The chemical reprogramming of unipotent adult germ cells towards authentic pluripotency and de novo establishment of imprinting
Received date: 23 May 2022
Accepted date: 13 Sep 2022
Published date: 15 Jul 2023
Copyright
Although somatic cells can be reprogrammed to pluripotent stem cells (PSCs) with pure chemicals, authentic pluripotency of chemically induced pluripotent stem cells (CiPSCs) has never been achieved through tetraploid complementation assay. Spontaneous reprogramming of spermatogonial stem cells (SSCs) was another non-transgenic way to obtain PSCs, but this process lacks mechanistic explanation. Here, we reconstructed the trajectory of mouse SSC reprogramming and developed a five-chemical combination, boosting the reprogramming efficiency by nearly 80- to 100-folds. More importantly, chemical induced germline-derived PSCs (5C-gPSCs), but not gPSCs and chemical induced pluripotent stem cells, had authentic pluripotency, as determined by tetraploid complementation. Mechanistically, SSCs traversed through an inverted pathway of in vivo germ cell development, exhibiting the expression signatures and DNA methylation dynamics from spermatogonia to primordial germ cells and further to epiblasts. Besides, SSC-specific imprinting control regions switched from biallelic methylated states to monoallelic methylated states by imprinting demethylation and then re-methylation on one of the two alleles in 5C-gPSCs, which was apparently distinct with the imprinting reprogramming in vivo as DNA methylation simultaneously occurred on both alleles. Our work sheds light on the unique regulatory network underpinning SSC reprogramming, providing insights to understand generic mechanisms for cell-fate decision and epigenetic-related disorders in regenerative medicine.
Yuhan Chen , Jiansen Lu , Yanwen Xu , Yaping Huang , Dazhuang Wang , Peiling Liang , Shaofang Ren , Xuesong Hu , Yewen Qin , Wei Ke , Ralf Jauch , Andrew Paul Hutchins , Mei Wang , Fuchou Tang , Xiao-Yang Zhao . The chemical reprogramming of unipotent adult germ cells towards authentic pluripotency and de novo establishment of imprinting[J]. Protein & Cell, 2023 , 14(7) : 477 -496 . DOI: 10.1093/procel/pwac044
| 1 |
Aibar S, Gonzalez-Blas CB, Moerman T et al. SCENIC: single-cell regulatory network inference and clustering. Nat Methods 2017;14:1083–+.
|
| 2 |
Alquicira-Hernandez J, Sathe A, Ji HP et al. scPred: accurate super-vised method for cell-type classification from single-cell RNA-seq data. Genome Biol 2019;20:264.
|
| 3 |
An J, Zheng Y, Dann CT. Mesenchymal to epithelial transition mediated by CDH1 promotes spontaneous reprogramming of male germline stem cells to pluripotency. Stem Cell Rep 2017;8:446–459.
|
| 4 |
Bian S, Hou Y, Zhou X et al. Single-cell multiomics sequencing and analyses of human colorectal cancer. Science 2018;362:1060–1063.
|
| 5 |
Butler A, Hoffman P, Smibert P et al. Integrating single-cell transcriptomic data across different conditions, technologies, and species. Nat Biotechnol 2018;36:411–+.
|
| 6 |
Byrne JA, Pedersen DA, Clepper LL et al. Producing primate embryonic stem cells by somatic cell nuclear transfer. Nature 2007;450:497–502.
|
| 7 |
Cao S, Yu S, Li D et al. Chromatin accessibility dynamics during chemical induction of pluripotency. Cell Stem Cell 2018;22:529–542.e525.
|
| 8 |
Choi J, Huebner AJ, Clement K et al. Prolonged Mek1/2 suppression impairs the developmental potential of embryonic stem cells. Nature 2017;548:219–223.
|
| 9 |
Costoya JA, Hobbs RM, Barna M et al. Essential role of Plzf in maintenance of spermatogonial stem cells. Nat Genet 2004;36:653–659.
|
| 10 |
Dobin A, Davis CA, Schlesinger F et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 2013;29:15–21.
|
| 11 |
Dong J, Hu Y, Fan X et al. Single-cell RNA-seq analysis unveils a prevalent epithelial/mesenchymal hybrid state during mouse organogenesis. Genome Biol 2018;19:31.
|
| 12 |
Gu C, Liu S, Wu Q et al. Integrative single-cell analysis of transcriptome, DNA methylome and chromatin accessibility in mouse oocytes. Cell Res 2019;29:110–123.
|
| 13 |
Guan K, Nayernia K, Maier LS et al. Pluripotency of spermatogonial stem cells from adult mouse testis. Nature 2006;440:1199–1203.
|
| 14 |
Gurdon JB, Byrne JA. Classic Perspective: The first half-century of nuclear transplantation. Proc Natl Acad Sci USA 2003;100:8048–8052.
|
| 15 |
Hou P, Li Y, Zhang X et al. Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds. Science 2013;341:651–654.
|
| 16 |
Jeong HS, Bhin J, Joon Kim H et al. Transcriptional regulatory net-works underlying the reprogramming of spermatogonial stem cells to multipotent stem cells. Exp Mol Med 2017;49:e315.
|
| 17 |
Kanatsu-Shinohara M, Inoue K, Lee J et al. Generation of pluripotent stem cells from neonatal mouse testis. Cell 2004;119:1001–1012.
|
| 18 |
Kanatsu-Shinohara M, Ogonuki N, Inoue K et al. Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol Reprod 2003;69:612–616.
|
| 19 |
Kanatsu-Shinohara M, Tanaka T, Ogonuki N et al. Myc/Mycn-mediated glycolysis enhances mouse spermatogonial stem cell self-renewal. Genes Dev 2016;30:2637–2648.
|
| 20 |
Kiselev VY, Yiu A, Hemberg M. scmap: projection of single-cell RNA-seq data across data sets. Nat Methods 2018;15:359–362.
|
| 21 |
Ko K, Tapia N, Wu G et al. Induction of pluripotency in adult unipotent germline stem cells. Cell stem cell 2009;5:87–96.
|
| 22 |
Konermann S, Lotfy P, Brideau NJ et al. Transcriptome Engineering with RNA-Targeting Type VI-D CRISPR Effectors. Cell 2018;173:665–+.
|
| 23 |
Korsunsky I, Millard N, Fan J et al. Fast, sensitive and accurate integration of single-cell data with Harmony. Nat Methods 2019;16:1289–1296.
|
| 24 |
Krueger F, Andrews SR. SNPsplit: Allele-specific splitting of alignments between genomes with known SNP genotypes. F1000Res 2016;5:1479.
|
| 25 |
La Manno G, Soldatov R, Zeisel A et al. RNA velocity of single cells. Nature 2018;560:494–+.
|
| 26 |
Li R, Liang J, Ni S et al. A mesenchymal-to-epithelial transition initiates and is required for the nuclear reprogramming of mouse fibroblasts. Cell Stem Cell 2010;7:51–63.
|
| 27 |
Li TD, Feng GH, Li YF et al. Rat embryonic stem cells produce fertile offspring through tetraploid complementation. Proc Natl Acad Sci USA 2017;114:11974–11979.
|
| 28 |
Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features. Bioinformatics 2014;30:923–930.
|
| 29 |
Liu L, Luo GZ, Yang W et al. Activation of the imprinted Dlk1-Dio3 region correlates with pluripotency levels of mouse stem cells. J Biol Chem 2010;285:19483–19490.
|
| 30 |
Ly CH, Lynch GS, Ryall JG. A metabolic roadmap for somatic stem cell fate. Cell Metab 2020;31:1052–1067.
|
| 31 |
Matoba S, Wang H, Jiang L et al. Loss of H3K27me3 imprinting in somatic cell nuclear transfer embryos disrupts post-implantation development. Cell stem cell 2018;23:343–354.e345.
|
| 32 |
Moore L, Cagan A, Coorens THH et al. The mutational landscape of human somatic and germline cells. Nature 2021;597:381–386.
|
| 33 |
Neri F, Krepelova A, Incarnato D et al. Dnmt3L antagonizes DNA methylation at bivalent promoters and favors DNA methylation at gene bodies in ESCs. Cell 2013;155:121–134.
|
| 34 |
Reik W, Dean W, Walter J. Epigenetic reprogramming in mammalian development. Science 2001;293:1089–1093.
|
| 35 |
Romanienko PJ, Camerini-Otero RD. The mouse Spo11 gene is required for meiotic chromosome synapsis. Mol Cell 2000;6:975–987.
|
| 36 |
Samavarchi-Tehrani P, Golipour A, David L et al. Functional genomics reveals a BMP-driven mesenchymal-to-epithelial transition in the initiation of somatic cell reprogramming. Cell stem cell 2010;7:64–77.
|
| 37 |
Schiebinger G, Shu J, Tabaka M et al. Optimal-transport analysis of single-cell gene expression identifies developmental trajectories in reprogramming. Cell 2019;176:928–943.e922.
|
| 38 |
Schmeier S, Alam T, Essack M et al. TcoF-DB v2: update of the database of human and mouse transcription co-factors and transcription factor interactions. Nucleic Acids Res 2017;45:D145–D150.
|
| 39 |
Schrans-Stassen BH, Saunders PT, Cooke HJ et al. Nature of the sper-matogenic arrest in Dazl -/- mice. Biol Reprod 2001;65:771–776.
|
| 40 |
Seisenberger S, Andrews S, Krueger F et al. The dynamics of genome-wide DNA methylation reprogramming in mouse primordial germ cells. Mol Cell 2012;48:849–862.
|
| 41 |
Shannon P, Markiel A, Ozier O et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res 2003;13:2498–2504.
|
| 42 |
Smallwood SA, Lee HJ, Angermueller C et al. Single-cell genome-wide bisulfite sequencing for assessing epigenetic heterogeneity. Nat Methods 2014;11:817–820.
|
| 43 |
Smith T, Heger A, Sudbery I. UMI-tools: modeling sequencing errors in Unique Molecular Identifiers to improve quantification accuracy. Genome Res 2017;27:491–499.
|
| 44 |
Stadtfeld M, Apostolou E, Akutsu H et al. Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells. Nature 2010;465:175–181.
|
| 45 |
Stuart T, Butler A, Hoffman P et al. Comprehensive integration of single-cell data. Cell 2019;177:1888–1902.e1821.
|
| 46 |
Szabó PE, Hübner K, Schöler H et al. Allele-specific expression of imprinted genes in mouse migratory primordial germ cells. Mech Dev 2002;115:157–160.
|
| 47 |
Takahashi K, Tanabe K, Ohnuki M et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007;131:861–872.
|
| 48 |
Takahashi K, Tanabe K, Ohnuki M et al. Induction of pluripotency in human somatic cells via a transient state resembling primitive streak- like mesendoderm. Nat Commun 2014;5:3678.
|
| 49 |
Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126:663–676.
|
| 50 |
Takashima S, Hirose M, Ogonuki N et al. Regulation of pluripotency in male germline stem cells by Dmrt1. Genes Dev 2013;27:1949–1958.
|
| 51 |
Takikawa S, Ray C, Wang X et al. Genomic imprinting is variably lost during reprogramming of mouse iPS cells. Stem Cell Res 2013;11:861–873.
|
| 52 |
Tarazona OA, Pourquie O. Exploring the influence of cell metabolism on cell fate through protein post-translational modifications. Dev Cell 2020;54:282–292.
|
| 53 |
Trapnell C, Cacchiarelli D, Grimsby J et al. The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells. Nat Biotechnol 2014;32:381–386.
|
| 54 |
Tucci V, Isles AR, Kelsey G et al; Erice Imprinting Group. Genomic imprinting and physiological processes in mammals. Cell 2019;176:952–965.
|
| 55 |
Van de Sande B, Flerin C, Davie K et al. A scalable SCENIC workflow for single-cell gene regulatory network analysis. Nat Protocols 2020;15:2247–2276.
|
| 56 |
Wang L, Zhang J, Duan J et al. Programming and inheritance of parental DNA methylomes in mammals. Cell 2014;157:979–991.
|
| 57 |
Wang M, Xu YW, Zhang YC et al. Deciphering the autophagy regulatory network via single-cell transcriptome analysis reveals a requirement for autophagy homeostasis in spermatogenesis. Theranostics 2021;11:5010–5027.
|
| 58 |
Wessels HH, Mendez-Mancilla A, Guo XY et al. Massively parallel Cas13 screens reveal principles for guide RNA design. Nat Biotechnol 2020;38:722–+.
|
| 59 |
Wolf FA, Angerer P, Theis FJ. SCANPY: large-scale single-cell gene expression data analysis. Genome Biol 2018;19:15.
|
| 60 |
Wolf FA, Hamey FK, Plass M et al. PAGA: graph abstraction reconciles clustering with trajectory inference through a topology preserving map of single cells. Genome Biol 2019;20:59.
|
| 61 |
Xie Y, Khan R, Wahab F et al. The testis-specifically expressed Dpep3 is not essential for male fertility in mice. Gene 2019;711:143925.
|
| 62 |
Yagi M, Kabata M, Ukai T et al. De Novo DNA methylation at imprinted loci during reprogramming into naive and primed pluripotency. Stem Cell Rep 2019;12:1113–1128.
|
| 63 |
Yagi M, Kishigami S, Tanaka A et al. Derivation of ground-state female ES cells maintaining gamete-derived DNA methylation. Nature 2017;548:224–227.
|
| 64 |
Yang XX, Breuss MW, Xu X et al. Developmental and temporal characteristics of clonal sperm mosaicism. Cell 2021;184:4772–+.
|
| 65 |
Ying QL, Wray J, Nichols J et al. The ground state of embryonic stem cell self-renewal. Nature 2008;453:519–523.
|
| 66 |
Zhang J, Zhang M, Acampora D et al. OTX2 restricts entry to the mouse germline. Nature 2018;562:595–599.
|
| 67 |
Zhang T, Oatley J, Bardwell VJ et al. DMRT1 is required for mouse spermatogonial stem cell maintenance and replenishment. PLoS Genet 2016;12:e1006293.
|
| 68 |
Zhao J, Lu P, Wan C et al. Cell-fate transition and determination analysis of mouse male germ cells throughout development. Nat Commun 2021;12:6839.
|
| 69 |
Zhao T, Fu Y, Zhu J et al. Single-cell RNA-Seq reveals dynamic early embryonic-like programs during chemical reprogramming. Cell stem cell 2018;23:31–45.e37.
|
| 70 |
Zhao XY, Li W, Lv Z et al. iPS cells produce viable mice through tetraploid complementation. Nature 2009;461:86–90.
|
| 71 |
Zhao Y, Zhao T, Guan J et al. A XEN-like state bridges somatic cells to pluripotency during chemical reprogramming. Cell 2015;163:1678–1691.
|
| 72 |
Zhou Q, Jouneau A, Brochard V et al. Developmental potential of mouse embryos reconstructed from metaphase embryonic stem cell nuclei. Biol Reprod 2001;65:412–419.
|
| 73 |
Zhou Q, Wang M, Yuan Y et al. Complete meiosis from embryonic stem cell-derived germ cells in vitro. Cell Stem Cell 2016;18:330–340.
|
| 74 |
Zhou Y, Zhou B, Pache L et al. Metascape provides a biologist-oriented resource for the analysis of systems-level datasets. Nat Commun 2019;10:1523.
|
| 75 |
Zhuang Q, Li WJ, Benda C et al. NCoR/SMRT co-repressors cooperate with c-MYC to create an epigenetic barrier to somatic cell reprogramming (vol 20, pg 400, 2018). Nat Cell Biol 2018;20:1227–1227.
|
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