Investigating the application of human induced neural precursor cells for generating dopamine neurons in vitro

Abstract

Parkinson’s disease (PD) is a common neurological disease characterised by progressive degeneration of substantia nigra dopamine neurons and the deposition of intracellular proteinacious aggregates called Lewy bodies. Reprogramming technology holds great promise for the study and treatment of PD, as patient-specific ventral midbrain dopamine (vmDA) neurons can be generated in a dish. This should facilitate the investigation of early changes occurring during PD pathogenesis, allowing for the identification of new drug targets and providing a platform for drug screening. To date, most research using reprogramming technology to study PD has been conducted on induced pluripotent stem cells. Research into PD using direct reprogramming has been limited, primarily due to an inability to generate high yields of authentic human vmDA neurons. Nevertheless, direct reprogramming offers a number of advantages, and development of this technology is warranted. A method to directly reprogram adult human dermal fibroblasts into induced neural precursors (iNPs) by non-viral SOX2/PAX6 transfection was developed in our laboratory. This thesis aimed to investigate if vmDA neurons could be generated from iNPs. First, the regional identity of iNPs was investigated by examining temporal changes in gene expression through quantitative real-time PCR and immunocytochemistry. Overall, iNPs showed a mixed regional identity, with widespread expression of the anterior neuroepithelial markers SOX1, PAX6 and FOXG1. There was limited expression of most of the vmDA markers examined, however some late markers were expressed, including NURR1 and PITX3. Next, iNPs were differentiated to a neuronal fate with or without exposure to the patterning molecules SHH/FGF8, and it was found that established iNPs did not respond to patterning cues. Subsequently, a series of experiments was performed to investigate if temporal exposure to a range of patterning factors during the reprogramming process, and/or exposure to vmDA-related transgenes, could induce a vmDA iNP fate. Ultimately, these strategies did not prove effective at inducing an authentic vmDA iNP fate. Nevertheless, this thesis reports for the first time that iNPs, which have been reprogrammed from adult human cells using non-viral expression of lineagespecific factors, can give rise to dopamine neuronal-like cells that express TH, AADC, VMAT2, DAT and GIRK2.