This study was approved by the Institutional Review Board of the Stanford University School of Medicine and the Stanford University Stem Cell Research Oversight Committee. Three human pluripotent stem cell (PSC) lines were used in this study for differentiation into pSMCs: 1) Huf5-iPSC line was reprogrammed from a 46yo female dermal fibroblasts via viral transduction of the transcription factors Oct3/4, Sox2, Klf4 and c-Myc ; 2) BIR-iPSC line was reprogrammed from a 18yo female dermal fibroblasts with modified miRNA reprogramming method . mRNA encoding Oct4, Klf4, Sox2, c-Myc and Lin28 were co-transfected with a mixture of several miRNAs into the fibroblasts to induce iPSCs; and 3) H9 human embryonic stem cell line was a gift from Dr. Joseph Wu, Stanford University. All PSC lines were cultured on Matrigel-coated dishes (BD Biosciences, San Diego, CA) in mTeSR medium (StemCell Technologies, Vancouver, BC).
Human bladder smooth muscle cells (bSMCs) were isolated from three female donors (30, 33 and 50-years old which were labeled as B1, B2, B3 respectively). IRB exemption and external approval from Donor Network West’s (federally designated organ procurement organization) Internal Research Council and Medical Advisory Board Research Subcommittee was obtained for collection of primary bladder tissue from the three female transplant donors. Patients with history of bladder pathology were excluded from study. The muscular layer of fresh bladders was cut into 2 mmx2mm pieces and cultured in 80% Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA) and 20% fetal bovine serum (Invitrogen) at 37 °C in an atmosphere of 95% air and 5% CO2. Tissue fragments were removed on day 14 and culture medium was changed to 90% DMEM and 10% FBS. Cells were passed to next passage with 0.05% Trypsin with EDTA (Thermo Scientific, Fremont, CA) when they were 80–90% confluent. SMC markers including αSMA and SM22 were detected by immunofluorescent staining to confirm cell types (data not shown).
Human vaginal fibroblasts were isolated from three female donors which were labeled as F1(66y), F2(69y), F3(65y) respectively. Fibroblasts cell explants were cut into approximately 1 mm x1mm fragments and cultured in 80% Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen) and 20% fetal bovine serum (Invitrogen) at 37 °C in an atmosphere of 95% air and 5% CO2 . Tissue fragments were removed on day 14 and culture medium was changed into 90% DMEM and 10% FBS. Cells were passed to next passage with 0.05% Trypsin with EDTA (Thermo Scientific, Fremont, CA) when they were 80–90% confluent.
Directed differentiation of human pluripotent stem cells to pSMCs and conditioned medium collection
Human pSMCs were differentiated from the three pluripotent stem cell lines using a modified, feeder cell-free, vascular progenitor protocol [20, 22, 23]. Briefly, the stem cells were seeded at a density of 5,000–20,000 cells per cm2 of a 10-cm dish precoated with Matrigel in mTeSR supplemented with Thiazovivin (2 µM; Cayman Chemical Company, Ann Arbor, MI). After 24–72 h in cultured in chemical defined medium (RPMI 1640 with 1 mM glutamax, 1% nonessential amino acids, 0.1 mM β-mercaptoethanol, 1% penicillin and streptomycin [Life Science Technology, Inc.], 1% ITS [Corning, Tewksbury, MA]) supplemented with Activin A (50 ng/mL), BMP4 (50 ng/mL; PeproTech, Rocky Hill, NY), and 2.5 µM/mL GSK 3 inhibitor, CHIR99021 on day 0, 2.0 µM/mL CHIR99021 on day 1 (Cayman Chemical Company), followed by basic fibroblast growth factor (50 ng/mL) and vascular endothelial growth factor (40 ng/mL; PeproTech) from day 2 to 12–14 days.
The differentiated cells were dissociated with Acutase (Innovative Cell Technologies, San Diego, CA) and prepared for magnetic-activated cell sorting (MACS) and fluorescence-activated cell sorting (FACS) of CD31+/CD34+ vascular progenitor cells (VPCs) . Briefly, the cells were first immunolabeled with CD34 microbeads for MACS (Miltenyi Biotec, Auburn, CA), and magnetic labeling was performed according to the manufacturer’s instruction. The magnetically labeled CD34+ VPCs were obtained by positive selection. The CD34+ VPCs sorted by MACS were immediately blocked with mouse IgG (R&d Systems, Inc., Minneapolis, MN) for 10 min and stained with FITC mouse anti-human CD31 and PerCP-Cy5.5 mouse anti-human CD34 antibodies (BD biosciences) for 30 min at 4 °C. The CD34+/CD31 + cells were sorted on a FACS Aria II (BD Biosciences). After sorting, the cells which were now labeled as passage 0 (P0), were expanded on mouse collagen IV-precoated plates (BD Biosciences) in smooth muscle cell growth medium (SMGS; Invitrogen) supplemented with recombinant human platelet-derived growth factor-BB (10 ng/mL, PDGF-BB; PeproTech) to yield pSMCs. The pSMCs were passed with 0.05% Trypsin with EDTA (Thermo Scientific, Fremont, CA) when they reached 80–90% confluency until they were harvested at passage 3 (P3). Conditioned medium was collected and frozen at -80 °C when medium was changed every other day from P0 to P3. The conditioned medium for each cell line was pooled at P0 and at P3, and a small amount of the pooled conditioned medium from each passage was frozen at -80 °C. The conditioned medium was collected and pooled at P4 for each cell line and concentrated 50 times by ultrafiltration using centrifugal filter units with 10-kDa cutoff (Sartorius Stedim SUS Inc., CA) for rodent periurethral injection.
In vitro treatment of human bladder and vaginal cells with pSMC-CM
Vaginal fibroblasts and bladder SMCs were passaged onto 6-well plates at passage 2 at a density of 10,000 cells per cm2 in 10% FBS and 90% DMEM. When they reached 90% confluency, medium was changed into basal medium 231 (Invitrogen) supplemented with 0.2% albumin (Sigma-Aldrich, St. Louis, MO) to synchronize for 24 h. The cells were then treated for 48 hours with non-concentrated CM collected from P0 and P3 pSMCs at 37 °C in an atmosphere of 95% air and 5% CO2, while cells in the control groups were treated with the baseline smooth muscle cell growth medium (SMGS; Invitrogen) supplemented with PDGF preincubated at 37 °C in an atmosphere of 95% air and 5% CO2 for 48 hours.
Animal care and generation of SUI rat model
Female immunodeficient Rowett Nude rats (RNU, Charles River Laboratories, Hollister, CA, USA, http://www.criver.com/) weighing 200–250 g were used. Animals were maintained at the Stanford University Research Animal Facility in accordance with Stanford University’s Institutional Animal Care and Use Committee guidelines. Animal experiments were approved by the Institutional Review Board of the Stanford University School of Medicine and the Stanford Administrative Panel of Laboratory Animal Care (APLAC).
The rat model of SUI was established via transabdominal urethrolysis as described by Rodriguez et al. . This SUI rat model showed significantly decreased urethral resistance (by leak point pressure measurements) and urethral smooth muscle damage for at least 8 weeks after surgery [17, 26]. Bilateral ovariectomy was done on the rodents to eliminate the influence of estrus cycle on the ECM metabolism and to simulate an estrogen-deficiency state of menopause . In brief, RNU rats were intraperitoneally anesthetized with ketamine (30 mg/kg) and xylazine (3 mg/kg). The ovaries were exteriorized through a lower abdominal incision. After ovarian vessels were ligated, bilateral ovaries were excised. The bladder and urethra were identified and circumferentially separated from anterior vaginal wall and pubic bone by sharp dissection, thus causing injury to the urethral sphincter and adjacent vagina.
Conditioned media peri-urethral injection and tissue collection
The rats were randomly divided into four treatment groups: 1. urethrolysis plus 50x concentrated SMGS (sham-SMGS group, n = 7), 2. urethrolysis plus 50x concentrated H9-pSMC-CM (H9 CM group, n = 6), 3. urethrolysis plus 50x concentrated Huf5-pSMC-CM (Huf5 CM group, n = 7), 4. urethrolysis plus 50x concentrated BIR pSMC-CM (BIR CM group, n = 8). Three weeks after urethrolysis, rats were anesthetized with 3–4% v/v isoflurane and 100 µL of the concentrated pSMC-CM or SMGS were injected in the peri-urethral area (once weekly for three weeks) at two sites using a 28.5-gauge insulin syringe. Researchers were blinded to the treatment group allocations. Leak point pressure (LPP) testing to evaluate urethral function was performed 5 weeks after initial injection. The rats were then euthanized and urethra and adjacent vagina were harvested. The proximal part of the urethra and vagina was embedded in Tissue-Tek O.C.T. compound (Sakura Finetek, Tokyo, Japan, http://www.sakura-finetek.com) for histologic study. The middle part of the urethra and vagina was used for RNA extraction, and the distal part of the urethra and vagina was used for protein extraction.
Leak point pressure (LPP) measurement
The LPP measurement was used to assess urethral sphincter function. LPP was performed as described by Conway et al. . Investigators performing LPP measurement were blinded to the group assignment of each animal. Briefly, 5 weeks after the injection, the rats were anesthetized with ketamine (30 mg/kg) and xylazine (3 mg/kg). A transvesical catheter with a fire-flared tip was inserted into the bladder dome through a small abdominal incision. The abdominal wall was closed, and the catheter was connected via a three-way stopcock to a 50-ml syringe for filling with methylene blue colored saline and to a pressure transducer (TSD 104A, BIOPAC Systems Inc., CA, USA, http://www.biopac.com) for monitoring bladder pressure. The bladder pressure was amplified and sampled by a biological signal acquisition system (BIOPAC MP 150) and digitalized for computer data collection using Acknowledge acquisition and analysis software (BIOPAC Systems Inc.).
Before LPP testing, the spinal cord was transected at the T8-T10 level to eliminate the voiding reflex mediated by spino-bulbo-spinal pathways. The urethral closure mechanism during urine storage remains intact because urethral contractile reflexes activated by sympathetic and somatic nerves responding to bladder distension are predominantly organized at the lumbosacral spinal cord level. The vertical tilt table/intravesical pressure clamp model was used to measure the LPP. The rat was taped to a board and placed in the vertical position. The 50 ml syringe (reservoir) which was connected to the bladder catheter via the three-way stopcock was then fixed onto a metered vertical pole. Bladder filling was done by manually raising the height of the reservoir by 2–3 cm increments for every 2 minutes starting from 0 cm, until urinary leakage (methylene blue saline) was observed at the urethral meatus. The bladder pressure (measured by the transducer) at which leakage was observed was recorded as the LPP. LPP is thus a measure of the urethral sphincter pressure against bladder filling. The mean of at least three consecutive LPPs was taken as a data point for each animal.
RNA extraction and quantitative reverse transcription-polymerase chain reaction
Total RNA of cells and rat tissue was extracted with the RNA-STAT-60 reagent (Tel-Test, Inc., Friendswood, TX, USA). RNA yield was determined using a Nanodrop 2000 spectrophotometer (Thermo Scientific). Total RNA (1 µg) was reverse transcribed into cDNA using the M-MLV reverse transcriptase system (Thermo Scientific). PCR primers were described previously [17, 24], except human TIMP2 (Sense: AAGCGGTCAGTGAGAAGGAA; Anti-sense: GATGTTCAAAGGGCCTGAGA), rat MMP2 (Sense: GTAAAGTATGGGAACGCTGATGGC; Anti-sense: CTTCTCAAAGTTGTACGTGGTGGA) and rat TIMP2 (Sense: ACACGCTTAGCATCACCCAGAA; Anti-sense: CAGTCCATCCAGAGGCACTCAT). Real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was carried out on the Mx3005P Multiplex Quantitative PCR System with MxPro QPCR software (Stratagene, La Jolla, CA, US). Brilliant SYBR Green QPCR Master Mix (Stratagene) was used to perform PCR. GAPDH was used as an endogenous reference against which the different template values were normalized. All PCR reactions were performed in duplicate. The cycle of threshold (Ct) method was used for quantification. Data were analyzed by MxPro QPCR software.
Western blot assay
Cell culture supernatant was collected and further concentrated (10x) by ultrafiltration using centrifugal filter units with 10-kDa cutoff (Sartorius Stedim SUS Inc., CA). Human bSMCs or vaginal fibroblasts were washed twice with cold PBS and homogenized on ice with a RIPA buffer (50 mM Tris, 150 mM NaCl, 1% NP40, 0.5% deoxycholate, 0.1% SDS, 4 mM EDTA, and 2 mM PMSF, PH 7.4) supplemented with proteinase inhibitor cocktail (Roche Diagnostics GmbH, Basel, Switzerland), and then rotated at 4 °C for 2 days to solubilize the protein more efficiently. Cell debris was removed by centrifugation at 14,000 RPM for 30 min. Protein extraction from rodent tissue was performed. Total protein concentrations of concentrated supernatants and cell lysates were determined using the Bradford method (Bio-Rad, Hercules, CA). The samples were reduced with a sodium dodecyl sulfate (SDS) sample buffer containing 5% of 2-mercaptoethanol and boiled for 7 min. The proteins (20 µg/lane for cell lysates and rat tissue lysates, 100 µg/lane for concentrated supernatants) were subjected to 8–10% (wt/vol) polyacrylamide gels (SDS-PAGE). The gels were blotted onto nitrocellulose membranes (Bio-Rad) in an electrophoretic transfer cell (Bio-Rad). Blots were blocked with 5% nonfat milk at 4 °C overnight, and then probed with mouse anti-TIMP-1 antibody (1:1000; Calbiochem, La Jolla, CA), mouse anti-TIMP-2 antibody (1:200; Calbiochem, La Jolla, CA), rabbit anti-collagen III antibody (1:1000; Abcam, Cambridge, MA), goat anti-alpha elastin antibody (1:1000; Abcam, Cambridge, MA), rabbit anti-human elastin antiserum (1:200; Elastin products company, Owensville, MO) at room temperature for 1 h. After washing three times with phosphate-buffered saline with 0.1% Tween-20, pH 7.4 (PBS-T), the membrane was then incubated with sheep anti-mouse IgG conjugated to HRP (1:5000, GE Healthcare, Pittsburgh, PA) or donkey anti-rabbit IgG conjugated to HRP (1:5000, GE Healthcare) or mouse anti-goat IgG conjugated to HRP (1:5000, GE Healthcare) for 1 h at room temperature, followed by three washes in PBS-T. Blots were developed by chemiluminescence. The blots were re-probed with goat anti-GAPDH polyclonal antibody (1:5000, Abcam) and then dilution of mouse anti-goat IgG conjugated to HRP (1:5000; Invitrogen). The band density was determined by Image Studio Software (LI-COR, Inc., Lincoln, Nebraska USA).
Elastin staining and qualitative examination of elastin morphology
Rodent tissues were embedded in OCT compound on liquid nitrogen and stored at -80 °C until cryosectioned. The cryosections were cut and mounted on superfrost slides (Thermo Fisher Scientific Life Sciences). The sections were warmed up for 15 minutes to room temperature before fixing in 4% wt/vol cold paraformaldehyde (Thermo Fisher Scientific Life Sciences) in PBS (pH 7.4) at room temperature for 15 minutes. The sections were washed three times in fresh PBS (5minutes per wash) to remove OCT and then rinsed in water once. The elastin fibers (black) were stained in Weigert’s Resorcin-Fuchsin solution for 2–4 hours according to the manufacturer’s instruction (Electron Microscope Sciences, Hatfield, PA, http://www.electronmicroscopy-sciences.com). The excess solution was removed with 95% vol/vol ethanol. The slides were differentiated with 1% vol/vol acid alcohol and then wash in water. Cell nuclei (dark blue) were stained with Weigert’s iron hematoxylin working solution (Poly Scientific R&D Corporation, Bay Shore, NY, http://www.polyrnd.com) for approximately 30 seconds. The slides were washed well in running water and then counterstained with van Gieson’s solution for 3–5 minutes for collagen fibers (red pink). The excess stain was rinsed from the slides with distilled water.
The slides were visually scored by four people separately for elastin length (1 = short, 2 = moderate, 3 = long), thickness (1 = thin, 2 = moderate, 3 = thick) and density (1 = sparce, 2 = moderate density, 3 = dense), collagen density (1 = sparce, 2 = moderate density, 3 = dense). The scorers were blinded to the group assignments. Scores were totaled for each characteristic and used for comparison to assess whether there were gross, visible differences in the morphologic characteristics.
Gelatinolytic activities of matrix metalloproteinases (MMPs) in cell culture supernatants and in cell lysates were assessed by gelatin zymography . In brief, samples were mixed with nonreducing sample buffer before being electrophoresed in 8% polyacrylamide gels containing 0.1% gelatin in the presence of SDS. After electrophoresis, the gels were washed twice with 2.5% Triton X-100 and were subsequently incubated overnight at 37 °C in the substrate buffer (containing 50 mM Tris-HCL, pH 8, 5 mM, CaCl2, 0.02% Azide). After staining with Coomassie blue, enzyme activity appeared as clear bands against the blue-stained background. The area of lysis for each band detected was analyzed using Bio-Rad Quality One Software (Bio-Rad). The bands density was determined by ImageJ Software (National Institutes of Health, Bethesda, MD, USA).
Statistical analyses were performed using SPSS version 21 (SPSS Inc., Chicago, IL). Results are expressed as mean ± SEM. Kruskal-Wallis one-way ANOVA test was used to compare multiple variables. The Student t-test was used for comparison between groups for the in vivo study. A value of p < 0.05 was considered significant.