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| Status |
Public on Nov 27, 2023 |
| Title |
Highly cooperative chimeric super-SOX induces naïve pluripotency across species [RRBS] |
| Organism |
Homo sapiens |
| Experiment type |
Methylation profiling by high throughput sequencing
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| Summary |
Our understanding of pluripotency remains limited: iPSC generation has only been established for a few model species, pluripotent stem cell lines exhibit inconsistent developmental potential, and germline transmission demonstrated only for mice and rats. By swapping structural elements between Sox2 and Sox17, we built a chimeric super-SOX factor, Sox2-17, that enhanced iPSC generation in five tested species: mouse, human, cynomolgus monkey, cow, and pig. A swap of alanine to valine at the interface between Sox2 and Oct4 delivered a remarkable gain-of-function by stabilizing Sox2/Oct4 dimerization on DNA, enabling generation of high-grade OSKM iPSCs capable of supporting the development of healthy all-iPSC mice. Sox2/Oct4 dimerization emerged as the core driver of naïve pluripotency with its levels diminished upon priming. Transient overexpression of Sox2-17 and Klf4 (S*K cocktail) restored the dimerization and boosted the developmental potential of pluripotent stem cells across species, providing a universal method for naïve reset in mammals.
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| Overall design |
To investigate the effects of SOX2AV and SOX2-17 on the fidelity of reprogramming, we generated and characterized 30 hiPSC lines using episomal reprogramming of new-born foreskin (young, Y) and 56-year-old male dermal (old, O, AG04148) fibroblasts. For episomal reprogramming 5x10^5 fibroblasts were nucleofected with 3 μg of plasmid DNA mix: pCXLE-SOX2-P2A-KLF4 (Addgene ID 193292), pCXLE-SOX2AV-p2a-KLF4 (Addgene ID193291) pCXLE-SOX2-17-P2A-KLF4 (Addgene ID 193290), pCXLE-L-MYC-F2A-LIN28 (ML, Addgene ID 27080), pCXLE-hOCT4-shTP53 (Addgene ID 27077), pCXWB-EBNA1 (Addgene ID 37624) using Lonza NHDF Nucleofector kit (U-23 program), and plated in ROCKi-containing fibroblast media on a CF1 feeder layer. All the selected clonal iPSC lines were integration-free with normal karyotypes. The hiPSC samples were collected at passage 10-12, at day 3 after plating on Matrigel (Cornig) in StemFlex media (Gibco). We performed reduced representation bisulfite sequencing (RRBS) to analyze the methylome of hiPSCs.
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| Contributor(s) |
Velychko S, Keshet G |
| Citation(s) |
38141611 |
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| Submission date |
Nov 06, 2023 |
| Last update date |
Feb 26, 2024 |
| Contact name |
Sergiy Velychko |
| E-mail(s) |
sergiy_velychko@hms.harvard.edu
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| Organization name |
Harvard Medical School
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| Department |
Genetics
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| Lab |
Schöler
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| Street address |
77 Avenue Louis Pasteur, Room 232
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platforms (1) |
| GPL24676 |
Illumina NovaSeq 6000 (Homo sapiens) |
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| Samples (34)
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| This SubSeries is part of SuperSeries: |
| GSE247051 |
Highly cooperative chimeric super-SOX induces naïve pluripotency across species |
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| Relations |
| BioProject |
PRJNA1036147 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE247050_RAW.tar |
450.0 Kb |
(http)(custom) |
TAR (of BED) |
SRA Run Selector |
| Raw data are available in SRA |
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