|Ph.D Student||Assady Suheir|
|Subject||Characterization of Differentiation Pathways in Human|
Embryonic Stem Cells
|Department||Department of Medicine||Supervisor||Professor Karl Skorecki|
|Full Thesis text|
In the current study, undifferentiated human embryonic stem cells (uhESCs) were utilized as a platform to study differentiation into lineage specific stem cells, as a potential starting point for the generation of insulin-producing b-cells.
uhESCs are pluripotent cells, derived from the blastocyst inner cell mass. Spontaneous in vitro differentiation was examined using both adherent and suspension cell culture conditions. At three weeks of differentiation, about 1% of cells in embryoid bodies stained positively for insulin. RT-PCR studies detected transcripts of insulin, key transcription factors in pancreatic differentiation, as well as GLUT2 and glucokinase that are involved in glucose-dependent insulin secretion, upon induction of differentiation, but not in uhESCs.
Next, we attempted to set strategies for enrichment of pancreatic cells. Transgenic, stably transfected uhESC clones were generated using a plasmid carrying two selectable markers: a neomycin resistance gene driven by the SV40 promoter and an enhanced green fluorescent protein gene driven by a modified human insulin promoter. The parental H9 uhESC line and these clones were utilized in several enrichment protocols via neuroectodermal pathway, claimed by other investigators to enrich for insulin containing cells. Electron microscopy revealed that the resultant differentiated clusters consisted of primitive epithelial cells. In parallel, microarrays showed an abundance of expression of genes related to nervous system rather than pancreatic development. Thus, we concluded that most of the insulin staining was not of endogenous origin, but probably endocytosed insulin trapped from culture media, and considered it unlikely that neuroectodermal differentiation would lead to the generation of b-cells. However, teratoma studies did reveal co-staining of insulin, C-peptide and Pdx1, thereby supporting the concept of the inherent capability of hESCs to differentiate into pancreatic b-cells.
Next we utilized uhESCs to study their potential to differentiate into endodermal derivatives. Exposure to high concentrations of activin directed cells to differentiate into cells positive for Sox17 and CXCR4 proteins, with minimal expression of a-fetoprotein and no expression of Sox7. This pattern is consistent with differentiation into definitive endoderm. Furthermore, we demonstrated no advantage of prior exposure of medium to feeder fibroblasts, thus induction was achieved mainly by the direct effect of activin on hESCs. Moreover, these endodermal progenitors retain telomerase activity, that was regulated at the promoter level.
In conclusion, recent work conducted using hESCs supports the rationale of considering them to be a potential platform to conduct studies regarding lineage commitment and differentiation, for potential future therapeutic applications.