Direct generation of region-specific induced neural Precursors from adult human fibroblasts using non-viral and viral methods

We previously demonstrated that adult human dermal fibroblasts (aHDFs) can be directly converted to neural precursor cells, and subsequently mature neurons and glia, by the introduction of the defined neural transcriptional factors SOX2 and PAX6. However, the understanding of the underlying gene changes that occur during reprogramming was limited, as gene characterisation in direct reprogramming of induced neural precursors (iNPs) often relies on the expression profile at the endpoint of reprogramming. This study will advance the understanding of underlying mechanisms during direct reprogramming. This thesis first aimed to characterise the temporal profile of proneural and region-specific markers during the generation of induced neural precursors (iNPs) using non-viral and viral methods to determine whether reprogrammed iNPs express the same sequential gene profile as that observed in human neural development. The expression of genes controlling neural induction and forebrain specification was examined by qPCR during iNP reprogramming. The plasmid-derived iNPs initially exhibited a gene profile representative of the anterior neuroectoderm, and then of a telencephalic glutamatergic and GABAergic phenotype. The lentiviral-derived iNPs appeared to bypass an anterior neuroectoderm fate and exhibited a gene profile representative of a telencephalic glutamatergic and GABAergic phenotype throughout the entire reprogramming. Hence, the gene profiles of plasmid- and lentiviral-derived iNPs are different and do not resemble the sequential gene profile observed in human neural development. Secondly, this thesis aimed to investigate the requirement of SOX2 and/or PAX6 for the direct reprogramming of iNPs. Induced NPs were generated with plasmid or lentiviral delivery of both SOX2 and PAX6, either SOX2 or PAX6, or reprogramming media alone. The neuronal differentiation ability of cells was assessed using neuronal markers NSE, VGLUT1, and GAD65/67 after differentiating in the neuronal media. Lentiviral-derived populations were further enriched by FACS, and NSE-positive neurons comprising either glutamatergic or GABAergic phenotypes were efficiently generated from SOX2/PAX6 and SOX2 only populations, while SOX2/PAX6 was the most effective condition for generating glutamatergic neurons. Moreover, glutamatergic and GABAergic neurons can both be effectively generated from the plasmidderived SOX2/PAX6 population. Together, these findings suggested that SOX2/PAX6 was the most effective condition for generating iNPs that can generate glutamatergic and GABAergic neurons representative of the telencephalic region of the developing human brain. The generation of feederfree region-specific neural precursors that retain the competence to generate mature neurons representative of a specific brain region provides a desirable approach for the study and the treatment of neurological diseases and neurodevelopmental disorders.

Name thesis:degree_name

PhD

Level thesis:degree_level

Doctoral

Discipline thesis:degree_discipline

Pharmacology

Grantor dc:publisher

ResearchSpace@Auckland

Year dc:date.issued

2016

Author dc:creator

• Liu, Rui

Advisors dc:contributor.advisor

• Connor, B
• Jones, K

Statement dc:rights

• Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. Previously published items are made available in accordance with the copyright policy of the publisher.

Licence dc:rights.uri

• https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm

Handle dc:identifier.uri

https://hdl.handle.net/2292/34408

OAI identifier oai:identifier

oai:researchspace.auckland.ac.nz:2292/34408