Biology of Reproduction, 2018, 99(5), 1057–1069 doi:10.1093/biolre/ioy121 Research Article Advance Access Publication Date: 20 June 2018 Partial regeneration of uterine horns in rats through adipose-derived stem cell sheets† Huijun Sun‡ , Jie Lu‡ , Bo Li, Shuqiang Chen, Xifeng Xiao, Jun Wang, Jingjing Wang and Xiaohong Wang∗ Department of Obstetrics and Gynecology, Tangdu Hospital, Fourth Military Medical University, 569 Xinsi Rd., Xian 710038, China ∗ Correspondence: Department of Obstetrics and Gynecology, Tangdu Hospital, Fourth Military Medical University, Xinsi Road 569#, Xian 710038, China. Tel: +86-029-84717269; Fax: +86-029-84777690; E-mail: wangxh-99919@163.com † Grant Support: The project was supported by grants from the National Natural Science Foundation of China (81370710) and the “Science and technology co-ordinating innovative engineering projects” of Science and Technology Department of Shaanxi Province (2013KTCL03-07). ‡ These authors contributed equally to this work. Edited by Dr. Romana Nowak, PhD, University of Illinois Urbana-Champaign. Received 22 October 2017; Revised 24 February 2018; Accepted 19 June 2018 Abstract Severe uterine damage and infection lead to intrauterine adhesions, which result in hypomenorrhea, amenorrhea and infertility. Cell sheet engineering has shown great promise in clinical applications. Adipose-derived stem cells (ADSCs) are emerging as an alternative source of stem cells for cell-based therapies. In the present study, we investigated the feasibility of applying ADSCs as seed cells to form scaffold-free cell sheet. Data showed that ADSC sheets expressed higher levels of FGF, Col I, TGFβ, and VEGF than ADSCs in suspension, while increased expression of this gene set was associated with stemness, including Nanog, Oct4, and Sox2. We then investigated the therapeutic effects of 3D ADSCs sheet on regeneration in a rat model. We found that ADSCs were mainly detected in the basal layer of the regenerating endometrium in the cell sheet group at 21 days after transplantation. Additionally, some ADSCs differentiated into stromal-like cells. Moreover, ADSC sheets transplanted into partially excised uteri promoted regeneration of the endometrium cells, muscle cells and stimulated angiogenesis, and also resulted in better pregnancy outcomes. Therefore, ADSC sheet therapy shows considerable promise as a new treatment for severe uterine damage. Key words: uterine regeneration, cell sheet engineering, adipose-derived stem cells, cell differentiation. Introduction The human endometrium is a highly regenerative tissue that endures cyclical episodes of proliferation, differentiation, and shedding more than 400 times during a woman’s reproductive years [1, 2]. Two layers compose the endometrium, the upper functionalis, which contains a large amount of glands, and the lower basalis, which is composed of branching glands and dense stroma [3]. The shedding of endometrium functional layer is followed by sequential changes in circulating sex steroid hormones during the menstrual cycle, and the basalis remains during menstruation and from which the new functionalis regenerates [4–6]. During the proliferative phase of the menstrual cycle, the basal layer of the endometrium functions in cell C Crown copyright 2018. renewal and tissue regeneration, and the endometrium regenerates during each cycle [7, 8]. Therefore, any endometrial dysfunction occurs following damage to the basal layer [1, 9]. A variety of diseases or traumas are capable of causing severe uterine damage that may lead to basal layer injury and, consequently, an inability of the functional layer of the endometrium to regenerate, resulting in scar formation [10, 11]. In severe cases, damage causes complete or partial scarring of uterine cavity, also known as intrauterine adhesions (IUA), followed by amenorrhea, infertility or abnormal placenta implantation, or recurrent miscarriage [12–14]. Several therapeutic methods, such as surgery, hormonal drugs, and cell therapy, have been adopted for 1057 Downloaded from https://academic.oup.com/biolreprod/article-abstract/99/5/1057/5040762 by Indian Institute of Technology Kharagpur user on 17 August 2019 Research Article 1058 Materials and methods Rats All experimental animals were provided by the Animal Center of The Fourth Military Medical University and treated in accordance with the guidelines of the Experimental Animals Management Committee (Xian Province, China). Ethical approval was obtained from the Ethics Committee of Military Medical Sciences (Shanxi, China) for this study. Two-day-old female Sprague Dawley (SD) rats were used for ADSC cell isolation. Six-to-eight-week-old female SD rats (250– 300g) were used to establish the model. SD rats were housed in the temperature-controlled environment at 23 ± 3◦ C under the humidity in the 44 ± 2% with a 12-h light and dark cycle. All the animals access food and water ad libitum. ADSC isolation and monolayer culture Two-day-old female rats were selected and sacrificed by cervical dislocation to excise the adipose tissue from the inguinal subcutaneous region (Supplementary Figure S1A). ADSCs were purified based on a previously described protocol [33–35], isolated and cultured in vitro. Briefly, after rinsing several times with phosphate-buffered saline (PBS, Gibco, Carlsbad, CA), the tissue was minced into fine pieces, digested with 0.75% collagenase for 60 min at 37◦ C, and centrifuged at 800 r for 5 min. Adipocytes were discarded. Subsequently, adipose-derived stromal vascular fraction (SVF) was obtained, which was a mixed cell source contains endothelial cells, immune cells, smooth muscle cells, stem cells, and other stromal cells [36]. The SVF pellet was resuspended in PBS. The suspension was filtered through an 80-μm nylon screen to remove tissue debris and then centrifuged at 800 r for 10 min. The cell suspension was incubated for 24 h in α-MEM (Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and penicillin/streptomycin (HyClone, Logan, UT) at 38◦ C under humidified 5% CO2 conditions. After 72 h of culture, the cell medium was exchanged to remove nonadherent cells. The medium was then changed every other day, and passaged cells were subcultured when they reached confluence (80– 100%). Third-generation cultured cells from a homogeneous cell population were ultimately obtained and then labeled with CM-DiI (CellTrackerTM CM-DiI; C7000, molecular probes, life technologies, Eugene, Oregon) (Supplementary Figure S1B). Cells at the same passage in culture were used for all experiments. Flow cytometric analysis Rat ADSCs were recognized via immunophenotyping using monoclonal antibodies as previously described [37, 38]. Third-passage ADSCs were identified by performing flow cytometry (FCM). Cells were harvested via trypsin enzyme digestion (Gibco) and then centrifuged, washed and resuspended. Resuspended cells at a concentration of 106 cells/ml were incubated for 30 min on ice with CD31 (abcam, Cambridge, UK), CD45 (abcam, Cambridge, UK), CD29 (abcam, Cambridge, UK), CD44 (abcam, Cambridge, UK) and CD90 (abcam, Cambridge, UK) monoclonal antibodies. The distribution of mesenchymal stem cells (MSCs) was determined by performing FCM (BD FACSVantage, USA). Preparation of ADSCs sheet Third-passage ADSCs were harvested for further experiments. To construct cell sheets, 5 × 105 ADSCs were cultured in 6-well plates for 14 days at 37◦ C in a humidified atmosphere with 5% CO2 . The inducing medium consisted of α-MEM (Gibco), 10% FBS, 1% antibiotic-antimycotic (HyClone), 10 nmol/L dexamethasone and 100 mg/ml ascorbic acid (Sigma). When the ADSCs sheet matured, they were detached from the culture dish by scraping and floated up in the medium as a membrane (Supplementary Figure S1C). The ADSC sheets were reshaped from the circle to the rectangle and layered according to uterine wounds in the rat model (Supplementary Figure S1D). Morphology of ADSCs sheet The surface morphologies of the ADSC sheets and metal complexes were investigated by performing scanning electron micrograph (SEM) analysis [39]. For the SEM study, glass slides were used as carriers for the cell sheets, which were rinsed twice with PBS, fixed with 2.5% glutaraldehyde, dehydrated in step-wise manner in increasing concentrations of ethanol, and dried with a critical point drier. The samples were then sputter-coated with platinum and examined using a scanning electron microscope (Hitachi Model S-3000N, Tokyo, Japan). For light microscopy studies, the samples were routinely processed, fixed in 4% formalin, dehydrated and paraffin-embedded. Specimen morphology was observed based on hematoxylin and eosin (H&E, Sigma) staining of conventional paraffin sections. Quantitative real-time PCR Total RNA from the ADSCs sheet and ADSCs suspension was isolated using TRlzol reagent (Invitrogen, MD, USA). Total RNA Downloaded from https://academic.oup.com/biolreprod/article-abstract/99/5/1057/5040762 by Indian Institute of Technology Kharagpur user on 17 August 2019 the treatment of endometrial fibrosis [12]. However, these strategies have not shown much benefit [15, 16]. Effective treatments for severe endometrial damage are limited. Therefore, tissue engineering may represent a new method for the repair of uterine function. Cell sheet engineering (CSE) is an innovative technology to regenerate injured or damaged tissues and has shown promising potential in the field of tissue regeneration. CSE involves cultured cells on thermo-responsive culture dishes that form dense cell sheets that detach when the temperature decreases. This technology was first developed by Okano in 1993 [17]. The primary benefit of this approach is that it retains extracellular matrix (ECM) proteins, growth factors, and larger amounts of cytokines without enzymolysis [18]. However, the entire process has been criticized as relatively complicated, timeconsuming, and costly because thermo-responsive-culture dishes are expensive [19, 20]. Thus, exploring novel approaches to improve cell sheet construction is imperative. One feasible method involves adding specific amounts of ascorbic acid to create cell sheets [19–22]. Ascorbic acid stimulates ECM production as well as DNA synthesis and subsequent cell proliferation [23–25]. Additionally, ascorbic acid supplementation has diverse effects on stem cells, and it could be used to maintain stem cell properties [26]. Ascorbic acid treatment plays key roles both ∗∗ in enhances the generation of induced pluripotent stem cells and increasing adipose-derived stem cell (ADSC) proliferation and upregulating Oct4 and Sox2 expression [27]. ADSCs are a type of adult stem cell characterized by self-renewal, multi-potential differentiation, immunosuppressive properties, and low immunogenicity [28–30]. In addition to these advantages, ADSCs secrete trophic factors that enforce therapeutic and regenerative outcomes for a wide range of applications [31, 32]. ADSCs are currently considered the most promising cell type in regenerative medicine. In this study, we sought to construct ADSCs sheet to evaluate the therapeutic effects of cell sheets on regeneration in a rat model. H. Sun et al., 2018, Vol. 99, No. 5 Regeneration of uterine horns in rats, 2018, Vol. 99, No. 5 Rat uterine horn damage model and ADSCs sheet transplantation To evaluate our animal model, vaginal smears were collected daily between 08:00 and 10:00 AM for estrous cycle determination. Sixtysix uterine horns from 33 rats (66 uterine horns) were randomly assigned to three groups after four estrous cycles: the ADSCs sheet transplant group (cell sheet group) (n = 23 uterine horns), the control group (n = 23 uterine horns), and the sham group (n = 20 uterine horns). Adult SD rats were anesthetized (Supplementary Figure S1E, first panel), and the uterine horns were exposed through an abdominal incision. The uterus of rat was opened along the side of nonmesometrium, and uterine walls comprise three layers—the serosa, the myometrium and the endometrium, with the myometrium consisting of circular smooth muscle near the lumen and subserosal longitudinal smooth muscle (Supplementary Figure S1E, second panel). A segment of circular smooth muscle and endometrium approximately 1.5 cm in length and 0.5 cm in width was excised from both sides of the uterine horn, leaving the subserosal longitudinal smooth muscle and the serosa intact (Supplementary Figure S1E, third panel). Immediately after resection surgery, the resected surface was pressed with hemostatic gauze for hemostasis, and the damaged area was confirmed to be thin. In a self-correlative study, rat uterine side-by-side comparisons were observed for diversity restoration effects between the cell sheet group and the control group. The cell sheets show strong adhesion, of which we take advantage to complete the transplant operation. In the cell sheet transplant group (the left uterine horn), triple-layer ADSCs sheet were implanted to replace the segmental uterine excised tissue (Supplementary Figure S1F), and the uterine surface wound with the ADSC sheets remained exposed for 30 min to ensure engraftment. A 100-mm culture dish covered the wound to prevent from water loss. The control group (the right uterine horn) had uterine lesions that were left untreated (Supplementary Figure S1F) and the sham group rats received abdominal incisions with the uterine horns intact. The uterine wound was closed by 7-0 absorbable sutures and abdominal incision was closed by 4-0 absorbable sutures. All rats received penicillin by intramuscular injection twice a day for three days. At 21, 30, and 60 days after surgery, all the uterine horns were tested by histological and immunofluorescence stain (Supplementary Figure S1F). Immunofluorescence analysis Single-cell ADSC suspensions were labeled with CM-DiI (CellTrackerT CM-DiI; C7000, molecular probes, life technologies, Eugene, Oregon) according to the manufacturer’s instructions before cell sheets were induced to trace the stem cells. Specimens from regenerating uteri were embedded in OCT (Leica, Nussloch, Germany), frozen, and serially sectioned at a thickness of 6 μm with a Leica cryostat (Leica, Nussloch, Germany). Frozen sections were incubated with primary anti-estrogen receptor beta (ER, 1:200, ab3576, abcam, UK), anti-progesterone receptor (PR, 1:200, ab2756, abcam, UK), anti-cytokeratin 18 (Ck, 1:200, ab133263, abcam, UK), anti- Vimentin (Vm, 1:1000, ab92547, abcam, UK), alpha smooth muscle (Sm, 1:200, ab32575, abcam, UK), and CD31 (1:100, ab64543, abcam, UK) antibodies at room temperature for 1 h in vivo. The sections were then washed three times with PBS, and FITC-conjugated goat anti-rat IgG secondary antibodies (Alexa Fluor 488 Conjugate, CST, USA) were applied for 30 min at room temperature. Additionally, all slides were incubated for 15 min with 4, 6-diamidino2-phenylindole (DAPI, Santa Cruz) for nuclear staining. Slides were observed with a confocal laser scanning microscope (Leica, Nussloch, Germany). Three visual fields at 200× magnification were randomly selected in each slide, and the average number of positive vessels was determined to perform statistical analysis. Histological analysis Samples from in vivo tissues were fixed with 4% paraformaldehyde for 24 h at 4◦ C, then embedded in paraffin blocks and cut into 8-μm-thick tissue sections using previously described methods [41]. Changes in uterine tissue structure were observed by performing H&E staining (Sigma), and the degree of fibrosis was evaluated with a Trichrome Stain (Masson) Kit (Sigma) according to the manufacturer’s protocols. Staining results were analyzed using an optical microscope (Leica, Nussloch, Germany). Functional testing Uterine function was assessed by determining whether the regenerating uterine horn was capable of implanting a fertilized ovum and providing adequate nourishment to the developing fetus. Sixty days postprocedure, male SD rats were cohabitated with females (n = 13 uteri each in the cell sheet group and the control group, n = 10 uteri in the sham group). Vaginal plugs were tracked every day at 8:00 AM to confirm whether mating had occurred. Animals were euthanized 18.5 days after the appearance of a vaginal plug, and uterine horns were examined to determine the number and position of embryos. Statistical analysis Data were graphed and analyzed with GraphPad Prism 5 (USA). Statistical analysis of endometrial thickness, CD31 density, the mean optical density of Sm-positive areas (AOD), and number of fetuses per uterine were performed with one-way ANOVA. A number of fetuses at scar site were calculated by independent sample t-tests between control and cell sheet group. All measurements are presented as the mean ± SEM. Fisher’s exact test was performed for comparison of pregnancy rate at scar site in cell sheet group versus control group. P-values < 0.05 were considered significant (∗ P < 0.05 or ∗∗ P < 0.01). Results Multipotent rat ADSCs The effects of enzyme digestion on phenotypic markers were assessed (Figure 1A), and rat ADSCs strongly expressed the stem cell markers CD29, CD44, and CD90 but were negative for CD31 and CD45 (Figure 1B–F). In addition, rat ADSCs demonstrated the potential to differentiate into adipocytes and osteoblasts (Figure 1G and H). CellTracker CM-DiI was used to mark and trace the growth of ADSCs that were transplanted into uteri (Figure 1I and J). The induction efficiency (%) of CM-Dil into ADSCs was 100%. Downloaded from https://academic.oup.com/biolreprod/article-abstract/99/5/1057/5040762 by Indian Institute of Technology Kharagpur user on 17 August 2019 was applied for cDNA synthesis using an iTaq Universal One-Step RT-qPCR Kit (Bio-Rad Laboratories, Hercules, USA). Quantitative real-time PCR (qRT-PCR) was performed using Platinum SYBR Green SuperMix (Invitrogen, USA) and an ABI Prism 7500 realtime PCR apparatus (Applied Biosystems). PCR primer pair (rat) sequences are summarized in Supplementary Table S1. The PCR reactions were carried out as previously described [40]. GAPDH was used as an internal control. 1059 1060 H. Sun et al., 2018, Vol. 99, No. 5 ADSC sheet formation and transplantation Scaffold-free ADSC sheets represent a new technology to regenerate injured or damaged tissues. After 10–14 days of cultivation in inducing medium, ADSCs sheets were harvested from 6-well plates in vitro. The first panel in Figure 2A shows the surface of a sheet that detached at the bottom with the edge cocked, while the second panel in Figure 2A was floating ADSCs sheet. The intact ADSC sheets were harvested and reshaped to the rectangle. The reshaped ADSC sheets were transplanted onto the damaged endometrium (Figure 2B). As observed by cross-section H&E staining, the ADSCs were embedded in their own secreted ECM. Moreover, ADSCs sheets were composed of several layers of cells and abundant ECM as determined by longitudinal section analysis (Figure 2C). Based on scanning electron microscopy examination, ADSCs sheets were compact overlayer films. Distinct secretory granules displaying spherical vesicles were present on ADSCs sheet surfaces (red arrow) (Figure 2D). Transforming growth factor β (TGFβ), collagen type I (Col I), vascular endothelial growth factor (VEGF), and fibroblast growth factor (FGF) secretion from ADSC sheets significantly increased, meanwhile this ADSC sheets construct significantly increased the expressions of stemness genes such as Nanog, and Sox-2 but did not impact gene Oct-4 expression compared to that in ADSCs in suspension (Figure 2E). Effects of the uterine resection model The anatomical structures of the normal uterine area were obvious and thick compared to those of the operative area which is observed by H&E and Masson’s trichrome staining. Based on H&E staining, the uterus comprised the endometrium, muscle layers, and serosa in normal areas. Abundant endometrial glands were observed Downloaded from https://academic.oup.com/biolreprod/article-abstract/99/5/1057/5040762 by Indian Institute of Technology Kharagpur user on 17 August 2019 Figure 1. Phenotype and multidifferentiation of rat ADSCs. (A) Rat ADSCs showed spindle-shaped fibroblastic morphology at the 3rd passage. (B–F) Flow cytometric characterization of rat ADSCs. ADSCs were positive for CD29 (B), CD90 (C), and CD44 (D), but did not express CD45 (E) and CD31 (F). (G and H) ADSCs were induced to differentiate into multiple lineages. (G) Rat ADSCs differentiated into adipocytes as indicated by cells stained with Oil red O. (H) ADSCs were induced to differentiate into an osteogenic lineage as observed by Alizarin red S staining. (I and J) CM-DiI was applied to trace, locate and recognize rat ADSCs. The appearance of labeled ADSCs under an optical microscope (I) and a fluorescent microscope (J). Regeneration of uterine horns in rats, 2018, Vol. 99, No. 5 1061 in the endometrial stromal layer (Figure 3A, left panel). Masson’s trichrome staining was performed on the uterine horns to characterize the extent of the muscle layers, which were composed of smooth muscle cells in circular and longitudinal patterns in the nonoperative area, leaving behind only the longitudinal muscle layer in the operative area (Figure 3A, right panel). Immunofluorescence staining with an anti-Ck antibody revealed Ck-positive epithelial cells on the normal uterine adluminal surface and the endometrial gland in the endometrial stromal layer (white arrow). No simple columnar epithelial cells were found on the endometrial surface of the resected tissue, and no endometrial glands were observed either (Figure 3B, first panel). Vm-positive stromal cells were distributed in region under the simple columnar epithelial cells (white arrow). Conversely, we did not find Vm-positive stromal cells in the resected area of the endometrium (Figure 3B, middle panel). Sm-positive muscle bundles were labeled with green fluorescence following immunofluorescence staining. As expected, no circular fibers were found in the muscle layer. Only Sm-positive longitudinal muscle bundles were observed in the resected area (yellow arrow). Conversely, Sm-positive circular and longitudinal fibers were observed in normal areas (white arrow) (Figure 3B, below panel). Based on these results, the endometrial epithelial layer, basal layer, and circular fibers were successfully ablated in the uterine damage model, laying the foundation for the regeneration of damaged uteri. Downloaded from https://academic.oup.com/biolreprod/article-abstract/99/5/1057/5040762 by Indian Institute of Technology Kharagpur user on 17 August 2019 Figure 2. Configuration of a 3D ADSCs sheet. (A) Visualization of the attachment and detachment of rat ADSCs sheet in vitro (bar: 5 mm). (B) H&E histological staining of cross-sections and longitudinal sections of ADSCs sheet. (C) SEM images of ADSCs sheet formation and distinct secretory granules with the appearance of spherical vesicles on cell sheet surfaces (red arrow). (D) Stemness and secretory cytokine gene expression by ADSCs sheet were detected by qRT-PCR. Data are presented as the mean relative density ± SEM; ∗ P < 0.05, ∗∗ P < 0.01, compared to ADSCs (n = 3). 1062 H. Sun et al., 2018, Vol. 99, No. 5 Histological structures of regenerated uterine horns Gross examination showed that luminal stenosis occurred in the surgical regions of the uteri in the control group at 30 days (Figure 4C), and adhesions or pathological stenosis formed at 60 days (Figure 4E). However, luminal stenosis and scars were not observed in the cell sheet group (Figure 4D and F), and the uterine anatomy was similar to the sham group (Figure 4A and B). Representative images show that more blood vessels were present in the cell sheet group at both 30 and 60 days after surgery (Figure 4D and F), while the control group exhibited pale serosa(Figure 4C and E). To evaluate the effects of ADSC sheets on the regenerative recovery of uterine horns, H&E and Masson’s trichrome staining were performed. As shown in Figure 4H, the cell sheet group exhibited greater numbers of endometrial glands in the endometrial stromal layer and more apparent neovascularization 30 days after surgery. There were significant differences in endometrial thickness between the cell sheet group (442.883 ± 28.185 μm) and the control group (120.9433 ± 13.676 μm) (P = 0.000) (Figure 4J). At 60 days, the specimens in the cell sheet group appeared near-normal and exhibited more angiogenesis. No epithelial-like cells were observed in uterine centers in the control group, but columnar epithelium lining was present in the cell sheet group (Figure 4H). Endometrial thickness in the cell sheet group (641.484 ± 58.284 μm) was significantly greater than that in the control group (300.048 ± 44.903 μm) (P = 0.000) but was thinner than the sham group (1044.226 ± 8.911 μm) (P = 0.000) (Figure 4K). According to Masson’s trichrome staining on postoperative day 30, uterine tissue in the sham group was present in the muscle layer, which consists of internal-circular muscle and external-longitudinal muscle (Figure 4I). At 30 days, positively stained smooth muscle cells turned light red and were arranged in a fascicular pattern in the cell sheet group but not the control group (Figure 4I). At 60 days, we observed the orderly arrangement of smooth muscle cell fascicles at near-normal levels in the cell sheet group. However, the endometrium in the control group exhibited fibrous tissue hyperplasia and collagen deposition and eventually formed scars (Figure 4I). Immunofluorescence staining of the regenerative uterine horns We assessed epithelial regeneration using an anti-Ck antibody. Ckpositive epithelial cells covered the surface of the excised uterine lumen after 30 days in the cell sheet group. In contrast, few epithelial cells were found at excision sites in the control group (Figure 5A). The lumenal surface of the uterus was a simple columnar epithelium, which became more organized and exhibited secretory glands after 60 days in the cell sheet group but lacked epithelial cells and secretory glands in the control group (Figure 5A). Uterine specimens from each group were tested for Vm expression by performing immunofluorescence microscopy to investigate stromal cell regeneration. Vm-positive stromal cells in excised sections were detected by the presence of a bright green fluorescent signal in the basal layer of the endometrium in the cell sheet group (Figure 5B). Although Vm expression was also observed in the same region in the control group, it was present at a lower frequency, and the endometrium was thinner than that in the cell sheet group (Figure 5B). At 60 days, greater numbers of endometrial glands were observed in the endometrial stromal layer in the cell sheet group, and the fluorescence intensities of Vm-positive stromal cells were enhanced compared with those at 30 days; however, the control group still exhibited lower frequencies of Vm-positive cells (Figure 5B). We used a panel of monoclonal rabbit anti-rat antibodies raised against smooth muscle actin (Sm) to explore smooth muscle regeneration at sites of injury. At 30 days, new circular muscle bundles formed a thin successive layer beneath the endometrium in the cell sheet group. However, muscle bundles were absent at injured sites in the control group with the exception of anastomosis sites (Figure 5C). AOD in the cell sheet group (0.035 ± 0.003) was higher than that in the control group (0.010 ± 0.001) (P = 0.000) but lower than that in the sham group (0.074 ± 0.003) (P = 0.000) (Figure 5E). At 60 days, some collagen deposition and scar tissue formation was observed in the control group, but no obvious circular muscle bundles had formed. The smooth muscle tissue converged to form thick muscle layers in the cell sheet group that were denser Downloaded from https://academic.oup.com/biolreprod/article-abstract/99/5/1057/5040762 by Indian Institute of Technology Kharagpur user on 17 August 2019 Figure 3. Experimental procedure for the rat uterine regeneration model. (A) Schematic illustration showed the experimental procedure. (B) Uterine horns were exposed, and half of the uterus, approximately 1.5 cm in length and 0.5 cm in width, was resected and removed. Triple-layer ADSCs sheets were transplanted onto the damaged inner uterine surface, and the uterine wound was left open for 30 min after transplantation to ensure engraftment. (C and D) To assess the uterine damage model, uteri were resected, and specimens were subjected to histological analysis after surgery. (C) Specimens stained with H&E and Masson’s trichrome stain were observed microscopically. Black arrows and blue arrows indicate endometrial glands and the circular muscle layer, respectively. (D) Immunofluorescence staining of specimen components using antibodies against Ck, Vm, and Sm. White arrows show the endometrial epithelial layer (first panel), endometrial basal layer (middle panel), and circular muscle layer, respectively (last panel). Yellow arrows indicate the presence only of the remaining longitudinal muscle layer in the regeneration model. Dotted lines indicate repair sites. Regeneration of uterine horns in rats, 2018, Vol. 99, No. 5 1063 Downloaded from https://academic.oup.com/biolreprod/article-abstract/99/5/1057/5040762 by Indian Institute of Technology Kharagpur user on 17 August 2019 Figure 4. Gross view and histological evidence for uterine horn regeneration among the groups. (A–F) Gross observations of surgical regions in the lower segment of the uterus at 30 days (A–D) and 60 days (E, F) in the sham group (A, B), control group (C, E), and cell sheet group (D, F). The appearance of surgical regions indicated superior vascularization for the repair of segmental defects in the rat uterus in the cell sheet group compared with the control group. H&E (H) and Masson’s trichrome staining (I) of the uterine horns after 30 or 60 days post-treatment in the sham group, control group, and cell sheet group. (J, K) Statistical analysis of endometrial thickness revealed a thicker endometrium in the cell sheet group than in the control group; however, the endometrium was thinner than that of the sham group. Dotted lines indicate repair sites. Black arrows indicate endometrial glands in the basal layer. Data are presented as the mean ± SEM. ∗ P < 0.05 and ∗∗ P < 0.01. 1064 H. Sun et al., 2018, Vol. 99, No. 5 Downloaded from https://academic.oup.com/biolreprod/article-abstract/99/5/1057/5040762 by Indian Institute of Technology Kharagpur user on 17 August 2019 Figure 5. Immunofluorescence staining indicated Ck (A), Vm (B), Sm (C), and CD31(D) levels in each group at 30 or 60 days after surgery. (E) We assessed AOD using IPP software and observed the continuous improvement of muscle bundle status over time. (D) CD31 expression, indicative of neovascularization in the regenerated endometrium, was detected at 30 and 60 days in the sham group, control group, and cell sheet group. (F) Based on a statistical analysis of capillary vessel number from at least three randomly selected fields at a magnification of 200×, cell sheet promoted blood vessel regeneration. Each experiment was repeated four times. Dotted lines indicate repair sites. Data are presented as the mean ± SEM. ∗ P < 0.05 and ∗∗ P < 0.01. Regeneration of uterine horns in rats, 2018, Vol. 99, No. 5 ADSC differentiation in vivo Third-passage ADSCs were incubated with CellTracker CM-DiI and exhibited red fluorescence (Figure 1J). Twenty-one days later, ADSCs fused with host cells and prompted local cells migration to the wound edges. Notably, clusters of cells labeled with red fluorescence were primarily distributed in the basal layer of the regenerated endometrium (Figure 6A). Based on further observations of uterine sections, some ADSCs may have directly differentiated into Vm-positive endometrial stromal cells (Figure 6D), while others expressed ER (Figure 6B) and PR (Figure 6C), which are also expressed in endometrial stromal cells. These typical images illustrate the differentiation of ADSC sheets during development in the uterine microenvironment. Functionality of regenerated uterine horns To investigate the effects of cell sheet treatment on the functional recovery of regenerated uterine horns, the ability to accept an embryo was tested. At 60 days, female rats were mated with male rats (the female: male ratio was 2:1) and were checked for pregnancy at 18.5 days after the appearance of the pessary. Fetuses implanted in all of the uterine horns in the sham group (Figure 7A and B). In the control group, embryos were rarely observed at scar sites, but the majority implanted at normal sites (Figure 7C). In contrast, fetuses were observed both in regenerated uterine tissue and in normal uterine tissue in the cell sheet group (Figure 7D). Pregnancy rate at the scar site was 69.23% for the cell sheet group and 15.38% for the control group (P = 0.005) (Table 1). In addition, the total number of implanted embryos in the control group (2.308 ± 0.634) was significantly reduced compared with that in the cell sheet group (5.230 ± 0.975) (P = 0.020) (Table 1). Furthermore, the number of embryos that implanted at the scar site was noted. In terms of scar site pregnancy, 0.846 ± 0.222 embryos demonstrated successful nidation, but in the control group only 0.154 ± 0.104 embryos showed successful scar site implantation (P = 0.009) (Table 1). Thus, cell sheets grafted onto injured endometrium in vivo reestablished maternal uterine functionality to support embryo implantation and fetal development. Discussion The clinical application of cell sheet therapy has been reported for the cornea [42], liver [43, 44], arteries [45], esophagus [46], bone [47], heart [48, 49], and brain [50]. Here, we revealed the feasibility of partial resection of the rat uterus to create a uterine regeneration model. This method permitted a transplantable ADSC sheet to firmly attach to a site of injury to maintain lumenal architecture. We also demonstrated remodeling of the 3D architecture of the rat uterus via engraftment of an ADSC sheet onto a partially excised uterus. Muscle bundles were rebuilt, and columnar epithelial-like cells were reconstituted. Furthermore, ADSCs were capable of differentiating into stromal-like cells when ADSC sheets were transplanted into the uterus. In regenerative medicine, other approaches have been reported to effectively regenerate injured uterine tissue, such as recellularization of a decellularized uterine matrix [51], collagen scaffolds in combination with collagen-binding human basic growth factor [52, 53] or the application of bone marrow MSCs [21]. However, inflammatory responses are induced by scaffold materials during biodegradation in vivo, and the resultant constructs exhibit low cell density [54]. The 3D tissue-like structures of cell sheets are formed by cell-to-cell junctions and deposited ECM in the absence of any scaffolds. Therefore, an immune response is not initiated, and inflammatory responses do not accompany transplantation [55]. Cell sheets are also easily shaped into different structures for different applications and are especially suitable for the narrow uterine lumen. With their capacity for self-renewal, multidirectional differentiation and low immunogenicity, ADSCs are emerging as an alternative stem cell source for cell-based therapies [56]. A previous study described the treatment of severe full-thickness uterine injury in rats using bone marrow MSCs for collagen scaffold transplantation [36]. MSCs primarily localized to the basal layer, and a small number differentiated into stromal cells. In the present study, we also observed the random distribution of most ADSCs onto the basal layer. Meanwhile, ADSCs were capable of differentiating into stromal cells. This preliminary analysis illustrates a possible mechanism to explain the effects of ADSC sheets on the injured uterus. The basal layer of the endometrium contains mesenchymal stemlike cells that express markers similar to bone marrow-derived stem cells, characterized by multiple differentiation potential [57]. Therefore, a new functional layer regenerates during each menstrual cycle [58]. Uterine endometrial cells adjacent to the wound margin have the potential to regenerate the wound site via uterine cell proliferation and migration. However, according to our study, uterine regeneration was insufficient in the control group. Therefore, the 3D structure of the ADSC sheets provided an efficient cell microenvironment that was critical for ADSC differentiation and uterine cell regeneration. In the present study, ADSC sheets demonstrated higher FGF, Col I, TGFβ, and VEGF expression compared to that in ADSCs in suspension. These bioactive factors not only promote growth and development but also inhibit apoptosis, benefit local cell proliferation, and are conducive to ADSC differentiation [36, 59, 60]. TGFβ is a pleiotropic cytokine that is important in the regulation of joint homeostasis and disease. TGFβ also regulates many of the processes common to cell differentiation, tissue repair and inflammation [61]. Angiogenesis, which is primarily mediated by the VEGF pathway, is crucial for tube formation and cell proliferation [62]. According to previous studies, collagen-binding VEGF remodels full-thickness uterine injury [53]. bFGF exerts many Downloaded from https://academic.oup.com/biolreprod/article-abstract/99/5/1057/5040762 by Indian Institute of Technology Kharagpur user on 17 August 2019 and arranged. However, at the site of anastomosis, only circular fibers were observed, rather than circular and longitudinal fibers (Figure 5C). The AOD in the cell sheet group (0.059 ± 0.031) was still significantly higher than the control group (0.022 ± 0.036) (P = 0.000), but no statistically significant difference was observed between the cell sheet group and the sham group (0.065 ± 0.035) (P = 0.341) (Figure 5E). Thus, cell sheets promoted the regeneration of injured muscle bundles, and this outcome became increasingly evident over time. Neovascularization is one of significant standards to evaluate the regeneration of the injured uterine regions. At 30 days after the implantation of cell sheet grafts, blood vessel density within the cell sheet group was higher, with neovascularization located near the site of injury (Figure 5D). Blood vessel density in injured uterine regions in cell sheets group (26.40 ± 1.806) was significantly higher than that in the control group (15.80 ± 2.634) (P = 0.011), and less than the sham group (54.40 ± 4.130) (P ≤ 0.001) (Figure 5F). At 60 days, the blood vessel was increased in all groups compared with 30 days, however, the cell sheet group (42.80 ± 3.262) showed a significant increase compared to control group (24.40 ± 2.112) (P = 0.002) and still less than the sham group (54.40 ± 2.227) (P = 0.019) (Figure 5F). 1065 1066 H. Sun et al., 2018, Vol. 99, No. 5 biological functions, such as stimulating the proliferation of fibroblasts, skeletal myoblasts, vascular endothelial and smooth muscle cells, and is also essential for cell survival, migration, and matrix production/degradation [63]. As reported by Li, collagen scaffolds loaded with collagen-binding FGF effectively repaired damaged endometrium through angiogenesis and epithelial regeneration [52]. Wound-healing synthesis processes, which include the rapid deposition of ECM, may be regulated by the ECM microenvironment [64]. A variety of components constitute the ECM, but the defining component is Col I [65]. After the transplantation of ADSC sheets into the injured uterus, different growth factors and extracellular vesicles interact to prompt wound healing and mediate cell regeneration. Our objective in evaluating regenerated uterine function was to assess pregnancy potential. In the present study, pregnancy rates at the scar site were 69.23% in the cell sheet group and 15.38% in the control group. In addition, the total number of implanted embryos in the control group was significantly reduced compared with that in the cell sheet group. Embryos were rarely observed in scar sites in the control group, but fetuses were observed in both the regenerated uterine tissue and the normal uterine tissue in the cell sheet group. Therefore, this therapeutic method not only remodeled the structure of the injured uterus by recovering the lumen-adjacent epithelial layer, basal layer, and smooth muscle layer but also restored functionality as evidenced by successful pregnancy. Conclusions The present study reviewed a novel approach for the regeneration of partial uterine defects. Thus far, tests have only been conducted on animals in a laboratory setting, but human testing is anticipated for the next phase. However, many challenges remain. Cell sheet therapy may be a new approach for treating partial uterine defects due to dilatation and curettage after abortion or postpartum, myomectomy for uterine fibroids, adenomyomectomy for uterine adenomyosis, severe IUA from endometrial tuberculosis, and conization in uterine cervical cancer. Our group is conducting further experiments with human subjects to benefit mankind. Downloaded from https://academic.oup.com/biolreprod/article-abstract/99/5/1057/5040762 by Indian Institute of Technology Kharagpur user on 17 August 2019 Figure 6. The location and differentiation of ADSCs in the newly regenerated uterine endometrium 21 days after cell sheet transplantation as determined by immunofluorescence staining. (A) ADSCs, labeled red, were tracked using a confocal microscope and were primarily located near the basal membrane of the regenerated rat endometrium. Representative fluorescent images demonstrate positive, ER (green) (B), PR (green) (C), and Vm (green) (D) protein expression (arrows) in some ADSCs after the transplantation of labeled cell sheets, indicating ADSCs may have differentiated into endometrial epithelial cells and stromal cells. ADSCs labeled with CM-DiI (red) were tracked using a confocal microscope, and cell nuclei were stained with DAPI (blue). Dotted lines indicate repair sites. Presence of double positive cell is indicated by white arrow. Regeneration of uterine horns in rats, 2018, Vol. 99, No. 5 1067 Table 1. Comparison of reproductive outcomes among different groups with regenerated uterine horns. Data are mean ± SEM or n (%). Pregnancy rate at scar site Number of fetuses per uterine horn: mean ± SEM Number of fetuses at scar site: mean ± SEM Sham (n = 10) Control (n = 13) Cell sheet (n = 13) 8.100 ± 0.314 2 (15.38%) 2.308 ± 0.634b 0.154 ± 0.104 9 (69.23%)a 5.230 ± 0.975b,c 0.846 ± 0.222d P < 0.01, vs. control group. P < 0.05, vs. sham group. c P < 0.05, vs. control group. d P < 0.01, vs. control group. a b Supplementary data Supplementary data are available at BIOLRE online. Supplementary Figure S1. Schematic illustration of experimental procedure. (A) Adipose tissues were resected from 2-day-old female rats. (B) The ADSCs were isolated from the tissue with enzymolysis, and then labeled with CM-DiI. (C) ADSCs were cultured with induced medium for 14 days at 37◦ C in a humidified atmosphere with 5% CO2 . (D) ADSCs sheet was harvested from the insert, reshaped, and layered according to the target tissue site. 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