Identification of novel transcripts involved in the haematopoietic differentiation of human embryonic stem cells

The holy grail of human embryonic stem cells (HESC) is the generation of a spectrum of differentiated cell types that may be used for clinical and therapeutic use in the treatment and cure of infinite diseases. Over recent years, research into the process of generation of specific cell types from HESC have advanced very rapidly and these advances appear only to be moving with greater speed as scientists sought to understand the specific mechanisms in the generation of these differentiated cells types. This study focused on furthering this understanding through the discovery of novel haematopoietic genes that may contribute to haematopoietic development by screening the entire human genome. This comparative microarray experiment relied heavily on the isolation and utilisation of haematopoietic-enriched cells acquired through the haematopoietic differentiation of HESC lines H9 and H1. The haematopoietic-enriched populations were generated using an approach that adapted published haematopoietic differentiation protocols, which was then optimised within this experiment for the generation of significantly greater quantities of haematopoietic progenitors compared with the standard foetal bovine serum differentiation approach. These differentiated cells were sorted for a haematopoietic-specific population by fluorescence-activate cell sorting (FACS) for KDR+ CD31+ (markers of early haematopoiesis). Microarray analysis revealed 3,162 transcripts that may play important roles in haematopoiesis, with pathway analysis supporting these findings revealing an overwhelming preference for vasculogenesis, angiogenesis, and erythropoiesis process networks within this gene list. A select number of transcripts were flagged for further investigation, and of these SOST, FLI1, EPO, and SCL were of particular interest and hypothesised to enhance in vitro haematopoietic differentiation from HESCs. The effect of SCL on haematopoietic differentiation was assessed using SCL over-expressing HESC lines, and it was shown to significantly enhance the efficiency of haematopoietic differentiation. The critical role of SCL in erythropoiesis has been well described in literature and within this study, it was discovered that SCL over-expression dramatically enhanced erythropoiesis of HESCs by over 25 fold compared to the control. The differentiation of SCL over-expressing HESCs within erythropoietic-optimised media may be the missing link in the efficient generation of therapeutic functional erythrocytes.EThOS - Electronic Theses Online ServiceGBUnited Kingdo