Ph.D Thesis

Ph.D StudentDamouni Kampania Rawan
SubjectRegulation of Cell Cycle during Maturation of Human Amniotic
Epithelial Cells, Directed to Express Germ Cells
and Primary Oocyte Markers
DepartmentDepartment of Medicine
Supervisor PROFESSOR EMERITUS Eliezer Shalev
Full Thesis textFull thesis text - English Version


Background: The increasing knowledge of primordial germ cells (PGC) specification in vivo has led to attempts to mimic the process in vitro. Several subsequent reports have demonstrated that PGCs, as well as sperm and oocyte-like cells, can be derived from mouse pluripotent stem cells. Recently the complex multistep pathway of germ cell development from mouse PSCs has been recreated and successfully generated healthy offspring from the derived sperm.

Recently we have reported that human amniotic epithelial cells differentiate into cells expressing germ cell specific markers when cultured in medium containing serum substitute supplement. Human amniotic epithelial cells (hAECs) develop from the epiblast. Due to the derivation of the amniotic epithelium before the time of gastrulation, the amniotic epithelium can provide an appropriate in vitro system for the investigation of some aspects of human oocyte maturation and gametogenesis. Most knowledge of the regulation of oocyte maturation comes from animal experiments, although similar concepts may be relevant to human oocytes. It has been shown that cyclinB-Cdc2/CDK1 is a common key controller in meiotic and mitotic M-phase. However, there are meiosis-specific modulations in the regulation of cyclinB-Cdc2.

The aim of the current study: was to establish an in vitro model, to study meiotic competent in human germ cell-like cells derived from hAECs induced by various growth factors and hormones and to explore the role of cell cycle regulation in these cells.

Methods: hAECs were cultured under floating conditions in medium containing various combinations of bone morphogenetic proteins (BMPs) BMP4, BMP8, stem cell factor (SCF) and epidermal growth factor (EGF) with and without retinoic acid (RA) induction. Stages of specific protein expression and cell cycle specific marker Cdc2 (Thr161) in the presence or absence of CDC25 phosphatase inhibitor was tested using, immunofluorescence, flow cytometry and chromatin spread analysis.

Results: The addition of RA induced the expression of germ cell-specific markers and meiotic markers, in particular VASA and DMC1, in a time-dependent manner in BMP4 treated hAECs. BMP4 and RA imply a microenvironment consistent of cues even more stimulatory and necessary for both differentiations of hAECs into cells expressing markers specific to primary oocyte and entry into meiotic prophase 1. Nuclear localization of the two meiosis specific markers DMC1 and SCP3 was observed in BMP4 treated hAECs at passage 5 after the induction of RA at passage 2, Whereas, putative PGCs differentiated spontaneously from hAECs, the nuclear distribution or expression of meiotic markers such as SCP3 and DMC1, were always highly abnormal in comparison to that observed in BMP4 treated RA-induced hAECs.

It appears that another cause of failure to differentiate hAECs to oocyte like cells was disruption of the activity of cell cycle regulator CDC2 assembly in BMP4 treated RA-induced hAECs, thereby preventing development of the CDC2 kinase activity required to trigger G2/M-phase transition by CDC25 phosphatase inhibitor.

Conclusions: These studies provide novel in vitro evidence for a link between meiotic cell cycle regulation and maturation of hAECs into cells expressing germ and primary oocyte cell specific markers.