Differentiation and characterization of MafA-GFP reporter iPS cells into beta cell lineage

Examensarbete för masterexamen

Please use this identifier to cite or link to this item: https://hdl.handle.net/20.500.12380/301119
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Type: Examensarbete för masterexamen
Title: Differentiation and characterization of MafA-GFP reporter iPS cells into beta cell lineage
Authors: Avijgan, Mahtab
Abstract: Diabetes is a chronic disease characterized by increased blood sugar because of dysfunctional glucose homeostasis by the beta cells in the pancreas. Functional beta cells can be generated by primary cells or stem cells. Primary cells are isolated directly from tissues (blood or bone marrow) and retain the morphological and functional characteristics of their tissue of origin. However, they have a finite period of cell culture, a limited potential for self-renewal and differentiation and are more sensitive than stem cells, they often require additional nutrients and growth factors. In contrast, stem cells allow to investigate basic biological processes, manipulate cellular functions, establish new methods, or perform preliminary screenings. Considering the limitations of the primary cells, stem cells can be an alternative source. Stem cells are at the forefront of research in cell therapy, drug discovery, and disease-modelling. Generation of pancreatic beta cells is possible by differentiating human induced pluripotent stem cells (hiPS cells), although in stem cell research, efficiency of mature beta cells generation is a key problem (optimal efficiency at 80%, current efficiency at 20%). This project first aims to improve efficiency of pancreatic beta cell generation from hiPS cells by performing six differentiation processes following the same protocol and later screening for beta cell production at different stages of the differentiation process. It uses a wild type and two MafA-GFP reporter iPS cell lines, where MafA as a critical beta-cell-specific transcription factor is tagged with GFP by using CRISPR/Cas9 technology. Second, flow cytometry and immunofluorescence analysis are used at different stages of the differentiation process to characterize the differentiated cells to confirm faithful expression of MafA. Expression of GFP corresponds to expression of MafA in adult beta cells, since MafA is only present in mature beta cells, which this confirms complete differentiation and maturation of iPS cells into beta cells. The results obtained from the differentiation processes showed low level of reproducibility, although this project was successful in showing the GFP and MafA expression of MafA reporter lines. The MafA reporter lines successfully expressed GFP signals (~11% efficiency) at stage 7 of beta cell differentiation.
Keywords: induced pluripotent stem cells;MafA;beta cells differentiation;pancreas;FACS
Issue Date: 2020
Publisher: Chalmers tekniska högskola / Institutionen för fysik
URI: https://hdl.handle.net/20.500.12380/301119
Collection:Examensarbeten för masterexamen // Master Theses



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