Paracrine effect of human stem cell-derived progenitor cells on remodeling of the vagina.

The human tissue studies were approved by the Institutional Review Board and the Research Compliance Office of Stanford University School of Medicine. The animal study was approved by the Institutional Review Board of the Stanford University School of Medicine and the Stanford Administrative Panel of Laboratory Animal Care (APLAC). Skin biopsies were collected from 5 female patients (ages 40-85). Dermal fibroblasts were cultured and reprogrammed into iPSCs using a mRNA/miRNA-based reprogramming approach. Briefly, fibroblasts were co-transfected with mRNA encoding Oct4, Klf4, Sox2, cMyc, and Lin28, as well as miR-302/367 cluster as described by McGrath, Diette et al. to induce the formation of iPSCs 44 , 45 . iPSC characterization was performed as previously described 42 . H9 human embryonic stem cell line was used as positive control. pSMCs were differentiated from 5 iPSC lines and H9 using a vascular progenitor cell protocol as previously described 42 , 46 – 48 . pSMCs were maintained on human placenta-derived collagen IV (Sigma) and expanded in DMEM/F12 HEPES supplemented with 5% fetal bovine serum (FBS, Invitrogen), recombinant human PDGF-BB (0.01 µg/ml) and FGF-basic (0.005 µg/ml) (Peprotech, Rocky Hill, NY). pSMCs were characterized by the analyses of smooth muscle cell markers, including smoothelin and α-SMA as described previously 42 . pSMC characterization was performed by FACS, PCR, and immunohistochemistry using SMA as smooth muscle cell marker and TRA 1-60 and TRA 1-81 at pluripotent cell markers. We used banked bSMCs obtained from the trigone area of a benign human bladder described previously 49 . The bSMCs were passaged up to passage 4 (P4) and served as positive controls for comparison with pSMCs. bSMCs were characterized with RT-PCR, immunocytochemistry and fluorescence-activated cell sorting (FACS) for SMC markers, including smoothelin and α-SMA (data not shown). CM were pooled at P4 SMCs for each cell line. CMs were concentrated (50 times) using a 3000-kDa cutoff centrifugal filter unit (Sartorius, Stedim, CA) for the animal study based on published concentrations for mesenchymal cell CM 41 . Pure media, the vehicle, was also concentrated and processed within the same timeline with this protocol to ensure same concentration and exposure time for both CM and vehicle treatment. Albumin in P4 CM concentrated to 20X was removed using Pierce™ Albumin Serum Depletion Kits (Thermo Scientific). The concentrated CM was sent to Stanford University Mass Spectrometry for label-free data-independent acquisition (DIA) proteomics. Identified proteins were mapped to Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) based on iDEP analysis ( http://bioinformatics.sdstate.edu/idep/ ) terms to determine their biological and functional properties. After the analysis was done, pathways were first filtered based on an FDR cutoff (0.05). Then the significant pathways were sorted by FDR. Only the top 10 pathways are shown. Nine patients consented for vaginal tissue harvest during surgery. The vaginal fibroblast isolation was done as previously described 50 . Women with a history of endometriosis, gynecologic malignancies, pelvic inflammatory conditions, connective tissue disorders, emphysema and prior pelvic surgery were excluded. Patients’ characteristics are as follows: three women in the proliferative phase of the menstrual cycle (ProF1, ProF2, ProF3; ages 40-50), three in the secretory phase (SecF1, SecF2, SecF3; ages 20-40), and three postmenopausal women (PostF1, PostF2, PostF3; ages 60-70). Vaginal fibroblasts at passage 3 (P3) were plated onto 6-well plates at a density of 10,000 cells/cm² in 90% DMEM and 10% FBS (Invitrogen). Once the cells reached 90% confluency, the medium was changed to DMEM/F12 supplemented with 0.2% albumin (Sigma) for 24 h. Then the cells were treated for 48 h with non-concentrated CM or the pSMC maintenance media (pure media, vehicle) described above as previously described 46 . Female immunodeficient Rowett nude rats (RNU, 150-200 g, Charles River Laboratories, Hollister, CA) were maintained at Stanford University Research Animal Facility in accordance with the guidelines of the Stanford University’s Institutional Animal Care and Use Committee (IACUC). The animal surgically injured model in rats was created as previously described 21 , 42 . Bilateral ovariectomy was performed to simulate the postmenopausal state since most POP occur in postmenopausal women 51 . Rats were divided into five treatment groups: (1) Pure media ( n  = 12); (2) A pSMC-CM ( n  = 15); (3) B pSMC-CM ( n  = 15); (4) D pSMC-CM ( n  = 13); (5) bSMC-CM ( n  = 14). Treatment injections were administered 7 weeks post-surgery after acute inflammation subsided. After anesthetizing the rats with 3–4% isoflurane, 100 μL of CM or pure media was injected into each side of the lateral vaginal wall using a 28.5-gauge insulin syringe once a week for 3 weeks (Fig. 10 ) 28 , 46 – 48 . The injection procedure was not randomized, as the CM for each group had to be freshly prepared before injection. Fig. 10 Vagina injection. a 100 µl of methylene blue saline solution was injected in each side of vagina wall; b Open the rat pelvic cavity with visualization of the vagina turning blue from the injection to confirm proper injection location. Brown arrow indicates the vagina injection site, black arrow indicates the urethra, red arrow indicates the bladder, blue arrow indicates the vagina, green arrow indicates the uterus. a 100 µl of methylene blue saline solution was injected in each side of vagina wall; b Open the rat pelvic cavity with visualization of the vagina turning blue from the injection to confirm proper injection location. Brown arrow indicates the vagina injection site, black arrow indicates the urethra, red arrow indicates the bladder, blue arrow indicates the vagina, green arrow indicates the uterus. Tissues were harvested 10–11 weeks post-surgery. Half of the proximal part of the vagina was fixed in paraffin for histology, while the other half was used for RNA extraction. The middle part of the vagina was first used for organ bath myography, followed by protein extraction. The bladder dome and trigone were also first used for organ bath myography, and then for histology, as well as protein and RNA extraction. The mid-urethra was used for RNA extraction, and the distal part for protein extraction. Carbon dioxide (CO 2 ) inhalation was used for euthanasia of the rats. The organ bath myography was performed as previously described 21 , 42 . Briefly, the middle portion of the vagina was mounted circumferentially, and the bladder dome and trigone were mounted longitudinally. Circumferential loading was selected for the vagina based on published data on its response compared to longitudinal loading 52 , 53 . Contraction responses were monitored using custom-built isometric force transducers, and signals were recorded with Lab Chart 7 (AD Instrument, Colorado Springs, Colorado). Tissue contraction was first assessed with potassium chloride, a muscle-mediated stimulant, at 40 mM for the vagina and 160 mM for the bladder. The tissue contractions were then evaluated using the non-selective muscarinic receptor agonist carbachol (Sigma-Aldrich) at cumulative concentrations of 0.625, 1.25, 2.5, 5, 10, and 20 µM. After washing three times, the contraction of the tissue was evaluated with 20 µM carbachol again after the addition of 1 µM atropine for 5 min to confirm the true carbachol-induced response. The contraction force data were normalized to tissue area and expressed as tension per unit area (g/cm²). mRNA expression levels of human elastin, collagen I, ROCK1, A2M , elastase and ITIH2 54 – 57 in cultured human fibroblasts and rat elastin, collagen I and III, ACTA2 and ROCK1 in the rodent tissues 21 , 42 , 58 were determined by qRT-PCR. The primers of human ITIH2 are 5’-CGGCTCAAGTCACGAATCAG; 3’-TTCCTGCATGAACTGGCCTA. RNA extraction and real time qRT-PCR were performed as previously described 46 . qRT-PCR was performed in duplicates on an Aria Mx Real-Time PCR system using Brilliant SYBR Green PCR Master Mix (Agilent Technologies) as described previously 46 . GAPDH was used as the endogenous reference. The amplifications were done following 10 minutes hot start at 95 °C in a three-step protocol with 30 sec denaturation (94), 1 min. annealing (55 °C–60 °C), and extension at 72 °C for 30 s. for forty cycles. Quantification was performed using the threshold cycle (Ct) method. Data was analyzed using the MxPro QPCR software, version 2.1. Protein extraction from the human fibroblasts and the rodent tissues was performed as previously described 46 , 57 . Samples were reduced with sodium dodecyl sulfate (SDS) sample buffer containing 5% of β-mercaptoethanol and boiled for 7 min. Proteins (20 μg/lane for the cell lysates and 30 μg/lane for the tissue lysates) were subjected to 10% polyacrylamide gel (SDS-PAGE) and blotted onto nitrocellulose membranes (Bio-Rad). Membranes were blocked with 5% non-fat milk at room temperature for 1 h, then incubated with the following primary antibodies: mouse anti-human elastin antiserum (1:100, Santa Cruz, CA), goat anti-human α2-macroglobulin (1:200, Affinity Biologicals, Ancaster, Canada), rabbit anti-human neutrophil elastase (1:500, Abcam company info) at room temperature for 1 h, or goat anti-rat α-elastin (1:250, Abcam), rabbit anti-rat SMA (1 μg/5 mL, Abcam) at 4 °C overnight. After three washes, the membranes were incubated with the following HRP-conjugated secondary antibodies: sheep anti-mouse IgG (1:2500, Amersham, Chicago, IL), donkey anti-rabbit IgG (1:10000, GE Healthcare, Chicago, IL), or mouse anti-goat IgG (1:10000, Sigma) for 1 h at room temperature. The blots were developed using chemiluminescence. The membranes were re-probed with rabbit anti-human GAPDH (1:2500, Abcam) or mouse anti-rat GAPDH (1:250, Abcam), followed by incubation with HRP-conjugated donkey anti-rabbit IgG (1:10000, GE Healthcare) or sheep anti-mouse IgG (1:2000, Amersham). The membranes were imaged with Odyssey Fc Imaging System (LI-COR Biotechnology, Lincoln, Nebraska). Band intensity was quantified using ImageJ Software Version 1.48 (NIH, Bethesda, MD). Tissues were paraffin-embedded and subjected to elastin staining, as previously described 46 , 59 . Briefly, elastin fibers were stained using the Weigert’s resorcin-fuchsin kit at room temperature for 2–3 h according to manufacturer’s instruction (Electron Microscopy Sciences, Hatfield, PA). Nuclei were counterstained with Weigert’s iron hematoxylin working solution (Poly Scientific R&D Corporation, Bay Shore, NY). Masson’s trichrome staining of the paraffin-embedded tissues was performed according to the manufacturer’s instructions (StatLab Empowering Anatomic Pathology, McKinney, TX). Immunohistochemical staining for ROCK1 was done on formalin-fixed, paraffin-embedded tissues. Endogenous peroxidase activity was blocked using 3% H 2 O 2 , and non-specific binding was blocked with 1% bovine serum albumin and 5% goat normal serum for 1 h at room temperature. The sections were then incubated overnight at 4 °C with rabbit anti-rat ROCK1 primary antibody (1:500, Abcam, the sections were then incubated with goat anti-rabbit biotin-conjugated secondary antibody (1:5000, GE Healthcare). The slides were further incubated at room temperature for 30 min using the Vectastain ABC Kit (Vector Laboratories, Newark, CA). To visualize the immunoreactivity, slides were incubated with the substrate solution containing levamisole to block endogenous alkaline phosphatase activity. The slides were counterstained with Mayer’s hematoxylin (Fisher Scientific). Visualization and imaging were performed using the AxioCam system (Zeiss, Oberkochen, Germany). A random area (20×) per slide was analyzed. Pixel measurements were obtained using ImageJ Software Version 1.48 (NIH, Bethesda, MD). Initially, background subtraction was applied to remove noise from the images. Color deconvolution was subsequently performed to separate the RGB channels for elastic fibers and average optical density of ROCK1. Masson’s trichrome channel was applied for collagen and smooth muscle area. The threshold for the blue channel was set to 0-250 60 . Elastin area % = elastin area/ total smooth muscle area. Average Optical Density (AOD) = integrated density/area. Because the primary focus of this manuscript was to test the in vivo effect of the pSMC-CMs in the surgically injured rat model, we used the average and standard deviation values from our previous publication on the surgical injury rat model (Ho T et al., 2023) for a sample size calculation. We determined to use elastin mRNA expression as primary outcome since this methodology provided a more reliable quantification of the changes induced by CM treatment. The sample size required per group was 12 when using the t -test to compare means of continuous variable (with a standardized effect size of 1.2) with two-tailed α set at 0.10 and β at 0.2. Statistical analysis was performed using JMP software version Pro 17 (SAS, Cary, NC). The results are expressed as mean ± SD. One way ANOVA followed with Tukey HSD test was used for multiple comparison of the vaginal fibroblast in vitro studies. The nonparametric Wilcoxon test was applied for statistical comparisons between groups for animal study because these data may not be normally distributed. qRT-PCR data from animal experiments, all data points, except for the highest outlier in each group, were included in the nonparametric Wilcoxon analysis. The exclusion of the highest outlier was due to the considerable variability observed among the maximum values. Statistical significance was set at P  < 0.05.

Proteomic analyses revealed that pSMC-CMs from premenopausal patients A (A pSMC-CM) and B (B pSMC-CM) contained 57 and 39 proteins, respectively. pSMC-CMs from postmenopausal patients C, D, E (C pSMC-CM, D pSMC-CM, E pSMC-CM) contained 13, 17, and 53 proteins, respectively. The positive controls, CM from primary culture of human bladder smooth muscle cells (bSMC-CM) and embryonic stem cell-derived pSMCs (H9-pSMC-CM), each contained 44 proteins, whereas no proteins were detected in the pure media (negative control). Gene Ontology (GO) analysis classified the proteins according to biological processes and molecular functions. With respect to biological processes, bSMC-CM proteins were enriched in biological processes involved in negative regulation of peptidase activity. A and D pSMC-CMs were enriched in biological processes of ECM disassembly, contractile actin filament bundle assembly, collagen catabolic processes, and metallopeptidase activity. pSMC-CM from patient A was enriched in tissue development processes. B, E, bSMC, and H9 CMs were enriched in wound healing processes. Regarding molecular functions, pSMC-CM proteins from patients A and D were enriched in metallopeptidase activity, while proteins from A, B, E, bSMC, and H9 CMs were enriched in structural molecule activity and serine-type endopeptidase inhibitor activity. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicated that proteins from A- and B pSMC-CM were enriched in the estrogen signaling pathway, whereas bSMC- and H9-CM proteins were involved in the Hippo signaling pathway (Fig. 1 ). Fig. 1 Protein profiles and bioinformatics analysis of pSMC-CMs and bSMC-CM. Heatmap of the protein expression in pSMC-CMs and bSMC-CM with cluster analysis, GO biological process and molecular function analysis, and KEGG analysis. Heatmap of the protein expression in pSMC-CMs and bSMC-CM with cluster analysis, GO biological process and molecular function analysis, and KEGG analysis. The heatmap showed that the CMs clustered into two groups with one that included pSMC-CMs from patients A, B, E and H9, and the other that included CMs from bSMCs and pSMC-CM from patients C and D. The heatmap patterns for the former group showed similarity in the GO molecular function categories of structural molecule activity, serine-type endopeptidase inhibitor activity, structural molecule activity, while the latter group showed similarity in the structural molecular activity and endopeptidase inhibitor activity. Because of the common heatmap patterns in serine-type endopeptidase inhibitor activity shown in the GO molecular function analysis, we focused on assessing the effects of pSMC-CM on ECM structural proteins (elastin, collagen I/III) and proteins involved in ECM protease activity [neutrophile elastase and elastase inhibitors, inter-alpha-trypsin inhibitor heavy chain H2 (ITIH2) and α2-macroglobulin (A2M)] in human vaginal fibroblasts. We also examined markers involved contractile actin filament bundle assembly, including alpha smooth muscle actin ( SMA ) and Rho-associated coiled-coil-containing protein kinase 1( ROCK1 ). To investigate the effects of pSMC-CM on human cells, we treated vaginal fibroblasts from different women with pSMC-CMs from 5 patients and compared the results to treatment with pure media (negative control). Primary culture fibroblasts from women with prolapse were grouped based on the hormonal status of the donor—specifically, proliferative and secretory phase of the menstrual cycle, as well as postmenopausal status —since reproductive hormone status can influence cellular ECM metabolism responses. For elastin mRNA expression, vaginal fibroblasts from proliferative, secretory phase, and postmenopausal patients showed increases in elastin mRNA when treated with the majority of the pSMC-CMs from 5 patients compared to treatment with pure media (negative control). The proportion of pSMC-CMs that induced statistically significant upregulation of elastin mRNA ( p  < 0.03) in the vaginal fibroblast groups (three patients per group, F1, F2, F3) were: proliferative phase vaginal fibroblasts (ProF1: 3/5, ProF2: 5/5, ProF3: 1/5), secretory fibroblasts (SecF1: 5/5, SecF2: 5/5, SecF3: 4/5), and postmenopausal fibroblasts (PostF1: 3/5, PostF2: 5/5, PostF3: 5/5). Interestingly, more pSMC-CMs increased elastin mRNA expression in secretory ( p  ≤ 0.02) and postmenopausal vaginal fibroblasts ( p  ≤ 0.03) (Fig. 2a and Table S1 ). Fig. 2 Gene expression of elastin and collagen I in pSMC-CM-treated vaginal fibroblasts. Gene expression levels of elastin ( a ), collagen I ( b ) were analyzed by quantitative real-time RT-PCR in vaginal fibroblasts from nine women with prolapse (Proliferative phase: ProF1, ProF2, ProF3; Secretory phase: SecF1, SecF2, SecF3; Post-menopause: PostF1, PostF2, PostF3). Data shown represent the mean ± SD of triplicates. Pure media: media only (vehicle), A pSMC-CM: conditioned medium from patient A iPSC-derived pSMCs, B pSMC-CM: conditioned medium from patieht B iPSC-derived pSMCs, C pSMC-CM: conditioned medium from patient C iPSC-derived pSMCs, D pSMC-CM: conditioned medium from patient D iPSC-derived pSMCs, E pSMC-CM: conditioned medium from patient E iPSC-derived pSMCs, bSMC-CM: conditioned medium from primary culture bladder smooth muscle cells (bSMCs), H9-CM :conditioned medium from H9 embryonic stem cell-derived pSMC. *◇†Significant difference between groups ( p  < 0.05): *Significant difference between CM groups compared to pure media group; ◇Significant difference between CM groups compared to bSMC-CM group; †Significant difference between CM groups compared to H9-CM group. Gene expression levels of elastin ( a ), collagen I ( b ) were analyzed by quantitative real-time RT-PCR in vaginal fibroblasts from nine women with prolapse (Proliferative phase: ProF1, ProF2, ProF3; Secretory phase: SecF1, SecF2, SecF3; Post-menopause: PostF1, PostF2, PostF3). Data shown represent the mean ± SD of triplicates. Pure media: media only (vehicle), A pSMC-CM: conditioned medium from patient A iPSC-derived pSMCs, B pSMC-CM: conditioned medium from patieht B iPSC-derived pSMCs, C pSMC-CM: conditioned medium from patient C iPSC-derived pSMCs, D pSMC-CM: conditioned medium from patient D iPSC-derived pSMCs, E pSMC-CM: conditioned medium from patient E iPSC-derived pSMCs, bSMC-CM: conditioned medium from primary culture bladder smooth muscle cells (bSMCs), H9-CM :conditioned medium from H9 embryonic stem cell-derived pSMC. *◇†Significant difference between groups ( p  < 0.05): *Significant difference between CM groups compared to pure media group; ◇Significant difference between CM groups compared to bSMC-CM group; †Significant difference between CM groups compared to H9-CM group. For collagen I mRNA expression, fibroblasts from the secretory phase and postmenopausal women showed upregulation of collagen 1 mRNA expression with most of the pSMC-CMs compared to controls ( p  ≤ 0.04), while the proliferative phase vaginal fibroblasts did not show consistent trends with pSMC-CM treatment. In PostF2 fibroblasts, collagen I expression was significantly increased after treatment with 4 out of the 5 pSMC-CMs. In PostF3 fibroblasts, significant upregulation of collagen I was observed in response to 3 out of the 5 pSMC-CMs. PostF1 vaginal fibroblasts showed inconsistent trends to pSMC-CM treatment compared to control (Fig. 2b ). For elastase mRNA expression, fibroblasts from the secretory and postmenopausal women exhibited significant upregulation in elastase mRNA expression. In secretory fibroblasts, SecF1 showed significant upregulation of elastase mRNA expression ( p  < 0.002) with B, C, and D pSMC-CM (3/5), SecF2 with B and D pSMC-CM (2/5), and SecF3 with C and D pSMC-CM (2/5). In postmenopausal fibroblasts, PostF2 responded to C and D pSMC-CM (2/5), while PostF3 responded to B, C, and D pSMC-CM (3/5). The effect of pSMC-CM on elastase expression in proliferative phase vaginal fibroblasts was inconsistent (Fig. 3a ). Fig. 3 Gene expression of extracellular matrix (ECM) metabolism proteins in pSMC-CM-treated vaginal fibroblasts. Gene expression levels of elastase ( a ), α2-macroglobulin ( A2M) ( b ), inter-alpha-trypsin inhibitor heavy chain H2 ( ITIH2) ( c ) were analyzed by quantitative real-time RT-PCR in vaginal fibroblasts from nine women (Proliferative phase: ProF1, ProF2, ProF3; Secretory phase: SecF1, SecF2, SecF3; Post-menopause: PostF1, PostF2, PostF3). Data shown represent the mean ± SD of triplicates. Pure media: media only (vehicle), A pSMC-CM: conditioned medium from A iPSC-derived pSMCs, B pSMC-CM: conditioned medium from B iPSC-derived pSMCs, C pSMC-CM: conditioned medium from C iPSC-derived pSMCs, D pSMC-CM: conditioned medium from D iPSC-derived pSMCs, E pSMC-CM: conditioned medium from E iPSC-derived pSMCs, bSMC-CM: conditioned medium from bSMCs, H9-CM: conditioned medium from H9 ESC-derived pSMC. *◇†Significant difference between groups ( p  < 0.05): *Significant difference between CM groups compared to pure media group; ◇Significant difference between CM groups compared to bSMC-CM group; †Significant difference between CM groups compared to H9-CM group. Gene expression levels of elastase ( a ), α2-macroglobulin ( A2M) ( b ), inter-alpha-trypsin inhibitor heavy chain H2 ( ITIH2) ( c ) were analyzed by quantitative real-time RT-PCR in vaginal fibroblasts from nine women (Proliferative phase: ProF1, ProF2, ProF3; Secretory phase: SecF1, SecF2, SecF3; Post-menopause: PostF1, PostF2, PostF3). Data shown represent the mean ± SD of triplicates. Pure media: media only (vehicle), A pSMC-CM: conditioned medium from A iPSC-derived pSMCs, B pSMC-CM: conditioned medium from B iPSC-derived pSMCs, C pSMC-CM: conditioned medium from C iPSC-derived pSMCs, D pSMC-CM: conditioned medium from D iPSC-derived pSMCs, E pSMC-CM: conditioned medium from E iPSC-derived pSMCs, bSMC-CM: conditioned medium from bSMCs, H9-CM: conditioned medium from H9 ESC-derived pSMC. *◇†Significant difference between groups ( p  < 0.05): *Significant difference between CM groups compared to pure media group; ◇Significant difference between CM groups compared to bSMC-CM group; †Significant difference between CM groups compared to H9-CM group. For A2M mRNA expression, secretory phase vaginal fibroblasts showed increase expression compared to control ( p  ≤ 0.02) when treated with 4–5 out of 5 CM’s. For postmenopausal vaginal fibroblasts, increases in A2M mRNA expression were observed in PostF1 fibroblasts (1/5 pSMC-CMs, p  = 0.002), PostF2 fibroblasts (3/5 pSMC-CMs, p  ≤ 0.007), and PostF3 fibroblasts (5/5 pSMC-CMs, p  ≤ 0.03) with the remaining pSMC-CMs showing an increased trend. Proliferative phase vaginal fibroblasts showed inconsistent changes with the five pSMC-CMs (Fig. 3b ). For ITIH2 mRNA expression, secretory phase vaginal fibroblasts responded to treatment with most of the five pSMC-CMs through upregulation of ITIH2 mRNA while there was no significant changes in the proliferative or postmenopausal phase vaginal fibroblasts (Fig. 3c ). For ROCK1 mRNA expression, secretory phase vaginal fibroblasts treated with the five pSMC-CMs showed increased ROCK1 mRNA expression ( p  ≤ 0.04). An overall increased trend was observed with pSMC-CM treatment in two out of three proliferative phase vaginal fibroblasts and in two out of three postmenopausal vaginal fibroblasts (Figure S1a ). Cell lysates from patient A and C pSMC-CM treated vaginal fibroblasts that demonstrated significant upregulations in elastin mRNA were analyzed with western blot to confirm protein expression of elastin, neutrophil elastase, and A2M (Fig. 4 ). Fig. 4 Elastin, neutrophil elastase and α2-macroglobulin (A2M) protein expression in vaginal fibroblasts. Elastin ( a ), neutrophil elastase (NE) ( b ) and α2-macroglobulin (A2M) ( c ) protein expression in vaginal fibroblasts from three women (Proliferation phase: ProF1; Secretory phase: SecF1; Post-menopause: PostF1) treated with C pSMC-CM. The expression levels for each bar can be compared with in each experiment, but not between experiments. Data shown represent the mean ± SD from triplicates. Pure media: media only (vehicle), C pSMC-CM: conditioned medium from C iPSC-derived pSMCs, bSMC-CM: conditioned medium from bSMCs, H9-CM: conditioned medium from H9 ESC-derived pSMC. *◇Significant difference between groups ( p  < 0.05): *Significant difference between CM groups compared to pure media group; ◇Significant difference between CM groups compared to bSMC-CM group. Elastin ( a ), neutrophil elastase (NE) ( b ) and α2-macroglobulin (A2M) ( c ) protein expression in vaginal fibroblasts from three women (Proliferation phase: ProF1; Secretory phase: SecF1; Post-menopause: PostF1) treated with C pSMC-CM. The expression levels for each bar can be compared with in each experiment, but not between experiments. Data shown represent the mean ± SD from triplicates. Pure media: media only (vehicle), C pSMC-CM: conditioned medium from C iPSC-derived pSMCs, bSMC-CM: conditioned medium from bSMCs, H9-CM: conditioned medium from H9 ESC-derived pSMC. *◇Significant difference between groups ( p  < 0.05): *Significant difference between CM groups compared to pure media group; ◇Significant difference between CM groups compared to bSMC-CM group. Compared to the pure media-treated group (controls), elastin (Fig. 4a ) and neutrophil elastase (Fig. 4b ) were upregulated in proliferative, secretory, and postmenopausal vaginal fibroblasts when treated with C pSMC-CM ( p  ≤ 0.03). Treatment with A pSMC-CM increased elastin expression in proliferative ( p  = 0.02) and secretory phase vaginal fibroblasts ( p  < 0.0001), and there was a trend for increased expression in postmenopausal vaginal fibroblasts. Neutrophil elastase levels were elevated only in secretory phase fibroblasts treated with A pSMC-CM ( p  = 0.02). In addition, A2M was elevated in premenopausal fibroblasts treated with C pSMC-CM ( p  ≤ 0.04), while no significant change was observed in postmenopausal fibroblasts (Fig. 4c ). There was no notable change in A2M expression in vaginal fibroblasts treated with A pSMC-CM (data not shown). Rock1 protein expression was not detected in vaginal fibroblasts. Potassium chloride (KCl)-stimulation, a muscle-mediated stimulant, of the vaginal tissue showed no contractile differences in pSMC-CM treated groups compared to the pure media (vehicle) -treated group (data not shown). One out of the three pSMC-CM treated groups, the D pSMC-CM group ( n  = 13, p  ≤ 0.047), and bSMC-CM group ( n  = 14, p  ≤ 0.03) showed significantly greater contraction responses to low doses of carbachol, a non-selective muscarinic receptor agonist, (at 0.625 and 1.25 μM). Vaginal contractile response of the A pSMC-CM group ( n  = 15) to carbachol stimulation also tended to be greater than that of the pure media (vehicle) treated group (Fig. 5a, b ). Fig. 5 Organ bath myography of the vagina stimulated with carbachol (concentrations: 0.625, 1.25, 2.5, 5, 10 and 20 μM). Measured contractions are normalized to tissue area. a Carbachol response curve of vagina; b Representative organ bath myography tracing of the vagina from each CM-injection group at different concentrations of carbachol stimulation. Data shown represent the mean ± SD. Pure media (vehicle) = vaginal-injury rats treated with concentrated medium only; A pSMC-CM = vaginal-injury rats treated with conditioned medium from A iPSC-derived pSMC; B pSMC-CM = vaginal-injury rats treated with conditioned medium from B iPSC-derived pSMCs; D pSMC-CM = vaginal-injury rats treated with conditioned medium from D iPSC-derived pSMCs; bSMC CM = vaginal-injury rats treated with conditioned medium from bSMCs. *Significant difference between CM groups compared to pure media group ( p  < 0.05). Measured contractions are normalized to tissue area. a Carbachol response curve of vagina; b Representative organ bath myography tracing of the vagina from each CM-injection group at different concentrations of carbachol stimulation. Data shown represent the mean ± SD. Pure media (vehicle) = vaginal-injury rats treated with concentrated medium only; A pSMC-CM = vaginal-injury rats treated with conditioned medium from A iPSC-derived pSMC; B pSMC-CM = vaginal-injury rats treated with conditioned medium from B iPSC-derived pSMCs; D pSMC-CM = vaginal-injury rats treated with conditioned medium from D iPSC-derived pSMCs; bSMC CM = vaginal-injury rats treated with conditioned medium from bSMCs. *Significant difference between CM groups compared to pure media group ( p  < 0.05). KCl stimulation contractile responses to in the bladder trigone were similar between the A pSMC-CM and the pure media (vehicle) group. The B pSMC-CM group ( n  = 15) exhibited a trend towards stronger contractions in the bladder trigone. The only significant difference was observed between the D- and bSMC-CM groups compared to the pure media group ( p  ≤ 0.007) (Fig. 6a, b ). Fig. 6 Organ bath myography of the bladder trigone stimulated with carbachol (concentrations: 0.625, 1.25, 2.5, 5, 10, and 20 μM). Measured contractions are normalized to tissue area. a Contraction response of the bladder trigone induced by KCl in different CM-injection groups; b Representative organ bath myography tracing of the bladder trigone from each CM-injection group of KCL stimulation; c Carbachol response curve of bladder trigone; d Representative organ bath myography tracing of the bladder trigone from each CM-injection group at different concentrations of carbachol stimulation. Data shown represent the mean ± SD. Pure media (vehicle) = vaginal-injury rats treated with concentrated medium only; A pSMC-CM = vaginal-injury rats treated with conditioned medium from A iPSC-derived pSMC; B pSMC-CM = vaginal-injury rats treated with conditioned medium from B iPSC-derived pSMCs; D pSMC-CM = vaginal-injury rats treated with conditioned medium from D iPSC-derived pSMCs; bSMC CM = vaginal-injury rats treated with conditioned medium from bSMCs. *Significant difference between CM groups compared to pure media group ( p  < 0.05). Measured contractions are normalized to tissue area. a Contraction response of the bladder trigone induced by KCl in different CM-injection groups; b Representative organ bath myography tracing of the bladder trigone from each CM-injection group of KCL stimulation; c Carbachol response curve of bladder trigone; d Representative organ bath myography tracing of the bladder trigone from each CM-injection group at different concentrations of carbachol stimulation. Data shown represent the mean ± SD. Pure media (vehicle) = vaginal-injury rats treated with concentrated medium only; A pSMC-CM = vaginal-injury rats treated with conditioned medium from A iPSC-derived pSMC; B pSMC-CM = vaginal-injury rats treated with conditioned medium from B iPSC-derived pSMCs; D pSMC-CM = vaginal-injury rats treated with conditioned medium from D iPSC-derived pSMCs; bSMC CM = vaginal-injury rats treated with conditioned medium from bSMCs. *Significant difference between CM groups compared to pure media group ( p  < 0.05). Unlike the vagina, B and D pSMC-CM, and bSMC-CM exhibited similar response curves to carbachol in the bladder trigone, which were higher than those of the pure media (vehicle) group at different concentrations ( p  ≤ 0.02) (Fig. 6c, d ). Carbachol-stimulated contractions in the A pSMC-CM group showed a higher trend than those in the pure media group. Similar to the vaginal response, the D pSMC-CM group showed greater carbachol-induced contractile responses in the bladder dome at a concentration of 1.25 μM ( p  = 0.01) (Figure S2 ). There was no difference in KCl-induced contractile responses in the bladder dome between the pure media (vehicle) group and any of the pSMC-CM groups (data not shown). Compared to the pure media (vehicle) group, mRNA expression of elastin, collagens I/III was significantly increased in the vaginal tissue of surgically-injured rats treated with all three pSMC-CM treatments ( p  ≤ 0.04). Additionally, smooth muscle actin mRNA ( ACTA2 ) expression in the vaginal tissue was higher in the D pSMC-CM and bSMC-CM groups ( p  < 0.0001) (Fig. 7a ). Fig. 7 Gene expression of rat elastin, collagen I, collagen III and ACTA2 in conditioned medium treated rat vagina, bladdertrigone and urethra. Gene expression of rat-elastin, rat-collagen I, rat-collagen III, rat- ACTA2 in conditioned medium treated rat vagina ( a ), bladder trigone ( b ), urethra ( c ) for each group. Data shown represent the mean ± SD. Pure media (vehicle) = vaginal-injury rats treated with concentrated medium only; A pSMC-CM = vaginal-injury rats treated with conditioned medium from A iPSC-derived pSMC; B pSMC-CM = vaginal-injury rats treated with conditioned medium from B iPSC-derived pSMCs; D pSMC-CM = vaginal-injury rats treated with conditioned medium from D iPSC-derived pSMCs; bSMC CM = vaginal-injury rats treated with conditioned medium from bSMCs. * Significant difference between CM groups compared to pure media group ( p  < 0.05). Gene expression of rat-elastin, rat-collagen I, rat-collagen III, rat- ACTA2 in conditioned medium treated rat vagina ( a ), bladder trigone ( b ), urethra ( c ) for each group. Data shown represent the mean ± SD. Pure media (vehicle) = vaginal-injury rats treated with concentrated medium only; A pSMC-CM = vaginal-injury rats treated with conditioned medium from A iPSC-derived pSMC; B pSMC-CM = vaginal-injury rats treated with conditioned medium from B iPSC-derived pSMCs; D pSMC-CM = vaginal-injury rats treated with conditioned medium from D iPSC-derived pSMCs; bSMC CM = vaginal-injury rats treated with conditioned medium from bSMCs. * Significant difference between CM groups compared to pure media group ( p  < 0.05). ROCK1 mRNA expression showed an increased trend in vaginal tissues of rats treated with all three pSMC-CMs; however, this increase did not reach statistical significance (Figure S1b ). Elastin protein expression in the vagina was higher in the B and D pSMC-CM and bSMC-CM groups ( p  ≤ 0.007), while smooth muscle actin (ACTA2) protein expression was increased in the A and D pSMC-CM, and bSMC-CM groups ( p  ≤ 0.002) (Fig. 8a ). Rock1 expression was not detected in the vagina tissue. Fig. 8 The protein expression of rat-elastin and SMA in conditioned medium treated rat vagina, bladder trigone and urethra. The protein expression of rat-elastin and rat-SMA in conditioned medium treated rat vagina ( a ), bladder trigone ( b ), urethra ( c ) for each group. Data shown represent the mean ± SD. Pure media (vehicle) = vaginal-injury rats treated with concentrated medium only; A pSMC-CM = vaginal-injury rats treated with conditioned medium from A iPSC-derived pSMC; B pSMC-CM = vaginal-injury rats treated with conditioned medium from B iPSC-derived pSMCs; D pSMC-CM = vaginal-injury rats treated with conditioned medium from D iPSC-derived pSMCs; bSMC CM = vaginal-injury rats treated with conditioned medium from bSMCs. *Significant difference between CM groups compared to pure media group ( p  < 0.05). The protein expression of rat-elastin and rat-SMA in conditioned medium treated rat vagina ( a ), bladder trigone ( b ), urethra ( c ) for each group. Data shown represent the mean ± SD. Pure media (vehicle) = vaginal-injury rats treated with concentrated medium only; A pSMC-CM = vaginal-injury rats treated with conditioned medium from A iPSC-derived pSMC; B pSMC-CM = vaginal-injury rats treated with conditioned medium from B iPSC-derived pSMCs; D pSMC-CM = vaginal-injury rats treated with conditioned medium from D iPSC-derived pSMCs; bSMC CM = vaginal-injury rats treated with conditioned medium from bSMCs. *Significant difference between CM groups compared to pure media group ( p  < 0.05). Histologic analyses revealed that elastin fiber pattern in the vagina was obliterated and difficult to localize in the pure media (vehicle) treated group. The histologic appearance of the pure media treated vagina is similar to that seen in our previous publication where we observed obliteration of the elastin fiber structure in the surgically injured vagina model compared to intact control rats 21 . In contrast, the vaginal tissue of rats treated with A, B, and D pSMC-CM, and bSMC-CM exhibited qualitative and quantitative changes in elastin fibers, with a significantly increased proportion of elastic fibers area ( p  ≤ 0.02), and the fibers appeared longer and thicker morphologically (Fig. 9a, b ). Compared to the pure media (vehicle) treated group, the total collagen area in the vagina treated with B and D pSMC-CM, and bSMC-CM was increased ( p  ≤ 0.004) (Fig. 9c , d ). No difference in Rock1 expression was observed in the vagina tissues between groups (data not shown). Fig. 9 Effect of pSMC-CM on the rat proximal vagina wall. Representative images of cross-section of the proximal vagina. a Weigert’s resorcin-fuchsin stains for elastin (black fibers) of proximal vagina; b Percentage of elastin fibers in proximal vagina; c Masson’s trichrome staining of proximal vagina; d Total collagen fiber area (blue) and smooth muscle area (red) in proximal vagina. Pure media (vehicle) = vaginal-injury rats treated with concentrated pure medium only; A pSMC-CM = vaginal-injury rats treated with conditioned medium from A iPSC-derived pSMC; B pSMC-CM = vaginal-injury rats treated with conditioned medium from B iPSC-derived pSMCs; D pSMC-CM = vaginal-injury rats treated with conditioned medium from D iPSC-derived pSMCs; bSMC CM = vaginal-injury rats treated with conditioned medium from bSMCs. *Significant difference between CM groups compared to pure media group ( p  < 0.05). The black arrows indicate the black elastin fibers. Scale bar = 50 μm. Representative images of cross-section of the proximal vagina. a Weigert’s resorcin-fuchsin stains for elastin (black fibers) of proximal vagina; b Percentage of elastin fibers in proximal vagina; c Masson’s trichrome staining of proximal vagina; d Total collagen fiber area (blue) and smooth muscle area (red) in proximal vagina. Pure media (vehicle) = vaginal-injury rats treated with concentrated pure medium only; A pSMC-CM = vaginal-injury rats treated with conditioned medium from A iPSC-derived pSMC; B pSMC-CM = vaginal-injury rats treated with conditioned medium from B iPSC-derived pSMCs; D pSMC-CM = vaginal-injury rats treated with conditioned medium from D iPSC-derived pSMCs; bSMC CM = vaginal-injury rats treated with conditioned medium from bSMCs. *Significant difference between CM groups compared to pure media group ( p  < 0.05). The black arrows indicate the black elastin fibers. Scale bar = 50 μm. In the bladder trigone, mRNA expression of elastin, collagen I, and ACTA2 was increased in rats treated with A, B, and D pSMC-CM, and bSMC-CM ( p  ≤ 0.049). Additionally, collagen III mRNA expression was increased in the B and D pSMC-CM, and bSMC-CM groups ( p  ≤ 0.02) (Fig. 7b ). ROCK1 mRNA expression was increased in the bladder trigone of surgically injured rats with all three pSMC-CMs ( p  ≤ 0.01) (Figure S1b ). The protein expression levels of elastin and ACTA2 in the bladder trigone were also higher in the A, B, and D pSMC-CM and bSMC-CM groups ( p  ≤ 0.03) (Fig. 8b ). We also assessed mRNA expression of elastin, collagens I/III, ACTA2 , and ROCK1 in the bladder dome. Compared to the pure media (vehicle) group, elastin, collagen III, ACTA2 , and ROCK1 mRNA expressions were increased in the bladder dome of rats treated with A, B, and D pSMC-CM and bSMC-CM (p ≤ 0.009), while collagen I mRNA expression was increased only in the B pSMC-CM group ( p  = 0.0005) (Figure S3a ). Elastin and smooth muscle actin (ACTA2) protein expression in the bladder dome was higher in the A, B, and D pSMC-CM, and bSMC-CM groups ( p  ≤ 0.002) (Figure S3b ). Rock1 was not detected in the bladder trigone or dome. Histologically, the bladder trigone and dome of rats treated with pSMC-CMs and bSMC-CM demonstrated both qualitative and quantitative changes in elastin fibers, with an increased proportion of elastic fiber area ( p  ≤ 0.01) (Figure S4 ). No difference was observed in the total collagen and smooth muscle areas of the bladder trigone and dome between pure media (vehicle) and pSMC-CM treated groups (data not shown). ROCK1 expression in the bladder dome was higher in rats treated with all three pSMC-CMs and bSMC-CM ( p  ≤ 0.02) (Figure S5 ). However, no difference in Rock1 expression was observed in bladder trigone (data not shown). Treatment with all three pSMC-CM and bSMC-CM led to increased mRNA expression of elastin, collagens I/III, and ACTA2 in the urethra ( p  ≤ 0.005) (Fig. 7c ). ROCK1 mRNA expression was notably elevated with A and D pSMC-CM ( p  ≤ 0.0005) (Figure S1b ). Elastin expression was higher in the urethra of rats treated with A pSMC-CM and bSMC-CM ( p  ≤ 0.007), and ACTA2 expression was elevated in all three pSMC-CM and bSMC-CM groups ( p  ≤ 0.04) (Fig. 8c ). ROCK1 was not detected in the urethra.

The lifetime risk of POP surgery in women with POP is one in eight 22 , 23 . The associated surgical risks and high recurrence rates (up to 29%) 24 underscore the need for novel non-surgical therapies to reduce recurrence after initial surgery. Several studies have demonstrated that the transplantation of adult MSCs can promote pelvic tissue repair via paracrine effects 25 – 27 . Because it is difficult to harvest and expand sufficient patient cells for therapeutic applications, patient derived induced pluripotent stem cells (iPSCs) is a promising stem cell source. Moreover, iPSCs can be differentiate into homogeneous populations of specific progenitor cells. Previously, we observed that CM collected from iPSC-dervied pSMCs induced new rat ECM protein deposition in the rat urethra 28 . This provided evidence that pSMCs could exert paracrine effects in vivo. In the present study, we aimed to evaluate whether pSMC-CM from patients can promote regeneration of the vagina following surgical intervention. Since the quality of the CM is dependent on the purity of the pSMC population, we evaluated the purity of the pSMCs through FACs, PCR, and immunohistochemistry to screen for cells that expressed pluripotency markers and smooth muscle cell markers. This revealed that pSMC populations from all five patients were >96.1% positive for smooth muscle cell markers and 8-17% positive for TRA 1–16 and 1–4% positive for TRA 1-81. We minimized batch to batch variability by pooling the CM from different batches of the same pSMC cell line for testing in both the in vitro vaginal fibroblasts and in vivo animal experiments. Proteomic analyses of pSMC-CM derived from five patients and controls, which included CM derived from primary bladder smooth muscle cells and pSMC-CM derived from the H9 human embryonic stem cell line, were done to investigate the cellular and molecular pathways affected by the proteins in the CM. Proteins related to serine protease activity/metallopeptidase regulation were enriched, such as A2M and ITIH2. A2M is a non-specific protease inhibitor of matrix metalloproteinases (MMPs) activity, thereby preventing tissue damage caused by excessive degradation 29 . ITIH2 stabilizes and protects ECM by binding to hyaluronic acid. During inflammation and tissue repair, ITIH2 helps maintain ECM structural integrity and prevents excessive protease degradation 30 , 31 . Proteins associated with contractile actin filament bundle assembly, smooth muscle actin (ACTA2) and ROCK1 32 , 33 , were enriched. The assembly of actin filament bundles affects cell motility, contraction, muscle contraction, cell migration, cell division, and ECM remodeling. The estrogen signaling pathway was also enriched. Estrogen has been implicated in the maintenance of pelvic floor structure and function by influencing collagen and elastin synthesis, regulating ECM stability, and enhancing pelvic floor muscle function 34 . These findings provide evidence that both pSMC-CMs produced from patient cells and controls contain numerous proteins related to ECM remodeling. Given the above proteomic findings, we used primary culture vaginal fibroblasts from POP women in different hormonal stages to examine the in vitro effect of pSMC-CMs derived from five patients on ECM proteins, protease metabolism proteins, and contractile proteins. Notably, patient pSMC-CMs and positive controls upregulated both gene and protein expression of elastin in all vaginal fibroblast groups. pSMC-CMs increased collagen I mRNA expression in proliferative vaginal fibroblasts, with more consistent and significant effects in secretory premenopausal and postmenopausal fibroblasts. pSMC-CMs upregulated the gene expression of elastase, A2M , and ITIH2 more consistently in secretory vaginal fibroblasts compared to proliferative and postmenopausal vaginal fibroblasts. Progesterone, secreted by the ovary during the secretory phase, affects the synthesis and degradation of ECM by regulating MMP metabolism 35 – 37 . It also influences the expression of genes involved in ECM stability, including A2M and ITIH2 38 . ITIH2 binds to hyaluronic acid, enhancing ECM stability and reducing protease-mediated matrix degradation 39 . Taken together, our data suggest that the effects of pSMC-CMs on ECM remodeling may be, at least in part, mediated by progesterone-responsive pathways. Overall, our in vitro data suggest that the effect of recipient hormonal status may be important in our outcomes, as secretory phase and postmenopausal vaginal fibroblasts showed enriched responsiveness to different CMs. In this in vitro study, we observed variability in responses within each group of vaginal fibroblasts. For example, the vaginal fibroblasts from one of the postmenopausal women showed no change in collagen I mRNA expression while the other two demonstrated significant responses. We believe that this may be due to patient-to-patient differences, variability in CM, and that the dose of the CM may not be maximized for all treatment groups. In this study, we chose a dose that has been previously used for CM derived from mesenchymal cells 40 , 41 . Because this study was not powered to address differences in patient vaginal fibroblasts, future studies should include larger numbers in each of the vaginal fibroblast groups and optimization of the CM dose. The in vivo animal study demonstrated that elastin and collagens I/III were significantly upregulated in the surgically injured vagina and in the surrounding tissues following treatment with pSMC-CMs. This study was powered to detect a difference based on elastin mRNA expression as outcome. Interestingly, in a recent study, we found no significant difference in elastin expression in the vagina of the surgically-injured rat model after a single-dose pSMC cell injection compared to controls 42 . This discrepancy is possibly due to insufficient cell dose or excessive cell death after transplantation into the vagina. Whereas, in the present study, treatment consisted of three weekly CM injections. This highlights the importance of a dose-response investigation in future studies and may account for the lack of response in vaginal contractility and ROCK1 expression in some groups. Taken together, both the human vaginal fibroblast and animal pSMC-CM studies suggest that pSMC-CM exert regenerative effects on vaginal ECM metabolism and that it may be more effective in secretory phase and postmenopausal women. POP is associated with significant changes in pelvic smooth muscle, such as reduced cell quantity, altered structure, decreased contractile capacity, and increased apoptosis and fibrosis 14 – 16 . These tissue deficiencies are further exacerbated by prolapse surgery and menopause 21 . In this study, vaginal contractility was enhanced with D pSMC-CM and bSMC-CM, with a trend towards improved contractility with A pSMC-CM, but not with B pSMC-CM, compared to pure media (vehicle). Interestingly, B pSMC-CM increased contractility of the bladder trigone suggesting that the lack of effect in the vagina may be due variability in CM diffusion into adjacent tissues post injection and/or variability due to tissue type. We found significant increases in smooth muscle actin (ACTA2) in 2 out of 3 pSMC-CM-treated groups. To explore whether Rho kinase or apoptosis pathways may be involved, we evaluated apoptosis markers ( Bad, Bax, Bcl-2, Bcl-xl ) in the vaginal fibroblasts treated with pSMC-CM, but the results were inconsistent (data not shown). Although ROCK1 mRNA expression, a downstream effector of RhoA associated coiled-coil-containing protein kinase that play a vital role in stress fiber and focal adhesion formation and smooth muscle contraction, increased with CM treatment in both vaginal fibroblast and animal experiments, the increase was not statistically significant. Hence, future studies with higher optimized doses of CM are needed to clarify potential molecular pathways. We assessed the bladder and urethra because the CM was injected into the vagina with likelihood of spillage into adjacent tissues/organs and these tissues/organs adjacent to the vagina were also injured during the surgery. In the bladder trigone and urethra, expression of elastin and collagens I/III was upregulated following pSMC-CM treatment. These observations further support the effect of pSMC-CMs and bSMC-CM on pelvic tissues. Bladder trigone contractility was enhanced in 2/3 pSMC-CM treated groups. Correspondingly, significant increases in SMA(ACTA2) expression were observed in the bladder trigone and dome with pSMC-CM treatment. ROCK1 mRNA expression, which promotes smooth muscle contraction via the RhoA/ROCK pathway, was significantly increased in the bladder trigone and dome. While an increase in bladder contractility is not the intended effect for this potential treatment, these data suggest that CM may improve smooth muscle function in pelvic tissues. Future directions should address evaluation on differences in tissue responses to CM and methods for more precise deliver of the CM to limit unwanted effects in surrounding organs and. Limitations of our studies include: 1) Proteomic analysis was performed without replicates, thus, only qualitative analysis of protein enrichment in the CM could be conducted. Further, due to the scope and resources for this study, we did not characterize the CM to fully understand batch-to-batch variability and confirm consistent bioactivity. Future studies are needed to fully characterize different CM batches. 2). Tissue sampling. Due to the small size of the rat vagina, the mid-vagina was used for organ bath studies followed by protein and histological examination, while the apex (proximal part) was used for RT-PCR. This may have introduced variability in the results. Small tissue size also restricted the protein loading amount for western blot analyses. 3). Small sample size for each menstrual group in the vaginal fibroblast study led to a lack of consistent, significant differences. Protein expression was analyzed in fibroblasts from each menstrual phase after treatment with A and C pSMC-CM to confirm the significant increases in elastin mRNA expression with A and C pSMC-CM treatment. We were unable to evaluate protein expression in all the CM treated cells due to lack of sufficient cells for western blot in some samples. While our approach provided meaningful insights into phase-dependent differences, we acknowledge that extending protein-level validation to all fibroblast populations would better capture overarching trends across the menstrual cycle and menopause. This represents an important direction for future research. 4). We did not analyze the exosomes in the CM, which may contain micro-RNAs, DNA, and proteins 43 . Future studies should focus on standardization of the CM and assessment of the exosomes to define their contribution to tissue regeneration. 5) Our surgical injury model differs from clinical prolapse surgery, as the vaginal lumen was not entered and the fibromuscular layer was allowed to heal without suture closure, which may influence tissue repair and ECM remodeling. We chose not to enter the vaginal lumen and add sutures in the small vagina because these procedures would have increased infectious and surgical morbidity in the immunodeficient rat. 6) The use of an immunodeficient rat model does not allow us to assess the role of the immune system in tissue remodeling. Our primary focus in this study was to assess whether the human proteins in the CM may have an in vivo effect, therefore we needed to use an immunodeficient rat model. Follow-up studies in immunocompetent models are needed. We will reprogram rat somatic cells into rat-iPSCs and differentiate these into rat-pSMCs to conduct the experiment in immunocompetent rats to examine long term immune tissue remodeling and cellular mechanisms. Despite these limitations, our studies provide important insights into the paracrine effect of induced pluripotent stem cell-derived progenitor cells on tissue regeneration after surgical injury. Specifically, iPSC-derived pSMC conditioned media exert a paracrine effect on the extracellular matrix metabolism and serine-protease activity in human vaginal fibroblasts and promote structural and functional recovery of the vagina in a rat model of surgical injury. These findings suggest a potential acellular therapeutic approach to improve vaginal smooth muscle function and tissue healing following pelvic reconstructive procedures.

Pelvic organ prolapse (POP) is a common condition, defined as the anatomical descent of the vaginal wall, including the uterus, to or beyond the level of the vaginal hymen 1 . POP incidence is 40% in women between the ages of 50-79, and it increases with advancing age 2 , 3 . POP is associated with lower genital, urinary, and gastrointestinal symptoms with negative impact on quality of life 4 . Surgical implantation of meshes or repair using patient’s tissues to reinforce prolapsed tissues are the standard approaches. The lifetime risk for women undergoing POP surgery is 11–19% 5 , 6 . Unfortunately, surgical risks, mesh-related complications, and high long-term recurrence rates (up to 29%) 7 – 12 make surgery suboptimal. Thus, there is a need for non-surgical therapies to restore supportive pelvic tissues following surgical interventions. The fibromuscular connective tissue of the vaginal wall, pelvic ligaments, and the levator ani muscles support the pelvic organs in the pelvic space. The smooth muscle component in these tissues contributes to vaginal support, as smooth muscle fibers from the vaginal wall attach to the levator ani muscle complex 13 . Women with uterine prolapse have significantly decreased smooth muscle content in the vaginal wall, uterosacral ligaments, and levator ani muscles 14 – 16 . In addition to reduced smooth muscle content, prolapsed pelvic tissues exhibit alterations in extracellular matrix (ECM) proteins such as decreased elastin and collagen, alterations in collagen subtypes, and changes in collagen metabolism, which lead to compromised tissue biomechanical properties 17 . Vaginal smooth muscle cell (SMCs) and fibroblasts contribute to maintaining ECM homeostasis, thus deficiencies in these cell types are associated with POP. Advances in regenerative medicine highlight the potential of stem cell-based therapies in tissue repair and regeneration. Mesenchymal stem cells (MSCs) mediate tissue regeneration and functional improvement through paracrine effects rather than cell differentiation 18 – 20 . This was demonstrated using factors secreted by MSCs into the conditioned media (CM) during cell culture. These secreted factors, which include proteins and exosomes, may be sufficient to enhance tissue repair without the need for MSCs 18 . Thus, CM is considered a promising acellular alternative to stem cell transplantation 19 , 20 . Because it is difficult to harvest and expand sufficient patient cells for therapeutic applications, induced pluripotent stem cells (iPSCs) is a promising autologous stem cell source. We hypothesize that CM derived from progenitors of smooth muscle cells (pSMCs) that are differentiated from iPSCs can restore the surgically injured vagina. In this study, we sought to characterize the effects of pSMC-CM on ECM metabolism on primary culture human vaginal fibroblasts and to examine the regenerative effects of pSMC-CM in a rat model of surgical injury to the vagina. These studies on the paracrine effects of pSMCs provide valuable insights for the design of non-surgical treatments to reduce recurrent prolapse after surgery.

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