The reprogramming of human somatic cells to primed or naive induced pluripotent stem cells recapitulates the stages of early embryonic development1,2,3,4,5,6. The molecular mechanism that underpins these reprogramming processes remains largely unexplored, which impedes our understanding and limits rational improvements to reprogramming protocols. Here, to address these issues, we reconstruct molecular reprogramming trajectories of human dermal fibroblasts using single-cell transcriptomics. This revealed that reprogramming into primed and naive pluripotency follows diverging and distinct trajectories. Moreover, genome-wide analyses of accessible chromatin showed key changes in the regulatory elements of core pluripotency genes, and orchestrated global changes in chromatin accessibility over time. Integrated analysis of these datasets revealed a role for transcription factors associated with the trophectoderm lineage, and the existence of a subpopulation of cells that enter a trophectoderm-like state during reprogramming. Furthermore, this trophectoderm-like state could be captured, which enabled the derivation of induced trophoblast stem cells. Induced trophoblast stem cells are molecularly and functionally similar to trophoblast stem cells derived from human blastocysts or first-trimester placentas7. Our results provide a high-resolution roadmap for the transcription-factor-mediated reprogramming of human somatic cells, indicate a role for the trophectoderm-lineage-specific regulatory program during this process, and facilitate the direct reprogramming of somatic cells into induced trophoblast stem cells.
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We developed an interactive online tool (http://hrpi.ddnetbio.com/) to facilitate exploration of the dataset, and for downloading all of the processed datasets. Raw and processed next-generation sequencing datasets have been deposited at the NCBI Gene Expression Omnibus (GEO) repository under accession numbers: GSE150311 (scRNA-seq experiments of intermediates during human primed and naive reprogramming); GSE150637 (scRNA-seq experiments of day 21 reprogramming intermediates cultured under fibroblast condition, naive pluripotent and trophoblast stem cell conditions); GSE147564 (snRNA-seq experiments of intermediates during human primed and naive reprogramming); GSE147641 (ATAC-seq experiments of intermediates during human primed and naive reprogramming); GSE150590 (ATAC-seq experiments of iTS cells); GSE149694 (bulk RNA-seq experiments of intermediates during human primed and naive reprogramming); and GSE150616 (bulk RNA-seq experiments of iTS cells and their derived placenta subtypes). Source data are provided with this paper.
All data were analysed with standard programs and packages as detailed. Scripts can be found at https://github.com/SGDDNB/hrpi.
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We thank staff at Monash Flowcore Facility for providing high-quality cell sorting services and technical input; S. Wang, T. Wilson and the University of Melbourne Centre for Cancer Research (UMCCR) core for assistance with next-generation library preparation and Illumina sequencing; J. Hatwell-Humble for assistance with the mouse work; and A. Purcell for providing the HLA antibodies. We acknowledge the use of the services and facilities of Micromon, Monash Micro Imaging and Monash Histology Platforms at Monash University. This work was supported by National Health and Medical Research Council (NHMRC) project grants APP1104560 to J. M. Polo and A. L. Laslett, APP1069830 to R.L., and a Monash University strategic grant awarded to C.M.N. X.L. was supported by the Monash International Postgraduate Research Scholarship, a Monash Graduate Scholarship and the Carmela and Carmelo Ridolfo Prize in Stem Cell Research. A.S.K. was supported by an NHMRC Early Career Fellowship APP1092280. J. M. Polo and R.L. were supported by Silvia and Charles Viertel Senior Medical Research Fellowships. J. M. Polo was also supported by an ARC Future Fellowship FT180100674. R.L was supported by a Howard Hughes Medical Institute International Research Scholarship. O.J.L.R. and J.F.O. were supported by a Singapore National Research Foundation Competitive Research Programme (NRF-CRP20-2017-0002). The Australian Regenerative Medicine Institute is supported by grants from the State Government of Victoria and the Australian Government.
O.J.L.R. and J. M. Polo. are co-inventors on a patent (WO/2017/106932) and are co-founders and shareholders of Mogrify Ltd., a cell therapy company. X.L., J.F.O., K.C.D., L.D., O.J.L.R. and J. M. Polo are co-inventors on a provisional patent application (application number: 2019904283) filed by Monash University, National University of Singapore and Université de Nantes related to work on derivation of iTS cells. The other authors declare no competing interests.
Peer review information Nature thanks Ashley Moffett, Samantha A. Morris and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 Experimental designs, analysis pipelines for snRNA-seq and scRNA-seq.
a, Morphological changes of cells undergoing reprogramming in fibroblast medium (FM); primed reprogramming (PR); naive reprogramming (NR). FM: D0, 3 and 7; PR: D13, D21 and iPS cells (iPSCs); and NR: D13, D21 and iPS cells, n > 10. Scale bar, 500 μm. b, Immunostaining at early stages (FM: D0, 3 and 7), during PR (D13 and D21) and NR (D13 and D21) with TRA-1-60 for primed colonies, KLF17 for naive colonies and DAPI for nuclei staining, n = 2. Scale bar, 50 μm. c, Experimental design for scRNA-seq libraries. iMEF, irradiated mouse embryonic fibroblasts. d, snRNA-seq and scRNA-seq data analysis strategy (Methods). e, Representation of integrated snRNA-seq and scRNA-seq experiments (43,791 cells) on FDL. f, g, Primed and naive libraries on FDL. h, FDL showing cells in predicted stages of the cell cycle. i, Reprogramming trajectories on FDL highlighting cells within each time point. j, Expression of genes associated with primed pluripotency (NLGN4X) and naive pluripotency (DPPA5) on FDL. k–r, PCA (k–p), diffusion maps (q) and UMAP (r) of snRNA-seq and scRNA-seq data. For more details on sample numbers and statistics, see ‘Statistics and reproducibility’ in Methods.
Extended Data Fig. 2 Resolving the molecular hallmarks of primed and naive reprogramming trajectories.
a, Unsupervised clustering projected onto the FDL shown in Fig. 1 (43,791 cells). fm1–fm6, fibroblast and early reprogramming intermediate cell clusters; mix, shared cell cluster; pr1–pr3, primed reprogramming cell clusters; nr1–nr4, naive reprogramming cell clusters; nic, novel intermediate cell cluster; re1–re6, refractory cell clusters. b, snRNA-seq time point and library contribution (composition and cell number) towards each cell cluster. c, PAGA trajectory inference on diffusion maps. d, snRNA-seq clusters, used to define gene signatures, on FDL. e, Dot plot showing the expression of mesenchymal and epithelial (MET)-associated genes across cell clusters. f, Jaccard similarity of snRNA-seq cluster-specific genes. Cluster-specific genes are then grouped to define the eight gene signatures, highlighted at the bottom. g, Defined gene signatures on FDL. h, Gene-expression heat map of the primed or naive pluripotency signatures across the cell clusters (coloured arrows indicate known marker genes). i, Area plots showing the transition and activation of the defined signatures during primed and naive reprogramming over time. For more details on sample numbers and statistics, see ‘Statistics and reproducibility’ in Methods.
Extended Data Fig. 3 Isolation and characterization of intermediates during reprogramming into several naive human induced pluripotent states.
a, Identification of cell-surface markers for the isolation of primed and naive reprogramming intermediates. b, PCA of bulk RNA-seq data of isolated intermediates during primed and naive reprogramming, n ≥ 2. c, Experimental designs for the generation, isolation and profiling of intermediates during reprogramming into several naive human induced pluripotent states. d, Morphological changes during reprogramming under naive 5iLAF, NHSM and RSeT culture conditions (Methods), n = 4. Scale bar, 500 μm. e, Visualization of flow cytometry profiles (SPADE tree) of intermediates during reprogramming, n = 2. f, PCA of RNA-seq of primed and several types of naive reprogramming intermediates (Methods), n ≥ 2. g, Heat map showing gene expression profiles of primed and naive pluripotency signatures genes (defined in snRNA-seq and scRNA-seq analysis) across reprogramming intermediates and iPS cells derived under all different culture conditions, n ≥ 2. For more details on sample numbers and statistics, see ‘Statistics and reproducibility’ in Methods.
Extended Data Fig. 4 Single-cell profiling of the reprogramming pathway into naive RSeT state.
a, FDL of fibroblast, primed, naive t2iLGoY and RSeT scRNA-seq libraries (9,852 cells) (Methods). b, Expression profile of genes associated with human fibroblasts (ANPEP), shared pluripotency (NANOG), primed pluripotency (ZIC2 and NLGN4X) and naive pluripotency (DNMT3L and DPPA5) on FDL. For more details on sample numbers and statistics, see ‘Statistics and reproducibility’ in Methods.
Extended Data Fig. 5 Dynamics of chromatin state transitions during reprogramming into primed and naive human induced pluripotency.
a, PCA plot of ATAC-seq nucleosome-free signals, PC1 versus PC3 related to Fig. 2c. ATAC-seq was performed using isolated reprogramming intermediates and iPS cells from FM (D0, D3 and D7), PR (D13, D21, P3 and P10), NR (D13, D21, P3 and P10), n = 2. FM, fibroblasts medium (black); PR, primed reprogramming (orange); NR, naive reprogramming (blue). b, c, PCA plot of the integration of RNA-seq and ATAC-seq experiments (n ≥ 2). d, e, ATAC-seq and corresponding RNA-seq tracks of primed and naive reprogramming intermediates for fibroblast marker, ANPEP; shared pluripotency marker, PRDM14; primed-specific pluripotency marker SOX11; naive-specific pluripotency marker DNMT3L. Model of each gene is shown: coding sequences, light blue boxes, and exons, dark blue boxes; introns are shown as light blue connecting lines. f, Naive-reprogramming-specific ATAC-seq signals (in light grey) around core pluripotency factors NANOG and POU5F1 (also known as OCT4), naive-reprogramming-specific KLF17 and ZNF729 in primed and naive reprogramming intermediates and iPS cells compared to human inner cell mass and primed ES cells (ESCs) ATAC-seq data58. For more details on sample numbers, see ‘Statistics and reproducibility’ in Methods.
Extended Data Fig. 6 Features of accessible chromatin landscape during reprogramming into primed and naive human induced pluripotency.
a, Proportion of genomic regions in each of the ATAC-seq clusters. b, Averaged chromatin accessibility (z-scaled, n = 2) and gene expression (z-scaled, n ≥ 2) of one representative gene from each of the ATAC-seq peak clusters. c, Standardized gene expression (averaged z-scaling) of genes associated with ATAC-seq cluster peaks (Methods). d, Transcription factor motif enrichment analysis of the ATAC-seq peak clusters. Motif enrichment (−log(P value)) heat map by colour and the size the percentage of sequences in the cluster featuring the motif. Red arrow points to OCT4, SOX2, NANOG and KLF4 motifs in transient ATAC-seq cluster (C3), Blue arrow = enrichment of TE-associated transcription factors TFAP2C and GATA2 (C7 and C8) are indicated by blue arrows. e, Gene-expression heat map transcription factors identified in the motif enrichment analysis in d. f, TFAP2C and GATA2 gene expression during primed and naive reprogramming. g, Reverse transcription qPCR analysis of shTFAP2C and shGATA2 compared to scrambled controls, n = 2. For more details on sample numbers and statistics, see ‘Statistics and reproducibility’ in Methods.
Extended Data Fig. 7 Uncovering the transcriptional programmes of human fibroblast reprogramming into naive induced pluripotency.
a, b, Primed and naive scores, using gene signatures defined in this study (Fig. 1g), on human preimplantation embryos at indicated embryonic stages based on scRNA-seq experiments from published studies24,25. c, EPI, PE and TE signatures score at indicated embryonic stages25. d, EPI, PE and TE gene signatures25 from embryonic (E) day 5, 6 and 7 on intermediates and iPS cells reprogrammed under primed and different naive culture conditions (Methods). e, Gene set enrichment analysis (GSEA) (Methods) of the EPI, PE and TE gene signatures in reprogramming intermediates and iPS cells reprogrammed under primed and several naive culture conditions. f, EPI, PE and TE gene signatures scores in reprogramming intermediates and iPS cells reprogrammed under primed and several naive culture conditions. We used a combined gene signature across E5 to E7 for each lineage (Methods). g, EPI and PE signatures on FDL with single-cell trajectories constructed using Monocle3 (43,791 cells), related to Fig. 3a. h, Scoring of novel-intermediate signatures defined in this study (Extended Data Fig. 2f, g) on human preimplantation embryos of different lineages at indicated embryonic stages based on scRNA-seq experiments from published studies24,25. For more details on sample numbers and statistics, see ‘Statistics and reproducibility’ in Methods.
Extended Data Fig. 8 Characterization of iTSCd21n.
a, Immunostaining of fibroblast, primed, naive t2iLGoY iPS cells with P63, TFAP2C, GATA2 and KRT7, n = 2. Scale bar, 100 μm. b, Gene expression of trophoblast genes in fibroblasts, primed, naive t2iLGoY iPS cells, iTSCd21n and TS cells (TSCs) derived from a human blastocyst (TSCblast)7 and first-trimester placental trophoblast (TSCCT)7, mean of replicates, n = 2. c, Phase-contrast image of ST and EVT cells differentiated from iTSCd21n, n = 4. Scale bar, 100 μm. d, Fusion index of iTSCd21n ST and iTSCd21n, n = 5, data are represented as mean ± s.e.m. P values by two-tailed unpaired Student’s t-test. e, Representative results for over-the-counter hCG pregnancy test for medium of ST cells differentiated from iTSCd21n and control medium, n = 6. f, hCG levels in iTSCd21n- and iTSCd21n-ST conditioned medium, detected by ELISA, n = 4. g, hCG level in mouse blood serum detected by ELISA, n = 4. h, Lesions collected from subcutaneously engrafted iTSCd21n in NOD-SCID mice, n = 4. i, Haematoxylin and eosin, and immunohistochemical staining of KRT7 in the lesions from h. No evident lesions were observed in vehicle controls, n = 4. Scale bar, 200 μm. j, Distinct level of CD70 expression in naive and TE populations (indicated by blue arrows) on FDL projection of snRNA-seq and scRNA-seq datasets. k, Quantification of KRT7+ colony clusters after 9 d of transitioning into TS cell medium of unenriched, CD70high and CD70low populations, n = 2 or 3 independent experiments, data are mean ± s.e.m. P values by two-tailed unpaired Student’s t-test. Representative images of whole-well scans (top panels) (scale bar, 1 mm) and KRT7 immunostaining (bottom panels) (scale bar, 100 μm). For more details on sample numbers and statistics, see ‘Statistics and reproducibility’ in Methods.
Extended Data Fig. 9 Cellular heterogeneity of fibroblast and iTSCd8 reprogramming intermediates revealed by scRNA-seq.
a, Experimental designs and preparation of scRNA-seq libraries of day-21 fibroblast, naive and TSCd8 reprogramming intermediates. b, Strength of EPI signatures on FDL (10,518 cells). The cell population not enriched for EPI signatures but enriched for TE signatures is indicated by a purple arrow, related to Fig. 4b. c, Representation of 13 cell clusters from unsupervised clustering projected onto the FDL, fibroblast medium cell clusters: D21fm1–D21fm7; naive reprogramming cell clusters: D21nr1–D21nr3; trophoblast reprogramming cell clusters: D21tr1–D21tr3. d, Contribution of each scRNA-seq library (%) to the composition of cell clusters. D21tr1 cluster is indicated by a purple arrow. e, Expression of genes associated with human fibroblasts (ANPEP), shared pluripotency (NANOG), primed pluripotency (ZIC2), naive pluripotency (DNMT3L) and trophoblast (GATA3) on FDL projection of day-21 fibroblast, naive and TSCd8 reprogramming intermediate scRNA-seq libraries (top panels). Defined fibroblast, early-primed, primed, novel-intermediate and naive signatures (Extended Data Fig. 2f) on the FDL projection (bottom panels). f, Experimental designs to validate the potential of day-21 fibroblast reprogramming intermediates for the derivation of primed, naive iPS cells and iTS cells. g, Phase-contrast images of primed, naive iPS cells and iTS cells generated from day 21 fibroblast reprogramming intermediates, n = 2. Scale bar, 50 μm. Immunostaining of primed, naive iPS cells and iTS cells with NANOG, KLF17, NR2F2, KRT7 and DAPI for nuclei staining, n = 2. Scale bar, 200 μm. h, Reverse-transcription qPCR analysis of NANOG, ZIC2, KLF17, DPPA3, GATA2 and KRT7 expression in primed, naive iPS cells and iTS cells generated from day-21 fibroblast reprogramming intermediates, n = 3. Data are mean ± s.e.m. For more details on sample numbers and statistics, see ‘Statistics and reproducibility’ in Methods.
Extended Data Fig. 10 Characterization of iTSCd8.
a, Sendai viral transgenes in iTS cell lines with positive and negative controls, n = 6. b, Gene expression of trophoblast genes in fibroblasts, primed iPS cells, naive t2iLGoY iPS cells, iTSCd8 and iTSCd21n compared to TSCs derived from a human blastocyst (TSCblast) and first-trimester placental trophoblast (TSCCT)7, data are presented as mean (n = 2). c, Cell fusion index of iTSCd8 ST and iTSCd8. n = 5, data are mean ± s.e.m. P values by two-tailed unpaired Student’s t-test. d, Representative results for hCG pregnancy test obtained from medium of ST cells differentiated from iTSCd8, n = 6. e, hCG levels of iTSCd8- and iTSCd8-ST-conditioned medium detected by ELISA, n = 4. f, Representative flow cytometry analysis of pan HLA-A, B, C class I marker (W6/32), HLA-Bw4 and HLA-G in fibroblasts and EVTs, n = 4. g, Representative flow cytometry analysis of pan HLA class I marker (W6/32) and HLA-G in iTSCd8 EVT and iTSCd21n EVT. h, Representative flow cytometry analysis of pan HLA class I marker (W6/32) in fibroblasts, primed iPS cells, naive t2iLGoY iPS cells, iTSCd8 and iTSCd21n, n = 4. i, j, Expression of ST genes in iTSCd8- and iTSCd21n-derived ST cells (i) and expression of EVT genes in iTSCd8 and iTSCd21n-derived EVT cells (j). k, l, Spearman correlation of the transcriptomes of fibroblast, primed and naive t2iLGoY iPS cells, iTSCd8 and iTSCd21n, iTSCd8 ST and iTSCd21n ST, iTSCd8 EVT and iTSCd21n EVT generated in this study with trophoblast organoids samples from refs. 28,29 (k) and single-cell fetal–maternal interface samples from ref. 27 (l), n ≥ 2, replicates are averaged before performing correlation. m, Lesions collected from subcutaneously engrafted iTSCd8 in NOD-SCID mice, n = 4. For more details on sample numbers and statistics, see ‘Statistics and reproducibility’ in Methods.
Supplementary Figure 1 Isolation of reprogramming intermediates using a panel of cell surface markers. a, Flow cytometry analysis of TRA-1-60 vs SSEA3, EPCAM vs SSEA3 over the reprogramming time-course into primed and naive induced pluripotency. b, Validation of the panel of cell surface markers for isolation of intermediates that carry the reprogramming potential across the reprogramming time-course. e.g., CD13+F11R- and CD13+F11R+ subpopulations were isolated on day 3 and reseeded for 5 days (for flow cytometry reanalysis) and for hiPSCs colony formation (AP staining), see Methods for details. Of note, SSEA3+EPCAM+ population on day 13 of NR showed negative for AP staining due to the substantial differentiation of these cells after reseeding. FM: Fibroblasts Medium (black); PR: Primed Reprogramming (orange); NR: Naive Reprogramming (blue).
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Liu, X., Ouyang, J.F., Rossello, F.J. et al. Reprogramming roadmap reveals route to human induced trophoblast stem cells. Nature 586, 101–107 (2020). https://doi.org/10.1038/s41586-020-2734-6
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Journal of Assisted Reproduction and Genetics (2023)
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