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Intraovarian injection of 3D-MSC-EVs-ECM gel significantly improved rat ovarian function after chemotherapy

Reproductive Biology and Endocrinology volume 22, Article number: 125 (2024) Cite this article

Abstract

Background

Restoring the function of the ovary is important for chemotherapy-induced ovarian failure (COF) patients. Stem cell and extracellular vesicles (EVs) therapy show promise but need further improvement.

Methods

Human umbilical cord mesenchymal stem cells (hUC-MSCs) were primarily cultured and further three-dimensional (3D) cultured using an ultra-low attachment surface method. The expression levels of nutritional cytokines and immunomodulatory and stemness-related genes of 3D-cultured hUC-MSCs were analyzed. EVs were isolated by ultracentrifugation and characterized. Ovaries were decellularized with sodium dodecyl sulfate to obtain extracellular matrix (ECM). Lyophilized EVs from three-dimensional (2D) or 3D hUC-MSCs were mixed with ECM to prepare the 2D/3D-MSC-EVs-ECM gels. The therapeutic effect of the MSC-EVs-ECM gel on cyclophosphamide (CTX) -treated rats was analyzed through various tests. RNA sequencing was used to analyze the expression changes of genes before and after treatment.

Results

After culturing in ultra-low attachment dishes, hUC-MSCs aggregated into spheroids and significantly upregulated the expression levels of immunomodulatory and stemness-related genes. The total EVs yield was also upregulated (5.6-fold) after 3D culture. The cell viability of CTX-treated ovarian granulosa cells (OGCs) was significantly rescued by coculture with the 3D-MSC-EVs-ECM gel. Hormones indicative of ovarian function, AMH, E2, and FSH, were recovered in both the CTX + 2D-MSC-EVs-ECM gel group and the CTX + 3D-MSC-EVs-ECM gel group, while the apoptosis-related protein Bax was significantly downregulated. The 3D-MSC-EVs-ECM gel was more effective than the 2D-MSC-EVs-ECM gel. Significantly differentially expressed genes, such as Hbb-b1, Gpd1, and Sirpa, were detected by RNA sequencing. Hbb-b1 was increased in the ovaries of CTX-treated rats, and this increase was attenuated by injecting the 2D/3D-MSC-EVs-ECM gel. Gpd1 was increased after CTX treatment, and this increase was reversed by the 3D-MSC-EVs-ECM gel. Sirpa was decreased in the ovaries of CTX-treated rats, and this decrease was attenuated by injecting the 3D-MSC-EVs-ECM gel.

Conclusions

Our study demonstrated that the 3D-MSC-EVs-ECM gel is an efficient strategy for the recovery of ovarian function in CTX-induced ovarian failure.

Introduction

In recent years, the burden of cancer has risen worldwide in adults, adolescents, and children [1, 2]. Current cancer treatments include chemotherapy, radiotherapy, surgery, and immunotherapy [3]. Chemotherapy is a common treatment option [4]. However, chemotherapy can cause many side effects, including myelosuppression, gastrointestinal reactions, and liver and kidney dysfunction. Among these adverse effects, the major concern of survivors and their parents are gonadal dysfunction and infertility [5]. Many chemotherapy drugs can cause ovarian failure, known as chemotherapy associated ovarian failure (COF) [6]. Cyclophosphamide (CTX) has a high risk of causing COF. CTX was the first chemotherapy drug to be associated with ovarian failure. It can induce ovarian granulosa apoptosis and follicular atresia and accelerate the activation of the primordial follicles [7, 8].

Currently, there are several available methods for ovarian protection from COF, such as embryo and oocyte cryopreservation, ovarian tissue cryopreservation, and gonadotrophin-releasing hormone analogs [6, 7, 9]. However, these methods have some limitations. For embryo and oocyte cryopreservation, oocytes need to be obtained through ovulation induction, which will delay tumor treatment and may increase the risk of invasion and metastasis of sex hormone-dependent tumors [6]. Also, this method cannot be performed on prepubertal girls. For ovarian tissue cryopreservation, the risk of tumor recurrence due to reintroduction of cancer cells by transplantation of cryopreserved ovarian tissue is unknown and more studies evaluating the safety of autologous ovarian transplantation in cancer patients should be needed in the future [6]. Besides, not all studies have confirmed that gonadotrophin-releasing hormone analogs can protect ovarian function, and gonadotrophin-releasing hormone analogs should be used with caution as an effective method to preserve fertility [6]. In addition, there are some novel strategies, such as stem cell therapy [6]. Mesenchymal stem cells (MSCs) are multipotent stem cells capable of cytokine secretion and immunomodulation. Stem cells have significant self-renewal capabilities and differentiate into specific tissues based on their surrounding environment and signals. Stem cells can regenerate and repair tissues by secreting cytokines and extracellular vesicles. They play an important therapeutic role in the fight against fibrosis in various organs[10]. Stem cells improve experimental colitis [11] and inhibit inflammation and fibrosis to prevent the progression of early diabetic nephropathy [12]. It has found that MSCs can reduce ROS accumulation and inhibit the AMP-dependent AMPK/mTOR signaling pathway to reduce the apoptosis of theca-interstitial cells and promote ovarian tissue repair [13]. Meanwhile, MSCs can also inhibit ovarian fibrosis through the AMPK/NR4A1 pathway [14]. However, stem cell therapy faces challenges, such as vascular embolism [15] and ethical concerns [16]. Therefore, studies have been conducted on extracellular vesicles (EVs). EVs are typically 30–200 nm in diameter and are released from multiple cell types. EVs are membrane-bound vesicles containing biomolecules such as microRNAs and proteins [17]. EV therapy can avoid many problems associated with stem cell therapy [18]. EVs have been widely used to treat multiple diseases, such as cerebral infarction [19], skin wound healing [20], and premature ovarian failure [21]. Some studies have found that EVs can upregulate the expression of Nrf2 in ovarian granulosa cells, promote the expression of oxidative stress-related proteins, inhibit cell apoptosis, and promote cell proliferation [22]. Other studies have found that human adipose-derived stem cells-EV can improve ovarian function, increase the number of follicles in premature ovarian insufficiency mice, and restore hormone levels to normal by regulating the SMAD signaling pathway [23]. However, conventional two-dimensional culture does not meet the requirements for optimal EV production. Three-dimensional (3D) culture can increase EV secretion and provide more efficacious EVs [24,25,26]. Therefore, we chose a 3D culture method to culture MSCs.

The ovaries have short blood flow, and the ovarian artery enters the ovarian hilus and branches within the ovary to supply both the cortex and medulla [27]. Thus, stem cells and EVs injected intravenously rarely reach the ovaries. Moreover, EVs injected intravenously, peritoneally, and subcutaneously are easily cleared by blood circulation [28], and EVs may accumulate in multiple non-target organs [29]. To solve these problems, some studies have used extracellular matrix (ECM) gels from the female reproduction system [30] or other gels [31, 32] to encapsulate EVs, concentrate EVs in the ovary, and sustain the release of EVs. However, no such studies have used a 3D-MSC-EVs-ECM gel to treat COF. Therefore, in the current study, we tested whether a 3D-MSC-EVs-ECM gel was effective in treating CTX-induced ovarian failure.

Materials and methods

Primary culture and identification of hUC-MSCs

The human umbilical cord (UC) was collected from healthy full-term fetuses that were born via cesarean delivery at Qilu Hospital of Shandong University. The use of the UC was approved by the Ethics Committee of the Qilu Hospital of Shandong University (KYLL-2021(KS)-086). Briefly, the UC was cut into small fragments (1–2 mm3) after the umbilical arteries and vein were dissected and placed in dishes with fresh MSC complete medium containing 90% α-MEM (Macgene Technology Ltd., Beijing, China), 10% fetal bovine serum (FBS; Bovogen Biologicals Pty Ltd, Keilor East, Victoria, Australia) and 1% penicillin and streptomycin (P/S; C125C5, New Cell & Molecular Biotech, Suzhou, China) in 5% CO2 at 37 ℃. When the cells reached 80%–90% confluence, they were trypsinized with 0.25% trypsin (C125C1, New Cell & Molecular Biotech) and passaged at a 1: 3 ratio. hUC-MSCs at passage three in the logarithmic growth period were collected to perform tri-lineage differentiation and phenotypic identification. The adipogenic, osteogenic, and chondrogenic abilities of the hUC-MSCs were detected after using the corresponding induction solution. After 4 weeks of induction, Oil Red O, Alizarin Red S, and Alcian Blue staining were used to detect adipogenic, osteogenic, and chondrogenic differentiation, respectively. In addition, hUC-MSCs were stained with antibodies against the human antigens CD29-PE (TS2/16, 303,004), CD31-PE (WM59, 303,106), CD44-PE (IM7, 25–0441-82), CD45-PE (2D1, 368,510), CD73-PE (AD2, 344,004), CD90-PE (eBio5E10,12–0909-42), CD105-PE (SN6, 12–1057-42), and CD271-PE (ME20.4, 12–9400-42). CD29, CD31, CD45, and CD73 antibodies were purchased from BioLegend (San Diego, CA, USA) and CD44, CD90, CD105, and CD271 antibodies were purchased from Invitrogen (Thermo Fisher Scientific, Inc.). The antibody dilution ratio was 1:20. The cells were then analyzed by flow cytometry (Guava easyCyte 6HT, EMD Millipore, Billerica, MA, USA), and the data were examined using FlowJo (version 10, Tree Star Inc., Ashland, OR, USA).

Three-dimensional culture of hUC-MSCs

hUC-MSCs (passages 3–6) were cultured in T75 cm2 flasks. For 3D culture, 5 × 106 hUC-MSCs (passages 3–6) were seeded in ultra-low attachment dishes (633,180, Greiner, Frickenhausen, Germany). 3D-cultured hUC-MSCs were stained with PKH26 (MIDI26, Sigma-Aldrich, USA) and 4′,6-diamidino-2-phenylindole (DAPI, ab104139, Abcam) and observed under a fluorescence microscope (BX53; Olympus, Tokyo, Japan).

Cell proliferation assay

Cell proliferation was determined using carboxyfluorescein diacetate succinimidyl ester for both 2D- and 3D-cultured cells (CFSE; BD Biosciences, San Jose, CA, USA). Cells were incubated for 5 min with 10 μM of CFSE in phosphate-buffered saline (PBS) at 37 ℃. All assays were performed using flow cytometry. Cell proliferation analyses were performed using ModFit LT software (version 3.2; Verity Software House, Topsham, ME, USA).

Quantitative real-time polymerase chain reaction (qRT-PCR) assay

Total RNA from 2D- and 3D-cultured cells was extracted using an RNA fast kit (Fastagen, Shanghai, China). Total RNA was reverse-transcribed using the ReverTra Ace qPCR RT Kit (Toyobo, Osaka, Japan). qRT-PCR was performed to detect the transcript levels of Oct4, Nanog, Igf, Sox2, Hgf, Tgfb1, Sdf, Vegf, Ido1, Il6 and Pge2 using an SYBR Green real-time PCR master mix (Toyobo, Osaka, Japan). Relative expression levels were normalized to those of GAPDH. The value for 2D-cultured cells was set to 1. Primer sequences used are listed in Supplementary Material 1.

Isolation and purification hUC-MSC-derived EVs

hUC-MSCs were cultured in flasks and ultra-low attachment dishes with EVs-depleted FBS. EVs-depleted FBS was prepared by separating EVs from FBS using ultracentrifugation at 100,000 × g for 18 h at 4 ℃ (Thermo Fisher Scientific, Sorvall Lynx 6000, Osterode, Germany) [33]. After 48 h, cell culture supernatants were collected to isolate the EVs. The 2D and 3D cell culture supernatants were centrifuged at 300 × g for 10 min, 2,000 × g for 20 min, and 10,000 × g for 30 min at 4 ℃ to eliminate the dead cells and cell debris. The supernatants were filtered using a 0.22-μm filter (Millipore). Then, the supernatants were centrifuged at 100,000 × g for 70 min, resuspended in PBS, and centrifuged again at 100,000 × g for 70 min at 4 ℃. Finally, EVs purified from the media of 2D- and 3D-cultured cells (2D-EVs and 3D-EVs, respectively) were resuspended in 100–200 μL of PBS and stored at -80 ℃.

Transmission electron microscopy (TEM)

The morphology of the EVs was assessed by TEM, which was conducted by Shanghai XP Biomed Co., Ltd. (Shanghai, China). Droplets were deposited onto a TEM copper grid for more than 1 min. The grid was then negatively stained with phosphotungstic acid for 5 min, blotted with filter paper, and dried at room temperature. Finally, the EVs were observed under a transmission electron microscope (JEM-1200EX, JEOL, Tokyo, Japan) and photographed.

Nanoparticle tracking analysis (NTA)

The size distribution and concentration of EVs were determined using NTA. The EV nanoparticle tracking analysis was conducted by Shanghai XP Biomed Co., Ltd. (Shanghai, China). Briefly, EVs were diluted in PBS and injected into the ZetaView particle tracker (ZetaView PMX 110, Particle Metrix, Meerbusch, Germany). NTA measurements were recorded and analyzed at 11 positions. ZetaView software (version 8.04.02 SP2) was used to analyze the data. We used the bicinchoninic acid assay (BCA) method to detect the protein concentration of EVs and combined it with NTA analysis to determine that there were 109 particles per μg of protein and 5 × 1011 particles per 0.5 mg of protein.

Preparation of ovarian ECM, lyophilized EVs, and gels

Five-week-old female Sprague–Dawley rats (Sibeifu Beijing Biotechnology Co. Ltd., Beijing, China) were euthanized for ovary collection. The ovaries were decellularized with 0.2% sodium dodecyl sulfate (SDS, Solarbio, Beijing, China) for 12 h at 18–20 ℃ on a horizontal shaker at 50 rpm. Then, the decellularized ovaries were washed in PBS eight times for 2 h. A Masson’s trichrome stain kit (G1340, Solarbio) was used to confirm that the ovaries were completely decellularized. The ovarian ECM was lyophilized using a Scientz − 12N (Ningbo, China). The lyophilized ovarian ECM was digested with pepsin (Solarbio) for 72 h at a final concentration of 10 mg/mL to obtain ovarian ECM solutions. A gel consisting of chitosan (419,419, Sigma-Aldrich, USA)/β-sodium glycerophosphate (Merck, Darmstadt, Germany)/gelatin (Amresco) loaded with ovarian ECM solutions was obtained as described by Xu et al. [31]. Then, 2D-EVs and 3D-EVs were lyophilized and mixed with the ECM gel at 0.5 mg/mL. The viscosity of the gel was measured using an NDJ-9S viscometer (Shanghai Performance Tai Electronic Technology Co., Ltd, Shanghai, China) at 60 rpm [34].

Isolation and characterization of rat ovarian granulosa cells (OGCs)

Rat OGCs were isolated from four-week-old female Sprague–Dawley rats as described previously [35]. The rat OGCs were cultured in DMEM/F12 (Macgene) medium with 10% FBS and 1% P/S at 37 ℃, 5% CO2. Immunofluorescence staining was used to identify the follicle-stimulating hormone receptor (FSHR, GB11275-1, Servicebio, Wuhan, China) specifically expressed in granulosa cells. The nuclei were stained with DAPI (ab104139, Abcam).

Labeling of EVs and cellular uptake in vitro

2D- and 3D-EVs were labeled with PKH26 (Sigma) according to the manufacturer’s instructions. These EVs were then co-cultured with OGCs for 24 h (the final concentration of EVs was 50 μg/mL). The nuclei of the OGCs were stained with DAPI (Abcam). The fluorescence signals of PKH26 and DAPI were observed under a fluorescence microscope.

Cell counting kit-8 assay (CCK-8) and cytocompatibility assay in vitro

Rat OGCs were cultured in 96-well plates to reach 70–80% confluence. Then, rat OGCs were divided into six groups: Control, ECM gel, CTX, CTX + ECM gel, CTX + 2D-MSC-EVs-ECM gel, and CTX + 3D-MSC-EVs-ECM gel groups (gel: media = 1:10; final concentration of CTX (PHR1404, Sigma-Aldrich, USA), 6 mg/mL; final concentration of EVs, 10 μg/well). After 48 h, cell viability was detected using a CCK-8 assay (C6005, New Cell & Molecular Biotech). Absorbance was measured with an enzyme-labeled analyzer (DNM-9602, Beijing Perlong New Technology Co., Ltd.) at a wavelength of 450 nm.

Establishment of COF rat model and therapeutic experiments

Five-week-old female Sprague–Dawley rats were obtained from Sibeifu Beijing Biotechnology Co., Ltd., Beijing, China. The use of rats was approved by the Ethics Committee of the Qilu Hospital of Shandong university (DWLL-2021–147). The rats were maintained at 22 ± 2 ℃ under a 12-h light/dark cycle, with free access to water and food. The rats were randomly divided into six groups (5–7 rats per group): Control, ECM gel, CTX, CTX + ECM gel, CTX + 2D-MSC-EVs-ECM gel, and CTX + 3D-MSC-EVs-ECM gel group. The Control group received no treatment. The first day of the CTX injection was designated as day 1. The CTX, CTX + ECM gel, CTX + 2D-MSC-EVs-ECM gel and CTX + 3D-MSC-EVs-ECM gel groups received intraperitoneal injections of CTX (50 mg/kg on day 1 and 8 mg/kg on days 2–14). The CTX group did not receive any treatment after 14 days. The ECM group received an intra-ovarian injection of ECM gel on day 15. The CTX + ECM gel, CTX + 2D-MSC-EVs-ECM gel, and CTX + 3D-MSC-EVs-ECM gel groups received intra-ovarian injections of ECM gel, 2D-MSC-EVs-ECM gel, and 3D-MSC-EVs-ECM gel, respectively, on day 15. Each ovary was injected with 25 μL of gel containing 0.5 mg/mL of EVs. All the rats were weighed daily. All vaginal smears were collected daily and stained with hematoxylin and eosin (H&E) to determine the estrous cycle phase. All rats were euthanized on day 30 for further analysis.

Enzyme-linked immunosorbent assay (ELISA)

Blood samples were obtained by cardiac puncture. Serum was collected by centrifuging at 1,200 × g for 15 min at 4 ℃, and it was stored at –20 ℃ for ELISA analysis. Serum levels of FSH (Cloud-clone Corp, CEA830Ra), estradiol (E2, Cloud-clone Corp, CEA461Ge), and anti-Mullerian hormone (AMH, Cloud-clone Corp, CEA228Ra) were detected using an ELISA kit according to the manufacturer’s instructions.

H&E staining

Rat ovaries were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned into 5-μm sections. Five discontinuous sections of each ovary were selected for follicle counting and morphological observation. The ovarian sections were dewaxed and rehydrated for H&E staining.

RNA sequencing

Ovary tissues (Control, CTX, CTX + 2D-MSC-EVs-ECM gel, and CTX + 3D-MSC-EVs-ECM gel groups) were chosen for RNA sequencing. Each group had one biological replicate. RNA sequencing and bioinformatics analyses were performed at Shandong Xiuyue Biotechnology Co. Ltd.

Western blotting

The protein lysates from exosomes or ovary tissues were prepared following standard protocols, and the protein content was determined using a BCA protein assay kit (P0010S, Beyotime, Shanghai, China). Equal amounts of proteins were electrophoresed on 7.5–15% SDS-PAGE gels and transferred onto polyvinylidene difluoride membranes (ISEQ00010, Merck Millipore, Darmstadt, Germany). Membranes were blocked for 2 h in blocking buffer composed of 5% non-fat dry milk in tris-buffered saline containing 0.1% Tween 20 at room temperature and were incubated with primary antibodies overnight at 4 ℃. The polyvinylidene difluoride membranes were then washed three times for 10 min each and incubated with secondary antibodies for 1 h at room temperature. A chemiluminescence detection system (Shanghai Jiapeng Technology Co., Ltd., Shanghai, China) was used to visualize the protein bands. Protein expression was expressed as relative abundance normalized to that of GAPDH (1:1000, 5174, Cell Signaling Technology). The primary antibodies used were anti-Alix (1:1000, 18269S, Cell Signaling Technology), anti-CD63 (1:1000, 25,682–1-AP, Proteintech), anti-CD9 (1:1000, 13174S, Cell Signaling Technology), anti-CD81 (1:1000, 52892S, Cell Signaling Technology), anti-TSG101 (1:1000, ab125011, Abcam, Cambridge, UK), anti-calnexin (1:1000, 2433S, Cell Signaling Technology), and anti-Bax (1:1000, 2772S, Cell Signaling Technology). The secondary antibody used was Goat Anti-Rabbit IgG (H + L) HRP (1:5000, AB0101, Abways).

Statistical analysis

All experiments were performed in triplicate. All data were analyzed using GraphPad Prism version 9 (GraphPad, San Diego, CA, USA). Comparisons between two groups or multiple groups were analyzed using Student’s t-test or one-way ANOVA. Quantitative data were expressed as mean ± standard deviation (SD). p < 0.05 was considered statistically significant.

Results

Morphology and identification of primary hUC-MSCs

After seven days, hUC-MSCs migrated out of the tissue and showed a typical fibroblast-like phenotype (Fig. 1A). Their ability to differentiate into adipocytes, osteoblasts, and chondrocytes was confirmed by specific staining. The lipid droplets of the adipocytes were stained red using Oil Red O, the calcified ECM of the osteoblasts was stained red using Alizarin Red S, and the proteoglycans of the chondrocytes were stained blue using Alcian Blue (Fig. 1B). Flow cytometry showed that isolated hUC-MSCs positively expressed CD29, CD44, CD73, CD90, and CD105 and negatively expressed CD31, CD45, and CD271 (Fig. 1C).

Fig. 1
figure 1

Identification of hUC-MSCs. A Microscopic image of hUC-MSCs. Scale bar: 200 μm. B hUC-MSCs differentiated into adipocytes, osteoblasts, or chondrocytes after corresponding induction, indicated by Oil Red O, Alizarin Red S, and Alcian Blue staining. Scale bars: 200 μm. C Detection of the specific markers of hUC-MSCs by flow cytometry illustrated that hUC-MSCs were positive for CD29, CD44, CD73, CD90, and CD105 and negative for CD31, CD45, and CD271

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Morphology and function of hUC-MSCs in 3D culture

A schematic diagram of hUC-MSCs cultured in an ultra-low attachment dish is shown in Fig. 2A. After 48 h, hUC-MSCs aggregated into spheroids, the cell membranes were stained red with PKH26, and the nuclei were stained blue with DAPI (Fig. 2B). The diameters of the spheroids were approximately 200 μm. Compared with hUC-MSCs in 2D culture, hUC-MSCs in 3D culture had a significantly higher proliferation index (50.522 ± 2.843 vs. 38.279 ± 1.036, p < 0.0001) (Fig. 2C). Otherwise, the expression levels of nutritional cytokines and immunomodulatory and stemness-related genes were significantly upregulated in hUC-MSCs in 3D culture. These included Oct4, Nanog, Igf, Sox2, Hgf, Tgfb1, Sdf, Vegf, Ido1, Il6 and Pge2 (Fig. 2D).

Fig. 2
figure 2

Morphology, proliferation, and gene expression of 3D-cultured hUC-MSCs. A The schematic diagram of hUC-MSCs cultured in an ultra-low attachment dish. B hUC-MSCs in 3D culture. The diameter of the spheroid is approximately 200 μm. Scale bars: 50 μm. C Cell proliferation of hUC-MSCs in 2D and 3D culture (n = 3, ****p < 0.0001, Student’s t-test). D Expression of nutritional cytokine, immunomodulatory, and stemness-related genes (n = 3; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, Student’s t-test)

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Characterization of 2D-EVs and 3D-EVs

EVs were isolated by ultracentrifugation (Fig. 3A). 2D-EVs and 3D-EVs showed teacup-like double-sided structures (Fig. 3B). NTA analysis showed that the median diameters of the 2D-EVs and 3D-EVs were 125.9 nm and 119.3 nm, respectively (Fig. 3C). This was consistent with the general diameter criteria (30–200 nm). Western blotting results showed that exosomal markers Alix, TSG101, CD9, CD63, and CD81 were positively expressed in hUC-MSCs, 2D-EVs, and 3D-EVs. However, calnexin, which is expressed in hUC-MSCs, was negatively expressed in 2D-EVs and 3D-EVs (Fig. 3D). We compared the relationship between cell number and extracellular vesicle secretion. The concentration of 2D-EVs from 2 × 107 cells was 2.5 × 1010 particles/mL (250 particles/cell), and the concentration of 3D-EVs from 2 × 107 cells was 1.4 × 1011 particles/mL (1,400 particles/cell) (Fig. 3E). The concentration of EVs was measured using a BCA kit, and the 2D- and 3D-EVs were approximately 1.0 × 109 particles/μg. The lyophilized EVs are shown in Fig. 3F.

Fig. 3
figure 3

Characterization of EVs and gels. A EVs at the bottom of the ultracentrifuge tube. B Morphology of EVs under TEM. Scale bars: 100 μm. C Particle size distribution of EVs in NTA. D Identification of EVs by western blot. E The yield of EVs from 2D-EVs and 3D-EVs. F Appearance of lyophilized EVs. G Decellularization of ovaries. H Ovarian ECM and control ovary were stained with Masson’s trichrome stain kit. Ovarian ECM was only left with collagen which was stained blue. Scale bars: 100 μm. I Appearance of lyophilized ovarian ECM. J ovarian ECM digestion. K ECM gel. L ECM gel after 37 ℃

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Preparation and characterization of ovarian ECM gel

The ovaries were decellularized using 0.2% SDS (Fig. 3G). Masson’s trichrome staining revealed only collagen fibers in the decellularized ovary, which stained blue (Fig. 3H). However, collagen fibers, muscle fibers, and nuclei were stained and observed in the control ovary, which was stained blue, red, and blue-black (Fig. 3H). This demonstrated that the ovaries were completely decellularized. The lyophilized and digested ovarian ECM are shown in Fig. 3I and J. Gel solutions were prepared using chitosan/β-sodium glycerophosphate/gelatin loaded with ovarian ECM solutions (Fig. 3K). The gel solutions formed gel in 37 ℃ (Fig. 3L). The viscosity of the gel was 98 mPa·s.

Identification of rat OGCs and enhanced therapeutic efficacy of 3D-MSC-EVs-ECM gel in CTX-treated OGCs

Rat ovarian granulosa cells were extracted from the rat ovaries. The morphology of the rat OGCs was fibroblastic (Fig. 4A). Immunofluorescence staining revealed that FSHR was specifically expressed in the cytoplasm of the OGCs (Fig. 4B). The cytoplasm was stained green, and the nuclei were stained blue. Moreover, 2D-EVs and 3D-EVs were successfully taken up by the OGCs (Fig. 4C). The nuclei of the OGCs were stained blue. The CCK-8 assay showed that the ECM gel did not affect normal rat OGCs, indicating that the ECM gel had good cytocompatibility. The viability of the CTX-treated OGCs was significantly enhanced by treatment with the 2D-MSC-EVs-ECM and 3D-MSC-EVs-ECM gels compared with the ECM gel, and the 3D-MSC-EVs-ECM gel was more effective (Fig. 4D).

Fig. 4
figure 4

Identification of rat OGCs and enhanced efficacy of treatment with the 3D-MSC-EVs-ECM gel. A Morphology of rat OGCs. Scale bars: 200 μm. B Identification of rat OGCs. Rat OGCs positively expressed FSHR. Scale bars: 200 μm. C EVs were effectively taken up by rat OGCs. Scale bars: 200 μm. D CCK-8 assay in rat OGCs (n = 3; *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t-test)

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Enhanced therapeutic efficacy of 3D-MSC-EVs-ECM gel in CTX-treated rat model

The procedures for animal experiments are described in Fig. 5A. Intraovarian injection of 25 μL of gel did not cause obvious tissue swelling or bleeding of the ovaries (Fig. 5B). At the beginning of the experiment, all rats in each group had similiar body weights; however, their body weights changed at day 30 (Fig. 5C). The Control and ECM gel groups had similar body weights, whereas the body weights of the other groups decreased. The CTX group showed the greatest decrease. The body weight of the CTX + ECM gel group recovered to a greater extent than that of the CTX group. The body weights of the CTX + 2D-MSC-EVs-ECM gel group and CTX + 3D-MSC-EVs-ECM gel group recovered more than those of the CTX + ECM gel group. However, the CTX + 3D-MSC-EVs-ECM gel group showed the best recovery. The estrus cycles of the rats were monitored, and they had regular estrus cycles at the beginning of the experiments (Fig. 5D). The CTX group had a longer estrous cycle for days after CTX injury. However, the CTX + 2D-MSC-EVs-ECM gel group and the CTX + 3D-MSC-EVs-ECM gel group recovered (Fig. 5E).

Fig. 5
figure 5

Animal experimental procedures and the weights and estrous cycle days in the rat model. A Schematic illustration of the procedures of animal experiments. B Intra-ovarian injection and after injection. C Body weight in each group before rats were sacrificed (n = 5–7; *p < 0.05, **p < 0.01, Student’s t-test). D Regular estrous cycles. a: Proestrus; b: Estrus; c: Metestrus; d: Diestrus. Scale bars: 200 μm (E) Estrous cycles in each group (n = 5–7; *p < 0.05, **p < 0.01, Student’s t-test)

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For primordial, primary, secondary, and preovulatory follicles, there were no differences between the Control and the ECM gel groups. There were a minimum number of primordial and secondary follicles in the CTX group. Furthermore, compared to the CTX group, the numbers of primordial follicles and secondary follicles had increased in the CTX + ECM gel, CTX + 2D-MSC-EVs-ECM gel, and CTX + 3D-MSC-EVs-ECM gel groups. The CTX + 3D-MSC-EVs-ECM gel group had the most primordial and secondary follicles. However, there were no differences in the number of primary follicles between the CTX + 2D-MSC-EVs-ECM gel and CTX + 3D-MSC-EVs-ECM gel groups (Fig. 6A).

Fig. 6
figure 6

H&E staining of ovaries, ELISA, and expression of Bax. A Representative images and analysis of H&E staining in the ovaries of each group. Scale bars: 200 μm. n = 3; *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t-test. B, C, D The serum levels of AMH, E2, and FSH in each group (n = 3; ***p < 0.001, ****p < 0.0001, Student’s t-test). E Western blotting analysis of Bax in each group (n = 3; *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t-test)

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The hormones indicative of ovarian function, AMH and E2, decreased, and FSH levels increased significantly with CTX treatment. In comparison, treatment with the CTX + 2D-MSC-EVs-ECM gel and the CTX + 3D-MSC-EVs-ECM gel gave less of a reduction in AMH and E2 and less of an increase in FSH than treatment with CTX alone. In particular, the CTX + 3D-MSC-EVs-ECM gel group recovered significantly (Fig. 6B–D).

Bax expression was detected by western blot analysis. There was no difference between the Control and ECM gel groups. The expression of Bax was the highest in the CTX group. The expression of Bax decreased in the CTX + ECM gel, CTX + 2D-MSC-EVs-ECM gel, and CTX + 3D-MSC-EVs-ECM gel groups compared to that in the CTX group (Fig. 6E).

RNA sequencing of rat ovaries

We named the Control group [Con], the CTX group [CTX], the CTX + 2D-MSC-EVs-ECM gel group [2D], the CTX + 3D-MSC-EVs-ECM gel group [3D]. Venn diagram software was used to analyze the upregulated and downregulated differentially expressed genes in CTX vs. Con and 2D vs. CTX (Fig. 7A) to detect the influence of the 2D-MSC-EVs-ECM gel on the CTX-treated ovaries, CTX vs. Con and 3D vs. CTX (Fig. 7B) to detect the influence of the 3D-MSC-EVs-ECM gel on the CTX-treated ovaries, and 2D vs. CTX and 3D vs. CTX to detect the difference in the effect between 2D-MSC-EVs-ECM gel and 3D-MSC-EVs-ECM gel (Supplementary Material 2) (Fig. 8C). Moreover, we compared the top 20 upregulated and downregulated genes in CTX vs. Con, 2D vs. CTX, and 3D vs. CTX, with counts ≥ 100 as a standard. We found that Hbb-b1 was upregulated in CTX vs. Con and downregulated in 2D vs. CTX and 3D vs. CTX; Gpd1 was upregulated in CTX vs. Con and downregulated in 3D vs. CTX; ENSRNOG00000013279 and Testin were upregulated in CTX vs. Con and downregulated in 2D vs. CTX; and Clec4a3 and Sirpa were downregulated in CTX vs. Con and upregulated in 3D vs. CTX. We also found that ENSRNOG00000062337, Irf7, Oasl2, Cxcl10, Lyz2, Mpeg1, Rt1-s3, and Lilrb4 were upregulated in 2D vs. CTX and 3D vs. CTX, and Col7al, Ptprn, and Nap1l5 were downregulated in 2D vs. CTX and 3D vs. CTX. Furthermore, we screened for differentially expressed genes between the CTX + 3D-MSC-EVs-ECM gel group and the CTX + 2D-MSC-EVs-ECM gel group with a criterion of |log2 (fold change)|≥ 1, p < 0.05, and counts ≥ 100. There were 233 differentially expressed genes, including 89 upregulated and 144 downregulated genes (Supplementary Material 3). The Kyoto Encyclopedia of Genes and Genomes pathway enrichment results showed that the top three pathways enriched for differentially expressed genes were cytokine-cytokine receptor interaction (map04060), neuroactive ligand-receptor interaction (map04080), and human papillomavirus infection (map05165) (|log2 (fold change)|≥ 1, p < 0.05) (Fig. 8A). The Gene Ontology (GO) analysis revealed that the top three pathways were enriched for developmental process (GO:0032502), membrane part (GO:0044425), and protein binding (GO:0005515) (|log2 (Fold change)|≥ 1, p < 0.05) (Fig. 8B–D).

Fig. 7
figure 7

Venn diagrams. The venn diagrams containing lists of upregulated and downregulated differentially expressed genes in the datasets of CTX vs. Con and 2D vs. CTX (A), CTX vs. Con and 3D vs. CTX (B), 2D vs. CTX and 3D vs. CTX (C)

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Fig. 8
figure 8

KEGG and GO analysis of differentially expressed genes between 3D vs. 2D. A KEGG enrichment analysis of differentially expressed genes between 3D vs. 2D. B GO classification of the biological processes. C GO classification of the cellular component. D GO classification of the molecular function

Full size image

Discussion

Chemotherapy-induced ovarian failure is a long-term adverse effect of chemotherapy drugs, especially alkylating agents [9].It disrupts endocrine and reproductive ovarian function and has become increasingly common in recent years. There are many methods to prevent and treat COF [6]; however, these methods have shortcomings. Stem cells have shown promise. However, owing to the ethical and safety issues of stem cells, EVs derived from mesenchymal stem cells have been used as an alternative therapy in regeneration medicine [36] and play important roles in many diseases. In our study, we used ultra-low attachment dishes to culture hUC-MSCs and produced 2D- and 3D-EVs. We then mixed these EVs with chitosan/β-sodium glycerophosphate/gelatin loaded with ovarian ECM solutions to obtain 2D- and 3D-MSC-EVs-ECM gels. We found that the 3D-MSC-EVs-ECM gel most significantly restored the cell viability of CTX-treated rat OGCs. In CTX-treated rats, the 3D-MSC-EVs-ECM gel increased the serum levels of AMH and E2 and decreased the serum levels of FSH. According to H&E staining, the 3D-MSC-EVs-ECM gel promoted an increase in the number of primordial follicles. The 3D-MSC-EVs-ECM gel decreased the expression levels of the apoptosis-related gene Bax, according to western blot analysis.

To increase the output of EVs, we used low attachment dishes to culture the hUC-MSCs. Methods for 3D culture are classified as scaffold-free or scaffold-based [37]. 3D culture can simulate the in vivo microenvironment [38]. 3D culture refers to a culture method that uses materials such as scaffolds and collagen to provide cells with a microenvironment that is closer to the living conditions in the body and can increase the communication between cells and between cells and the extracellular matrix. 3D culture can simulate the biochemical and biomechanical environment in the body, make the cytoskeleton more consistent with the reality of cells in vivo, promote the self-renewal of stem cells and inhibit their differentiation [39, 40]. 3D culture enables cells to grow and migrate in all three-dimensional spaces, which is beneficial for the molecular transfer and material exchange between cells and between cells and the extracellular matrix, and is beneficial for promoting the secretion of exosomes by cells. Ultra-low attachment dishes are scaffold free. After 48 h of 3D culture, hUC-MSCs aggregated into spheroids, and the proliferation index of the hUC-MSCs increased compared to that in 2D culture. Surprisingly, nutritional cytokines, immunomodulatory, and stemness-related genes were significantly upregulated in hUC-MSCs after 3D culture. This reflected that the immunomodulatory function, antiapoptotic function, and angiogenesis of hUC-MSCs in 3D culture were upregulated. It has been demonstrated that exosomes from 3D-cultured human embryonic stem cell can improve liver fibrosis by targeting the TGFβRII-SMADS pathway [41] and improve memory and cognitive deficits in mice with Alzheimer’s disease [42]. For EVs, the particles/cell in 3D culture were 5.6 times more than in 2D culture. The yield of EVs increased after 3D culture, which was consistent with previous studies [25, 26]. The ovary is a reproductive organ, and it has better safety than DNA-free EVs in treatment to avoid foreign DNA contamination.

Recently, it was shown that bone marrow MSC-derived exosomes can inhibit the apoptosis of cisplatin-induced ovarian granulosa cells [43], recover the estrus cycle, and increase the number of basal follicles in premature ovarian failure [35]. However, the use of EVs has many risks, as previously mentioned. Hydrogels can prolong the release of exosomes and increase their retention in vivo [32]. Organ-specific ECMs have been widely used in recent years. The ECM is a non-cellular 3D network that consists of collagen, elastin, and laminin and provides structural and functional support [44]. ECMs have emerging applications in gynecology, such as uterine ECM [45], ovarian ECM [46], oviduct ECM [47], and vagina ECM [48]. In our study, we mixed chitosan/β-sodium glycerophosphate/gelatin with ovarian ECM solutions to prepare a gel. Chitosan has been widely used for gel preparation [49]. Gelatin was used to minimize gelation time. Finally, we demonstrated that the gel itself was not harmful to rats, and the 3D-MSC-EVs-ECM gel improved multiple endpoints in CTX-treated rats.

We used ovarian tissue (Control, CTX, CTX + 2D-MSC-EVs-ECM gel, CTX + 3D-MSC-EVs-ECM gel groups) for RNA sequencing. As shown in Fig. 7A, 502 genes were differentially expressed between CTX_Con_up and 2D_CTX_down, and 1021 genes were differentially expressed between CTX_Con_down and 2D_CTX_up. As shown in Fig. 7B, 622 genes were differentially expressed between CTX_Con_up and 3D_CTX_down, and 1017 genes were differentially expressed between CTX_Con_down and 3D_CTX_up. As shown in Fig. 7C, 973 genes were differentially expressed between 2 and 3D_CTX_up, and 630 genes were differentially expressed between 2 and 3D_CTX_down. These overlapping genes need to be further analyzed and studied.

In our study, we found that the expression of Hbb-b1 increased in CTX-treated rats but decreased in CTX + 2D-MSC-EVs-ECM gel and CTX + 3D-MSC-EVs-ECM gel groups. It has been demonstrated that capsaicin can stimulate the expression of erythroid-specific genes, such as Hbb-b1, to induce the development of the erythroid lineage from bone marrow cells [50]. Hbb-b1 was downregulated in mice with intratracheal instillation of silver nanoparticles (AgNPs) because AgNPs may induce Th2-type dominant inflammatory responses and tissue damage in the lungs of mice [51]. In addition, chronic stress can affect the expression of genes involved in vascular system function, such as Hbb-b1, Hba-a2 [52], and Hbb-b1, and these can be potential markers of chronic social stress in mice [53]. Hba-a2 expression was also downregulated in the CTX + 3D-MSC-EVs-ECM gel group as compared to that in CTX treatment alone. Thus, we speculated that Hbb-b1 and Hba-a2 might be related to changes in ovarian vascular stress. Testin, a Sertoli cell secretory protein, was upregulated in the CTX group and downregulated in the CTX + 2D-MSC-EVs-ECM gel group. Some studies had shown that testin is involved in the epithelial-mesenchymal transition of endometrial cancer [54].

Gpd1 is a key enzyme regulating glycolysis and glycerol metabolism, and Gpd1 was upregulated in the CTX group and downregulated in the CTX + 3D-MSC-EVs-ECM gel group. Gpd1 may play an important role in hypoxia and lipid metabolism pathways in clear cell renal cell carcinoma [55], and Gpd1 may be a novel tumor suppressor in bladder cancer [56]. Sirpa was downregulated in the CTX group and upregulated in the CTX + 3D-MSC-EVs-ECM gel group. The CD47-SIRPA axis can mediate cancer cell immune escape and immunotherapy [57] and inhibit inside-out activation of integrin signaling in macrophages to suppress phagocytosis [58]. According to the enriched pathways of differentially expressed genes between the CTX + 3D-MSC-EVs-ECM gel group and CTX + 2D-MSC-EVs-ECM gel group, a cytokine-cytokine receptor interaction is involved in the estrous cycle in the ovaries at proestrus and estrus [59], and the neuroactive ligand-receptor interaction pathway is enriched in the differentially expressed genes between the low and the high egg production groups [60]. The differentially expressed genes could help us to identify novel targets and mechanisms for COF and how EVs function. This merits future research.

In summary, we have developed a new approach to use EVs to treat multiple diseases. Ovarian-related diseases could be treated by the injection of a 3D-MSC-EVs-ECM gel in the posterior fornix under the guidance of B-ultrasound in the future. However, several aspects of this approach could benefit from further research. Novel 3D culture methods need to be developed to improve the yield and performance of the EVs derived from them. In addition to gels, new carries need to be advanced to improve the release profile and preserve the functionality of EVs.

Conclusions

In our study, the output of EVs derived from mesenchymal stem cells increased in 3D culture compared with that in conventional 2D culture. The expression of nutritional cytokines and immunomodulatory and stemness-related genes were significantly upregulated in hUC-MSCs after 3D culture. We demonstrated that a 3D-MSC-EVs-ECM gel could recover the cell viability of CTX-treated OGCs in vitro and improve ovarian function by regulating the serum levels of AMH, E2, and FSH and the number of follicles in CTX-treated rats. Our study provides a novel cell-free therapy to treat COF and warrants further research on other diseases. Also, our study provides new evidence for the widespread development of 3D stem cell culture in the future. We chose hydrogel to encapsulate EVs, which is beneficial to protect EVs from rapid clearance, and can use the 3D grid structure and degradation process of hydrogel to regulate the sustained release of EVs, providing a new way for the targeted application of EVs. The differential genes and pathways we discovered through sequencing are helpful to find the differences between EVs derived from 3 and 2D cultured UC-MSCs, providing new targets and treatments for the treatment of COF. The in-situ injection method also provides a new way to treat ovarian-related diseases in the future, for example, injecting EV and ECM into the uterine cavity can be used to treat thin endometrium. This method can be performed under B-ultrasound guidance to avoid damage caused by surgery.

Data availability

No datasets were generated or analysed during the current study.

Abbreviations

COF:

Chemotherapy-induced ovarian failure

CTX:

Cyclophosphamide

EVs:

Extracellular vesicles

hUC-MSCs:

Human umbilical cord mesenchymal stem cells

MSCs:

Mesenchymal stem cells

3D:

Three-dimensional

2D:

Two-dimensional

UC:

Umbilical cord

ECM:

Extracellular matrix

FBS:

Fetal bovine serum

P/S:

Penicillin and streptomycin

OGCs:

Ovarian granulosa cells

DAPI:

4′,6-diamidino-2-phenylindole

CFSE:

Carboxyfluorescein diacetate succinimidyl ester

PBS:

Phosphate-buffered saline

qRT-PCR:

Quantitative real-time polymerase chain reaction

TEM:

Transmission electron microscopy

NTA:

Nanoparticle tracking analysis

BCA:

Bicinchoninic acid assay

SDS:

Sodium dodecyl sulfate

FSHR:

Follicle-stimulating hormone receptor

H&E:

Hematoxylin and eosin

ELISA:

Enzyme-linked immunosorbent assay

CCK-8:

Cell counting kit-8 assay

AMH:

Anti-Mullerian hormone

E2:

Estradiol

FSH:

Follicle-stimulating hormone

α-MEM:

α-Minimal essential medium

DMEM/F12:

Dulbecco's modified eagle medium/nutrient mixture F-12

Gapdh:

Glyceraldehyde-3-phosphate dehydrogenase

Alix:

Apoptotic linked-gene-product 2 (ALG-2) interacting protein X

TSG101:

Tumor susceptibility gene 101

Bax:

BCL2 associated X

KEGG:

Kyoto encyclopedia of genes and genomes

GO:

Gene ontology

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Acknowledgements

The authors sincerely thank Mr. Shizhe Liu and Mr. Peifeng Ji for their guidance in the analysis of gene sequencing data.

Funding

Natural Science Foundation of Shandong Province, No. ZR2023MH079; JiNan Science and Technology Bureau, No. JNKCJN202201; Natural Science Foundation of Shandong Province, No. ZR2020MH063; Shandong Province Enterprise Innovation Ability Enhancement Project, No. 2022TSGC2001.

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Author notes

  1. Yaping Zhang and Dong Li contributed equally to this work.

Authors and Affiliations

  1. Department of Pediatrics, Qilu Hospital of Shandong University, No. 107 Wenhua West Road, Jinan, Shandong Province, 250012, China

    Yaping Zhang, Yi Han, Min Wu, Shule Zhang & Xiuli Ju

  2. Laboratory of Cryomedicine, Qilu Hospital of Shandong University, Jinan, Shandong Province, 250012, China

    Dong Li, Yi Han, Min Wu, Shule Zhang, Huixian Ma, Linghong Liu & Xiuli Ju

  3. Department of Anesthesiology, Shanghai Jiaotong University First People’s Hospital (Shanghai General Hospital), Shanghai, China

    Yaping Zhang

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  1. Yaping Zhang
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Contributions

Yaping Zhang and Dong Li: Investigation, Methodology, Visualization, Writing - Original Draft. Yi Han, Min Wu and Shule Zhang: Investigation, Methodology. Huixian Ma and Linghong Liu: Validation, Project administration, Conceptualization, Supervision, Writing - Review & Editing. Xiuli Ju: Conceptualization, Funding acquisition, Writing - Review & Editing.

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Correspondence to Xiuli Ju.

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Ethics approval and consent to participate

The use of the umbilical cords was approved by the Ethics Committee of the Qilu Hospital of Shandong University (Title: Study on the treatment of chronic inflammatory diseases by mesenchymal stem cells; Approval document of Ethics Committee: Qilu Hospital of Shandong university; IACUC Issue No.: KYLL-2021(KS)-086; Approval date: February 20, 2021). The use of rats was approved by the Ethics Committee of the Qilu Hospital of Shandong University (Title: Study on the treatment of premature ovarian failure by mesenchymal stem cells in rats; Approval document of Ethics Committee: Qilu Hospital of Shandong university; Approval No.: 2021-147; IACUC Issue No.: DWLL-2021-147; Approval date: December 15, 2021).

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Zhang, Y., Li, D., Han, Y. et al. Intraovarian injection of 3D-MSC-EVs-ECM gel significantly improved rat ovarian function after chemotherapy. Reprod Biol Endocrinol 22, 125 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12958-024-01299-3

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  • DOI: https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12958-024-01299-3

Keywords

Reproductive Biology and Endocrinology

ISSN: 1477-7827

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