Adikibi, Tonye T (2011) Investigation into the functional role of the stem cell marker CD133. (PhD thesis), Kingston University, .
Abstract
CD133 is a pentaspan membrane protein found on pseudopodia, microvilli and other plasma protrusions irrespective of cell type in both humans and mice. CD 133 has been classified as a marker of primitive haemopoietic and neural stem cells. At the molecular level it interacts with cholesterol and is located within lipid micro domains known as lipid rafts. Using cellular and molecular techniques we investigated the functional role CD 133 plays in stem cell biology. Prior to commencing the functional experiments, we determined that MUTZ-2, Caco-2 and primary CD34+ cells provided the best characteristics to investigate CD133 function. We established growth conditions, patterns and CD 133 expression for all 3 cell types and concluded that Caco-2 was the preferential cell line based on CD133 expression and cell stability. A number of approaches were used to knock down CD133 usmg a variety of RNAi oligonucleotides. Plasmid vectors were used in an attempt to produce a permanent CD 133 knockout Caco-2 cell line. However, after successfully inserting the plasmid, the cell line failed to proliferate. Ultimately, a 73% CD 133 phenotypic knockdown was achieved using RNAi technology from Santa Cruz, with 50% CD133 re-expression within 5 days, confirmation by both PCR and flow cytometry. Knockdown of CD 133 in Caco-2 cells. resulted in no change in proliferation or adhesive properties to plastic, however a slight increase in cell cycle activity was observed. Gene profiling of CD 133 knocked down Caco-2 and control cells was carried out using microarray technology. This was also applied to cells incubated with monoclonal antibodies against epitopes of CD133, as these are often used as a means of cell isolation for CD133 functional studies. A variety of genes were up and down regulated in both groups when compared to the control cells. CD 133 knockdown caused an up-regulation of genes associated with cell migration, motility, cell cycle, Wnt and tyrosine kinase pathway inhibitors and lipid transport across the membrane and down-regulated cell adhesion genes and apoptotic related genes. Caco-2 cells incubated with monoclonal antibodies against CD 133 showed up regulation in genes associated with cell cycle, migration and DNA replication and down regulation of genes associated with regulation of cell proliferation and apoptosis. This result is significant due to the extensive use ofCD133 antibodies in functional CD133 and CD133+ cell population studies. Confocal studies showed partial co-localisation between the lipid rafts and CD133, removal of the lipid rafts using the drug Beta Methyl Cyclodextrine caused loss of CD 133. However, lipid raft expression remained relatively constant on CD133 knockdown cells. Examining the distribution of CD 133 on Caco-2 cells adhered to fibronectin compared to glass via confocal analysis, showed there is no direct involvement of CD133 with anchorage type cell adhesion. However, considering the confocal analysis showing the association of CD 133 within lipid rafts and the results of microarray, this would imply a more indirect role of CD 133 within the processes of cell adhesion. This study has revealed that that CD133 plays a suppressive role in stem cell biology and play a regulatory role in maintaining quiescence, keeping the early stem and progenitor cells in a non proliferating, non motile state. A number of studies in recent years have attempted to determine the function of CD133 which still remains relatively elusive. However, this study has contributed to a greater understanding of CD133 function and identified key areas for further investigation.
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