Transplantation of human amniotic epithelial cells promotes morphological and functional regeneration in a rat uterine scar model

Abstract

Background: Cesarean scar defect (CSD) is characterized by the presence of fibrotic tissue and decreased muscular density which is induced by cesarean section. Serious CSD may eventually result in infertility or obstetrical complications. Human amniotic epithelial cells (hAECs) have shown great promise in tissue regeneration. This study aims to investigate whether hAEC transplantation has the therapeutic effects on the rat uterine scar following full-thickness injury.

Methods: A rat uterine scar model was established by excising the full-thickness uterine wall of about 1.0 cm in length and 1/2-2/3 of the total circumference in width. At day 30 post-surgery, hAECs were transplanted into the uterine scar. At day 30 and 60 post-transplantation, hematoxylin and eosin (H&E) staining, Masson staining, and IHC staining for vWF, VEGFA, α-SMA, and MMP-8 were performed to evaluate the regeneration of the scarred uterus and the underlying mechanism. Pregnancy outcomes were assessed at day 60 after hAEC transplantation. Finally, hAECs were incubated with hydrogen peroxide to verify the paracrine effect of hAECs.

Results: Collagen deposition, thin myometrium, and injured endometrium were observed in the rat uterine scar model. After hAEC transplantation, collagen deposition in the uterine scar decreased, and myometrial and endometrial recovery was facilitated. hAEC transplantation also increased the fetus number implanted within the scarred area. Moreover, we found hAECs promoted angiogenesis via upregulation of VEGFA and decreased collagen deposition by upregulating MMP-8 in the uterine scar. The in vitro studies further demonstrated an increase in the expression level of MMP-8 in hAECs cultured with hydrogen peroxide.

Conclusions: These results suggested that hAEC transplantation may be efficacious in the functional repair and collagen degradation of uterine scars, which provides a new therapeutic strategy to CSD.

Keywords: Cesarean scar defect; Fertility; Human amniotic epithelial cells; Matrix metalloproteinase-8; Uterine scar; Wound healing.

Fig. 1

hAECs express specific surface markers and have stem cell characteristics with low immunogenicity. a hAECs presented an epithelial morphology under bright-field microscopy. Scale bar = 100 μm. b hAECs were labeled with CFSE to track implanted cells. The expression rate of green fluorescence staining was nearly 100%. Scale bar = 100 μm. c-f By flow cytometry, hAECs were positive for stem cell marker SSEA-4 (c) and epithelial marker CD324 (d) and were negative for mesenchymal markers CD146 (e) and HLA-DR (f). g Immunofluorescence staining for CK18 (an epithelial marker) expression and vimentin (a mesenchymal marker) in hAECs. Scale bar = 200 μm

Fig. 2

Schematic representation of experimental design and establishment of the animal model. A The timeline and design of the experimental flow of the uterine scar rat model. The red area of the timeline indicates different interventions and the gray area indicates the same procedures. B Diagram of establishment and treatment of the uterine scar rat model. A segment of around 1.0 cm in length and 0.5 cm in width (one-third to half of the uterine circumference) of the full-thick uterine wall was excised and removed while the mesometrium was retained. After 30 days, hAECs were transplanted into the uterine scar. C Gross image of the uterine scar rat model. D Hematoxylin and eosin (H&E)-stained cross-sections of the uterus in the uterine scar model group (a) and sham group (b). Scale bar = 500 μm. E Masson’s trichrome-stained cross-sections of uterine segments in the uterine scar model group (a) and sham group (b) 30 days after surgery. Scale bar = 200 μm. hAECs, human amniotic epithelial cells

Fig. 3

hAEC transplantation improved the recovery of endometrium and myometrium in scarred uteruses. a H&E staining of uterine scars at day 30 and 60 post-transplantation in the sham group, PBS group, and hAECs group (n = 8 uterine horns per group). Red arrows indicated repair sites. Scale bars = 500 μm. b Statistical analysis of the endometrial thickness in the uterine scars. c Statistical analysis of the number of endometrial glands per cross-section of the uterus. d Immunohistochemical (IHC) staining of α-smooth muscle actin (α-SMA) for smooth muscle abundance in uterine scars at day 30 and 60 post-transplantation in the sham group, PBS group, and hAECs group (n = 8 uterine horns per group). Red arrows indicated repair sites. Scale bars = 500 μm. e Statistical analysis of the percent of α-SMA-positive areas (α-SMA-positive area in the selected region/total α-SMA-positive area) measured by Image-Pro Plus software (*P < 0.05; **P < 0.01; ***P < 0.001; NS, P ≥ 0.05)

Fig. 4

hAECs promoted the collagen degradation through increasing the expression of MMP-8. A Masson staining results showed less collagen deposition (stained blue) around the uterine scar in the hAECs group (i, l) compared with the PBS group (e, k). Scale bar = 200 μm. B IHC staining was used to detect the expression of MMP-8 in the uterine scars at day 30 and 60 post-transplantation in the sham group, PBS group, and hAECs group (n = 8 uterine horns per group). Scale bar = 25 μm. C The MMP-8 expression was semi-quantified by calculating the positive cells per field under a magnification of × 400. D Western blot analysis showed the MMP-8 expression of the uterine scars in the hAECs group and PBS group at day 5 after injection of hAECs or PBS. E The grayscale values of the western blots were evaluated. The protein level of MMP-8 was normalized to that of β-tubulin (n = 5; **P < 0.01; ***P < 0.001)

Fig. 5

Effects of hAECs on angiogenesis in the injured uteruses. a IHC staining of von Willebrand factor (vWF) reflected the blood vessel density in the uterine scars of different groups at day 30 and 60 after injection of hAECs or PBS (n = 8 uterine horns per group). Scale bars = 25 μm. b IHC staining of VEGFA in the uterine scars at day 30 and 60 after injection of hAECs or PBS in different groups (n = 8 uterine horns per group). Scale bars = 25 μm. c Statistical analysis of the blood vessel density indicated by vWF-positive staining. d The VEGFA expression level was semi-qualified by calculating the percentage of positive cells per field under a magnification of × 400. e Western blot analysis showed that VEGFA expression of the uterine scars in the hAECs group and PBS group at day 5 after injection of hAECs or PBS. f The grayscale values of the western blots were analyzed. The protein level of VEGFA was normalized to that of β-tubulin (n = 5) (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, P ≥ 0.05)

Fig. 6

Localization of transplanted hAECs in the injured uteruses. CFSE-labeled hAECs were transplanted into uterine scars. At day 1, day 2, and day 3 after hAEC or PBS transplantation, uterine tissues containing the scarred areas were removed, embedded, and sectioned. Cell nuclei were stained with DAPI (blue). hAECs were tracked by the green fluorescence of CFSE under a fluorescence microscope. Scale bar = 200 μm

Fig. 7

The expression levels of MMP-8 increased in hAECs treated with H₂O₂ in vitro. a The cell viability of hAECs treated with the indicated concentrations of H₂O₂ for 12 h by CCK-8 assay. The cell viability of hAECs increased after exposure to 25 μM H₂O₂ for 12 h; however, the viability of hAECs decreased significantly with the increase of H₂O₂ concentration (n = 3; *P < 0.05, ***P < 0.001, ****P < 0.0001 vs 0 μM H₂O₂). b Total MMP-8 levels in the supernatant derived from hAECs stimulated by H₂O₂ (25 μM) for 12 h were quantified by ELISA (n = 3; **P < 0.01). c The expressions of MMP-8 in hAECs exposed to H₂O₂ (25 μM) for 12 h were measured by western blot. d The relative expression level of MMP-8 normalized to β-tubulin (n = 3; **P < 0.01). Control, cultured with a normal medium; H₂O₂, cultured with a medium containing 25 μM H₂O₂

Fig. 8

hAEC transplantation improved pregnancy outcomes in the uterine scar rat model. a Pregnancy outcome in uterine horns of female rats 90 days after injury in different groups. Similar size and shape of implanted fetuses were observed in the sham group; however, in the PBS group, there was no fetus implanted in the uterine scar. After hAEC transplantation, implanted fetuses were observed in the scarred area and were of similar size and shape to the fetuses implanted in the healthy area. Black arrows showed implanted fetuses and white arrows indicated the pre-marked margins of the uterine scar. b The total fetus number per uterine horn in the PBS group and hAECs group were smaller significantly than that in the sham group. c The number of fetuses implanted within the scarred area in the hAECs group was significantly larger than that in the PBS group. Data were presented as median, minimum, maximum, and individual data points (***P < 0.001; ****P < 0.0001; NS, P ≥ 0.05)