Ph.D Thesis

Ph.D StudentGerecht-Nir Sharon
SubjectGrowth and Differentiation of Human Embryonic Stem Cells
within Three-Dimensional Cultures
DepartmentDepartment of Biotechnology
Supervisors PROFESSOR EMERITUS Joseph Itskovitz
PROF. Smadar Cohen


In vitro differentiation of human embryonic stem cells (hESCs) can be initiated in two main ways- the formation of human embryoid bodies (hEBs) in suspension and 2-D culturing on a differentiation-inducing layer.

This thesis aimed to examine whether the differentiation of hESCs can be affected and manipulated by the cultivation conditions of the cells. To this end, three in vitro differentiation systems were established and studied. The formation of differentiating hEBs was established in rotating bioreactors. Using quantitative analyses for cell proliferation and differentiation and metabolic indices, this study defined the culture conditions in which control over hEBs aggregation.  The dynamic culture yielded a more efficient formation of hEBs, in terms of number of particles and cell proliferation and differentiation. 

Another model examined the generation of hEBs within 3-D porous alginate scaffolds. The confining environments of the alginate scaffold pores enabled a physical control over hEBs agglomeration resulting in an efficient formation of hEBs, with a relatively high degree of vascularization.

Human neovasculogenesis was explored in early developing human embryos and teratomas generated from hESCs. The involvement of vascular smooth muscle cells as well as the remodelling kinetics of endothelial cells during the early stages of developing human embryonic blood vessels and teratomas was demonstrated.

In a sequential study, early human vasculogenesis was explored in vitro using spontaneous and directed differentiation of hESCs.  Developing hEBs were found to spontaneously form vasculature structures and networks. Establishment of directed differentiation system was achieved by strict 2-D culture conditions which enhance vascular cell generation from hESCs.

Overall, this thesis shows that hEB formation and differentiation can be enhanced by physical constraints of 3-D culture systems, in addition to chemical cues of specific 2-D culture systems.