Programming and reprogramming neural cell types using synthetic transcription factors

Abstract

Production of large numbers of desirable human cell types in the laboratory is one of the major goals of stem cell research. Current experimental approaches have focused on the strategy of recapitulating the events of normal embryogenesis in culture, by treating cells – either tissue stem cells or pluripotent stem cells (iPS/ES cells) – with cocktails of growth factors, matrix proteins or pharmacological agents. This is challenging and often requires weeks or months of elaborate cell culture regimes. An alternative approach is the forced expression of master regulatory transcription factors; this can bypass developmental programs and drive conversion to the target cell type. Each of these strategies is inefficient and unreliable. Recently a new opportunity has arisen to exploit synthetic transcription factors (sTFs) to program and reprogram cell fate. To create such sTFs the CRISPR/Cas9 system is repurposed through tethering of catalytically dead Cas9 to various transcriptional regulatory effector domains (e.g. VP16, KRAB). In this thesis, we have explored sTFs as tools to reset transcriptional regulatory networks in neural stem cells and mouse embryonic fibroblasts. We tested transcriptional activation of key neural lineage target genes (e.g Olig2, Sox10 and Nkx6.2). We designed and validated a series of sTFs that could effectively activity these. We have found that activation of Sox10 by dCas9-VP160 in mouse neural stem cells can increase the amount of arising oligodendrocyte and oligodendrocyte precursors cells during the differentiation. The activity of sTFs strongly depends on cellular context: i.e. a specific sTF might work well in one cell type but not another. Importantly, these biological barriers are not easily overcome by increasing the strength of the sTF – either through levels or types of effector domains used. Our data inspecting single cells suggests that multiplex delivery of sTFs can indeed cooperate by both increasing the number of cells that activated the gene of interest and increasing the level of transcriptional activation in a given cell. To fully exploit these new technologies, we therefore developed a new construction pipeline that allows easy and efficient assembly of multiple sTFs. Using this approach, we were able to successfully activate three different target genes from a single expression plasmid (Olig2, Sox10 and Nkx6.2) in fibroblasts. These sTFs we able to force fibroblast transdifferentiation towards oligodendrocyte lineage. Future studies will explore further how to exploit these sTFs to augment or replace current reprograming strategies.