Human Pluripotent Stem Cell Models of Huntington's Disease

Abstract

Huntington’s disease (HD) is a late-onset, autosomal, dominant and progressive neurodegenerative disorder for which there is no disease-modifying therapy. An abnormal trinucleotide CAG repeats expansion (>36 CAG repeats) in exon 1 of the gene coding for the Huntingtin (HTT) protein is the causative mutation for the disease. Animal models of HD, such as the R6/2 mouse model, already exist. Yet, in order to understand the disease at a molecular level, cellular models of HD are also needed. At the beginning of this project, existing models had been developed from tumour cell lines using genome-integrating lentiviral delivery system carrying a mutated HTT exon 1. This model was not optimal, as the cells will always have noninnate extra copies of HTT, which expression is driven by exogenous promoter. In order to circumvent this, we were aiming to develop two human stem cell models of HD and use them as tools in drug discovery and further understanding of molecular mechanisms of the disease. The first model is human embryonic stem cells (hESCs) isolated from clinically unsuitable embryos, donated by consenting couples. The embryos carrying a mutation in HTT gene, as determined by Preimplantation Genetic Diagnosis (PGD), were used to derive HD-specific mutation-carrying hESC lines (HD-hESC). Seven HD- hESCs lines were derived at the Assisted Conception Unit (ACU) at Guy’s Hospital, King’s College London (KCL). The second model is induced pluripotent stem cells (iPSCs) that I would derive from keratinocytes obtained from plucked hairs of consenting HD patients. Their greater availability means that we would be able to derive lines representing a larger spectrum of HD phenotype due to variations in CAG repeats expansion. Reprogramming will be done using a combination of modified mRNAs and/or a lentiviral vector encoding the transcription factors needed for reprogramming. The overall goal of the project was to optimize culture conditions for the HD-hESC and HD-iPS cell lines, fully characterise them, and differentiate them in vitro. Epidemiology studies suggest that cardiac failure is the second cause of death in HD patients. For this reasons, HD-ESCs were differentiated into cardiac cells (cardiomyocytes) in order to define and characterize the cardiac HD phenotype. Here, I am describing thorough characterization of normal and HD-hESC lines and their differentiation into cardiomyocytes. I am also presenting my data on reprogramming of human fibroblasts into iPSC using synthetic modified mRNA. Due to time limit and several technical issues outlined in the text, I was unable to successfully reprogram cells from keratinocytes obtained from plucked hairs of consenting HD patients.

Date of Award	2014
Original language	English
Awarding Institution	King's College London