Testicular Cell Conditioned Medium Supports Differentiation of Embryonic Stem Cells into Ovarian Structures Containing Oocytes

Abstract

Previous reports and the current study have found that germ cell precursor cells appear in embryoid bodies (EBs) formed from mouse embryonic stem cells as identified by positive expression of specific germ cell markers such as Oct-3/4, Mvh, c-kit, Stella, and DAZL. We hypothesized that if exposed to appropriate growth factors, the germ cell precursor cells within the EBs would differentiate into gametes. The source for growth factors used in the present study is conditioned medium collected from testicular cell cultures prepared from the testes of newborn males. Testes at this stage of development contain most growth factors required for the transformation of germ stem cells into differentiated gametes. When EBs were cultured in the conditioned medium, they developed into ovarian structures, which contained putative oocytes. The oocytes were surrounded by one to two layers of flattened cells and did not have a visible zona pellucida. However, oocyte-specific markers such as Fig-␣ and ZP3 were found expressed by the ovarian structures. The production of oocytes using this method is repeatable and reliable and may be applicable to other mammalian species, including the human. STEM CELLS 2006;24:266 -273

The promoter of the oocyte-specific gene, Gdf9, is active in population of cultured mouse embryonic stem cells with an oocyte-like phenotype

“…In addition, eGFP-positive cell viability was increased since less degeneration was seen by day 2 in the EB culture when compared to the ES cells differentiated in monolayer. Several other investigators have demonstrated the ability of EBs to give rise to germ cells (both male and female) in culture [4,10,41,42]. This suggests that the three-dimensional structure of the EB may preserve greater intercellular communication between the cells, allowing for activation f the transcriptional machinery for oocyte-specific genes [43].…”

“…When the transfected ES cells were allowed to form embryoid bodies (EBs) in culture [4,10], the appearance of eGFP-positive cells was again seen by d1. The eGFP-positive cells were seen clustered around the EBs and their structure was similar to that of the eGFP-positive cells that formed during in vitro differentiation of ES cells (Fig 5A).…”

“…However, the precise mechanisms by which specification occurs remains unclear, especially in humans, where research limitations make similar studies done in animals difficult. While the ability of ES cells to give rise to germ cells in vitro was once thought to be implausible, recently several studies have demonstrated the ability of ES cells to give rise not only to PGCs, but also to oocyte and sperm-like cells in vitro [4][5][6][7][8][9][10]. In vivo, several growth factors are responsible for the differentiation of PGCs into oogonia after the arrival at the genital ridge.…”

“…These include kit ligand (KL), bone morphogenic proteins (BMPs), stem cell factor (SCF), fibroblast growth factor (βFGF) and members of the transforming growth factor β (TGFβ) superfamily [11]. Several studies have established conditions by which ES cells generate germ cells in vitro through the use of PGC and germ cell enrichment techniques [4][5][6][7][8][9][10]. These in vitro systems of germ cell generation have helped to establish the mechanisms by which germ cells are specified.…”

Productionin vitrode cellules germinales murines et humaines à partir de cellules souches pluripotentes

“…[5, 7,13,16,19,22,24,25,27] Cellules Sertoli, cellules Leydig, cellules de la granulosa, cellules de la thèque…”