In vitro and in vivo analysis of stem cells on the basis of stem cell parasitism in the urochordate Botryllus schlosseri
Zusammenfassung der Projektergebnisse
Stem cells play a crucial and conserved but not well-understood role as biological units of development and regeneration in all metazoan organisms. Botryllus schlosseri a marine colonial urochordate offers, thanks to its high regeneration potential, its weekly occurring asexual reproduction and its special life history trait of naturally occurring stem cell transplantation and stem cell parasitism between related colonies, a tantalizing model to study stem cell behavior. In a first project we used a candidate gene approach to further characterize stem cells in B. schlosseri. Therefore we identified and annotate in a first step conserved stem cell genes in a recently completed EST project from Sanger sequencing (JGI) and pyrosequencing. Additionally we used a recently developed technique to enrich stem cells via FACS sorting. Combining both approaches we analyzed the expression of conserved stem cell genes in enriched stem cell populations and colonies from different developmental stages. In this study we were able to show that all analyzed stem cell genes are upregulated in the enriched stem cell population and differentially expressed during asexual development. In addition our results show that all investigated stem cell genes are much high expressed in stem cells isolated from sexual immature animals pointing together with a recent publication (Brown et al., 2009) to the fact that stem cells from sexually immature colonies have probably a higher stem cell activity. Thus allowing the stem cells of young colonies to parasitize other colonies more efficiently. We also indentified differences in the expression of stem cell genes between two previously described stem cell populations which were considered in an earlier study to have comparable biological activities (Laird et al., 2005). By identifying differences in the stem cell gene expression we could show that further refined biological studies with improved stem cell enrichment procedures are necessary to identify probable differences in their biological activity. In addition we were able to identify two predicted transmembrane proteins (CD133 and IFITM 3), which are highly expressed in the enriched stem cell population compared to non BAAA+ cells enabling future attempts to improve the enrichment and localization of stem cells in Botryllus schlosseri. In a second project we isolated the promoter of two housekeeping genes (β-Actin / Ef-1 α), generated and tested reporter gene construct. By this we were able to show in preliminary experiments that lentivirus in combination with the enrichment of stem cells could provide a method to establish transgenisis in B. schlosseri.
