Exosomes derived from human adipose mesenchymal stem cells improve ovary function of premature ovarian insufficiency by targeting SMAD

Abstract

Background: Although many reports show that various kinds of stem cells have the ability to recover the function of premature ovarian insufficiency (POI), few studies are associated with the mechanism of stem cell treatment of POI. We designed this experimental study to investigate whether human adipose stem cell-derived exosomes (hADSC-Exos) retain the ability to restore ovarian function and how hADSC-Exos work in this process.

Methods: A POI mouse model was established and human ovarian granule cells (hGCs) collected from individuals with POI were prepared to assess the therapeutic effects and illuminate the mechanism of hADSCs in curing POI. The hematoxylin and eosin assay method was employed to assess the number of follicles. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum levels of sex hormones. The proliferation rate and marker expression levels of hGCs were measured by flow cytometry (fluorescence-activated cell sorting). Real-time PCR and western blot assays were used to determine the mRNA and protein expression levels of SMAD2, SMAD3, and SMAD5. Western blot assays were used to test the protein expression levels of apoptosis genes (Fas, FasL, caspase-3, and caspase-8).

Results: After the hADSC-Exos were transplanted into the POI mice model, they exerted better therapeutic activity on mouse ovarian function, improving follicle numbers during four stages. ELISA results showed that hADSC-Exos elevated the hormone levels to the normal levels. In addition, after hADSC-Exos were cocultured with POI hGCs, our results showed that hADSC-Exos significantly promoted the proliferation rate and inhibited the apoptosis rate. Furthermore, hADSC-Exos also increased the marker expression of hGCs to the normal level. Besides, mRNA and protein assays demonstrated that hADSC-Exos downregulated the expression of SMAD2, SMAD3, and SMAD5 in vivo and in vitro. Western blot assay demonstrated that hADSC-Exos inhibited expression of the apoptosis genes in POI hGCs, and SMAD knockdown increased the protein expression of apoptosis genes.

Conclusions: These findings demonstrate for the first time the molecular cascade and related cell biology events involved in the mechanism by which exosomes derived from hADSCs improved ovarian function of POI disease via regulation of the SMAD signaling pathway.

Keywords: Exosome; Human adipose stem cells; Premature ovarian insufficiency; SMAD pathway.

Conflict of interest statement

The use of human ovarian granular cells and human peripheral blood mononuclear cells was 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 of 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.

Not applicable.

The authors declare that they have no competing interests.

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

Figures

Fig. 1

Characterization of hADSC-Exos. a Phenotype of hADSC-Exos detected by electron microscopy. b Expression levels of CD9, CD63, and CD81 in hADSC-Exos detected by flow cytometry. c Expression levels of CD9, CD63, and CD81 in hADSC-Exos detected by western blot. Scale bars = 100 nm. GAPDH glyceraldehyde 3-phosphate dehydrogenase, hADSC human adipose mesenchymal stem cells

Fig. 2

hADSC-Exos improved function of POI mouse model. a Number of primordial follicles counted over 4 weeks after hADSC-Exos injection. b Number of primary follicles counted over 4 weeks after hADSC-Exos injection. c Number of secondary follicles counted over 4 weeks after hADSC-Exos injection. d Number of antral follicles counted over 4 weeks after hADSC-Exos injection. All experiments carried three times; error bars indicate SD. **p < 0.01, ***p < 0.001 (compared with POI group). POI premature ovarian insufficiency, PBS phosphate buffered saline

Fig. 3

hADSC-Exos improved hormone level of POI mouse model. a E2 levels measured by ELISA over 4 weeks after hADSC-Exos injection. b FSH levels measured by ELISA over 4 weeks after hADSC-Exos injection. c AMH levels measured by ELISA over 4 weeks after hADSC-Exos injection. All experiments carried three times; error bars indicate SD. ***p < 0.001 (compared with POI group). E2 estradiol, FSH follicle-stimulating hormone, AMH anti-Mullerian hormone, POI premature ovarian insufficiency

Fig. 4

hADSC-Exos improve proliferation rate and inhibit apoptosis rate in POI hGCs. a Schematic overview of hGC filter procedures. b hADSC-Exos improve proliferation in hGCs more significantly than using PBS. c hADSC-Exos inhibit apoptosis in hGCs more effectively than using PBS. Error bars indicate SD. ***p < 0.001 (compared with normal group). GC granulosa cell, POI premature ovarian insufficiency, hGC human granulosa cell, PBS phosphate buffered saline

Fig. 5

hADSC-Exos improve marker expression in POI hGCs. a hADSC-Exos increase number of FSHR+AMH+ hGCs. b hADSC-Exos increase number of FOXL2+CYP19A1+ hGCs. Error bars indicate SD. **p < 0.01, ***p < 0.001 (compared with normal group). POI premature ovarian insufficiency, PBS phosphate buffered saline, EXO exosome, FSHR follicle-stimulating hormone receptor, AMH anti-Mullerian hormone

Fig. 6

hADSC-Exos upregulated SMAD pathway in POI hGCs. a qPCR analysis of mRNA expression levels of SMAD2, SMAD3, and SMAD5 after hADSC-Exos coculture with POI hGCs. b Western blot analysis of protein expression levels of SMAD2, SMAD3, and SMAD5 after hADSC-Exos coculture with POI hGCs. Error bars indicate SD. ***p < 0.001 (compared with normal group); ###p < 0.001 (compared with hADSC-Exos treatment group). POI premature ovarian insufficiency, PBS phosphate buffered saline, hADSC human adipose mesenchymal stem cells, GAPDH glyceraldehyde 3-phosphate dehydrogenase, EXO exosome

Fig. 7

hADSC-Exos upregulated SMAD pathway in POI mouse model. a qPCR analysis of mRNA expression levels of SMAD2, SMAD3, and SMAD5 after hADSC-Exos injection into POI mouse model. b Western blot analysis of protein expression levels of SMAD2, SMAD3, and SMAD5 after injection into POI mouse model. Error bars indicate SD. ***p < 0.001 (compared with normal group); ###p < 0.001 (compared with hADSC-Exos treatment group). POI premature ovarian insufficiency, PBS phosphate buffered saline, hADSC human adipose mesenchymal stem cells, GAPDH glyceraldehyde 3-phosphate dehydrogenase, EXO exosome

Fig. 8

hADSC-Exos repress apoptosis genes through SMAD pathway. a Western blot analysis of protein expression levels of Fas, FasL, Caspase-8, and Caspase-3 after hADSC-Exos coculture with hGCs. b Western blot analysis of protein expression levels of Fas, FasL, Caspase-8, and Caspase-3 after SMAD2, SMAD3, and SMAD5 knockdown in normal hGCs. c Proposed model for exosomes derived from hADSCs improved ovaries of POI mice through regulating SMAD pathway. Error bars indicate SD. **p < 0.01, ***p < 0.001 (compared with normal group). GAPDH glyceraldehyde 3-phosphate dehydrogenase, POI premature ovarian insufficiency, PBS phosphate buffered saline, EXO exosome, siRNA small interfering RNA, hADSC human adipose mesenchymal stem cells