Inducible forward programming of human induced pluripotent stem cells to skeletal myocytes
Typ
Examensarbete för masterexamen
Master's Thesis
Master's Thesis
Program
Biotechnology (MPBIO), MSc
Publicerad
2024
Författare
Löfgren, Emil
Modellbyggare
Tidskriftstitel
ISSN
Volymtitel
Utgivare
Sammanfattning
Differentiation of human induced pluripotent stem cells into a desired phenotype is
challenging. Previous attempts of hiPSC differentiation into skeletal myocytes using
an in-house protocol has resulted in heterogeneous populations of cells. Resolving
the heterogeneity of Skeletal myocyte differentiation is key for acquiring tissues and
cell types suited for exploring novel therapies and mechanisms within muscle related
diseases. Thus the main objective of this thesis was to investigate if alterations
in gene expression could resolve the heterogeneity of the differentiation process of
Skeletal Myocytes. In this thesis, differentiation of hiPSCs into skeletal myocytes
was explored through modulation of the transcription factors MYOD1 and OCT4
using the genome editing enrichment method "Xential".
The addition of the HBB IVS2 intron sequence led to a 2-fold increase in MYOD1
expression, furthermore adjustments to the seeding density led to an additional 2-
fold increase of MYOD1. Knockdown efficiency of shRNA targeting POU5F1 is
correlated with the seeding density. The downstream marker of myogenesis DES
had a 15-fold increase when seeding density was adjusted. This correlation between
differentiation efficiency and seeding density highlights the interplay between envi-
ronmental pressures and gene expression. The HBB IVS2 intron sequence led to
higher homogeneity in the cultures, this provides insights into future strategies to
mitigate heterogeneity in hiPSC differentiation.
Beskrivning
Ämne/nyckelord
Skeletal Myocytes , hiPSC , iPSC , MYOD1 , OCT4 , Seeding Density , Xential , Diphtheria Toxin , differentiation , HBB ISV2