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

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

Quantitative Comparison of Constitutive Promoters in Human ES cells

BACKGROUND: Constitutive promoters that ensure sustained and high level gene expression are basic research tools that have a wide range of applications, including studies of human embryology and drug discovery in human embryonic stem cells (hESCs). Numerous cellular/viral promoters that ensure sustained gene expression in various cell types have been identified but systematic comparison of their activities in hESCs is still lacking. METHODOLOGY/PRINCIPAL FINDINGS: We have quantitatively compared promoter activities of five commonly used constitutive promoters, including the human β-actin promoter (ACTB), cytomegalovirus (CMV), elongation factor-1α, (EF1α), phosphoglycerate kinase (PGK) and ubiquitinC (UbC) in hESCs. Lentiviral gene transfer was used to ensure stable integration of promoter-eGFP constructs into the hESCs genome. Promoter activities were quantitatively compared in long term culture of undifferentiated hESCs and in their differentiated progenies. CONCLUSION/SIGNIFICANCE: The ACTB, EF1α and PGK promoters showed stable activities during long term culture of undifferentiated hESCs. The ACTB promoter was superior by maintaining expression in 75-80% of the cells after 50 days in culture. During embryoid body (EB) differentiation, promoter activities of all five promoters decreased. Although the EF1α promoter was downregulated in approximately 50% of the cells, it was the most stable promoter during differentiation. Gene expression analysis of differentiated eGFP+ and eGFP- cells indicate that promoter activities might be restricted to specific cell lineages, suggesting the need to carefully select optimal promoters for constitutive gene expression in differentiated hESCs

FGF4 and Retinoic Acid Direct Differentiation of hESCs into PDX1-Expressing Foregut Endoderm in a Time- and Concentration-Dependent Manner

BACKGROUND: Retinoic acid (RA) and fibroblast growth factor 4 (FGF4) signaling control endoderm patterning and pancreas induction/expansion. Based on these findings, RA and FGFs, excluding FGF4, have frequently been used in differentiation protocols to direct differentiation of hESCs into endodermal and pancreatic cell types. In vivo, these signaling pathways act in a temporal and concentration-dependent manner. However, in vitro, the underlying basis for the time of addition of growth and differentiation factors (GDFs), including RA and FGFs, as well as the concentration is lacking. Thus, in order to develop robust and reliable differentiation protocols of ESCs into mature pancreatic cell types, including insulin-producing beta cells, it will be important to mechanistically understand each specification step. This includes differentiation of mesendoderm/definitive endoderm into foregut endoderm--the origin of pancreatic endoderm. METHODOLOGY/PRINCIPAL FINDINGS: Here, we provide data on the individual and combinatorial role of RA and FGF4 in directing differentiation of ActivinA (AA)-induced hESCs into PDX1-expressing cells. FGF4's ability to affect endoderm patterning and specification in vitro has so far not been tested. By testing out the optimal concentration and timing of addition of FGF4 and RA, we present a robust differentiation protocol that on average generates 32% PDX1(+) cells. Furthermore, we show that RA is required for converting AA-induced hESCs into PDX1(+) cells, and that part of the underlying mechanism involves FGF receptor signaling. Finally, further characterization of the PDX1(+) cells suggests that they represent foregut endoderm not yet committed to pancreatic, posterior stomach, or duodenal endoderm. CONCLUSION/SIGNIFICANCE: In conclusion, we show that RA and FGF4 jointly direct differentiation of PDX1(+) foregut endoderm in a robust and efficient manner. RA signaling mediated by the early induction of RARbeta through AA/Wnt3a is required for PDX1 expression. Part of RA's activity is mediated by FGF signaling

Fiska efter läsförståelse med metakognitiva strategier

Iden här kunskapsöversikten har vi studerat litteratur som behandlar metakognitiva strategier som hjälpmedel vid läsförståelseundervisningoch dess inverkan på elevernas förståelse. Studien innehåller en bearbetning avsex artiklar som motsvarar syftet med litteraturstudien som är att lyfta fram vadaktuell forskning säger om olika metakognitiva strategiers möjliga inverkan påelevers utveckling av läsförståelse, samt att se om det i forskningen går atturskilja skillnader i utfall och vad dessa i så fall beror på. Denna studievisar att undervisning om metakognitiva strategier generellt har en positivinverkan på elevers läsförståelse samt flera olika förmågor som kopplas tillläsförståelse. De förmågor som behandlas i forskningen om läsförståelse är: Medvetenheti läsningen, Kopplingar i läsning, förstå syftet med texten och Att förutspåvad som ska ske i texten. Skillnader i resultat kan dels bero på socioekonomiskstatus, attityder och tidigare kunskaper