Human Embryonic Stem Cells - Constitutive Gene Expression and Differentiation Towards Definitive Endoderm and Posterior Foregut Endoderm

Abstract: Abstract More than 300 million people are suffering from diabetes worldwide and the numbers of diabetic patients are increasing. Today´s treatments mainly rely on daily insulin injections. This help people to control blood glucose levels but still many patients suffer from the many complications associated with diabetes. Transplantations of islet cells are an alternative treatment that could help patients to become insulin independent and presumably also delay the development of complications. However, the increasing number of diabetic patients would require large amounts of transplantable insulin producing cells. Due to the fact that islet donor material is a limiting factor for transplantations, human embryonic stem cells (hESCs) could be an alternative source of transplantable insulin producing cells. hESCs have the unique potential to self-renew and to differentiate to many cell types, presumably also beta cells. In order to generate therapeutically relevant beta cells, the scientific community focuses on recapitulating the embryonic processes behind beta cell development. In order to develop protocols that direct differentiation of hESCs along the developmental program of pancreas development, cellular markers and methods that allow the identification and isolation of cells that displays a correct phenotype of each developmental stage are fundamental research tools. Identification of definitive endoderm (DE) - from where pancreas originates, requires analysis methods that can detect multiple markers within individual cells, since many markers expressed in DE are also detected in extraembryonic endoderm. In this thesis, we approached single cell gene expression analysis of hESCs differentiated towards DE to provide insight about expression of a panel of DE markers at the cellular level. Some of these markers have conventionally been measured by gene expression analysis at the population level and little information is available about expression at the cellular level in differentiating hESCs. To differentiate hESCs towards DE three different methods of activin A treatment were used. Single-cell gene expression analysis identified distinct gene expression signatures both between the activinA treated populations and within each population. Within the SOX17+ population, the DE markers CER1 and FOXA2 were co-expressed in the majority cells independent of activin A treatment. By contrast, HHEX, CXCR4, FOXA2, MIXL1 and LIM1/LHX1 were expressed to various extents within the SOX17+ populations of each activin A treatment. These data provides novel insight in DE gene expression at the cellular level of vitro differentiated hESCs and illustrates the usefulness of single-cell gene expression analysis in to identify the molecular signature of in vitro differentiated hESCs. Thus, this technique could be of great help to develop protocols that mimics pancreas development in vivo. Furthermore, this thesis includes work that test the ability of RA and FGF4 alone or in combination to direct differentiation of hESCs towards PDX1+ foregut endoderm. The rationale for this was that both RA and FGF signaling exhibit a patterning effect during endoderm patterning and also supports pancreas specification. By optimizing the timing and concentration of RA and FGF4, it was shown that RA is required to convert activin A-induced hESCs into PDX1+ cells and that part of the underlying mechanism involves FGF signaling. Characterization of the PDX1+ cells suggests that they represent posterior foregut endoderm not yet commited to pancreatic, posterior stomach or duodenal endoderm. Directed differentiation of hESCs would greatly benefit from a deeper understanding of the molecular mechanism that regulate growth and differentiation. To approach these questions, efficient genetic engineering techniques are advantageous tools for controlled expression of genes or to introduce fluorescent reporter genes. Constitutive promoters are useful tools due to their high level of expression in most cell types. Different eukaryotic/mammalian and viral constitutive promoters have been reported to ensure high level and sustained activity in hESCs but a comprehensive study was lacking. In this thesis, we performed a comparative study the activity and stability of five commonly used promoters in undifferentiated hESCs and during differentiation. These data suggested ACTB, EF1α and PGK promoters as the most stable promoters during long term culture of undifferentiated hESCs. During EB differentiation, activities of all five promoters were downregulated and EF1α was the most stable promoter although it was downregulated in 50% of the cells. Gene expression analysis of differentiated cells indicated that promoter activities might be restricted to specific cell lineages, indicating the need to carefully select optimal promoters for constitutive gene expression in differentiated hESCs.