Ph.D Thesis

Ph.D StudentFine Amir
SubjectLocalized Stimulation of Growth Factors in Human Stem
Cells as a Model for Early Embryonic
DepartmentDepartment of Biotechnology
Supervisor PROF. Shulamit Levenberg


Embryogenesis is one of the most exciting fields in biology and medicine. However, apart from the very early stages of pre-implantation development, human embryos are inaccessible for research and are limited to section studies of diseased aborted fetuses. One approah to overcome this obstacle is to use animal models, usually mice, taking advantage of their well-defined genetics and reproductive characteristics. The use of mice as a model for human development has been justified by the observation that there is strong conservation throughout evolution of developmental processes and control genes.

Moreover, the relative ease by which their genome can be genetically manipulated and used for the introduction of specific mutations has made them extremely important for studying specific genes and pathways that are involved in embryonic development. Yet, despite the similarities between mouse and human, there are still major differences between species. These crucial discrepancies emphasize the need for a more powerful system for studying early human embryo development.

Embryonic stem (ES) cells have the ability to differentiate into all germ layers, holding great promise not only for a model of early embryonic development but also for a robust cell source for cell-replacement therapies. Embryoid body (EB) formation from ES cells is a common method for producing different cell lineages for further applications, nevertheless, the 3D organization and structure of EBs also presents unique challenges to effectively direct the differentiation of the embryonic cells.  ES cells differentiation is strongly influenced by physical and chemical signals comprising the local extracellular microenvironment, thus current methods to engineer EB differentiation have focused primarily on spatially controlling EB size, adding soluble factors to the media, or culturing EBs on or within natural or synthetic extracellular matrices. In order to investigate the mechanism that control ES differentiation and the cues that determine germ layers differentiation, we developed a new approach for control differentiation of human ES cells in the three dimensional system of the EB. Incorporating fluorescent microspheres coupled with the BMP4 growth factor into human EBs allowed us to induce differentiation from inside the hEB and localize this induction , relative to the fluorescent coupled microsphere by following the differentiation of the early mesoderm marker Brachyury.  Overall, our results clearly demonstrate the potential of the new approach for directing controlled differentiation of human ES cells  in a three dimensional system and a novel system for following differentiation at the single cell level in the hEB.