The experimental protocols were in compliance with the Helsinki Declaration and approved by the Ethics Committee of the Second Affiliated Hospital of Chongqing Medical University.
Dulbecco’s modified eagle medium low-glucose (L-DMEM) and fetal bovine serum (FBS) were purchased from Gibco Co. (Grand Island, NY, USA). Cell Counting Kit-8 (CCK-8), penicillin, streptomycin, TUNEL apoptosis assay kit, Bradford Protein Assay Kit, RIPA lysis buffer and BeyoECL Plus kit were purchased from Beyotime Institute of Biotechnology (Haimen, China). Adipogenic differentiation medium (ADM), osteogenic differentiation medium (ODM), chondrogenic differentiation medium (CDM), Oil Red O, Alician blue and Alizarin Red S were purchased from Cyagen Biosciences Inc. (Suzhou, China). 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) and phosphate-buffered saline (PBS) were purchased from Boster Biological Technology Co. Ltd (Wuhan, Hubei, China). CXCR4 antibody was purchased from Novus Biologicals (Littleton, CO, USA). Recombinant human SDF-1 was purchased from PeproTech Inc. (Cranbury, NJ, USA). SDF-1, anti-Müllerian hormone (AMH), estradiol (E2) and follicle-stimulating hormone (FSH) ELISA kits were purchased from Uscn Life Science Inc. (Wuhan, Hubei, China). Bax, LY294002 and the secondary antibodies were purchased from Cell Signaling Technology Inc. (Boston, MA, USA). DyLight549 was purchased from Abbkine Scientific Co., Ltd (Liyang, Jiangsu, China). AMD3100 was purchased from MedChemExpress (Monmouth Junction, NJ, USA). Cleaved-Caspase 3, Bcl-2, vascular endothelial growth factor (VEGF) and vascular endothelial growth factor receptor 2 (VEGFR2) were purchased from Affinity Biosciences (Wuhan, Hubei, China). Cyclophosphamide was purchased from Hengrui Medicine Co., Ltd. (Lianyungang, Jiangsu, China). All other chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA).
Isolation and culture of hAD-MSCs
Primary hAD-MSCs were isolated from term amnions according to our previous protocols. Term placentas were collected from healthy donors who received cesarean section at the Second Affiliated Hospital of Chongqing Medical University, Chongqing, China. Written informed consent was obtained from all these donors before collection. hAD-MSCs were cultured in L-DMEM supplemented with 12% FBS, 100 U/mL penicillin and 0.1 mg/mL streptomycin. The third passage of hAD-MSCs were used for the subsequent experiments.
Identification and characterization of hAD-MSCs
hAD-MSCs were identified according to our previous published protocols. The morphological characteristics and growth of hAD-MSCs were observed and imaged by an inverted microscope (Olympus Corporation, Tokyo, Japan). The expression of MSC surface markers on hAD-MSCs was detected by flow cytometry. To identify the multipotent differentiation of hAD-MSCs, hAD-MSCs were cultured in ADM, ODM and CDM for 21 days, respectively. After staining with Oil Red O, Alizarin Red S or Alician blue, the cells were observed and imaged under an inverted microscope (Olympus Corporation, Tokyo, Japan).
The growth curve of hAD-MSCs was detected by CCK-8 assay according to the manufacturer’s instructions. Cells were seeded at a concentration of 5×103 cells/well in 96-well plates and cultured for 24h. Then, the optical density (OD) value at 450 nm was measured every day for 5 continuous days, using a plate reader (1510model; Thermo Fisher Scientific Oy, Vantaa, Finland).
Transwell migration assay
A cell migration model in vitro was constructed by transwell chamber (Corning, NY, USA) composed of a membrane filter with 8 μm diameter pores suspended in a 6-well plate according to the manufacturer’s instructions. hAD-MSCs were placed in serum-free medium at a density of 1×105/cm2 into the upper chamber, and the lower chamber was filled with the same medium containing 2% FBS. For the inhibitor treatment, hAD-MSCs were preincubated with AMD3100 (44nM, specific inhibitor of CXCR4) or LY294002 (50 μmol/L, specific inhibitor of PI3K/Akt pathway) for 1 h. For the SDF-1 treatment, SDF-1 with the concentration of 0, 50, 100 or 150 ng/mL was added into the lower chamber. After 24 h, migration assays were terminated by retrieving the membrane filter from each group, and hAD-MSCs on the underside of the filter were stained with crystal violet staining solution and counted in 6 visual fields (200×) randomly chosen under microscope (Olympus Corporation, Tokyo, Japan).
For exploring the CXCR4 expression in hAD-MSCs, hAD-MSCs were collected from four different persons. For exploring the mechanisms associated with hAD-MSC migration mediated by SDF-1/CXCR4 axis, hAD-MSCs were pre-treated with AMD3100 (44nM) or LY294002 (50 μmol/L) for 1 hour followed by the treatment of SDF-1 (100 ng/mL). After treatment, hAD-MSCs were lysed in RIPA lysis buffer and proteins were isolated after centrifugation. The protein concentrations were determined by the Bradford Protein Assay Kit. The protein samples were separated by SDS-PAGE and subsequently electro-transferred to PVDF membranes (Millipore, USA). After washing, the membranes were blocked with 5% skim milk for 1 h and incubated with specific primary antibodies for CXCR4, Akt, phospho-Akt, ERK1/2 or phospho-ERK1/2 overnight at 4°C. After washing, the membranes were incubated with the secondary antibodies for 1 h at room temperature, and BeyoECL Plus kit was used for color development according to the manufacturer’s instructions.
For further confirming the expression of CXCR4 in hAD-MSCs, cells were detected by immunofluorescence assay. The cells were fixed, washed and permeabilized in PBS containing 0.5% Triton X-100 for 30 min. After washing, the cells were blocked with 5% BSA for 2 h and incubated with specific primary antibody for CXCR4 overnight at 4°C. After washing, the cells were incubated with secondary antibodies conjugated with DyLight549 for 1 h at 37°C. Then, the cells were counterstained with DAPI and imaged under a laser scanning confocal microscope (Nikon Corporation, Tokyo, Japan).
Labeling and tracking of hAD-MSCs
To track and locate the transplanted hAD-MSCs in ovarian tissue, the cells were pre-labeled with the PKH26 Red Fluorescent Cell Linker Kits according to our previous published protocols before transplantation. At 24 h after transplantation of PKH26-labeled hAD-MSCs, ovaries were made into fresh sections and incubated with DAPI, and the sections were imaged under a laser scanning confocal microscope (Nikon Corporation, Tokyo, Japan).
The quantification of homing efficiency of hAD-MSCs within ovarian tissue is typically assessed by averaging the number of PKH26-labeled cells present in 800× microscopic fields randomly chosen from per tissue sample under confocal microscope according to the protocol which has been published.
Female Sprague-Dawley (SD) rats aged 10~12 weeks were purchased from the Experimental Animal Center of Chongqing Medical University.
To investigate whether SDF-1/CXCR4 axis mediates the homing of hAD-MSCs to chemotherapy-induced POI ovaries in rats, 72 of female SD rats were randomly divided into 4 groups as follows: control, POI, hAD-MSCs and hAD-MSCs+AMD3100 groups (n=18 in each group). Firstly, the rats were intraperitoneally injected with cyclophosphamide to establish POI models in the POI, hAD-MSCs and hAD-MSCs+AMD3100 groups according to our previous published protocols. For the inhibitor treatment, hAD-MSCs were incubated with AMD3100 (44nM) for 1 h before cell transplantation in the hAD-MSCs+AMD3100 group. Then, at 24 h after chemotherapy, the rats from the hAD-MSCs and hAD-MSCs+AMD3100 groups were injected with 0.6 ml of PBS containing 4×106 of hAD-MSCs labeled with PKH-26 via the tail vein, while the rats in the control and POI groups were injected with 0.6 ml of PBS. At 24 h, 3 w and 6 w after cell transplantation, 6 rats in each group were sacrificed under sodium pentobarbital anesthesia, and samples were collected for the subsequent experiments.
The estrous cycles of rats in each group were recorded by vaginal smears observation as described in our previous published protocols. Regular estrous cycles consist of 4 sequential stages as follows: proestrus, estrus, metestrus and diestrus (Fig. 6a). Irregular estrous cycles were also defined as described in our previous published protocols.
To further investigate molecular mechanisms involved in the homing of hAD-MSCs mediated by SDF-1/CXCR4 axis in vivo, 24 of female SD rats were randomly divided into 4 groups as follows: control, POI, hAD-MSCs and hAD-MSCs+ LY294002 groups (n=6 in each group). The rats were intraperitoneally injected with cyclophosphamide to establish POI models in the POI, hAD-MSCs and hAD-MSCs+ LY294002 groups. For the inhibitor treatment, hAD-MSCs were incubated with LY294002 (50 μmol/L) for 1 h before cell transplantation in the hAD-MSCs+ LY294002 group. At 24 h after chemotherapy, the rats from the hAD-MSCs and hAD-MSCs+ LY294002 groups were injected with 0.6 ml of PBS containing 4×106 of hAD-MSCs labeled with PKH-26 via the tail vein, while the rats in the control and POI groups were injected with 0.6 ml of PBS. At 24 h after cell transplantation, rats in each group were sacrificed under sodium pentobarbital anesthesia, and samples were collected for the tracking tests.
Enzyme-linked immunosorbent assay (ELISA)
To detect the SDF-1 levels in the ovaries and serum of POI rats at 24 h after chemotherapy, ovarian tissue and serum were collected from the rats in the control and POI groups. Ovarian tissue was homogenized and the supernatant was collected. The SDF-1 levels were detected by ELISA kit according to the manufacturer’s instructions.
To detect the serum levels of AMH, FSH and E2 of rats in each group, serum was collected at 0, 3 and 6 w after cell transplantation and analyzed using ELISA kits according to the manufacturer’s instructions.
Ovarian morphology analysis and follicle counts
Ten ovaries from each group were collected at 6 w after hAD-MSC transplantation. Ovaries were fixed, dehydrated, paraffin embedded and cut into 5 μm sections. The sections were stained with hematoxylin and eosin (HE). The ovarian morphology was observed by an optical microscope (Olympus Corporation, Tokyo, Japan). The follicles in ovaries were classified as primordial, primary, secondary, preovulatory and atretic follicles. The number of follicles was counted as described previously[3, 11].
Ovarian cell apoptosis was tested by the TUNEL apoptosis assay kit according to the manufacturer’s instructions. Sections were observed and imaged by an optical microscope (Olympus Corporation, Tokyo, Japan). Nuclei of ovarian apoptotic cells were stained dark brown.
Ovarian tissue sections were incubated with specific primary antibodies for Bcl-2, Bax, cleaved-caspase-3, VEGF and VEGFR2, and then the corresponding secondary antibodies followed by horseradish peroxidase. After that, the sections were stained with 3,3’-diaminobenzidine and counterstained using hematoxylin. Then, the sections were observed and imaged under an optical microscope (Olympus Corporation, Tokyo, Japan). The sections were analyzed as described in our previous published protocols: the number of positive cells graded as 4 (>75%), 3 (51~75%), 2 (25~50%), 1 (5~25%) or 0 (<5%), and the staining intensity graded as 3 (brown), 2 (light brown), 1 (light yellow) or 0 (no color). The total score was the sum of the two grades, which was named immunoreactivity score (IS). Ten high-power fields (HPFs, 400×) were randomly chosen from five sections in each group for scoring. The median and range of ISs for each group was calculated.
For all assays, at least three independent experiments were performed. Statistical analysis of data was processed by SPSS 22.0 software (IBM, NY, USA). Data with normal distribution was presented as mean ± standard deviation, and independent samples t-test was used for comparisons between groups. Data with skewed distribution was presented as median and range, and nonparametric Wilcoxon rank test was used for comparisons between groups. Statistical significance was set at P<0.05.