% paraformaldehyde for 15 min at room temperature. Cells were blocked with 10% normal serum blocking solution (3% bovine serum albumin and 0.1% Triton X-100 and 0.05% Tween-20) for 2 h at room temperature. After being washed, cells were incubated overnight at 4 °C with anti-AQP1 (Proteintech) and p16INK4A (Abcam), followed by a mixture of Alexa Fluor 594-conjugated secondary antibodies (Molecular Probes) was incubated 2 h at room temperature. Immunofluorescence was visualized with a Nikon Ts2R fluorescence microscope (×20 or ×40 objectives). For EdU detection, the BeyoClick™ EdU Cell Proliferation Kit with Alexa Fluor 647 was used according to the manufacturer’s protocol (Beyotime Biotechnology). Immunofluorescence was visualized with an Olympus FV1000 confocal microscope (×40 objectives).

TSPCs migration assay

TSPCs were plated on six-well plates and grown to confluence. Then the medium was removed, and the monolayer was scratched with a sterile plastic pipette tip. The TSPCs were washed with PBS and incubated for 16 h before being imaged under an inverted microscope. The initial scratch length and scratch bridging time were measured and used for the calculation of cell velocity. Images were captured by an Olympus CKX53 inverted phase-contrast microscope (×4 objectives).

Investigation of actin dynamics

Actin dynamics analysis was performed similarly to previous study4. Briefly, the young, aged and AQP1-overexpressing aged TSPCs were plated on six-well plates and incubated for 48 h. Then cells were treated with 0.4 μM Latrunculin A (Sigma-Aldrich) in a time-dependent manner (0, 5, 10, 15, 30 and 60 min). Cells were fixed in 4% paraformaldehyde and permeabilized with 0.1% Triton X-100, then the cells were stained with Alexa Flour 546 phalloidin (Thermo Scientific). Immunofluorescence was visualized with a Nikon Ts2R fluorescence microscope (×20 or ×40 objectives). For quantification of the F-actin amount, the fluorescence images were analyzed using ImageJ software (NIH). The mean fluorescence intensity was recorded.

Western blotting

TSPCs were washed in cold PBS buffer, then the cell proteins were extracted by homogenizing the tissue in lysis buffer. The supernatant was then collected for measurement of protein concentration by BCA protein assay (Thermo Scientific). Thirty micrograms of protein was denatured, fractionated by electrophoresis on SDS-PAGE and electrophoretically transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore). The blots were blocked with 5% nonfat dry milk in PBST solution, incubated with primary antibody against AQP1 (Proteintech), cyclin A2 (Proteintech), cyclin B1 (Proteintech), cyclin D1 (Bioworld), p16INK4A (Abcam), JAK2 (Proteintech), p-JAK2 (Abcam), STAT3 (Proteintech), p-STAT3 (Abcam) and GAPDH (Proteintech) at 4 °C overnight. After incubating with secondary antibody, immunoreactive bands were detected by ECL reagents (Keygen Biotech). The gray value of each band was measured and data are presented as a ratio to GAPDH.

β-galactosidase staining

The β-galactosidase (β-gal) assay was performed using the SA-β-gal staining kit (Sigma). Cells were plated on 12-well plates and incubated for 48 h. Cells were incubated with the kit’s staining mixture for 16 h at 37 °C. The percentages of β-gal-positive cells were calculated by counting 300 cells in six microscopic fields. Images were captured by an Olympus CKX53 inverted phase-contrast microscope (×4 or ×10 objectives).

CCK-8 assay

The Cell Counting Kit-8 (CCK-8, Keygen Biotech) assay was used to measure cell proliferation. Cells were plated into 96-well culture plates at an optimal density of 3000 cells/well in 200 μl complete culture medium. The cells were observed under a microscope and the CCK-8 assay was performed at 0, 24, 48 and 72 h. Then the 10 μl CCK8 solution was added to each well and incubated for 2 h at 37 °C; the absorbance of each well was read by the microplate reader at 450 nm.

Quantitative RT-PCR

TSPCs were harvested and homogenized for RNA extraction with the MiniBEST universal RNA extraction kit (Takara). The mRNA was reverse transcribed to cDNA by the First-Strand cDNA kit (Promega). One microliter of total cDNA of each sample was amplified in the final volume of 20 μl of reaction mixture containing Power SYBR Green PCR Master Mix (Invitrogen) and specific primers using the ABI Step One Plus system (all from Applied Biosystems). Cycling conditions were denaturation at 95 °C for 10 min, 45 cycles at 95 °C for 20 s, optimal annealing temperature for 20 s, 72 °C for 30 s, and finally at 60–95 °C with a heating rate of 0.1 °C/s. The expression of the target gene was normalized to that of the β-actin gene. Relative gene expression was calculated as fold change over control and calculated as 2−ΔΔCt. The primer sequences used in this study are listed in Table S3.

Cell cycle analysis

TSPCs was cultured on 10-cm dishes in 2% FBS/DMEM for 48 h. Then cells were trypsinized and detached, washed with PBS, and then fixed in 70% ethanol overnight at 4 °C. Then, the cells were washed with PBS and incubated with RNase (Keygen biotech) and propidium iodine (Keygen Biotech) for 30 min. The percentage of cells in the three phases of the growth cycle (G1, S and G2/M phase) was measured by flow cytometry (Becton Dickinson) using Cell Quest software.

Colony-forming unit (CFU) assays

For the CFU assay, 2, 5 and 10 cells/cm2 of TSPCs were plated in six-well plates respectively for 10 days in complete media. The cells were stained with 0.5% crystal violet for counting the number of cell colonies. CFU efficiency was estimated as percentage of counted colonies to the number of plated cells.

Population doubling time (PDT) assay

Population doubling time (PDT) assay was performed as described previously35. PDT was calculated from the formula log2 [Nc/N0], where N0 refers to the total cell number during seeding, and Nc is the total cell number at confluence.

Statistical analysis

All data are plotted as mean ± standard deviation (SD). Differences in mean values between groups were tested using one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc multiple comparison test. P < 0.05 was considered statistically significant. Each experiment consisted of at least three replicates per condition. Variance was similar between the groups that were being statistically compared.