Identification and characterization of multipotential stem cells in immortalized normal ovarian surface epithelial cells

Abstract

The ovarian surface epithelium (OSE) is a single layer of squamous-to-cuboidal epithelial cells that experience repetitive ovulatory rupture and subsequent repair. However, the characteristics of human immortalized ovarian surface epithelial cells (IOSE80) remain elusive. This study aims to determine whether IOSE80 cells have the characteristics of stem cell proliferation and multilineage differentiation and their application in regenerative medicine. IOSE80 cells are sequenced by high-throughput transcriptome analysis, and 5 sets of public data are used to compare the differences between IOSE80 cells and bone marrow mesenchymal stem cells, pluripotent stem cells, and oocytes in transcriptome profiling. The IOSE80 cells present a cobblestone-like monolayer and express the epithelial cell marker KRT18; the stem cell markers IFITM3, ALDH1A1, and VIM; lowly express stem cell marker LGR5 and germ cell markers DDX4 and DAZL. In addition, the GO terms “regulation of stem cell proliferation”, “epithelial cell proliferation”, etc., are significantly enriched (P<0.05). IOSE80 cells have the potential to act as mesenchymal stem cells to differentiate into adipocytes with lipid droplets, osteoblasts, and chondroblasts in vitro. IOSE80 cells express pluripotent stem cell markers, including OCT4, SSEA4, TRA-1-60, and TRA-1-81, and they can be induced into three germ layers in vitro. IOSE80 cells also form oocyte-like cells in vitro and in vivo. In addition, IOSE80 cells exhibit robust proliferation, migration, and ovarian repair functions after in vivo transplantation. This study demonstrates that IOSE80 cells have the characteristics of pluripotent/multipotent stem cells, indicating their important role in tissue engineering and regenerative medicine.

1. Introduction

The ovary is covered by a single layer of squamous, cuboidal, or columnar ovarian surface epithelium (OSE), and it plays an important role in normal ovarian physiology [1‒4]. During the lifespan, these epithelial cells can encounter a series of cell cycle events, including quiescence, mitosis, epithelial rupture, repair, apoptosis, and degeneration [5,6]. As follicles are selected to develop during the follicular phase of the cycle, they are juxtaposed with the ovarian surface, and OSE cells in this region undergo mitosis to accommodate the rapid increase in follicle size. A key mechanism involved in wound repair is epithelial-mesenchymal transition (EMT) [7,8]. EMT is necessary for wound repair, enabling cells to migrate and secrete ECM proteins into the injured area [9]. The OSE for morphological EMT during wound repair in rhesus monkeys and rats has been studied, in which the OSE layer is flat on the developing corpus luteum [10,11]. After ovulation, cells left in the postovulatory follicle and epithelial cells undergo luteinization and form the corpus luteum (CL), a transient endocrine structure that produces progesterone or degenerates without pregnancy [12]. The recovery of OSE is very fast and effective, and the wound is completely closed within 12 h to 3 days after rupture in mice [13]. The remarkable circulation and regeneration abilities of OSE indicate the existence of resident stem cells.

In recent years, the stemness characteristics of ovarian epithelial cells have been reported [14,15]. The gene expression profile supports the hypothesis that human ovarian epithelial cells are pluripotent [16]. Bowen et al. [16] conducted comparative gene expression profiling analysis on normal human ovarian surface and ovarian cancer epithelial cells isolated from human serous papillary ovarian adenocarcinoma. The genes related to the maintenance and pluripotency of adult stem cells are highly expressed in healthy ovarian surface epithelial cells, while they are not expressed or are expressed at low levels in serous papillary ovarian adenocarcinoma [16]. OSE was found to express several well-established markers of stem cells, including NANOG, CD44, ALDH1A1, ALDH1A2, SFRP1, and LIM homeobox proteins LHX2 and LHX9 [16‒18]. In addition, they express stem cell markers, such as LY6A (also known as SCA1), Lgr5, Procr (protein C receptor), and c-KIT (KIT ligand) [19‒22]. Moreover, OSE has the potential to differentiate into multiple cell lineages. Many previous studies demonstrated that small round-shaped cells in the upper cortex of the ovarian surface derived from women are positive for markers of pluripotent embryonic stem cells, and they could differentiate into oocyte-like cells or parthenogenetic blastocyst-like structures in vitro [23‒28]. In addition, our research team reported for the first time that ovarian epithelial cells could be obtained from human follicular fluid. In the in vitro culture system, these epithelial cells spontaneously form oocyte-like cells and express specific germ cell markers (DAZL, STELLA, etc.). Furthermore, these ovarian epithelial cells e