Abstract
Purpose
Cell therapy is a promising strategy for the treatment of Asherman’s syndrome (AS), but the origin of these cells and injection route influence the therapeutic effect and complications of cell therapy. Herein, we compared the effects of systemic or local intrauterine injection of bone marrow or adipose-derived mesenchymal stem cells (BMSCs/AMSCs) on the endometrium in a rat model of AS.
Methods
After induction of AS in adult Wistar rats, the CM-Dil-positive BMSCs or AMSCs were injected either locally or intravenously. After 3 weeks, endometrial thickness, collagen deposition, cell migration, and VEGF expression were evaluated using histochemistry/immunofluorescence studies.
Results
In all stem cell-treated groups, an ameliorative effect on the damaged endometrium was noted. Collagen deposition diminished in both groups (IV and local injection) compared to the AS model. In rats injected locally with MSC, fibrosis decreased compared to the other groups. Moreover, endometrial thickness increased in the groups that received local injection of BMSCs and AMSCs more than the IV-transplanted AMSCs group. Immunofluorescent staining demonstrated that although the systemic transplantation of BMSCs was more effective than the other groups on VEGF expression, it led to the lowest number of CM-Dil+ stem cells in the damaged endometrium.
Conclusion
Stem cell transplantation may reconstruct the damaged endometrium, but it is recommended to select the most effective stem cells and injection route. Because the removal of the fibrosis and the replacement of the epithelia cells is an effective therapeutic strategy for AS, in this study, we conclude that the local injection of AMSCs is more appropriate than BMSCs to treat AS.
Keywords: Asherman’s syndrome, Bone marrow-derived mesenchymal stem cell, Adipose-derived mesenchymal stem cell, Endometrium
Introduction
Asherman’s syndrome (AS) is a typical gynecological disorder that is characterized by intrauterine adhesion, fibrosis, and loss of luminal epithelial cells [1]. The incidence of AS is up to 30% in women who have undergone dilation and curettage after a late spontaneous abortion, and 13% in women who have had a pregnancy termination in the first trimester [2]. The removal of the adhesions and fibrosis and replacement of the epithelial cells may serve as an effective therapeutic strategy for AS. Hysteroscopic adhesiolysis and hormonal therapy are commonly used to reconstruct and restore the uterine lining in the AS [3]. However, these therapies are not effective and the recurrence of adhesions is likely in those patients with severe AS [4]. The human endometrium contains intrinsic stem cells that show similar properties to bone marrow-derived mesenchymal stem cells (BMSCs) and are supported by them during cyclic endometrial regeneration [5].
Therefore, the endometrium has an appropriate niche for transplants of stem cells and stem cell-based therapy can be a promising approach for the damaged uterus [6]. Recent studies have demonstrated the therapeutic effects of stem cells for the regeneration of the damaged endometrium not only in rodents but also in clinical settings [7–9]. Gao et al. found that BMSC transplantation regenerated the damaged endometrial lining in the rat model of severe AS and increased endometrial receptivity marker, resulting in an improvement of reproductive outcomes [10]. In another study, the application of adipose-derived mesenchymal stem cells (AMSCs) caused an improvement in the endometrium of the rat model of AS by increasing vascular endothelial growth factor (VEGF), proliferating cell nuclear antigen (PCNA) and Ki-67, and decreasing fibrosis [11]. Zhao et al. reported that the intrauterine injection of autologous bone marrow mononuclear cells normalized the stemness alterations in the AS patients [12]. In another pilot cohort study, autologous cell therapy with systemic transplantation of BMSC in AS led to the improvement of the uterine cavity and an increase in endometrial thickness and vessel density [8]. Although AMSCs and BMSCs have the same biological characteristics, they also reportedly have notable differences [13, 14]. Stem cell origin and route of injection seem to be one of the most important factors for the efficacy and complications of cell therapy [15]. To the best of our knowledge, no study has yet been conducted to compare the healing effects of adipose- and bone marrow-derived stem cells in a model of AS. The present study aimed to compare the regenerative effects of AMSCs or BMSCs on the endometrium in a rat model of AS. More clearly, we aimed to investigate whether intrauterine injection of stem cells results in better recruitment and angiogenesis than the systemic administration and whether adipose-derived stem cells have an advantage over bone marrow-derived stem cells.
Methods and materials
Ethics approval and study design
The entire procedure of this study was reviewed and approved by the Ethics Committee of Hamadan University of Medical Sciences (HUMS, IR.UMSHA.REC.1397.865). Female Wistar rats (180–200 g) were procured from the Animal Center of Hamadan University of Medical Sciences and kept in the standard condition (12:12 h light-dark cycle, 20 ± 2 °C, and 50% relative humidity) with free access to food and water. Twenty-five rats were randomly divided into the following groups:
Asherman (AS) group underwent abdominal surgery and uterine traumatization using a 27 gauge needle [16]. Briefly, following exposure of the uterus, a needle was inserted into the lumen of right horn and scratched on the walls. Only the rats in the diestrus phase were used to induce the model. The left horn remained intact and was used as control (n = 5).
AS group that was transplanted with BMSC or AMSC intravenously(n = 10).
AS group that was injected with BMSC or AMSC locally (n = 10).
Isolation and transplantation of BMSCs and AMSCs
BMSCs were obtained from the tibia and femur of the male Wistar rats using the flashing method, and the cells were plated in DMEM-low glucose supplemented with FBS 10%.
Inguinal adipose tissue was collected from male Wistar rats and digested using 0.1% collagenase I at 37 °C for 1 h. The digestion was centrifuged and passed through a 40-μm cell strainer. The liquid portion was plated in DMEM-F12 media containing 10% FBS. Both BMSCs and AMSCs were incubated at 37 °C in 5% CO2. Cells at passages four to six were used for transplantation. Surface CD markers of the stem cells were then analyzed for characterizing the MSC phenotype by flow cytometry. Both stem cells were incubated in a green fluorescent CM-Dil dye at 37 °C for 20 min and locally transplanted with a density of 5 × 105 or 1 × 106 via the tail vein. We used a fluorescent CM-DiI dye for monitoring of the injected cells. It is well retained in living cells through several generations, allowing for multigenerational tracking of cellular movements.
Cell transplantation was performed simultaneously with model induction [17]. To perform local intrauterine transplantation, a syringe containing the labeled cells was inserted into the right horn and injected into all four walls of the horn.
Histopathological investigations
Fourteen days after model induction and cell transplantation, the uterine horns were collected and fixed in 4% paraformaldehyde. A total of 15 sections of 5-μm thickness were prepared from the horns of each rat and processed for histological and immunofluorescence staining (five sections of each horn for any staining). Epithelial thickness and collagen deposition were assessed using hematoxylin-eosin and Masson’s trichrome stainings. For both stainings, the slides were first processed in xylene and descending ethanol. Next, they were incubated in the hematoxylin-eosin stains or solutions and reagents of the Masson’s trichrome staining kit (Sigma-Aldrich, St Louis, MO, USA). Finally, the slides were mounted and imaged using a digital camera connected to a light microscope. The thickness of the endometrium was assessed using ImageJ software. In accordance with the protocol of Hadi et al., collagen deposition was analyzed using the batch mode of the ImageJ Macro [18]. Briefly, the percentage of fibrotic area was determined and compared to the total amount of tissue within the image.
Immunofluorescence staining
VEGF is a member of plasma-derived growth factors that is secreted by cells and stimulates angiogenesis. The VEGF expression in the endometrium and the recruitment of CM-Dil+ cells were assessed using the immunostaining method. Briefly, after antigen retrieval in a 10 mM citrate buffer (pH 6.0, 15 min), the sections were permeabilized and blocked in 0.5% Triton 100× and 1% bovine serum albumin, respectively. Next, the slides were incubated in a rabbit polyclonal VEGF antibody (1:100, Abcam, Cambridge, UK) and Alexa Fluor anti-rabbit secondary antibody (1:200, Abcam, Cambridge, UK). Nuclei were distinguished using DAPI staining. After taking the images using fluorescent microscopy, 10 fields were randomly selected from five sections of each specimen, and then 100 cells were selected to evaluate the expression of VEGF and CM-Dil. The VEGF and CM-Dil indexes were expressed as the percentage of positive cells.
Data analysis
The data were presented as mean ± SEM. Data analysis was conducted in GraphPad Prism software using one-way ANOVA and Tukey’s multiple range test. P < 0.05 was considered significant.
Results
Verification of MSCs with surface markers and flow cytometry data
Our flow cytometry data showed that both types of MSCs were CD45- and CD34-negative, but CD29- (AMSC), CD90- (BMSC), and CD44-positive (Fig. 1).
Fig. 1.
Flow cytometry analysis of CD45, CD34, CD90, CD44, and CD29 in BMSCs (a) and AMSCs (b)
Evaluation of the thickness of the epithelium following transplantation of stem cells
Endometrial thickness was evaluated using hematoxylin-eosin staining (Fig. 2). The data analysis showed the greatest endometrial thickness in the control group (p < 0.001). Transplantation of both sources of cells remarkably increased endometrial thickness in both IV and local injection compared to the AS group (p < 0.001).
Fig. 2.
Light micrographs of endometrium (E) thickness of the control (a), Asherman (b), and local BMSC (c), IV BMSC (d), local AMSC (e), and IV AMSC (f) groups using H&E staining. The arrow shows the endometrial thickness (a and b, p < 0.001 vs. the other groups; c, p < 0.05 vs. IV AMSC; d, p < 0.01 vs. local BMSC; e, p < 0.001 vs. local AMSC). Each value is mean ± SEM
Local injection of BMSCs and AMSCs significantly increased endometrial thickness compared to IV-transplanted AMSCs (p < 0.001 for AMSC; p < 0.01 for BMSC). There was no significant difference in endometrial thickness between the two locally injected groups and the IV BMSC-transplanted group.
Intravenous transplantation of BMSC was more effective than intravenously injected AMSCs (p < 0.05). Out of the two routes of AMSC transplantation, the local administration was more efficient than IV injection (p < 0.001). The IV injection of BMSCs was as effective as local injection of these cells.
Evaluation of collagen deposition percentage in the endometrium by Mason’s trichrome staining
Due to increased fibrosis in AS, in this study, we investigated the rate of collagen deposition by Masson’s trichrome staining. Our results showed the greatest amount of fibrosis in the AS group (p < 0.001) that was attenuated following cell transplantation (Fig. 3).
Fig. 3.
Light micrographs of collagen deposition of the control (a), Asherman (b), and local BMSC (c), IV BMSC (d), local AMSC (e), and IV AMSC (f) groups using Masson trichrome staining. The blue shows the collagen deposition (a, p < 0.001, and d, p < 0.05 vs. the control; b, p < 0.001 vs. Asherman’s group; c, p < 0.0.01, and e, p < 0.001 vs. IV BMSC; f, p < 0.001 vs. IV AMSC; g, p < 0.05 vs. local AMSC). Each value is mean ± SEM. Part of this figure (a and b) has been published in our previous study [19]
Although AMSC transplantation significantly decreased the collagen deposition in the endometrium compared to the AS group (p < 0.001), the difference was more pronounced in the group that received intrauterine injection compared to the group that received intravenous injection (p < 0.05). In addition, local injection of BMSCs showed a greater effect than IV transplantation (p < 0.001). Further investigations revealed that the local AMSC injection was more effective than other groups. As seen in Fig. 3, the local injection of AMSCs showed the greatest but insignificantly reducing effect on collagen deposition than the intrauterine injection of BMSCs. Furthermore, there was a significant difference between the local AMSC group and IV BMSC group (p < 0.001).
Regarding IV transplantation, collagen deposition was significantly lower in the IV AMSC-transplant group compared to the IV BMSC-transplant group (p < 0.001).
Evaluation of CM-Dil-positive cells and VEGF expression in the endometrium
Immunofluorescence staining was used to assess the expression of VEGF and the recruitment of CM-DIL+ cells in the uterus (Fig. 4).
Fig. 4.
Immunofluorescence of VEGF expression and CM-Dil-positive cells of the control (a), Asherman (b), local BMSC (c), IV BMSC (d), local AMSC (e), and IV AMSC (f) groups. h (a, p < 0.001, and d, p < 0.01 vs. the control; b, p < 0.001 vs. Asherman’s group; c, p < 0.01 vs. local AMSC; e, p < 0.05 vs. local BMSC; f, p < 0.001 vs. local BMSC and AMSC; g, p < 0.001 vs. local AMSC). f (a, p < 0.001 vs. the control and Asherman groups; b, p < 0.001 vs. other groups). Each value is mean ± SEM. Part of this figure (a and b) has been published in our previous study [19]
The results showed a decrease in VEGF expression after AS induction relative to the control group (p < 0.001). Injection of both types of stem cells resulted in a significant increase in the VEGF in both local and systemic transplantation (p < 0.001). We found that the systemic transplantation was more effective than local injection (p < 0.001 for AMSC; p < 0.05 for BMSC). Further investigations showed the most pronounced increase in VEGF expression after the systemic administration of BMSCs. Although there was no significant difference to IV-injected AMSCs, systemic administration of BMSCs caused a more pronounced increase in VEGF expression than the local injection of cells (p < 0.001). Furthermore, the local injection of BMSCs increased the VEGF more pronouncedly than locally-injected AMSCs (p < 0.01).
As shown in Fig. 4, in all treated groups, some of the injected cells were localized in the uterus that appears in red. Local administration of BMSCs resulted in a greater increase in the recruitment of CM-DIL+ cells to the injured area than systemic transplantation (p < 0.001). Furthermore, the study of different cell sources and transplantation routes showed the lowest recruitment of CM-DIL+ cells in the systemic transplantation of BMSCs compared to other groups (p < 0.001).
Discussion
Recent studies have shown that MSCs have therapeutic potential in AS [10, 11]. Although these cells are mostly derived from bone marrow, recently adipose-derived stem cells have drawn attention because of easier access. Despite definitely established similarities between stromal cells derived from bone marrow and adipose tissue, notable cell-specific differences have also been reported [13, 14]. Therefore, the present study aimed to comparatively investigate the regenerative effects of BMSCs or AMSCs on the endometrium following local or systemic injection in an AS model.
The results of our study showed that both local and systemic transplantation of AMSCs or BMSCs resulted in endometrial regeneration by increasing epithelial thickness and VEGF expression, and decreasing the percentage of collagen deposition. Collagen acts as an agent between the cell and the extracellular matrix and maintains the cell morphology, proliferation, migration, and differentiation through focal adhesion and certain signaling pathways such as mitogen-activated protein kinase (MAPK) [20]. The pathological deposition of collagen leads to an alteration in the differentiation environment of endometrial stem cells, which is responsible for the regeneration of the endometrium [21]. The deregulation of the proliferation and differentiation of these stem cells leads to endometrial diseases [22].
We found that although the local injection of adipose-derived stem cells caused the most pronounced reduction in collagen deposition, systemic transplantation of BMSCs was more effective in increasing VEGF expression. Regarding endometrial thickness, local injection of AMSCs or BMSCs was more effective than the systemic transplantation. In the study of Domnina et al., bone marrow and endometrial MSC were administered intravenously or locally to an AS model, and reported that the different cell sources exhibited similar positive effects on fertility and litter size [23]. They also observed that the treatment efficacy did not depend on the transplantation route of the cells, but local injection was more effective than systemic transplantation in the conception rate.
The study of Santamaria et al. showed that although transplantation of MSC-CD133+ cells improved endometrial regeneration in the Asherman model, the injected cells accounted for only 0.6% of total uterine cells, which is consistent with our study in which the percentage of cells reaching the injured area was low. The uterine cavity can be repaired by endometrial remnants even in severe damage [8], and our results suggested that even a small number of transplanted cells that recruit into the uterus can regenerate the uterine tissue.
In the present study, more cells were injected in the systematic route than in the local transplantation, but the percentage of CM-DIL+ cells was higher in the local injection. In the systemic transplantation, the injected cells travel a farther distance to reach the target area. In addition to migrating to other tissues, these cells are more likely to die due to exposure to blood toxic factors. Our results confirmed the study by Liu et al. that transplantation of 1 × 106 stem cells resulted in a lower percentage of injected cells in the damaged endometrium compared to the transplantation of 1 × 105 stem cells [15].
They concluded that the mortality occurred when the number of injected cells exceeded a certain threshold. We found that the cell migration to the uterus was lower in the IV BMSC group compared to the other transplanted groups.
A comparative study of biological characteristics of BMSCs and AMSCs showed that expression of CD106 was present on BMSC but absent on AMSC [14]. CD106, also known as VCAM-1, is a cell adhesion molecule that also mediates cell migration, which may be the reason for the weaker migratory ability of BMSCs compared to AMSC [14]. Several studies have reported lower VEGF expression in the endometrium of AS model and its relationship with impaired angiogenesis [10, 24, 25]. Gao et al. reported that systemic administration of BMSCs restored the functional endometrium in the rat model of severe AS by increasing VEGF and bFGF [10]. We confirmed the results of their study that VEGF expression and angiogenesis increased in the endometrium after the administration of all cell types.
By binding to VEGFR and stimulating the intracellular pathway of FAK/paxillin, VEGF enhances cell migration and proliferation and plays role in the growth of vascular endothelial cells and angiogenesis [26]. Angiogenesis helps to transport nutrients and oxygen to the damaged area and is essential for the growth of capillaries throughout the wound healing process [27]. Our results showed that some CM-DIL+ cells expressed VEGF, indicating involvement of the transplanted cells in vessel formation. In agreement with our study, Liu et al. reported that some of GFP-labeled stem cells expressed vimentin following the administration of these cells. Vimentin is a marker of stromal cells, suggesting that the transplanted cells not only were recruited to the endometrium but also differentiated into stromal cells [15]. They detected the transplanted cells in the glandular and epithelial compartments 8 months after cell injection [28]. It seems that the stroma is the primary target for stem cell recruitment and uterine tissue regeneration after the injury [15]. The study of different injection routes and cell types showed the highest expression of VEGF in intravenous BMSC-treated AS group. Furthermore, the number of CM-DIL+ cells that committed the vessel wall was higher in the intrauterine BMSC-injected groups. A proteomic and genomic study revealed that AMSCs expressed the genes mostly involved in cellular communication, but BMSCs upregulated the WNT signaling and differentiation pathway genes. Furthermore, markers such as TGFβ1 and VEGF were more expressed in BMSC than in AMSC [13].
In the present study, we made a comprehensive comparison of the effects of different cell types and the route of injection on collagen deposition, and demonstrated that although some reduction was observed in all groups, the greatest improvement in the removal of fibrosis was noticed in the local AMSC group.
It seems that AMSCs produce a comparably more regenerative impact with varied effects on collagen integrins and receptors, as well as the MAPK signaling pathway. This argument deserves further investigation.
Taken together, both local and systemic transplantation of cells exhibited regenerative effects on the endometrium, but the local injection of AMSCs caused the most pronounced reduction in collagen deposition and increase in the endometrial thickness. Systemic BMSC was also more effective to increase VEGF expression. Because the fibrosis and replacement of the epithelial cells serve as an effective therapeutic strategy for AS, AMSCs may be more appropriate to restore the damaged uterine than BMSCs.
Acknowledgments
This study was supported by the Hamadan University of Medical Sciences (9804042566).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no conflicts of interest.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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