Abstract
Cellular reprogramming can manipulate the identity of cells to generate the desired cell types1,2,3. The use of cell intrinsic components, including oocyte cytoplasm and transcription factors, can enforce somatic cell reprogramming to pluripotent stem cells4,5,6,7. By contrast, chemical stimulation by exposure to small molecules offers an alternative approach that can manipulate cell fate in a simple and highly controllable manner8,9,10. However, human somatic cells are refractory to chemical stimulation owing to their stable epigenome2,11,12 and reduced plasticity13,14; it is therefore challenging to induce human pluripotent stem cells by chemical reprogramming. Here we demonstrate, by creating an intermediate plastic state, the chemical reprogramming of human somatic cells to human chemically induced pluripotent stem cells that exhibit key features of embryonic stem cells. The whole chemical reprogramming trajectory analysis delineated the induction of the intermediate plastic state at the early stage, during which chemical-induced dedifferentiation occurred, and this process was similar to the dedifferentiation process that occurs in axolotl limb regeneration. Moreover, we identified the JNK pathway as a major barrier to chemical reprogramming, the inhibition of which was indispensable for inducing cell plasticity and a regeneration-like program by suppressing pro-inflammatory pathways. Our chemical approach provides a platform for the generation and application of human pluripotent stem cells in biomedicine. This study lays foundations for developing regenerative therapeutic strategies that use well-defined chemicals to change cell fates in humans.
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Data availability
All data are available in the Article and its Supplementary Information. Raw and processed next-generation sequencing datasets have been deposited at the NCBI GEO under accession numbers GSE178325 (scRNA-seq data), GSE178324 (scATAC-seq data), GSE176381 (bulk RNA-seq data), GSE178323 (bulk ATAC-seq data) and GSE178966 (whole-genome bisulfite sequencing data). The public datasets we used can be accessed under accession numbers GSE143753 (human limb bud data), GSE106269 (axolotl data), GSE165901 (frog data), GSE135985 (mouse data), GSE83765 (human naive pluripotency stem cell data) and GSE36552 (human embryo data) from the GEO, and E-MTAB-3929 (human embryo data) from ArrayExpress. Source data are provided with this paper.
Code availability
The computational methods used to generate results reported in this study are described in the Methods. Computer code files have been uploaded to GitHub (https://github.com/sajuukLyu/Guan_et_al._2022).
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Acknowledgements
We thank T.-T. Sun, J. Xu, S. Liao, Y. Du, C. Wang, Z. Yang, Y. Ma and W. Chen for critiquing the manuscript; F. Tang and P. Lu for data discussion; N. Li, Y. Luo, A. Xue, Z. Zhou, Z. Zhao and H. Zhou for technical assistance; the staff at the flow cytometry Core at National Center for Protein Science at Peking University, particularly H. Yang, for technical help; and S. Qin for assistance with confocal imaging. This work was supported by the National Key R&D Program of China (2017YFA0103000) and the National Natural Science Foundation of China (31521004, 31730059 and 32025006) and the Beijing Science and Technology Major Project (Z191100001519001). J.G. and Y.F. were supported in part by the Postdoctoral Fellowship of Peking-Tsinghua Center for Life Sciences.
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J.G., G.W., J.W., Z.Z., Y.F., L.C., G.M., J.Z., Y. Li, Y.W., S. Liuyang, B.L., Z.Y., H.H., X. Zhong, Q.C., X. Zhang, W.L., Y.S., L.L., L.W. and S. Lu performed experiments. Y. Lyu performed sequencing data analysis and C.L. provided guidance. H.D., J.G., J.W. and S.S. drafted the manuscript. H.D. supervised the entire work.
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Competing interests
H.D., J.G., J.W., G.W., Z.Z., Y.F., L.C. and G.M. have filed a patent for the small-molecule combinations used in the chemical reprogramming reported in this paper (application no. PCT/CN2021/085936; patent organization, Peking University). The other authors declare no competing interests.
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Extended data figures and tables
Extended Data Fig. 1 Morphological changes, gene expression during hCiPSCs induction and effect of small molecules in each stage.
a, Small molecules used in hCiPSCs reprogramming and their targets. b, Morphological changes of HEFs after treatment of different chemical conditions. Scale bar, 100 μm. Representative of three independent experiments. c, Representative images of cells at the end of each stage during hCiPSCs induction. Scale bar, 100 μm. Representative of three independent experiments. d-e, RT-qPCR validations of gene expression during hCiPSC induction. (d) Relative expression of fibroblast-related genes, (e) epithelial-related genes and LIN28A at the end of stage I. Data presented as mean; n = 3. Representative of three independent experiments. f, Distribution of the different DNA CpG methylation statuses in fibroblasts, cells at the end of stage I and cells at the end of stage II with or without JNKIN8. g, Number of the cells treated with the indicated conditions at stage II. Data presented as mean ± SD; n = 3. h-i, RT-qPCR validations of gene expression during hCiPSC induction. (h) Relative expression of LIN28A and SALL4 at the end of stage II. (i) Relative expression of LIN28A, SALL4, and OCT4 at the end of stage III. Data presented as mean; n = 3. Representative of three independent experiments. j-m, The numbers of hCiPSCs colonies at the end of reprogramming after withdrawing individual chemicals from each stage. Data presented as mean ± SD; (n = 3 for panel j; n = 3 for –ABT869 and –Tranyl in panel k; n = 4 for other conditions in panel k; n = 8 for Stage III in panel l; n = 4 for other conditions in panel l; n = 4 for panel m).
Extended Data Fig. 2 Characterization of HEF-derived hCiPSCs.
a, Calculated doubling time of hCiPSCs and hESCs. Data are represented as mean ± SD; n = 3. b, Immunofluorescence analysis of pluripotency markers in the HEFs-derived hCiPSCs. Scale bar, 100 μm. Representative of three independent experiments. c, Immunofluorescence analyses of pluripotency markers in HEF-derived hCiPSCs induced from different donors. Scale bar, 100 μm. Representative of three independent experiments. d, RT-qPCR analysis of pluripotency markers in the indicated hCiPSCs and hESCs. Data are represented as mean; n = 3. e, Scatter plots comparing the global gene expression in hCiPSCs, hESCs and HEFs. f, Hierarchical clustering of global transcriptional profiles of hCiPSCs, hESCs and HEFs. Distance was calculated by 1-spearman correlation coefficient. g, Heat maps showing the expression of pluripotency genes and fibroblast genes in hCiPSCs, hESCs and HEFs. h, Hierarchical clustering of hCiPSCs, hESCs, HEFs, and hADSCs based on their global DNA methylation statuses. Distance was calcultated by 1-pearson correlation coefficient. i, DNA CpG methylation status of the OCT4, NANOG and DPPA4 promoter loci in hCiPSCs, hESCs and HEFs. j, Chromatin accessibilities of pluripotency genes in hCiPSCs, hESCs, HEFs, and hADSCs.
Extended Data Fig. 3 Developmental potential of HEF-derived hCiPSCs.
a, Haematoxylin and eosin staining of hCiPSCs-derived teratoma sections (hCiPSCs-1117-1#, hCiPSCs-1117-2# and hCiPSCs-1117-3#). For each hCiPSCs clone, images which contained features representing tissues of endoderm, mesoderm and ectoderm were obtained from the same teratoma. Representative of three independent experiments. b, Immunofluorescence analysis of differentiation markers for the three germ layers in embryoid body-mediated differentiation of hCiPSCs (hCiPSCs-1117-1#). Scale bars, 100 μm. c-e, Directed differentiation of hCiPSCs (hCiPSCs-1117-2#) to haematopoietic lineage cells. The percentages of CD34/CD144 double-positive and CD34/CD31 double-positive cells counted by FACS analysis (c and d). Data are represented as mean ± SD; n = 3. The percentage of hCiPSC-derived T progenitor cells counted by FACS analysis. PC7 means PE-CY7 (e). f, Immunofluorescent analysis of the markers of neural stem cells derived from hCiPSCs (hCiPSCs-1117-2#) by in vitro directed differentiation. Scale bar, 100 μm. Representative of three independent experiments. g-k, In vitro directed differentiation of hCiPSCs (hCiPSCs-1117-2#) to hepatocytes. FACS analysis of AFP and ALB double-positive hCiPSC-derived hepatic progenitor cells (HPCs) (g). hCiPSC-derived hepatocyte (hdHeps) morphology (h). Scale bar, 100 μm. Representative of three independent experiments. Quantitative analysis of urea (i) and ALB (j) secretion by hdHeps and primary human hepatocytes (PHHs) Data presented as mean ± SD; n = 3. RT-qPCR analyses of functional hepatocyte marker genes in hiPSCs, HPCs, hdHeps, and PHHs (k). Data presented as mean; n = 3. l, Karyotype analysis showing HEF-derived hCiPSCs with a normal, diploid chromosomal content.
Extended Data Fig. 4 Small molecules that promote reprogramming efficiency of hADSCs.
a-b, Identification of the small molecules and their combinations at stage I (a) and stage II (b) that promote the generation of hCiPSCs from hADSCs. Data presented as mean ± SD; (n = 6 for stage I condition in panel a; n = 3 for other conditions in panel a; n = 6 for stage II condition in panel b; n = 3 for other conditions in panel b. c, Numbers of hCiPSC colonies generated from hADSCs under different time courses. “8+8+8+7” represents a sequential duration for each stage, respectively. Data presented as mean ± SD; n = 3. d, Small molecule combination at stage II promotes the generation of hCiPSCs from HEFs. Data presented as mean ± SD; (n = 10 for stage II condition; n = 5 for +U2+R+Sg condition). All the results above are representative of three independent experiments. e, Small molecules used to promote the reprogramming efficiency and their targets. f-g, Schematic diagram of chemical reprogramming of hADSCs and HEFs to hCiPSCs with additional small molecules promoting efficiency respectively. h, Representative images of cells at the end of each stage during hCiPSC induction from hADSCs. Scale bar, 100 μm. Representative of three independent experiments. i, The reprogramming efficiency at the end of each stage detected by the indicated markers using flow cytometry. j, Immunofluorescence analysis of pluripotency markers in hCiPSCs derived from hADSCs obtained from different donors. Scale bar, 100 μm. Representative of three independent experiments. k, RT-qPCR analysis of pluripotency markers in the indicated hCiPSCs and hESCs. l, Bisulfite genomic sequencing of the OCT4 and NANOG promoter regions in hCiPSCs, hESCs and hADSCs. m, Immunofluorescence analysis of differentiation markers for the three germ layers in embryoid body-mediated differentiation of hCiPSCs. Scale bars, 100 μm. Representative of three independent experiments. n, Karyotype analysis showing normal, diploid chromosomal content for hADSC-derived hCiPSCs.
Extended Data Fig. 5 Characterization of hCiPSCs derived from hASFs.
a, Representative images of hASFs and the cells at end of reprogramming each stage. Representative of three independent experiments. b, Representative image of hASF-derived hCiPSCs. c, RT-qPCR analyses of pluripotency markers in the indicated hCiPSCs and hESCs. Data presented as mean; n = 3. d, Immunofluorescence analyses of pluripotency genes in hASF-derived hCiPSCs. Representative of three independent experiments. e, Hierarchical clustering of transcriptomic profiles of hCiPSCs, hESCs, and adult somatic cells. Distance was calculated by 1-spearman correlation coefficient. f, Karyotype analysis showing normal, diploid chromosomal content of hASF-derived hCiPSCs. g, Haematoxylin and eosin staining of hCiPSCs-derived teratoma sections (hCiPSCs-0408-1#). Images were from the same teratoma. Scale bars, 100 μm. Representative of three independent experiments. h, Summary of reprogramming efficiencies of hADSCs and hASFs from different donors.
Extended Data Fig. 6 Dissection of chemical reprogramming from human somatic cells to pluripotent stem cells.
a, Schematic diagram of single-cell RNA-sequencing analysis strategy during chemical reprogramming. b, UMAP layout visualization of single-cell RNA-sequencing profiles of chemical reprogramming process from hADSCs to hCiPSCs. c, UMAP layout visualization of reprogramming right trajectory coloured by cluster identities. d, UMAP layout visualization of single-cell RNA-sequencing profiles at the reprogramming each stage. e, ForceAtlas layout showing activity of the indicated gene signatures during chemical reprogramming. f, Heatmap showing the dynamics of accessibility gene scores that detected by single-cell ATAC sequencing in hADSCs and intermediate plastic state at stage II. g, GO analyses of significantly opened genes of intermediate plastic state at stage II. The p value is determined by a one-sided hypergeometric test without adjustments. h, Distribution of the different DNA CpG methylation statuses in hADSCs and cells at the end of stage I and stage II. i, Venn diagrams showing the number of methylated gene promoters in hADSCs and cells at the end of stage I and stage II. j, Dot plot showing the relative gene expression level, chromatin accessibility and DNA methylation status of the genes in the indicated GO terms.
Extended Data Fig. 7 Chemicals inducing the human somatic cells dedifferentiated to an intermediate plastic state at early stages.
a, Gene expression heatmap of fibroblast, XEN, pluripotency, and embryonic development and regeneration related genes. b, GO analyses of genes downregulated in stage I (371 genes, Fold change > 4) and upregulated in stage II (1,724 genes, fold change > 4). Four batches of independent experiments using HEFs and hADSCs were used to analyse the differentially expressed genes. p value is calculated by a one-sided hypergeometric test without adjustments. c, Heatmap showing the dynamics of accessibility gene scores that detected by single-cell ATAC sequencing in the fibroblasts and LIN28A+SALL4+ cells in stage II. Representative genes are noted on the right side. d, GO analyses of significantly opened genes in the LIN28A+SALL4+ cells in stage II. The p value is calculated by a one-sided hypergeometric test without adjustments. e, Distribution of the different DNA CpG methylation statuses in fibroblasts and cells at the end of stage I and stage II. f, Venn diagrams showing the number of methylated gene promoters in fibroblasts and cells at the end of stage I and stage II. g, The relative gene expression level, chromatin accessibility and DNA methylation status of the genes in the indicated GO terms. h, RT-qPCR analyses of the fibroblast and epithelial related genes during reprogramming early stage. Data presented as mean; n = 3. i, UMAP visualization of integrated single-cell RNA-sequencing profiles of the reprogramming early stage samples from HEFs, hADSCs and hASFs and the human embryonic limb bud cells. Representative expression of the indicated genes in these samples showed on UMAP. j, Venn diagram showing the overlapped key transcriptional factors that enriched in the intermediate plastic state and downregulated after JNKIN8 removed. k, The expression of SALL4 and LIN28A at the end of stage II with knockdown measured by RT-qPCR. Data presented as mean; n = 3. l, Cell numbers at the end of stage II after the knockdown of SALL4 or LIN28A. Data presented as mean ± SD; (n = 4 for shLIN28A 2#; n = 3 for other conditions). m, GSEA analysis of the intermediate plastic state related features in LIN28A or SALL4 knockdown cells at the end of stage II. p value is calculated by a one-sided permutation test without adjustments. n, Heatmap showing the expression pattern of embryonic development related genes at the end of stage II after knocking down SALL4 or LIN28A. o, Relative expression of the embryonic development related genes at the end of stage II after knocking down MSX1, MSX2 or HOXB9. Data presented as mean; n = 3.
Extended Data Fig. 8 XEN-like state was induced in stage III.
a, RT-qPCR analysed the dynamic expression change of XEN-related markers during the chemical reprogramming process. Data presented as mean; n = 3. Representative of three independent experiments. b, Immunofluorescence of typical colonies co-expressing XEN-related markers and OCT4 at the end of stage III. Scale bar, 50 μm. Representative of five independent experiments. c, Percentages of OCT4-positive cells generated from the inside (black) and outside (red) the XEN-like colonies in 5 batches of independent experiments. More than 3 wells were counted for each experimental batch. d, UMAP visualization of single-cell RNA sequencing profiles of cells (3,759 cells total) at stage III and the expression of XEN-related markers. e, Dot plot showing the expression pattern of primitive endoderm related genes in human pre-implantation embryo and the cell clusters in stage III. f, GSEA analysis of primitive endoderm related features in the XEN-like cells. p value is calculated by a one-sided permutation test without adjustments. g, The expression of HNF1B and SOX17 at the end of stage III with knockdown measured by RT-qPCR. Data presented as mean; n = 3. h, Numbers of hCiPSC colonies at the end of reprogramming with HNF1B or SOX17 knockdown. Data presented as mean ± SD; (n = 3 for shControl and shHNF1B 2#; n = 4 for other conditions). p values were calculated through a two-tailed unpaired t-test. i, Immunofluorescence of typical colonies co-expressing EPCAM and SOX17 at the end of stage III. Scale bar, 100 μm. Representative of three independent experiments. j, Number of hCiPSC colonies generated from EPCAM-positive (+) and EPCAM-negative (-) cell population sorted at the end of stage III. Data presented as mean ± SD; n = 3. p values were calculated through a two-tailed unpaired t-test. k, PHATE visualization of single-cell RNA sequencing profiles of stage III and Stage IV (left). The reprogramming trajectory coloured by hCiPSC scores and coloured in the order of pseudotime (middle). hCiPSC score of cells were defined as the probability of converting to hCiPSCs cells at SIV D10, which were calculated using WOT approach. PHATE visualization of the indicated gene expression at the late stage (right). l, The expression dynamics of the SOX17, HNF1B, OCT4 and NANOG based on the pseudotime analysis of the cells on the right trajectory at reprogramming late stage (SIII and SIV). Data presented as mean ± standard error. The cell numbers of each time point were indicated in the Method. m, Immunofluorescence analysis of the expression of SOX17, OCT4 and NANOG at day 2, 4 and 8 in the stage IV. Scale bar, 100 μm. Representative of three independent experiments. n, The percentage of the indicated cell clusters in the total OCT4 positive clusters at day 2, 4 and 8 in the stage IV. Data are represented as mean; n = 2. o, Numbers of XEN-like colonies with LIN28A or SALL4 knockdown. Data presented as mean ± SD; n = 3. p, Numbers of XEN-like colonies with MSX1, MSX2 or HOXB9 knockdown. Data presented as mean ± SD; n = 3. q, Fold change of XEN-like colonies at the end of stage III after withdrawing individual chemicals from stage III. Data presented as mean ± SD; n = 3. r, Fold change of XEN-like colonies with OCT4 positive cells at the end of stage III after withdrawing individual chemicals from stage III. Data presented as mean ± SD; n = 3. For panel o-r, p values were calculated through a two-tailed unpaired t-test.
Extended Data Fig. 9 hCiPSCs undergo a naive state at late stage of chemical reprogramming.
a, Immunofluorescence analysis of naive state human pluripotent stem cells markers in primary hCiPSCs at stage IV. Scale bar, 100 μm. Representative of three independent experiments. b, PHATE visualization of the expression pattern of human naive pluripotency related genes at the late stage (stage III and stage IV). c, Dot plot showing the expression pattern of human naive and prime pluripotency related genes in human pre-implantation embryo, primary hCiPSCs, mTESR cultured hCiPSCs (hCiPSCs-0618-4#), hESCs (H1) and hADSCs. d, Principal component analysis (PCA) of H1, primary hCiPSCs, mTESR cultured hCiPSCs and hESCs and other indicated samples. e, The global CpG methylation level of primary hCiPSCs, mTESR cultured hCiPSCs and hESCs, ICM in human pre-implantation embryo and published primed and naive pluripotent stem cell lines. f, Hierarchical clustering of the primary hCiPSCs, mTESR cultured hCiPSCs and hESCs, epiblasts in human pre-implantation embryo and published primed and naive pluripotent stem cell lines based on their global DNA methylation statuses. g, Dot plot showing the expression pattern of pluripotent genes in mTESR cultured hCiPSCs and H1, hADSCs, hASFs and HEFs. h, Hierarchical clustering of global transcriptomic profiles of mTESR cultured hCiPSCs, H1, hADSCs, hASFs and HEFs. Distance was calculated by 1-spearman correlation coefficient. i, Schematic diagram for key molecular events during the reprogramming process.
Extended Data Fig. 10 The effects of withdrawing individual chemicals from the stage II condition.
a, RT-qPCR analysis of SALL4 expression under indicated stage II conditions. Data presented as mean; n = 3. Representative of three independent experiments. b, UMAP visualization of single-cell RNA sequencing data across fibroblasts and indicated stage II conditions. 3,838, 3,865, 3,383, 2,961 and 1,903 cells were analysed for fibroblasts, stage II, stage II-Tranyl, stage II-5azaC, and stage II-JNKIN8, respectively. c, Percentage of SALL4+LIN28A+ cells in fibroblasts and indicated stage II conditions. d, Frequency of cells in different cell-cycle phases under the indicated stage II conditions defined by single-cell RNA sequencing analysis. e, GO analysis of the differently expressed genes after removal of JNKIN8. p value is calculated by a one-sided hypergeometric test without adjustments. f, Histogram showing normalized signal intensity of the opened loci in stage II after withdrawing individual chemicals. g-h, Promoter methylation levels of XEN (g) and pluripotency (h) related genes in fibroblasts, stage I and indicated stage II conditions. i, Schematic diagram showing key molecular events during the reprogramming early stages.
Supplementary information
Supplementary Fig. 1
Flow cytometry gating strategy singlets were gated (P1) from all captured events by forward scatter (FSC) and side scatter (SSC). P1 events were then gated based on clustering of subpopulations and their marker expression levels. All flow cytometry results are shown as pseudocolour plots.
Supplementary Table 1
Small molecules or cytokines used in human CiPS cell reprogramming. Detailed information (target, working concentration, stock solution, source, molecular mass and structure) of the small molecules or cytokines used in human CiPS cell reprogramming.
Supplementary Table 2
Donor information of human fibroblasts and ADSCs. Summary of the donor information of human fibroblasts and ADSCs reprogrammed in this study.
Supplementary Table 3
Karyotype analysis of hCiPS cell lines. Summary of the karyotype information of all hCiPS cell lines analysed in this study.
Supplementary Table 4
STR analysis. Information regarding the results of STR analysis of all hCiPS cell lines analysed in this study and their corresponding parental somatic cells.
Supplementary Table 5
Summary of pluripotency analyses for hCiPS cell lines. Summary of hCiPS cell lines and all the assays of pluripotency analyse we performed in this study.
Supplementary Table 6
Primers used for RT–qPCR and bisulfite genomic sequencing. Information regarding the list of RT–qPCR primers used in this study.
Supplementary Table 7
Antibody information. Information regarding the list of antibodies used for immunostaining and FACS sorting in this study.
Supplementary Table 8
Gene list of regeneration program identified by scRNA-seq and scATAC-seq. Specific activated 216 genes in SII intermediate plastic state identified by scRNA-seq and scATAC-seq.
Supplementary Table 9
Gene lists of the signatures during human chemical reprogramming. Gene signatures during human chemical reprogramming. Gene lists used to define different programs (signatures) during the chemical reprogramming of human somatic cells.
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Guan, J., Wang, G., Wang, J. et al. Chemical reprogramming of human somatic cells to pluripotent stem cells. Nature 605, 325–331 (2022). https://doi.org/10.1038/s41586-022-04593-5
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DOI: https://doi.org/10.1038/s41586-022-04593-5
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