Comparative Analysis of Embryonic Stem Cells and Multipotent Adult Germline Stem Cells at the Level of Transcriptome and Proteome

Abstract

Pluripotency of different stem cell types is a complex biological state which allows the cells to continuously proliferate (self-renewal) but also primes them for differentiation into all germ layers and germ cells. Regulation of pluripotency involves the presence of transcriptional regulatory networks, which components are, however, until now incompletely defined. In the present study, we aimed at the identification of similarities and differences in gene expression patterns of embryonic stem cells (ESCs) and multipotent adult germline stem cells (maGSCs) with a special focus on genes known to be involved in the regulation of pluripotency. Another goal was the identification of putative new pluripotency-regulating factors. Therefore, we first performed whole genome microarray analysis to compare undifferentiated and differentiated ESCs and maGSCs with each other at RNA-level. It could be shown that the undifferentiated cell lines are not only indistinguishable from each other based on their expression of known pluripotency-regulating factors but also based on their global gene expression pattern. We could find that, as expected, the cell types change their gene expression profile during differentiation. However, after differentiation both cell types again show a very similar gene expression pattern. Furthermore, the comparison of ESCs and maGSCs at protein level is described. Herewith, it was possible to confirm the similarities between both cell types in their undifferentiated state. However, differences in protein abundance could be found after differentiation of the cell lines. Additionally, we could show that the post-translational modification hypusination of Eif5a has an effect on the proliferation potential of ESCs and maGSCs, whereas it did not influence the pluripotency of the cells. Finally, the functional characterization of the putative pluripotency-regulating factor Stra8 is described. It was found that Stra8 fulfills all the criteria for a protein involved in transcriptional regulation of pl uripotency. We could show that a change in protein level of Stra8, that are siRNA-mediated knockdown and stable overexpression, results in a change of expression level of known pluripotency regulators as well as marker genes for differentiation into the three germ layers. In addition, the identification and characterization of further putative pluripotency-regulating factors are shown. Therefore, a reanalysis of the results of the transcriptional profiling experiments described in the first part of the thesis was performed to identify transcription factors whose expression is downregulated during differentiation of ESCs and maGSCs. These candidate genes were further analyzed according to their expression pattern in pluripotent cell lines and adult organs.