Abstract
Recent advances have demonstrated the power of small molecules in promoting cellular reprogramming. Yet, the full potential of such chemicals in cell fate manipulation and the underlying mechanisms require further characterization. Through functional screening assays, we find that mouse embryonic fibroblast cells can be induced to trans-differentiate into a wide range of somatic lineages simultaneously by treatment with a combination of four chemicals. Genomic analysis of the process indicates activation of multi-lineage modules and relaxation of epigenetic silencing programs. In addition, we identify Sox2 as an important regulator within the induced network. Single cell analysis uncovers a novel priming state that enables transition from fibroblast cells to diverse somatic lineages. Finally, we demonstrate that modification of the culture system enables directional trans-differentiation towards myocytic, glial or adipocytic lineages. Our study describes a cell fate control system that may be harnessed for regenerative medicine.
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Acknowledgements
We thank L Yan, Z Zhong, Y Wang, X Tian, M Nguyen, HE Benjamin, E Macro, Z Shao, M Jiang, L Sun for help on experiments. We thank EpigenDx for DNA methylation analysis and RiboBio for ChIP-seq analysis. We thank Y Zhou, J Ji, W Cai, J Chen, Z Li for insightful discussions on the project. This work was supported by funding from Fundamental Research Funds for the Central Universities (GG), 1000 Youth Talent Plan (GG) and Harvard Stem Cell Institute Pilot Grant (SHO). This work was supported by Zhejiang University Stem Cell Institute. Patent applications have been filed related to the chemical reprogramming method reported in this paper.
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( Supplementary information is linked to the online version of the paper on the Cell Research website.)
Supplementary information
Supplementary information, Table S1
Small molecule combinations activate lineage specific regulators in the mouse embryonic fibroblast (MEF) cells. (XLSX 15 kb)
Supplementary information, Table S2
Phenotypic data from screened chemicals or chemical combinations. (XLSX 37 kb)
Supplementary information, Table S3
List of antibodies used for molecular characterization of lineage specific cell types. (XLSX 9 kb)
Supplementary information, Table S4
Microarray data for 6TCF and SGCF treatment time course. (XLSX 4560 kb)
Supplementary information, Table S5
Analysis of differentially expressed genes during iMT process. (XLSX 816 kb)
Supplementary information, Table S6
MACS peak files from H3K4me3 or H3K27me3 ChIP-seq experiments for 6TCF treated or control MEF cells. (XLSX 2317 kb)
Supplementary information, Table S7
Annotation of peaks from H3K4me3 or H3K27me3 ChIP-seq experiments for 6TCF treated or control MEF cells. (XLSX 5526 kb)
Supplementary information, Table S8
Single cell qPCR data during iMT process as well as used qPCR primer. (XLSX 283 kb)
Supplementary information, Movie S1
Spontaneous contraction of trans-differentiated cardiac myocytes from 6TCF treated MEF. (MP4 2936 kb)
Supplementary information, Movie S2
Spontaneous contraction of trans-differentiated cardiac myocytes from SGF treated MEF. (MP4 6810 kb)
Supplementary information, Movie S3
Spontaneous Ca2+ waves observed in trans-differentiated cardiac myocytes from 6TCF treated MEF. (MP4 4312 kb)
Supplementary information, Figure S1
Immunocytochemistry identifies trans-differentiated cells from MEF and TTF cells. (PDF 1132 kb)
Supplementary information, Figure S2
Analysis with differentially expressed genes during iMT process reveals involvement of several key pathways. (PDF 1662 kb)
Supplementary information, Figure S3
iMT process is associated with changes in chromatin structure. (PDF 250 kb)
Supplementary information, Figure S4
A unique gene expression module marks the iMT priming state before cell fate decision. (PDF 128 kb)
Supplementary information, Figure S5
Modification of the iMT chemical combinations enables efficient lineage specific trans-differentiation. (PDF 1125 kb)
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Han, X., Yu, H., Huang, D. et al. A molecular roadmap for induced multi-lineage trans-differentiation of fibroblasts by chemical combinations. Cell Res 27, 386–401 (2017). https://doi.org/10.1038/cr.2017.17
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DOI: https://doi.org/10.1038/cr.2017.17
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