Use of Induced Neural Precursor Technology to model Huntington's Disease

Abstract

Recent advances in cellular reprogramming have evolved a promising approach for the study, treatment and prevention of neurological diseases. Establishment of cell models relevant to a disease requires generation of high yields of the specific neuronal phenotypes affected by neurological disorders. The novel approach of directly reprogramming adult human dermal fibroblasts (HDF) into neural precursor (iNP) cells through transient ectopic expression of the neural genes SOX2 and PAX6 using non-viral plasmid transfection has been recently established in the Connor laboratory. We hypothesised that this novel technology could generate a cell model for the study of Huntington‟s disease (HD). We aimed to reprogram adult HDFs into iNP cells, and subsequently differentiate the cells into GABAergic mediumspiny- neurons (MSNs). In addition, we aimed to reprogram transgenic HD rat embryonic fibroblasts (REFs) into iNP cells. Neural genes SOX2 and PAX6 were transiently over-expressed in adult human dermal fibroblasts and transgenic HD REFs. The cells were cultured until colony formation was observed. The human iNP cells were examined for expression of multiple neural precursor cell (NPC) and embryonic forebrain markers using quantitative polymerase chain reaction (qPCR). The iNP cells were then cultured in differentiation media and neuronal phenotype was determined by immunocytochemistry using markers of GABAergic MSNs (DARPP32, GAD65/67 and calbindin) and neurons (TUJ1 and NSE). The transfected HD REFs were characterised by reverse-transcriptase PCR for detection of multiple NPC markers. Human iNP cells showed increased expression of self-renewal and pro-neural NPC genes including ventral telencephalon lineage-specific transcription factors DLX2 and GSX2. In addition, many ventral forebrain markers were detected. Transfected transgenic HD REFs showed no increased expression of NPC markers. Differentiation of human iNP cells demonstrated a considerable increase in the number of GABAergic MSN generated from iNP cells (40-60%) compared to that reported for induced pluripotent stem cells (5-10%). While reprogramming of transgenic HD REFs into iNP cells requires further investigation before development of a cellular model will be possible, adult human-derived iNP cells can generate high yields of GABAergic MSN. Therefore they may provide a cell source for HD modelling, providing high yields of the specific neuronal phenotype affected in the disease.