Different therapeutic effects of cells derived from human amniotic membrane on premature ovarian aging depend on distinct cellular biological characteristics
- Chenyue Ding†1,
- Hong Li†1,
- Yun Wang3,
- Fuxin Wang1,
- Huihua Wu1,
- Rulei Chen4,
- Jinghuan Lv4,
- Wei Wang1 and
- Boxian Huang1, 2Email author
© The Author(s). 2017
Received: 23 May 2017
Accepted: 16 June 2017
Published: 27 July 2017
Many reports have shown that various kinds of stem cells have the ability to recover premature ovarian aging (POA) function. Transplantation of human amniotic epithelial cells (hAECs) improves ovarian function damaged by chemotherapy in a mice model. Understanding of how to evaluate the distinct effects of adult stem cells in curing POA and how to choose stem cells in clinical application is lacking.
To build a different degrees of POA model, mice were administered different doses of cyclophosphamide: light dose (70 mg/kg, 2 weeks), medium dose (70 mg/kg, 1 week; 120 mg/kg, 1 week), and high dose (120 mg/kg, 2 weeks). Enzyme-linked immunosorbent assay detected serum levels of sex hormones, and hematoxylin and eosin staining allowed follicle counting and showed the ovarian tissue structure. DiIC18(5)-DS was employed to label human amniotic mesenchymal stem cells (hAMSCs) and hAECs for detecting the cellular retention time in ovaries by a live imaging system. Proliferation of human ovarian granule cells (ki67, AMH, FSHR, FOXL2, and CYP19A1) and immunological rejection of human peripheral blood mononuclear cells (CD4, CD11b, CD19, and CD56) were measured by flow cytometry (fluorescence-activated cell sorting (FACS)). Distinction of cellular biological characteristics between hAECs and hAMSCs was evaluated, such as collagen secretory level (collagen I, II, III, IV, and VI), telomerase activity, pluripotent markers tested by western blot, expression level of immune molecules (HLA-ABC and HLA-DR) analyzed by FACS, and cytokines (growth factors, chemotactic factors, apoptosis factors, and inflammatory factors) measured by a protein antibody array methodology.
After hAMSCs and hAECs were transplanted into a different degrees of POA model, hAMSCs exerted better therapeutic activity on mouse ovarian function in the high-dose administration group, promoting the proliferation rate of ovarian granular cells from premature ovarian failure patients, but also provoking immune rejection. Meanwhile, our results showed that the biological characteristics of hAMSCs were superior to hAECs, but not to expression of immune molecules.
These results suggest that hAMSCs are a more effective cell type to improve ovarian function than hAECs. Meanwhile, this distinct effect is attributable to cellular biological characteristics of hAMSCs (telomerase activity, expression level of pluripotent markers, cytokine and collagen secretion) that are superior to hAECs, except for immunological rejection. Sufficient consideration of cell properties is warranted to move forward to more effective clinical therapy.
The ovary is a unique and complex organ responsible for the generation of an appropriate number of developmentally competent oocytes through folliculogenesis . The major function of the ovary is to regulate female fertility and keep women healthy during a definite life stage. However, premature ovarian aging (POA) affects 1% of women in the general population . A loss of oocytes and fertility potential is correlated with exposure to genetic abnormalities, pelvic irradiation, and systemic chemotherapy .
Ovarian reserve (OR) is widely deemed to be the sum of all remaining available follicles in ovaries. Therefore, it is a predictive criterion for the outcome of infertility treatment. Various standards have been proposed to assess OR. Estradiol and inhibin A are largely produced by the dominant and preovulatory follicle and thus largely reflect ovulatory activity . The FSH level is a key factor to affect the selection of a limited number of follicles at the stage of cyclic recruitment [5, 6]. Meantime, AMH is an excellent marker for the number of small, growing follicles and is produced by the granulosa cells of follicles from the time at which follicle growth is first initiated, which might aid in the diagnosis and follow-up of women at risk of POA . Consequently, the hormone level of E2, FSH, and AMH is a classical test criterion for POA. Additionally, a key point of POA is the decrease in oocyte quality as well as quantity. Therefore, the number of antral follicles and the viability of granulosa cells are regarded as the evaluation standard for POA .
Many research studies indicate that the most severe form of decreased ovarian reserve (DOR) is represented by premature ovarian failure (POF) in young females [9–11]. Therefore, according to the aforementioned criteria, two different degrees of POA are defined: DOR and POF. Currently, POA cannot be reversed and although treatments are available using long-term hormone replacement, the therapy relieves menopausal symptoms but cannot restore ovarian aging in women. There is an urgent need to improve treatment strategies. Regenerative medicine research suggests that due to the self-renewal capacity and multiplex differentiation potential of stem cells, they could be used to cure various human diseases . Recent interest in the therapeutic potential of stem cells has grown and multipotent stem cells could be developed from different sources, such as bone marrow , adipose tissue , amniotic fluid, and the amnion [15, 16], and all have been shown to have potential to restore ovarian function and rescue long-term infertility in chemotherapy-treated female mice. However, little is still known about which kind of stem cells will exert the best therapeutic activity and how to choose the stem cells for POA, especially in clinical therapy.
The human amniotic membrane (hAM) is a kind of superior biomaterial that could be suitable for allotransplantation and regenerative medicine. hAM includes two different stem cell populations: human amniotic epithelial cells (hAECs) and human amniotic mesenchymal stem cells (hAMSCs). There are several advantages which make these suitable for clinical therapy: acquirement of a high cell number, easy to obtain because hAM is usually discarded after delivery, and their use is within the legal and ethical framework. Meanwhile, compared to adipose tissue-derived stromal cells (ADSCs) and bone marrow mesenchymal stem cells (BMSCs), stem cells derived from hAM possess characteristics of a less invasive procedure.
Although hAMSCs and hAECs are all derived from hAM, the characters of both cell types not only show similar aspects but also demonstrate distinction. Therefore, the purpose of the present study was to determine whether hAMSCs and hAECs exhibit different therapeutic potential in a mice model of ovarian aging disease and cell samples from DOR and POF patients.
Establishment of a mice model with different degrees of POA
Female ICR mice between 7 and 8 weeks of age were obtained from Nanjing Medical University with Institutional Animal Care and Use Committee approval in accordance with institutional guidelines. To build the mice model with different degrees of ovarian aging, different doses of CTX (Sigma, USA) were classified into three groups: light-dose treatment (70 mg/kg, 2 weeks), medium-dose treatment (70 mg/kg, 1 week; 120 mg/kg, 1 week), and high-dose treatment (120 mg/kg, 2 weeks). The animals were divided into four groups (per group, n = 10) respectively after CTX treatment: control group (saline injection), light-dose CTX treatment group, medium-dose CTX treatment group, and high-dose CTX treatment group. Follicle numbers, weight of the ovary, hormone level, and litter size were estimated after cell transplantation.
Assessment of ovarian function by comparison of ovarian follicle counts and litter sizes
After cell transplantation, mice (cell translated and nontransplanted) were euthanized from 0 to 4 weeks, ovaries on both sides were removed and fixed by 10% formalin, paraffin embedded, serially sectioned at 5-mm thickness, mounted in order on glass microscope slides, and stained with hematoxylin and eosin (HE). Four stages of follicles (primordial, primary, secondary, and antral follicles) were detected and classified. The ratio of the number of follicles from the ovary on both sides at four stages was calculated and compared between each group (per group, n = 10). Three representative sections from each ovary were selected. Only follicles containing an oocyte were counted to avoid counting any follicle twice. To test the safety of cell transplantation and improvement in ovarian function, we compared the litter sizes obtained by natural mating after the transplantation of hAMSCs and hAECs into the ovaries on both sides. Eight weeks after cell transplantation, cell transplanted and nontransplanted model mice with different degrees of ovarian aging (per group, n = 10) were found to be in proestrus based on a vaginal smear test, and were then put into the same cage with male mice for natural mating at a ratio of 1:2. The number of offspring was then recorded.
Enzyme-linked immunosorbent assay analysis
Mouse plasma was harvested to evaluate the expression level of E2, AMH, or FSH using an enzyme-linked immunosorbent assay (ELISA) kit (Mybiosource, USA) according to the manufacturer’s guide. Briefly, 50 μl of serum sample was added to each well. The test plate was wrapped with membrane, and incubated for 30 min at 37 °C. Thereafter, wells on the plate were dried and washed with Wash Buffer five times (10 s per time). Then 50 μl HRP-conjugate reagent was added into each sample well and incubated for 60 min at 37 °C. Samples were washed with Wash Buffer five times (10 s per time). Subsequently, 50 μl substrate A Solution followed by 50 μl substrate B Solution was added and incubated for 15 min at 37 °C. Then 50 μl Stop Solution was added to each control and sample well. Finally, the light absorbance was measured and recorded by a spectrophotometer (Varian Company, Australia).
Preparation and culture of hAECs and hAMSCs
Human placentas were obtained from term pregnancy during uncomplicated caesarean sections. Written and informed consent was obtained from each woman who tested negative for HIV-I and hepatitis B and C. The institutional ethics committee approved the use of human amnions for this project. According to the protocol reported previously , the amniotic membrane was mechanically separated from the chorion, and cut with a razor to yield 1.5–2.0 cm2. The amniotic membrane segments were digested with 0.25% Trypsin/EDTA at 37 °C for 45 min to isolate hAECs. After repeating this treatment several times, the epithelial cells were removed completely. The rest of the tissue pieces were placed in DMEM containing collagenase I (1 mg/ml; Gibco, USA) and DNase I (1 mg/ml; Sigma, USA) and were incubated at 37 °C for 60 min to isolate hAMSCs. The isolated hAECs and hAMSCs were cultured with DMEM medium supplemented with 10% FBS (Gibco, USA), penicillin/streptomycin (Gibco, USA), glutamine (Gibco, USA), EGF (R&D, USA), and bFGF (R&D, USA) and incubated at 37 °C, 5% CO2. When cells reached 80–90% confluence, adherent cells were trypsinized and passaged.
hAMSC and hAEC phenotypic characterization
hAMSC and hAEC specific surface antigens were stained with PE-conjugated antibodies of anti-human-CD-105, anti-human-CD-29, anti-human-CD-44, anti-human-CD-73, anti-human-CD-90, anti-human-CD-34, anti-human-CD-45, anti-human-EpCam, or anti-human-CD-166 (BD, USA) or their corresponding isotype control. The stained cells were then analyzed respectively by fluorescence-activated cell sorting (FACS). The detailed instruction is the same as the FACS analysis already described. Differentiation ability of hAMSCs was tested using hAMSC differentiation kits for culture (Thermo, USA).
Isolation of primary human ovarian granulosa cells and human peripheral blood mononuclear cells from DOR and POF patients
Patients with tubal occlusion served as the control group. Patient selection followed a standard as follows. DOR was defined as antral follicle count < 5 or AMH < 1.1 ng/ml and FSH ≥ 10 mIU/ml. Women aged > 40 years were not included in this study in order to exclude patients with physiological ovarian aging. POF patients were recruited according to inclusion criteria that consisted of primary amenorrhea or secondary amenorrhea for at least 4 months, younger than 40 years of age, and at least two recordings of serum concentrations of FSH measurements exceeding 40 IU/L. Women with known normal karyotype, previous chemotherapy or radiotherapy, autoimmune diseases, or ovarian surgery were excluded. Primary human ovarian granulosa cells (hGCs) were obtained following informed consent from tubal occlusion (TO; n = 28), DOR (n = 31), and POF (n = 43) patients respectively after approval from the Suzhou Hospital Affiliated to Nanjing Medical University Research Ethics Board. All patients were treated with recombinant FSH (Puregon; Schering Plough, NJ, USA) and GnRH antagonist Ganirelix (Merck, Frosst, Montreal, Canada). Vaginal ultrasound examination was performed to monitor follicular development. Final follicular maturation was induced by administering 10,000 IU of human chorionic gonadotropin (hCG) (Pregnyl; Merck). hGCs were purified using density centrifugation from follicular aspirates collected from women undergoing oocyte retrieval as described previously . Primary hGCs were cultured in six-well plates in DMEM/F12 media (Thermo, USA) containing 1% penicillin/streptomycin, 10% fetal bovine serum (complete medium), 100 mg/ml of streptomycin sulfate (Thermo, USA), and 1× GlutaMAX (Thermo, USA). The culture medium was changed every other day in all experiments. We collected human peripheral blood mononuclear cells (hPBMCs) from TO (control group, n = 41), DOR (n = 48), and POF (n = 58) patients in our reproductive center, and then hPBMCs were filtered for conditions such as Addison disease, autoimmune oophoritis, and few cells. Finally, hPBMCs were obtained from TO (n = 20), DOR (n = 29), and POF (n = 36) patients respectively after informed consent was obtained. Women with known Addison disease and autoimmune oophoritis were excluded. Venous blood was collected from healthy volunteers into BD sodium-heparin tubes. hPBMCs were separated by density-gradient centrifugation using a Percoll separation medium (PSM; Sigma, USA). In brief, 20 ml of twofold diluted peripheral blood from healthy donors were layered on 15 ml of PSM and centrifuged at 400 × g for 30 min at room temperature. hPBMCs were collected and were cultured in six-well plates in RPMI-1640 media (Thermo, USA).
Live imaging of transplanted hAMSCs and hAECs in a mice model
According to the protocol instruction, probe DiIC18(5)-DS (Life Technologies, USA) was employed to label hAMSCs and hAECs for detection. Next, after tail-vein injection of Dil-labeled hAMSCs and hAECs from 6 h to 14 days respectively, cell transplanted mice (n = 15) and nontransplanted mice (n = 15) were screened with a live imaging system (Xenogen IVIS 100 in vivo Imaging System; PerkinElmer, USA) to characterize, quantify, and visualize Dil-labeled cells. To avoid a very weak detecting signal caused by fur, mice were anesthetized and the abdominal fur shaved which was convenient for signal detection.
Western blot analysis
hAMSCs and hAECs from three pregnant women were harvested and dissociated in a lysis buffer. Protein was extracted from each sample, which was then loaded onto 10% gels and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Next, the separated proteins were transferred to polyvinylidene difluoride membrane (PVDF; Millipore, USA). Thirdly, the proteins were incubated with the primary antibodies (Abcam, USA) of anti-human-collagen I, II, III, IV, and VI, anti-human-telomerase reverse transcriptase, anti-human-OCT4, anti-human-NANOG, anti-human-Gapdh, anti-human-β-Tubulin, anti-human-SSEA4, or anti-human-TRA-1-81 and appropriate secondary antibodies (goat anti-rabbit HRP conjugates; Jackson Immunoresearch, West Grove, PA, USA) separately. The specific signals were detected by enhanced chemiluminescence (Pierce ECL Western blotting Substrate; Thermo). Finally, the membrane was checked by a chemiluminescence detection system (Tanon, China) and the signal intensity of each band was analyzed using Imaging J Software (National Institutes of Health, USA). Experiments were repeated three times, results are presented as fold change ± SD, and p < 0.05 is determined as significant difference.
hAMSCs and hAECs were digested separately by Trypsin/EDTA for 3 min and blown gently into single cells, which were fixed and permeated by the Cytofix/Cytoper Fixation/Permeabilization Solution Kit (BD, USA) following the manufacturer’s instruction. Treated cells were then stained with PE-conjugated or FITC-conjugated antibodies of anti-human-ki67 (Abcam, USA), anti-human-AMH (Thermo, USA), anti-human-FSHR (Thermo, USA), anti-human-FOXL2 (Thermo, USA), anti-human-CYP19A1 (Abgent, USA), anti-human-CD8 (BD, USA), anti-human-CD4 (BD, USA), anti-human-CD11b (BD, USA), anti-human-CD19 (BD, USA), or anti-human-CD56 (BD, USA) or their corresponding isotype control, for 30 min at 4 °C as already described. The stained cells were analyzed on fluorescence-activated cell sorter (Beckman, USA). Experiments were repeated three times, results are presented as fold change ± SD, and p < 0.05 is determined as significant difference.
The primary antibodies of anti-human-BrdU (Abcam, USA), anti-human-AMH (Abcam, USA), anti-human-FSH (Abcam, USA), anti-human-inhibin α (Abcam, USA), and anti-human-inhibin β (Abcam, USA) were selected to characterize ovarian granular cells. For the staining procedure, ovarian sections were fixed with 4% (w/v) paraformaldehyde (PFA; Sigma, USA) at room temperature for 10 min and then washed three times for 5 min with phosphate buffer solution (PBS), permeated with 0.1% Triton X-100 (Sigma, USA)/PBS on ice for 10 min, and blocked with fresh 4% bovine serum albumin (BSA; Sigma, USA)/PBS at room temperature for 30 min. The treated cells were washed with PBS three times for 5 min and then incubated with primary antibodies overnight at 4 °C. After rinsing with PBS for 5 min, the cells were stained by Cy2-conjugated or FITC-conjugated secondary antibodies (Jackson Immunoresearch) in the dark at room temperature for 30 min. The stained cells were mounted with 4′,6-diamidino-2-phenylindole (DAPI; Vector Lab, USA) after washing with PBS for 5 min, and then photographed under a fluorescence microscope (Olympus, Japan).
Antibody microarray analysis
Cytokines were measured by a protein antibody array methodology (RayBio Human Cytokine Antibody Array, RayBiotech G Series 2000; RayBiotech, Inc., Norgross, GA, USA) that contains antibodies targeted to detect protein expression levels which are differentially expressed in hAMSC-conditioned media (CM) and hAEC-CM. One hundred micrograms of CM was used according to the manufacturer’s instructions.
All results were shown as means ± SD. Statistically significant difference was determined by one-way ANOVA with SPSS 17.0 software, and p < 0.05 was regarded as statistical significance.
Established mice model with different levels of POA
As a whole, a mice model with different levels of POA was established successfully by CTX treatment.
hAMSCs restored ovary function more powerfully than hAECs in the high-dose CTX treatment mice
In summary, hAMSCs exhibited more powerful ability to restore ovarian function than hAECs.
hAMSCs revealed more powerful ability to improve the proliferation rate of patients’ human ovarian granular cells (hGCs) than hAECs
In summary, hAECs exhibited less recovery effects for hGCs than hAMSCs, especially in the POF group.
hAECs showed less immune rejection in patients’ PBMCs than hAMSCs
In summary, hAMSCs caused stronger immune rejection than hAECs, particularly in the POF group.
hAMSCs exhibited more dominant cellular biological characteristics than hAECs
In summary, hAMSCs revealed more powerful cellular biological characteristics than hAECs, except for the expression level of immune molecules.
A variety of stem cells has been used to restore chemotherapy-induced ovarian failure, such as ADSCs, hAECs, human amniotic fluid stem cells, and human menstrual blood stem cells [14–16, 19]. However, understanding of how to evaluate the different effects of adult stem cells in curing POA and how to choose stem cells in clinical application is still lacking.
Many previous studies employed a single dose of CTX to inhibit ovarian function ; it is hard to investigate the effects of stem cell therapy for different degrees of POA. Therefore, in order to assess the different therapy effects between hAMSCs and hAECs, we successfully established a mouse model with different degrees of ovarian aging by combining doses of CTX. In this mice model, we observed that the number of follicles, level of hormones, weight of ovaries, and number of offspring were decreased significantly dose-dependently (Fig. 1). Our chemotherapy protocol is also supported by a recent study indicating that a different degree of male infertility mouse model was established after injection of busulfan with different doses . Subsequently, hAMSCs and hAECs were transplanted into this model separately. Our results demonstrate that hAMSCs could restore ovarian function more forcefully than hAECs, especially in the high-dose CTX mice model group (Fig. 2). The present results hint that hAMSCs may be perceived as a more suitable cell resource in curing POA compared to hAECs.
The ultimate goal of research is to treat disease. To bridge the bench-to-bedside gap, preclinical efficacy of hAMSCs and hAECs for treating DOR and POF disease were evaluated; both of these cells were cocultured with hGCs derived from DOR and POF patients. Our findings indicated that hAMSCs increased the proliferation rate of hGCs more effectively than hAECs (Fig. 4). In the present study, the possible reason for the higher therapeutic activity of hAMSCs could be related to enormous secretion of collagen (Fig. 6B). In the meantime, we have found that activation of telomerase in hAMSCs was higher than in hAECs (Fig. 6C). These current findings are consistent with results of a previous study that collagen could maintain longer engraftment and survival of the cells in vivo . A previous study revealed that the paracrine effect may play a key role in restoration of ovarian function . In the present study, we found that hAMSCs excreted a much larger number of growth factors than hAECs (Fig. 6F). Moreover, the distinction of the therapeutic effect may be attributed to the fact that hAMSCs exhibited a higher expression of transcription factor OCT4 and NANOG than hAECs (Fig. 6E). These findings are similar to previous studies that upregulation of NANOG could improve production of cytokines via the JAK/STAT pathway [23, 24].
In addition, assessment of immune rejection is a necessary process for a preclinical trial . Thereby, to evaluate the preclinical safety of hAMSCs and hAECs for treating DOR and POF patients, both of these cells were cocultured with hPBMCs respectively. Our study indicated that hAMSCs induced a greater immune response in hPBMCs than hAECs (Fig. 5). In the present research, one possible reason for such a distinction is that immune molecules were more strongly expressed in hAMSCs than in hAECs (Fig. 6d). Another possible reason for immune response could be related to a larger expression of OCT4 and NANOG in hAMSCs (Fig. 6e). This upregulated expression may explain the reason for initiating immune rejection in vivo. These findings are consistent with those reported previously and also support the idea that overexpression of transcription factor could induce immune rejection .
This study is the first to assess the different therapy potential between hAMSCs and hAECs. Meanwhile, our findings indicated that hAMSCs are a more effective cell type to improve ovarian function than hAECs. Furthermore, our present study revealed that cellular biological characteristics of hAMSCs are superior to hAECs, except for immunological rejection. This discovery has important implications for understanding that cellular biological characteristics play a pivotal role in the distinction of stem cell therapy effects. Additionally, this study suggests that hAECs were suitable only for DOR disease. Meanwhile, a combination of hAMSCs and immunosuppressant was considered to treat POF disease better.
This work was supported by grants from a project funded by China Postdoctoral Science Foundation (2017 M611871), Wu Jieping research funds (320.6755.15027), Suzhou Key Medicine Center (SZZX201505), Jiangsu projects (BE2012653), Suzhou Health Bureau fund (lczx201411), and a maternal and health care project of Jiangsu Province Assisted Reproduction Medicine Center (FKX201224). Jiangsu medical innovation team in prevention and control of birth defect (CXTDB2017013).
Availability of data and materials
All data generated or analyzed during this study are included in this published article.
CD performed cellular and molecular assays in vivo and in vitro. FW and HW contributed to hGC and hPBMC collection, purification, and culture. RC and JL established the different levels ovarian aging mice model. YW and WW contributed to hAMSC and hAEC collection, purification, and culture. HL and BH planned the experiments and wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
The use of human ovarian granular cells and human peripheral blood mononuclear cells was in accordance with relevant guidelines and regulations and the experimental protocols were approved by the Medical Ethics Committee of the Suzhou Hospital Affiliated to Nanjing Medical University (NJMU-2015-014). All patients provided written informed consent prior to participation in this study.
Our investigation using experimental animals was conducted on the basis of the Nanjing Medical University Animal Center’s specific guidelines and standards.
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