Ovarian surface epithelium in patients with severe ovarian infertility: a potential source of cells expressing markers of pluripotent/multipotent stem cells

Abstract

The aim of this study was to confirm the presence of stem cells in the ovarian surface epithelium of patients with premature ovarian failure and no mature follicles and oocytes. In these patients, small round cells of unknown origin expressing SOX-2 marker of pluripotency were observed among the epithelial cells just after the ovarian surface epithelium scraping. These cells were an integral part of the ovarian surface epithelium. When the scraped cells were cultured in a medium with added follicular fluid to provide some ovarian niche, primitive oocyte-like cells and typical round-shaped cell clusters positively stained on alkaline phosphatase, and markers of pluripotency, such as SOX-2 and SSEA-4, were developed. These markers were expressed early and also later in the culture. Single oocyte-like cells expressed genes OCT4A, SOX-2, NANOG, NANOS, STELLA, CD9, LIN28, KLF4, GDF3, and MYC, characteristic for pluripotent stem cells. The results of this study confirmed the presence of putative stem cells in the ovarian surface epithelium of these patients and provided some basis to create a stem cell line in the future.

Figure 1

Histology of ovarian cortex sections in patients. (a) Ovarian cortex with primordial follicles after haematoxylin-eosin staining in the first patient (magnification 100-times). (b–d) Ovarian surface epithelium (arrow) and primordial follicles positively (brown) stained on cytokeratin in the first patient (magnifications 100-times and 200-times). (e, f) Fibrous ovarian cortex without follicles after haematoxylin-eosin staining and ovarian surface epithelium (arrow) positively (brown) stained on cytokeratin in the second patient (magnification 100-times). (g, h) Fibrous ovarian cortex without follicles after haematoxylin-eosin staining in the third patient (magnification 100-times). (Light microscope.)

Figure 2

Small round cells (arrows)—putative stem cells with diameters of 2 to 4 μm in the ovarian surface epithelium scrapings. (a, b) Among the epithelial cells. (Inverted microscope, Hoffman illumination, magnifications 100 and 200-times.)

Figure 3

Small round cells (arrows)—putative stem cells in the ovarian surface epithelium. (a–d) Among the epithelial cells in the ovarian surface epithelium scrapings (magnification 6,000-times). (e) Among the erythrocytes in a scraped population of cells (magnification 200-times). (Inverted microscope, dic-Nomarski illumination, immersion objective for magnification 6,000-times, and Hoffman illumination for magnification 200-times.) e: epithelial cells, er: erythrocytes.

Figure 4

Small round cells (arrows)—putative stem cells in a population of cells scraped from the ovarian surface epithelium. (a) Just after scraping (magnification 100-times). (b) After DAPI staining (magnification 100-times). (c) Just after scraping (magnification 100-times). (d) After DAPI staining (magnification 100-times). (e) Weakly-stained small round cell-putative stem cell after Giemsa staining (magnification 200-times). (f) Blue-stained lymphocyte (arrow) after Giemsa staining (magnification 200-times). (Light, fluorescent, inverted microscopes.)

Figure 5

Small round cells (arrows)-putative stem cells in a population of cells scraped from the ovarian surface epithelium. (a) Just after scraping (magnification 400-times). (b) Just after scraping (magnification 1,000-times). (c) After DAPI staining (magnification 400-times). (d) Positively (red) stained on the SOX-2 marker of pluripotency (magnification 400-times). (Light and fluorescent microscope.)

Figure 6

In vitro culture of cells scraped from the ovarian surface epithelium. (a) Oocyte-like cell developed in vitro on autologous ovarian fibroblasts and small round cells (arrow) with diameters of 2 to 4 μm proliferating in the surroundings as attached to fibroblasts on day 4 of the culture (magnification 100-times). (b) Oocyte-like cell developed in vitro on autologous ovarian fibroblasts on day 10 of the culture (magnification 200-times). (c, d) Cell clusters developed in vitro on autologous ovarian fibroblasts on day 4 of the culture (magnification 100-times). (Inverted microscope, Hoffman illumination.) f: autologous ovarian fibroblast, o: oocyte-like cell.

Figure 7

Alkaline phosphatase staining of cells and cell clusters during the culture of cells scraped from the ovarian surface epithelium. (a) Nonstained cell cluster (magnification 40-times). (b) Positively stained oocyte-like cell (arrow) (magnification 200-times). (c, d) Positively stained small round cells with diameters of 2 to 4 μm proliferating in a cell culture (magnification 200-times). (e, f) Positively stained cell clusters (magnification 40-times). (g) Positively stained cell cluster developing on the layer of autologous ovarian fibroblasts (magnification 100-times). (h) Small round cells (arrow) with diameters of 2 to 4 μm proliferating in the near surroundings (magnification 200-times). (inverted microscope, Hoffman illumination.)

Figure 8

Immunocytochemical staining of cell clusters and oocyte-like cells developed during the in vitro culture of cells scraped from the ovarian surface epithelium on markers of pluripotency SOX-2 and SSEA-4. (a, b) Non-stained cell clusters (negative control) (magnifications 40-times and 100-times). (c, d). Cell clusters positively (brown) stained on SOX-2 (magnification 40-times). (e) SOX-2 non-stained round cell (negative control) (magnification 200-times). (f) Round cell positively (brown) stained on SOX-2 (magnification 200-times). (g, h) Cell clusters positively (brown) stained on SSEA-4 surface antigen (magnification 100-times). (Inverted microscope, Hoffman illumination.)

Figure 9

Single-oocyte-like cell gene expression analyses in comparison with human embryonic stem cells and human fibroblasts. (a) Heatmap clustering. (b) Hierarchical clustering (Ward's Algorithm, Euclidean Distance Measure). (c). Principal component analysis (PCA). Legend: OLC: single oocyte-like cells, H1 hESC: human embryonic stem cells (H1 cell line), F161: human fibroblasts (F161 cell line), 1: single cell, 5: group of five cells, 10: group of ten cells, and 20: group of twenty cells.

Figure 10

Cell culture of human embryonic stem cells as positive control. (a,b) Early cell clusters (magnification 100-times). (c) Developing cell cluster (magnification 100-times). (d) Developed cell cluster (magnification 100-times). (e,f) Cell clusters positively stained on alkaline phosphatase activity (magnification 100-times). (g) Cell cluster surrounded by small cells positively (brown) stained on the SOX-2 marker of pluripotency (magnification 100-times). (h) Cell cluster positively (brown) stained on surface antigen SSEA-4 characteristic for pluripotent stem cells (magnification 100-times). (Inverted microscope, Hoffman illumination.)