Identification and characterization of epithelial cells derived from human ovarian follicular fluid

Abstract

Introduction: Follicular fluid is important for follicular development and oocyte maturation. Evidence suggests that follicular fluid is not only rich in proteins but cells. Besides oocytes, the follicular fluid contains granulosa, thecal, and ovarian surface epithelial cells, and both granulosa and thecal cells are well-characterized. However, epithelial cells in follicular fluid are poorly studied. This study aims to isolate and characterize in vitro epithelial cells that originate from human ovarian follicular fluid retrieved in the assisted fertilization program.

Methods: Follicular fluid samples were collected from 20 women in the assisted reproduction program. Epithelial cells were characterized by flow cytometry assay, immunofluorescence staining, real-time PCR, and time lapse photography.

Results: Epithelial cell cultures were established from 18 samples. A small population of epithelial cells expresses germ-line stem cell markers, such as octamer-binding transcription factor 4 (OCT4), NANOG, and DEAD box polypeptide 4 (DDX4). In the epithelial cell culture system, oocyte-like cells formed spontaneously in vitro and expressed the following transcription markers: deleted in azoospermia-like (DAZL), developmental pluripotency associated protein 3 stella-related protein (STELLA), zona pellucida gene family C (ZPC), Syntaptonemal complex protein (SCP), and growth and differentiation factor 9 (GDF9). Some of the oocyte-like cells developed a zona pellucida-like structure. Both the symmetric and asymmetric division split of epithelial cells and early developing oocytes were observed using time lapse photography. Cell colonies were formed during epithelial culturing, which maintained and proliferated in an undifferentiated way on the feeder layer and expressed some pluripotency markers. These colonies differentiated in vitro into various somatic cell types in all three germ layers, but did not form teratoma when injected into immunodeficient mice. Furthermore, these epithelial cells could be differentiated directly to functional hepatocyte-like cells, which do not exist in ovarian tissues.

Conclusions: The epithelial cells derived from follicular fluid are a potential stem cell source with a pluripotent/multipotent character for safe application in oogenesis and regenerative medicine.

Figure 1

Morphology of epithelial cells in follicular fluid. (A) Epithelial cells (inside dotted line) were found from one sample of follicular fluid after 3 days of culturing. (B) Epithelial cells had cobblestone-like morphology and grew rapidly after 7 days of culturing. (C) Epithelial cells were found from another sample of follicular fluid after 5 days of culturing. (D) To the left of the dotted line were epithelial cells and to the right were granulosa cells. (E) Human embryonic stem cell-like colonies were growing in the epithelial cell culture. (F) Contrasting with epithelial cells, granulosa cells appeared irregularly polygonal, and the pseudopodia were long and tightly adherent. Scale bars: 200 μm (A, D) and 100 μm (B, C, E, F).

Figure 2

Stem cell characteristics of follicular fluid-derived epithelial cells. (A) Typical cell growth curve of epithelial cells after seeding 2.5 × 10⁴ cells in each well of 24-well culture plates, compared with granulosa cells. (B) Cell-surface expression of DDX4 in epithelial cells was detected by fluorescence-activated cell sorting analysis after 14 days of propagation. (C), (D), (E), (F) Assessment of epithelial cells proliferation by dual detection of DDX4 expression (green) and bromodeoxyuridine (BrdU) incorporation (red) in vitro cultures. (G), (H) Part of the epithelial cells was OCT4-positive or NANOG-positive. (I) Double staining of OCT4 and cytokeratin 18 in epithelial cells. Scale bars: 100 μm (C, D, E, F, G, H) and 20 μm (I). DAPI, 4′,6-diamidino-2-phenylindole.

Figure 3

Larger oocyte-like structures developed spontaneously from epithelial cells derived from human follicular fluid. (A) The volume of primary epithelial cells increased after 2 weeks of culturing. (B) Prominent nucleus and perinuclear accumulation of cytoplasmic organelles were present in large oocyte-like cells. Different sizes of oocyte-like structures after they were detached by trypsin after (C) 2 weeks , (D) 3 weeks , and (E) 4 weeks of culturing. (F) Blastocyst-like structures stained with 4′,6-diamidino-2-phenylindole developed after 6 weeks of culturing (arrowhead). Zona pellucida-like structures (arrow) were observed in epithelial cells post culture, indicating different stages of oocyte-like structure maturation. Scale bars: 200 μm (A), 100 μm (B, C), and 50 μm (D, E, F).

Figure 4

Real-time PCR analysis of human skin fibroblast, human ovarian cortex, epithelial cells cultured for 1 week, and epithelial cells cultured for 1 month. (A) Gene expression of transcripts for pluripotency markers showed higher expression levels for OCT4, Nanog, TERT, and Sox-2 in earlier epithelial cells, and decreased after 1 month of culturing. Human ovarian cortex also showed the presence of transcripts for pluripotency markers. (B) Transcripts for germ cell markers DAZL, STELLA, BLIMP1, STRA8, VAZA, GDF9, SCP3, ZPA, and ZPC in epithelial cells increased significantly after culturing for 1 month. Comparatively, human ovarian cortex also showed different expression levels of transcripts for germ cell markers. Human skin fibroblast cells served as negative controls, and 18s RNA as a house-keeping control gene was detected in all samples. Data represent mean ± standard error of three independent experiments, *P <0.05, **P <0.01. F, human skin fibroblast; OV, human ovarian cortex; E1, epithelial cells cultured for 1 week; E2, epithelial cells cultured for 1 month.

Figure 5

Characterization of oocyte-like structures observed post culture of epithelial cells by immunolocalization of germ cell markers. The oocyte-like structures stained positive after 4 weeks culturing for DAZL, ZPC, STELLA, SCP, and GDF-9, respectively. All markers are specific to ooplasm. 4′,6-Diamidino-2-phenylindole (DAPI) was used to counterstain and observe the nuclei. Scale bar = 50 μm.

Figure 6

Self-renewal of epithelial cells and in vitro development of oocytes. (A) Epithelial cells were split symmetrically. (B) Epithelial cells were split asymmetrically. (C) Cross-talk between epithelial cells with other cells. Scale bar: 25 μm (A, B) and 50 μm (C).

Figure 7

Pluripotent characteristics of cell colonies derived from epithelial cell cultures using human amnion epithelial cells as a feeder layer. (A) Cell colonies showed positive immunofluorescence staining for pluripotency markers, namely OCT-4, SSEA4, Tra-1-60, and Tra-1-82. All cell colonies were from the same 2-month-old cell culture. (B) Cells were pluripotent, as demonstrated by their potential to differentiate in vitro into progeny representing the three germ lineages. Immunofluorescence staining showed differentiated cells expressing nestin (ectoderm), brachyury (mesoderm), and sox17 (endoderm). Scale bars: 200 μm (A) and 100 μm (B). DAPI, 4′,6-diamidino-2-phenylindole.

Figure 8

Differentiation of epithelial cells to functional hepatocyte-like cells. Glycogen storage by hepatocyte-like cells was confirmed by Periodic Acid Schiff (PAS) staining and indocyanine green (ICG) uptake in hepatocyte-like cells (green). Scale bar, 100 μm.