2.1 Collection of umbilical cord
Umbilical cord samples from women with healthy pregnancies were retrieved at the time of cesarean section from the Department of Obstetrics and Gynecology, the first affiliated hospital of USTC. Umbilical cord samples were collected in phosphate-buffered saline (PBS) containing 500 IU heparin (Sigma–Aldrich, St. Louis, MO, USA), 300Units/mL penicillin, and 300mg/mL streptomycin (Invitrogen, Grand Island, NY, USA) and were immediately transferred to the laboratory. Umbilical cord samples were processed within 12 hours of collection.
2.2 Isolation of WJ from umbilical cord
Each human umbilical cord was transferred into a sterile laminar flow hood and washed twice in PBS to remove contaminating blood cells. The umbilical cord was cut into approximately 5-cm long segments, which were subsequently cut longitudinally to expose the umbilical vein. Blood vessels (two arteries and one vein) were removed from each segment, and Wharton’s jelly was carefully separated from the amniotic membrane34.
2.3 Six widely used WJ-MSCs isolation methods
M1 (explant method): 2g WJ was excised to about 1-2mm size pieces, and they were transferred to 100mm dishes (Corning) coated with 0.2% gelatin. 2g weight of tissues were plated per dish and covered entirely with the culture medium. Half of the medium was changed on day 5 without disturbing the tissue pieces. On day 8, all the tissue pieces were removed, and the medium was completely replaced with fresh medium.
M2(explant method): 2g WJ was excised to about 1-2mm size pieces, then homogenized in DMEM/F12 + 20%FBS and grinded crosswise for about 40 times using a homogenizer into a fine granular shape (1–2mm in diameter). Then the sample was rinsed repeatedly with DMEM/F12 + 20%FBS and placed as tissue pieces on the 100mm dishes (Corning) coated with 0.2% gelatin. Half of the culture medium was replaced on day 5 without disturbing the tissue pieces. On day 8, all the tissue pieces were removed, and the medium was completely replaced with fresh medium. Every 2 days thereafter, the cells were observed and passaged according to their growth.
M3(enzymatic-explant method): 2g of WJ was excised to about 1-2mm and incubated with 1mg/mL collagenase II (Sigma–Aldrich) at 4°C overnight. The next day, the tissue was filtered through a 100-mesh sieve to filter out large pieces of tissue and washed three times by PBS and one time by culture medium (DMEM/F12 + 20%FBS). Then, the tissue was plated to a 100mm dish coated with 0.2% gelatin and covered by 5ml medium. The next day, cells started to dissociate from the tissue, and another 2-3ml medium was added to the plate. After two days, more cells were dissociated from the tissue, and another 2-3ml medium was added. The medium was changed every three days until day 8, or the cells grew to approximately 80–90% confluence.
M4 (enzymatic-explant method): WJ was excised to about 1-2mm pieces, and about 2g tissue pieces were transferred to a 15ml falcon tube to which 3ml enzymes mix (1mg/mL collagenase II and 0.25mg/mL trypsin) was added. The sample was incubated for 15min in the shaker at 37°C, shaking at 200 rpm. The reaction was neutralized by 7ml culture medium. The dissociated cells and tissue pieces were collected by centrifugation at 750g and transferred to a 10cm dish coated with 0.2% gelatin at the density of 0.5-1.5pieces/cm2. 5mL culture medium was added to the plate, and the sample was placed in a 5% CO2 incubator for 4–5 days until the cells were attached. The culture medium was replaced on Day 5 and changed every 2 days thereafter.
M5 (enzymatic method): 2g of WJ was soaked in culture medium and excised to about 1-2mm. The tissue pieces were washed three times by PBS to remove the culture medium completely. Then, the tissue pieces were transferred to a 15ml falcon tube to which 1.6ml enzymes mix (0.4mL collagenase I (10mg/mL), 0.4mL collagenase IV (10mg/mL), 0.4mL hyaluronidase (3mg/mL), 0.4mL DNAse I (1mg/mL) and 2.4mL trypsin (0.25%)) was added. The sample was incubated for 2hrs in the incubator shaker at 37°C, with agitation at 200rpm. The isolated cells were washed three times with PBS and the cell pellet was resuspended in the culture medium. The cells were cultured in 100mm tissue culture plate and incubated in a 37°C incubator with 5% CO2, with a medium change every three days.
M6 (enzymatic method): 2g WJ was soaked in culture medium and excised to about 1-2mm. The tissue pieces were washed three times by PBS to completely remove the culture medium. Then, the tissue pieces were transferred to a 15ml falcon tube and subjected to two steps of digestion. In the first step, 0.5mL collagenase II (10mg/mL), 4.5mL trypsin (0.25%) and 0.5% ethanol was used. In the second step, 0.5mL collagenase II (10mg/mL), 0.5mL hyaluronidase (3mg/mL), 0.5mL DNAse I (1mg/mL), and 3.5mL culture medium was used. The sample was incubated in the incubator shaker at 37°C, with agitation at 200rpm, for 90min. The cells were collected by centrifugation at 700g and resuspended in 10ml culture medium. Then, the cells were cultured in 100mm tissue culture plate and incubated in a 37°C incubator with 5% CO2, with a medium change every three days.
2.4 Isolation of WJ-MSCs by “Mince-Soak-Digest (MSD)”
2g WJ tissue sample was minced to about 1-2mm and soaked in culture medium for 7 days on 100mm tissue culture dish. Then, the tissue pieces were collected into a 15ml falcon tube and subject to enzymatic digestion with a combination of collagenase II, III, and VI at 2mg/ml and DNAse I (0.1mg/mL) at 37°C for 1.5hours, under 200rpm agitation. The digestion mixture was filtered through a 40um filter to remove undigested tissue, and cells were washed three times by PBS to completely remove the digestion enzymes. Cells were then collected by centrifugation at 700g and resuspended in 10ml culture medium. Cells were cultured in 100mm tissue culture dish coated with 0.2% gelatin and incubated in a 37°C incubator with 5% CO2, with a medium change every three days for additional 7 days.
2.5 Culture of WJ-MSCs
WJ-MSCs were maintained in low-glucose Dulbecco’s Modified Eagle’s Medium (DMEM-LG; Invitrogen), supplemented with 10% fetal bovine serum (FBS; Sigma), 100Units/mL penicillin, and 100mg/mL streptomycin. Cultures were grown at 37°C in a humidified atmosphere containing 5% CO2. The medium was changed every three days until 80% confluence was reached. To passage the cells, 0.05% Trypsin-EDTA (Invitrogen) was used to dissociate the cells and neutralized them with the culture medium. The cells were then washed once with PBS and collected by centrifugation. The cells were seeded at 1×104 cells/cm2.
2.6 Cell growth curves
MSCs generated with different methods at passage 3 were seeded at 1000 cells/well in 96-well plates in 100 µL of culture medium. Cell numbers with each method were counted for 14 days with each sample in triplicates, using a hemocytometer. Doubling time was estimated, and Typan Blue staining assay (Invitrogen) was used to check the viability of the cells.
2.7 Flow cytometry analysis
Flow cytometry analysis was performed using Human MSC Analysis Kit (BD Biosciences, Cat. No. 562245) based on the manufacture’s protocol. Briefly, cells were dissociated using Accutase (BD Biosciences, Cat. No 561527) at 37°C for 5 mins and collected by centrifugation at 200g. Then, the cells were passed through 70 µm strainers to ensure they were digested as single cells before they were subject to flow cytometry analysis. Dissociated cells were incubated in 1% BSA in PBS containing primary antibodies on ice for 20 min. Cells were then analyzed using a BD FACSAriaII cell sorter. Analysis was performed using FlowJo software (Tree Star).
2.8 BCA (Bicinchoninic Acid) protein assays
BCA protein assays were performed to quantify the total protein concentration using Pierce BCA Protein Assay Kit (Prod #23225) based on the manufacture’s protocol.
2.9 Differentiation assays for WJ-MSCs
WJ-MSCs were differentiated into multiple mesenchymal lineages (adipogenic, osteogenic, and chondrogenic) using Human MSC Functional Identification kit (Catalog # SC006, R&D Systems) based on the manufacture’s protocol.
The animals were housed in an environment-controlled room at 22°C with a 12hrs light and dark cycle. Animal handlers were blinded to the experimental group. Tissues obtained were examined in a blinded fashion.
2.11 Staging of the estrous cycle of the rats
8 to10-week-old Sprague Dawley female Rats were used for this study. Vaginal smear/cytology is applied to stage the estrous cycle of the rats as previously described35. Briefly, a cotton-tipped swab wetted with saline was introduced into the vagina of the rat. The swab was gently twisted against the vaginal wall to scratch the cells. The cells were collected onto a dry glass slide by gently rub the swab across the slide. The slide is air-dried, stained, and viewed under a microscope. The estrus phase is determined by the presence of abundant anucleated cornified epithelial cells. Rats at the estrus phase were used for establishing the endometrium-injury model.
2.12 Establishment of the rat intrauterine adhesions (IUA) model
The rat model of endometrium-injury was established based on previously published protocols36–38 with a few major modifications. Essentially, to increase the survival rate and reduce the complication rate due to the surgery, we exposed a relatively small incision (3-5mm versus 3cm in published protocols) and inserted a tweezer to scrape the inner uterine surface instead of a surgical scalpel blade. Briefly, 10% chloral hydrate (300mg/kg) was injected in the lower left abdominal quadrant of the rat to induce anesthesia. After anesthesia, the left uterine endometrium was exposed by a 3-5mm longitudinal incision on the uterine horns. A tweezer was inserted through the incision and the inner uterine surface was scratched by pulling the tweezer back and forth, left and right for 10 times at each direction. Then, wash the uterine surface with saline and close the uterine horn by 3–4 intermittent stitches using absorbable sutures.
2.13 Transplantation of WJ-MSCs (MSD) to the rat uterus
Twelve rats were randomly assigned to two groups: the control (IUA-model) group and the transplantation group (transplanted with WJ-MSCs (MSD)). The left uterine horns of all rats were incised open and then closed by 3–4 intermittent stitches using absorbable sutures as a sham control. For the control group, the inner uterine surface on the right was injured as described above, and MSC culture media was injected as control. For the transplantation group, immediately after the uterine horn was exposed and injured, 2×106 WJ-MSCs (MSD) in 200 ul media were injected inside of the uterine horn, then the uterine horn was sutured. Two weeks post-surgery, half of the rats in each group were used for fertility tests, and the other half were used for H&E staining and immunofluorescence analysis.
2.14 Fertility test
Rats in estrus were mated with 10-week-old fertile male Sprague-Dawley rats. Day 0 of pregnancy was defined as the day a vaginal plug was found. Rats were then euthanized on gestation day 15–19, and uterine horns were examined to record the number of embryos.
2.15 Hematoxylin and Eosin (H&E) and Masson’s trichrome staining
Formalin-fixed rat uterine tissues were paraffin-embedded and sectioned into serial cross-sections of 5–10 µm thickness each. Samples were stained as described previously [Animals 2020, 10(4), 683; https://doi.org/10.3390/ani10040683]. From the images of H&E staining, the mean thickness of the endometrium was determined from four measurements at 0 °, 90 °, 180 °, and 270 °in horizontal sections, and the number of glands was counted from horizontal sections under a 40×magnification.
2.16 Immunofluorescent staining
Rat uterine sections were deparaffinized in xylene, rehydrated through an ethanol-graded series. For all samples, antigen retrieval was performed by boiling the sections in 0.01 M sodium citrate buffer (pH 6.0) for 20 min, followed by incubation at room temperature for 30 min. A 10% solution of normal donkey serum (Jackson ImmunoResearch) in PBS was used as a blocking buffer. Sections were incubated with the following primary antibodies diluted in blocking solution (1.0% Normal Donkey Serum, 0.1% Triton X-100, and sterile PBS) overnight at 4°C. The sections were washed and labeled with Alexa dye-conjugated secondary antibodies. Sections were mounted in ProLong Gold Antifade mounting media containing DAPI (Life Technologies). Negative controls included incubation with rabbit immunoglobulin G antibodies and omission of the primary antibody for all samples.
2.17 RNA extraction, reverse transcription, and quantitative real-time PCR
Total RNA was extracted using TRIzol Reagent (Invitrogen) and purified with the RNeasy Mini Kit (Qiagen). Reverse transcription was performed using the SuperScript II Kit (Invitrogen). Quantitative PCR analyses were performed in real-time using an ABI PRISM 7900 sequence detection system and SYBR green master mix (Life Technologies). The primer information is provided in the Supplementary Table. Data are presented as the mean ± SEM and derived from three independent experiments.
2.18 Data Analysis
Data are presented as mean ± SD for at least three independent experiments (different patient samples) performed in triplicate unless stated otherwise. Statistical significance is determined by analysis of variance and set at p < .05.