Ph.D Thesis

Ph.D StudentKreizman-Shefer Hila
SubjectDifferentiation and Histone Modifications in Human Amniotic
Epithelial Cells Expressing Germ Cells and
Gametogenesis Markers
DepartmentDepartment of Medicine
Supervisor PROFESSOR EMERITUS Eliezer Shalev
Full Thesis textFull thesis text - English Version


Epigenetic patterning is believed to be crucial for prevention, at least in part, of some heritable diseases and reproductive failure. Primordial germ cells (PGCs) transfer genetic and epigenetic information between generations and unique epigenetic modifications are necessary for PGCs to progress properly to the establishment of the gametes totipotent state. Human amniotic epithelial cells (hAECs) were recently suggested to serve as an in vitro culture system that may allow us to study germline differentiation pathway. In the current study, the potential of bone morphogenetic protein (BMP) 4 and retinoic acid (RA) to induce epigenetic patterning in hAECs expressing germ cell markers was studied. The sex determination process in hAECs expressing germ cell markers was characterized as well.

BMP4 and RA administration in the context of serum free media was found to create a stimulatory microenvironment for PGCs differentiation, which was studied using quantitative (q) PCR, flow analysis, immunofluorescence, elisa assays as well as fluorescence in-situ hybridization (FISH) and karyotype analyses. In addition, EZH2 activity was inhibited in order to determine its physiological significance in epigenetic reprogramming in hAECs expressing germ cell markers.

BMP4 and RA stimulation in hAECs was found to induce both germ cells markers expression and epigenetic state modification. While showing oocyte-like morphology, hAECs were found to express stage-specific markers for the specification (PRDM1 and PRDM14), post-migration (DAZL and VASA) and gametogenesis and meiosis (GDF9, DMC1 and SCP3) stages of PGCs development. Epigenetic patterning of specific epigenetic marks (H3K9me2, H3K27me3, H3K9ac, H3K4me3 and H3S10phosph.) was examined in hAECs expressing OCT-3/4 or VASA, as well as the expression of DNMT3A and total methylation level in hAECs expressing germ cells markers. OCT-3/4- and VASA expressing cells were shown to have different expression profiles of the examined epigenetic marks and their susceptibility to BMP4 and RA induction was found to be diverse. PGCs epigenetic reprogramming was found to occur against a background of both germ cells markers expression and EZH2 activity, as they may have a crucial role in early germ cell fate determination.

The current study provides information on the dynamics in specific histone H3 modifications expression and general epigenetic state. These modifications may represent, along with germ cells markers, the stage of hAECs differentiation towards cells expressing germ cells markers. Following the induction of hAECs with BMP4 and RA, the cells were directed to express germ cells markers in an epigenetic state which characterizes post-migratory PGCs.