טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentEvron Ayelet
SubjectThe Use of Amnion Epithelial Stem Cells in Reproduction
DepartmentDepartment of Medicine
Supervisor Professor Emeritus Eliezer Shalev
Full Thesis textFull thesis text - English Version


Abstract

Background: The common applied culture medium in which human amniotic epithelial cells (hAEC maintain their stem cell characteristics contains fetal calf serum (FCS) and thus is not compatible with possible future clinical applications due to the danger of animal derived pathogens. We replaced FCS with serum substitute supplement (SSS) in the common applied medium and cultured hAECs in this substitute serum medium (SSM). Due to the limited in vitro self-renewal potential of hAECs it is of importance to develop an appropriate cryopreservation method. We used TEST-Yolk Buffer as the cryoprotective agent during slow freezing and rapid thawing of hAECs.

The differentiation of hAECs into germ cell like cells has never been described before. In the present study, hAECs were cultured in SSM in order to induce differentiation of hAECs into oocyte like cell

Methods: Purity validation and characterization of freshly isolated and cultured hAECs was assessed through the expression of stem cell and germ cell specific markers by RT-PCR, by immunofluorescence staining and FACS. Karyotype was performed at passage four in order to exclude possible chromosome anomalies in hAECs cultured in SSM. The differentiation potential of hAECs into the cardiomyogenic lineage was tested through cardiac Troponin T expression by immunohistochemistry.

Results: hAECs cultured in SSM maintained expression of all the major pluripotent genes Sox-2, Oct-4  and Nanog as well as the expression of the stem cell specific surface antigens SSEA-4, SSEA-3 and TRA-1-60 over four passages. Using cardiac differentiation medium, hAECs differentiated into cardiac troponin T expressing cells.

FACS analysis revealed that cryopreserved in TEST-Yolk Buffer, between 17%-60% of hAECs expressed SSEA-4, SSEA-3 and TRA-1-60, expressed mRNA of Oct-4, Sox-2 and Nanog and maintained their ability to differentiate into cardiac troponin T expressing cells.

When cultured in SSM, already at passage 1, hAECs express germ cell specific genes and at passage 5 Large, round cells, resembling oocytes appeared, expressing germ cell and oocyte specific markers at the protein level. Gene and protein expression of meiosis specific markers revealed that the oocyte like cells entered meiosis. Yet, oocyte like cells failed to propagate through prophase of the first meiotic division.

Conclusions: hAECs maintain their stem cell characteristics when cultured in SSM for up to 4 passages and after cryopreservation in TEST-Yolk Buffer. This increases the safety of hAECs in possible future clinical applications. From our preliminary results we can conclude that hAECs have the potential to differentiate into oocyte like cells.