| 研究実績の概要 |
The key motivation of this research is to generate a diverse population of neurons at will. Together with Joachim, we have identified - based on literature and FANTOM5 - a set of transcription factors that are well-defined in brain tissues and neurons. These transcription factors were cloned into lentivirus vector expressing yellow fluorescent proteins. Transduction together with carefully tested and designed cocktail of growth factors, small molecules and matrixes led to a morphological diversity that reveals the complexity of neurons. Based on morphological, qRT-PCR and immuo-histochemistry analyses, neuron-specific markers start to express as early as four days and continue to increase up to 14 days post transduction. Because we introduced a unique set of transcription factors that have not been published before, we could observe new subtypes of induced-neuron. Subsequently, these induced neurons were subjected to single cell RNA sequencing. While the data is still preliminary, we could detect genes that are involved in neurogenesis, nervous system development, neuron projection development, cell morphogenesis involved in neuron differentiation, etc (all p-value less than 0.01).
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| 今後の研究の推進方策 |
The single cell RNA sequencing should be repeated with additional transcription factors in order to capture the wide diversity of induced neurons in our system. Furthermore, the data analysis of single cell RNA sequencing is not trivial. Several tests should be conducted to confirm that the variation we observe is derived from biology and not from technical aspect of the experiment. Furthermore, the analysis should reveal a novel set of transcription factors multiple subtypes of neurons. The validation work will be imperative. We have established a collaborator who can validate the neuronal activities of the induced neurons.
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