Diabetes is rapidly becoming a global epidemic, with the International Diabetes Federation noting that close to 9% of the global adult population suffers from the disorder. Over the past few decades, work has begun to combat this as many protocols have been developed to in vitro differentiate viable pancreatic cells for cell therapy. However, this process is currently not cost effective and other avenues for production may be optimal. Transdifferentiation is one such avenue, as overexpression of key transcription factors can shift an initial cell type toward that of a target cell type. Here transdifferentiation was employed to generate pancreatic progenitors, which could express markers from all key lineages of the pancreas. Specific transcription factors used for this process were determined by two powerful and pioneering bioinformatic analyses, Lineage-guided Principle Component Analysis (LgPCA) and Mogrify. LgPCA investigation was undertaken to deduce the most potent transcription factors from this novel genome-wide analysis of 19,362 genes. Mogrify complemented this data set with transdifferentiation specific factors. The combination of which provided a highly optimised cohort of genes to study. A comprehensive study of known regulatory networks in mouse and human pancreas development was combined with expression profiling during the in vitro differentiation of human embryonic stem cells (hESCs) to early pancreatic beta-cells. Having selected a prioritised cohort of 8 transcription factors an enhanced adeno-associated viruses (AAVs) generation protocol was developed for their delivery into cells in vitro. Transcription factors were applied to human fetal fibroblasts to assess their capacity to induce transdifferentiation toward pancreatic progenitors. This was evaluated by gene expression profiling, including the expression of the endogenous genes encoding the transduced factors. This approach identified the best trio of transcription factors for transdifferentiation toward the phenotype of pancreatic progenitor. While further optimisation is needed, including in vivo transplantation, the data indicates that cell phenotype can be reproducibly altered for at least 1-2 weeks by the transduction of a very limited set of transcription factors. The conclusion is that transdifferentiation could offer a viable route to the ultimate large-scale production of insulin-secreting beta-cells from fibroblasts.
|Date of Award||1 Aug 2020|
- The University of Manchester
|Supervisor||Neil Hanley (Supervisor) & Karen Piper Hanley (Supervisor)|
- adeno-associated virus
Transdifferentiation toward pancreatic progenitors
Wearne, S. (Author). 1 Aug 2020
Student thesis: Phd