2023 Fiscal Year Final Research Report

Inverse genetics: A new methodology for the identification of key genes of somatic cell differentiation

Research Project

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Project/Area Number	21K19603
Research Category	Grant-in-Aid for Challenging Research (Exploratory)
Allocation Type	Multi-year Fund
Review Section	Medium-sized Section 57:Oral science and related fields
Research Institution	Okayama University
Principal Investigator	Kubota Satoshi 岡山大学, 医歯薬学域, 教授 (90221936)
Co-Investigator(Kenkyū-buntansha)	西田崇岡山大学, 医歯薬学域, 准教授 (30322233) 服部高子岡山大学, 医歯薬学域, 助教 (00228488) 高江洲かずみ (河田かずみ) 岡山大学, 医歯薬学域, 助教 (10457228) 青山絵理子岡山大学, 医歯薬学域, 助教 (10432650) 滝川正春岡山大学, 医歯薬学域, 教授 (20112063) 大野充昭岡山大学, 医歯薬学域, 准教授 (60613156)
Project Period (FY)	2021-07-09 – 2024-03-31
Keywords	reprogramming / iPS cell / chondrocyte / transcription factor / lncRNA
Outline of Final Research Achievements	Among the somatic cells being reprogrammed, only a limited population of the cells eventually metamorphose into iPS cells. In this study, under a hypothesis that the cells that followed their proper differentiation pathway backwards to pluripotent stem cells only could become iPS cells, we chased the transcriptomic transition of each cell under reprogramming by time-coursed single cell RNA-seq analyses. A minor population of chondrocytes being reprogrammed turned off SOX9, a master gene of chondrocyte differentiation, and then moved forward to iPS cells. The other off-targeted cells formed a major population and followed a single pathway to PRG4-positive articular surface zone chondrocyte-like cells. These findings indicate only the cells that retrospectively followed their proper differentiation pathway become iPS cells. This hypothesis was further confirmed by the fact that overexpression of SOX9 interfered with the iPS cell conversion of chondrocytes (iPS interference).
Free Research Field	硬組織分子生物学
Academic Significance and Societal Importance of the Research Achievements	本研究では軟骨細胞にリプログラミング因子を発現させ、iPS細胞となる細胞の中で起こるトランスクリプトームの変化をシングルセル解析で追うことで、リプログラミングでは分化過程で起こる現象を時間を逆転させた形で再現されることを示すことができた。この反転遺伝学(インバース・ジェネティクス)的手法を用いれば、幹細胞からの分化のプロセスが明らかではない体細胞の分化経路を、体細胞から幹細胞までのリプログラミング過程を辿ることで知ることができる。つまりこの新手法はあらゆる体細胞分化のマスター遺伝子の探索に有用と考えられ、さまざまな細胞、組織、臓器の医学生命科学的研究の進歩に大きく貢献する可能性がある。