| 研究課題/領域番号 |
14F04775
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| 研究機関 | 国立研究開発法人理化学研究所 |
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研究代表者 |
SHIN JAE・WOO 国立研究開発法人理化学研究所, ライフサイエンス技術基盤研究センター, ユニットリーダー (60553849)
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| 研究分担者 |
LUGINBUEHL JOACHIM 国立研究開発法人理化学研究所, ライフサイエンス技術基盤研究センター, 外国人特別研究員
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| 研究期間 (年度) |
2014-04-25 – 2017-03-31
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| キーワード | Induced neurons / Cell Reprogramming / Single cell sequencing / Gene network |
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| 研究実績の概要 |
Aim of this research is to infer gene regulatory networks controlling the direct conversion of fibroblasts into different neuronal subtypes from single cell analysis. After having performed proof of principle experiments using single cell RNA sequencing of induced neurons generated by the overexpression of a small set of transcription factors (TFs), we identified several hurdles that had to be overcome in order for the project to succeed. First, further optimization of the differentiation protocol using additional sets of small molecules, growth factors and epigenetic modifiers raised conversion efficiency to ~60%. Second, extensive analysis of conversion efficiencies of different sets of these candidate TFs in combination with a mathematical model yielded a final list of 18 TFs. Based on morphological, qRT-PCR and immunofluorescence analyses, transduction of these 18 TFs successfully generated a heterogeneous population of induced neurons consisting of different neuronal subtypes. Third, combination of these methods with the pseudo-alignment software Kallisto allowed to simultaneously acquire endogenous and exogenous gene expression signatures of single induced neurons. Forth, while this work is still preliminary, we performed different statistical tests such as correlations and clustering analysis which identified potential combinations of exogenous TFs with implications into neuronal subtype specification.
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| 現在までの達成度 (区分) |
現在までの達成度 (区分)
2: おおむね順調に進展している
理由
To a large degree, the work is in line with the proposed research plan. Particular importance was given to the analysis of induced neurons prior to performing single cell sequencing, with the aim of reducing costs. Therefore, the main run of single cell sequencing analyses will be performed with a slight delay. However, we believe that careful evaluation of the experimental outline and the cell types to be analyzed prior to sequencing can significantly reduce costs and time. Many hurdles that were identified last year could be overcome this year, which is why we think that the project is at a good stage and likely will lead to a publication within this fiscal year.
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| 今後の研究の推進方策 |
Currently we are performing some additional analysis of the single cell RNA sequencing and C1-CAGE data. Shortly, this should give us definitive answers on how to design the final experiments. After analysis of the data of the final run, we should be able to propose a new screening method to identify transcription regulatory networks regulating the sub-classification of neurons derived from fibroblasts. Aim is to use additional experiments - for example by additional transduction of fibroblasts with small sets of candidate TFs and with the help of a collaborator who can analyze the neuronal activities – to validate the screen and obtain interesting biological data. Finally, we aim to publish a manuscript within this fiscal year.
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