Human amniotic epithelial cells can differentiate into granulosa cells and restore folliculogenesis in a mouse model of chemotherapy-induced premature ovarian failure

Abstract

Introduction: Ovarian dysfunction frequently occurs in female cancer patients after chemotherapy, but human amniotic epithelial cells (hAECs) that can differentiate into cell types that arise from all three germ layers may offer promise for restoration of such dysfunction. Previous studies confirmed that hAECs could differentiate into cells that express germ cell-specific markers, but at this time hAECs have not been shown to restore ovarian function.

Methods: To model premature ovarian failure, hAECs infected with lenti-virus carrying green fluorescent protein were injected into the tail vein of mice sterilized with cyclophosphamide and busulphan. hAECs migrated to the mouse ovaries and overall ovarian function was measured using immunohistochemical techniques.

Results: Seven days to two months after hAECs transplantation, ovarian cells were morphologically restored in sterilized mice. Hemotoxylin and eosin staining revealed that restored ovarian cells developed follicles at all stages. No follicles were observed in control mice at the same time period. Immunostaining with anti-human antigen antibodies and pre-transplantation labeling with green fluorescent protein (GFP) revealed that the grafted hAECs survived and migrated to mouse ovary, differentiating into granulosa cells. Furthermore, the ovarian function marker, anti-Müllerian hormone, was evident in treated mouse ovaries after hAEC transplantation.

Conclusions: Intravenously injected hAECs reached the ovaries of chemotherapy-treated mice and restored folliculogenesis, data which suggest promise for hAECs for promoting reproductive health and improving the quality of life for female cancer survivors.

Figures

Figure 1

Human amniotic epithelial cells (hAECs) did not express germ-cell-specific genes. (A) Quantitative PCR was used to analyze germ-cell-specific expression in hAECs. CT values were expressed as a percentage of 18S RNA (18S = 100%) and used to calculate mean normalized expression relative to 18S. Results are shown as mean and standard deviation of three experiments. (B) Immunofluorescence analysis of germ-cell-specific genes in hAECs. Note that hAECs expressed OCT4 (red), NANOG (green) and CD117 (green), but did not express DAZL, STELLA or VASA. DAPI staining for nuclei. Scale bars = 50 μm.

Figure 2

Transplantation of GFP-transfected hAECs into sterilized recipient mice and the follicle activation induced by hAECs transplantation. (A) hAECs cells grown to 85% density. (B) GFP-transfected hAECs. Representative H&E micrographs of ovary sections from: non-sterilized normal control mice (C, D); sterilized non-transplanted mice after a 7-day recovery period (E), a 14-day recovery period (F), a 21-day recovery period (G) and a 2-month recovery period (H) showing stroma, and atretic primordial or primary follicles; sterilized recipient mice 7 days (I), 14 days (J), 21 days (K) and 2 months (L) after transplantation of hAECs. No obvious follicles were observed in recipients seven days after hAEC transplantation (I); however, the hollow structure destroyed by chemotherapy was reduced and obviously compared with (E). Primordial follicles are visible in J, primary follicles were visible in K and large antral follicles are shown in L. Arrows indicated follicles at various stages of maturational development. Scale bars = 100 μm. (M). Differential follicle counts of primordial, primary, secondary and atretic follicles in ovaries of each groups. NC, Normal control; UC, Untreated control; hAECs, hAECs transplantation group. GFP, Green fluorescent protein; hAECs, Human amniotic epithelial cells.

Figure 3

Macroscopic appearance of recipient ovaries seven days to two months after transplantation with GFP-transfected hAECs. (A-F) GFP staining dots shown in recipient ovaries 7 to 14 days after transplantation with GFP-transfected hAECs, whereas no GFP signal was observed in follicles. Follicles containing GFP-positive (green) cells were shown in recipient ovaries 21 days (G-I), 28 days (J-L) and 2 months (M-R) after transplantation with GFP-transfected hAECs. (S-U) Oocytes in recipients without hAECs transplantation had no GFP signal after a two-month recovery period. Arrows indicate the GFP distribution pattern in ovaries. Blue, DAPI immunofluorescence. Scale bars, 200 μm (S-U), 100 μm (A-R). GFP, Green fluorescent protein; hAECs, Human amniotic epithelial cells.

Figure 4

Double-staining with GFP and human nuclear antigen to observe GFP-positive cells after hAECs transplantation. (A-F) Ovarian sections stained with GFP and anti-human nuclear antibody reveal grafted hAECs 28 days after hAEC transplantation. (G-L) GFP staining co-localized with human nuclear antigen in antral follicles of recipient ovaries two months after hAEC transplantation. Arrows indicate a double-staining pattern in ovaries. Scale bars, 200 μm (G-L), 100 μm (A-F). GFP, Green fluorescent protein; hAECs, human amniotic epithelial cells.

Figure 5

Grafted cells detected by immunochemistry against human antigens. (A) Human specific nuclear antigen expression was negative in recipient ovaries without hAECs transplantation. (B) Human nuclear antigen was not detected in some recipient ovaries two months after hAEC transplantation. (C, D) Human nuclear antigen was expressed in antral follicles in recipient ovaries two months after hAEC transplantation. (E) Human FSHR was not detected in recipient ovaries without hAEC transplantation. (F) Human FSHR was not detected in some recipient ovaries two months after hAEC transplantation. (G, H) Human FSHR were detected in recipient ovaries two months after hAEC transplantation. Arrows indicate human antigen expression in granulosa cells surrounding the ovum, whereas other granulosa cells were negative controls. Scale bars = 100 μm. hAECs, human amniotic epithelial cells.

Figure 6

AMH expression in recipient mouse ovaries. (A) AMH was expressed in all granulosa cells of primary, preantral and small antral follicles in normal ovaries. (B) AMH expression disappeared in stromal and atretic follicles in recipients ovaries without hAECs transplantation two months after chemotherapy. (C) AMH expression was negative in recipient ovaries seven days after hAEC transplantation. However, AMH expressions were detected in recipient ovaries after hAEC transplantation for 14 days (D) and 21 days (E). (F) AMH expression patterns are strong in recipient ovaries two months after hAEC transplantation; data are consistent with that from control normal ovaries. Scale bars = 100 μm. AMH, Anti-Müllerian hormone; hAEC, human amniotic epithelial cell.