Human amniotic mesenchymal stem cells improve ovarian function in natural aging through secreting hepatocyte growth factor and epidermal growth factor

Abstract

Background: Although many reports show that various kinds of stem cells have the ability to recover function in premature ovarian aging, few studies have looked at stem cell treatment of natural ovarian aging (NOA). We designed this experimental study to investigate whether human amniotic mesenchymal stem cells (hAMSCs) retain the ability to restore ovarian function, and how hAMSCs work in this process.

Methods: To build the NOA mouse model, the mice were fed for 12-14 months normally with young fertile female mice as the normal control group (3-5 months old). Hematoxylin and eosin staining permitted follicle counting and showed the ovarian tissue structure. An enzyme-linked immunosorbent assay was used to detect the serum levels of the sex hormones estradiol (E2), anti-mullerian hormone (AMH), and follicle-stimulating hormone (FSH). The proliferation rate and marker expression level of human ovarian granule cells (hGCs) (ki67, AMH, FSH receptor, FOXL2, and CYP19A1) were measured by flow cytometry (FACS). Cytokines (growth factors) were measured by a protein antibody array methodology. After hepatocyte growth factor (HGF) and epidermal growth factor (EGF) were co-cultured with hGCs, proliferation (ki67) and apoptosis (Annexin V) levels were analyzed by FACS. After HGF and EGF were injected into the ovaries of natural aging mice, the total follicle numbers and hormone levels were tested.

Results: After the hAMSCs were transplanted into the NOA mouse model, the hAMSCs exerted a therapeutic activity on mouse ovarian function by improving the follicle numbers over four stages. In addition, our results showed that hAMSCs significantly promoted the proliferation rate and marker expression level of ovarian granular cells that were from NOA patients. Meanwhile, we found that the secretion level of EGF and HGF from hAMSCs was higher than other growth factors. A growth factor combination (HGF with EGF) improved the proliferation rate and inhibited the apoptosis rate more powerfully after a co-culture with hGCs, and total follicle numbers and hormone levels were elevated to a normal level after the growth factor combination was injected into the ovaries of the NOA mouse model.

Conclusions: These findings provide insight into the notion that hAMSCs play an integral role in resistance to NOA. Furthermore, our present study demonstrates that a growth factor combination derived from hAMSCs plays a central role in inhibiting ovarian aging. Therefore, we suggest that hAMSCs improve ovarian function in natural aging by secreting HGF and EGF.

Keywords: EGF; HGF; Human amniotic mesenchymal stem cells; Natural ovarian aging.

Conflict of interest statement

Ethics approval and consent to participate

The use of human ovarian granular cells and human peripheral blood mononuclear cells were in accordance with the 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 the 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.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1

Human amniotic mesenchymal stem cells (hAMSCs) improve the follicle numbers in the ovaries of a natural ovarian aging (NOA) mouse model at four stages. a The number of primordial follicles was counted over 4 weeks after hAMSC transplantation. b The number of primary follicles was counted over 4 weeks after hAMSC transplantation. c The number of secondary follicles was counted over 4 weeks after hAMSC transplantation. d The number of antral follicles was counted over 4 weeks after hAMSC transplantation. All the experiments were repeated three times; the error bars indicate SD. **p < 0.01, ***p < 0.001, versus the NOA group. PBS phosphate-buffered saline

Fig. 2

Human amniotic mesenchymal stem cells (hAMSCs) improve the hormone levels in the ovaries of an NOA mouse model. a The level of estradiol (E2) was measured by ELISA over 4 weeks after hAMSC transplantation. b The hormonal level of follicle-stimulating hormone (FSH) was measured by ELISA over 4 weeks after hAMSC transplantation. c The hormonal level of anti-mullerian hormone (AMH) was measured by ELISA over 4 weeks after hAMSC transplantation. All the experiments were repeated three times; the error bars indicate SD. ***p < 0.001, versus the aging mice group

Fig. 3

Human amniotic mesenchymal stem cells (hAMSCs) improve the proliferation rate of human granulosa cells (hGCs) and upregulate the expression of hGC markers. a A schematic overview of the hGC filter procedures. b The number of ki67+AMH+ hGCs was evaluated after a co-culture with hAMSCs. c The expression level of the ki67+FSHR+ hGCs was tested after a co-culture with hAMSCs. d The expression level of the ki67+FOXL2+ hGCs was tested after a co-culture with hAMSCs. e The number of ki67+CYP19A1+ hGCs was evaluated after a co-culture with hAMSCs. The experiments were carried out after 7 days of co-culture, n = 3; error bars indicate SD. **p < 0.01, ***p < 0.001, versus the NOA group. AMH anti-mullerian hormone, FOXL2 forkhead box L2, FSHR follicle-stimulating hormone receptor, NOA natural ovarian aging, PBS phosphate-buffered saline

Fig. 4

Epidermal growth factor (EGF) and hepatocyte growth factor (HGF) derived from the human amniotic mesenchymal stem cells (hAMSCs) were higher than other growth factors. HGF and EGF derived from the hAMSCs exhibited a higher level of secretion than other growth factors. All the experiments were repeated three times; error bars indicate SD. BDNF brain-derived neurotrophic factor, TGF transforming growth factor

Fig. 5

Immunofluorescence analysis of the cytokines hepatocyte growth factor (HGF), epidermal growth factor (EGF), brain-derived neurotrophic factor (BDNF), Osteoprotegerin, and transforming growth factor (TGF)-β2 in the ovaries after human amniotic mesenchymal stem cell (hAMSCs) transplantation. HGF and EGF were highly expressed in the ovary tissue of the NOA mice after hAMSC transplantation

Fig. 6

A combination of epidermal growth factor (EGF) and hepatocyte growth factor (HGF) improves proliferation and inhibits the apoptosis level to a greater degree than using them alone after a co-culture with human granulosa cells (hGCs). a A schematic diagram of the EGF and HGF co-culture with hGCs. b A combination of EGF and HGF improved proliferation in the hGCs more significantly than using them singly. c A combination of EGF and HGF inhibited apoptosis in the hGCs more effectively than using them singly. All the experiments were repeated three times; error bars indicate SD. **p < 0.01, ***p < 0.001, versus the HGF group; ##p < 0.01, ###p < 0.001, versus the EGF group. NOA natural ovarian aging, PBS phosphate-buffered saline

Fig. 7

A combination of epidermal growth factor (EGF) and hepatocyte growth factor (HGF) increases the follicle numbers and elevates the hormone levels to a greater extent than using them alone after an injection into a natural ovarian aging (NOA) mouse model. a A combination of EGF and HGF increased the follicle numbers to a higher level than using them singly. b A combination of EGF and HGF increased the level of estradiol (E2) to a higher level than using them singly. c A combination of EGF and HGF increased the level of follicle-stimulating hormone (FSH) to a higher level than using them singly. d A combination of EGF and HGF increased the level of anti-mullerian hormone (AMH) to a higher level than using them singly. All the experiments were repeated three times; error bars indicate SD. *p < 0.05, **p < 0.01, versus the HGF group; #p < 0.05, ##p < 0.01, versus the EGF group