Differentiation and characterization of MafA-GFP reporter iPS cells into beta cell lineage
Typ
Examensarbete för masterexamen
Program
Biotechnology (MPBIO), MSc
Publicerad
2020
Författare
Avijgan, Mahtab
Modellbyggare
Tidskriftstitel
ISSN
Volymtitel
Utgivare
Sammanfattning
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.
Beskrivning
Ämne/nyckelord
induced pluripotent stem cells , MafA , beta cells differentiation , pancreas , FACS