Human endometrial mesenchymal stem cells restore ovarian function through improving the renewal of germline stem cells in a mouse model of premature ovarian failure

Abstract

Background: Human endometrial mesenchymal stem cells (EnSCs) derived from menstrual blood have mesenchymal stem/stromal cells (MSCs) characteristics and can differentiate into cell types that arise from all three germ layers. We hypothesized that EnSCs may offer promise for restoration of ovarian dysfunction associated with premature ovarian failure/insufficiency (POF/POI).

Methods: Mouse ovaries were injured with busulfan and cyclophosphamide (B/C) to create a damaged ovary mouse model. Transplanted EnSCs were injected into the tail vein of sterilized mice (Chemoablated with EnSCs group; n = 80), or culture medium was injected into the sterilized mice via the tail vein as chemoablated group (n = 80). Non-sterilized mice were untreated controls (n = 80). Overall ovarian function was measured using vaginal smears, live imaging, mating trials and immunohistochemical techniques.

Results: EnSCs transplantation increased body weight and improved estrous cyclicity as well as restored fertility in sterilized mice. Migration and localization of GFP-labeled EnSCs as measured by live imaging and immunofluorescent methods indicated that GFP-labeled cells were undetectable 48 h after cell transplantation, but were later detected in and localized to the ovarian stroma. 5'-bromodeoxyuridine (BrdU) and mouse vasa homologue (MVH) protein double-positive cells were immunohistochemically detected in mouse ovaries, and EnSC transplantation reduced depletion of the germline stem cell (GSCs) pool induced by chemotherapy.

Conclusion: EnSCs derived from menstrual blood, as autologous stem cells, may restore damaged ovarian function and offer a suitable clinical strategy for regenerative medicine.

Fig 1

Morphology, phenotype and pluripotency of human EnSCs. a Cultured EnSCs appear to have stromal cell morphology. b GFP-transfected EnSCs. c Normal chromosomes expressed by cells as measured by karyotype analysis at passage 20. d Mesenchymal stem cell marker expression in EnSCs as measured by flow cytometry. e EnSCs can differentiate into adipocytes (oil red), chondroblasts (alcian blue) and osteoblasts (alizarin red) under standard in vitro differentiating conditions (original magnification, 100x). Scale bars = 200 μm (a, b)

Fig. 2

Human EnSCs transplantation increased animal weight and restored cyclicity in sterilized mice. a Weight of untreated mice did not increase over the study period. Mice of EnSCs-treated groups weighed significantly more compared with chemoablated mice from the 4th week onward (*P <0.01); however, there was no significant difference between untreated control and EnSCs-treated groups (P > 0.05). b The percentage of substages of diestrus (DE), proestrus (PE), estrus (E) and metestrus (ME) in different groups. Cyclicity was similar to normal animals 4 weeks after cell transplantation, whereas changes in untreated control animals were maintained at diestrus throughout the experiment. U, Untreated control; E, Chemoablated with EnSCs transplantation group, C, Chemoablated group

Fig. 3

Vaginal exfoliative cell smear and cervical mucus crystallization indicating estrous cycles of mice in different groups and at different observed time points. At 2, 4, and 8 weeks after cell transplantation, the typical cornified epithelial (black arrow) and typical ferning patterns (white arrow) were observed in EnSCs-treated mice, whereas cyclicity in Chemoablated animals remained unchanged

Fig. 4

EnSCs transplantation restores fertility in mice treated with chemotherapy. Reproductive outcomes were assessed over three successive mating rounds in different mouse groups. a Offspring obtained by mating after EnSCs transplantation into mice sterilized by chemotherapy compared with chemoablated group and untreated control. Note the stillbirth in a chemoablated mouse (Arrow). b Mean litter size per pregnant mouse for three litters in each group. Data represent means ± standard error. n = 10 per group. **P < 0.001. c Midline histological sections of ovaries removed after the mating rounds and stained with H&E. Scale bars = 100 μm

Fig. 5

Grafted human EnSCs were detected in vivo. a Sterilized mice after tail vein cell transplantation were screened by live imaging (eXplore Optix, GE Company) for the identification of GFP-positive cell tracking in vivo. GFP (+) cells first entered the pelvic organs 6 to 12 h after transplantation, then migrated to chest organs 24 h after transplantation. However, the signal was too weak to be detected 48 h after cell transplantation and few signals were collected in pelvic organs 7 days after cell transplantation. Chemoablated mouse as negative control. b Human nuclear antigen was expressed in antral follicles in recipient ovaries and co-localized with GFP staining 2 months after EnSCs transplantation. c GFP staining was co-localized with human FSHR staining in antral follicles of recipient ovaries 2 months after EnSCs transplantation. Scale bars: (b, c) 100 μm; (b, c insets) 10 μm

Fig. 6

EnSCs transplantation improves GSCs proliferation in sterilized ovaries. Dual immunostaining for BrdU (green) and MVH (red) of GSCs (arrow) were observed near the surface epithelium of mouse ovaries in: (a) Untreated control (b) Chemoablated group (negative control) (c) EnSCs transplantation for 2 weeks (d) EnSCs transplantation for 2 months. A, C, D: Scale bars = 50 μm; insets = 10 μm; B: Scale bars = 100 μm

Fig. 7

a Comparison of GSCs in ovaries of untreated control, chemoablated group or EnSCs-treated mice (mean ± standard error, n = 4–5 mice per data point, *P <0.01). b A schematics showed timing of chemotherapy, transplantation, cyclicity resumed, GFP (+) cells, GSCs (+) cells and FSHR (+) cells detected