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| Status |
Public on Jun 27, 2024 |
| Title |
Inverse genetics tracing the differentiation pathway of human chondrocytes |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Objective: Mammalian somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) via the forced expression of Yamanaka reprogramming factors. However, only a limited population of the cells that pass through a particular pathway can metamorphose into iPSCs, while the others do not. This study aimed to clarify the pathways that chondrocytes follow during the reprogramming process. Design: The fate of human articular chondrocytes under reprogramming was investigated through a time-coursed single-cell transcriptomic analysis, which we termed an inverse genetic approach. The iPS interference technique was also employed to verify that chondrocytes inversely return to pluripotency following the proper differentiation pathway . Results: We confirmed that human chondrocytes could be converted into cells with an iPSC phenotype. Moreover, it was clarified that a limited population that underwent the silencing of SOX9, a master gene for chondrogenesis, at a specific point during the proper transcriptome transition pathway, could eventually become iPS cells. Interestingly, the other cells, which failed to be reprogrammed, followed a distinct pathway toward cells with a surface zone chondrocyte phenotype. The critical involvement of cellular communication network factors (CCNs) in this process was indicated. The idea that chondrocytes, when reprogrammed into iPSCs, follow the differentiation pathway backward was supported by the successful iPS interference using SOX9. Conclusions: This inverse genetic strategy is expected to be utilized to identify the master genes for the differentiation of various somatic cells. The utility of CCNs in articular cartilage regeneration is also supported.
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| Overall design |
Human normal articular chondrocytes were commercially available (Lonza, Basel, Switzerland). The chondrocytes in the original stock were collected via centrifugation and resuspended in the basal medium (Lonza) containing human recombinant fibroblast growth factor 2 (rhFGF-B: Lonza), R3-insulin-like growth factor-I (R3-IGF-1: Lonza), fetal bovine serum (FBS), gentamicin/amphotericin-B (GA), and insulin and transferrin (chondrocyte growth medium: CGMTM). Cells were plated, expanded in the monolayer culture at 37 °C in a 5% CO2 and detached for subcultures using 0.05% trypsin/0.02% EDTA (Biowest, Nuaillé, France). The chondrocytes were infected with SRV iPSC-3 recombinant virus, incubated for 2 hours at room temperature and further incubated in a CO2 incubator overnight at 37 °C. The following day, cells were collected and seeded on an iMatrix-511-coated (Takara Bio, Kusatsu, Japan) plate with a Stemfit Basic 02 medium (Ajinomoto, Tokyo, Japan). The medium was removed and replenished every 2 days until day 8. Thereafter, the medium was changed to the basal medium containing rhFGF-2, and cells were cultured until day 10.
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| Web link |
https://s3.okayama-u-bsg.org?appName=GSE261806
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| Contributor(s) |
Do HT, Ono M, Wang Z, Kitagawa W, Dang AT, Yonezawa T, Kuboki T, Oohashi T, Kubota S |
| Citation(s) |
38925474 |
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| Submission date |
Mar 18, 2024 |
| Last update date |
Aug 28, 2024 |
| Contact name |
Ziyi Wang |
| E-mail(s) |
wangziyi@s.okayama-u.ac.jp
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| Organization name |
Okayama University
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| Department |
Molecular Biology and Biochemistry
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| Street address |
2-5-1 Shikata-cho, Kita-ku
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| City |
Okayama |
| State/province |
Okayama |
| ZIP/Postal code |
700-8558 |
| Country |
Japan |
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| Platforms (1) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
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| Samples (10)
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| Relations |
| BioProject |
PRJNA1089120 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE261806_RAW.tar |
122.5 Mb |
(http)(custom) |
TAR (of ZIP) |
SRA Run Selector |
| Raw data are available in SRA |
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