1. Developmental Biology and Stem Cells
Download icon

Unique molecular events during reprogramming of human somatic cells to induced pluripotent stem cells (iPSCs) at naïve state

  1. Yixuan Wang  Is a corresponding author
  2. Chengchen Zhao
  3. Zhenzhen Hou
  4. Yuanyuan Yang
  5. Yan Bi
  6. Hong Wang
  7. Yong Zhang
  8. Shaorong Gao  Is a corresponding author
  1. School of Life Sciences and Technology, Tongji University, China
Research Article
Cite as: eLife 2018;7:e29518 doi: 10.7554/eLife.29518
6 figures, 1 table, 4 data sets and 2 additional files

Figures

Figure 1 with 1 supplement
Establishment of the secondary naïve iPSC induction system.

(A) Representative bright field images of hiF-Ts, niPSC-Ts and reprogramming cells at the indicated time points during reprogramming. Scale bar, 100 μm. (B) Phase and OCT4-ΔPE-GFP images of niPSC-Ts and reprogramming cells at the indicated time points during reprogramming. Scale bar, 100 μm. (C) Flow cytometry analysis of the proportion of GFP+ cells in OCT4-ΔPE-GFP niPSC-Ts. (D) Immunostaining images of pluripotency-related marker expression in niPSC-Ts. Scale bar, 50 μm.

https://doi.org/10.7554/eLife.29518.002
Figure 1—figure supplement 1
Optimization of secondary human naïve iPSCs reprogramming system.

(A) Schematic overview of secondary naïve reprogramming strategy to generate human naïve iPSCs. 1° HEF, primary human embryonic fibroblasts; 1° piPSCs, primary primed iPSCs; 2° hiF, inducible fibroblasts; 2° hiF-T, immortalized inducible fibroblasts; 2° niPSCs, secondary naïve iPSCs. (B) Growth curves of hiF and hiF-T cells at different passages. (C) Alkaline phosphatase (AP) staining representing the naïve reprogramming efficiencies of HEFs, hiFs and hiF-Ts at different passages. (D) Representative images showing the senescent cells in hiFs and hiF-Ts at different passages by senescence-associated-beta-galactosidase (SA-β-GAL) assay. Scale bar, 50 μm. Senescent cells are stained in blue and indicated with red arrowheads.

https://doi.org/10.7554/eLife.29518.003
Figure 2 with 2 supplements
Transcriptional profiling of cells during naïve reprogramming.

(A) Schematic representation of reprogramming intermediate collection at different time points, as indicated. hiF-T cells were first cultured in conventional hESM with dox for 6 days and then switched to 5iLAF culture medium supplemented with dox until day 20. Cells with or without dox treatment for four additional days were collected. (B) MDS analysis of RNA-seq data during the naïve reprogramming process. (C) Line plots showing transcriptional dynamics of differentially expressed genes during the naïve reprograming process. Genes were grouped by k-means clustering. Gray shades represent a 95% bootstrap confidence interval around the mean value. (D) Heatmaps showing the expression patterns of genes with pre-implantation signatures in both the naïve and primed reprogramming process. (E) Correlation analysis of transcriptional profiles between naïve reprogramming and the embryonic development process, with the Pearson correlation coefficient of each pair shown on each cell of the heatmap. (F) Immunostaining images of pluripotency-related marker expression in the reprogramming cells at indicated time points during naïve and primed reprogramming. Scale bar, 50 μm.

https://doi.org/10.7554/eLife.29518.004
Figure 2—figure supplement 1
Transcriptional profiling of naïve pluripotency reprogramming cells.

(A) Correlation analysis (left panel) ad cell clustering analysis (right panel) of transcriptional profiles at different time points during naïve reprograming. (B) Bar plot showing dynamic changes in differentially expressed (DE) gene numbers of two adjacent time points during naïve reprogramming. (C) Expression dynamics of epiblast-specific genes and primed-specific genes during naïve reprogramming relative to those in the primed reprogramming process. (D) Bar plot showing the absolute expression values of OTX2 and ZIC2 genes in the naïve and primed reprogramming processes. Error bars represent a 95% confidence interval around the mean value.

https://doi.org/10.7554/eLife.29518.005
Figure 2—figure supplement 2
Expression dynamics of gene clusters in Figure 2C.

Detailed description of Figure 2D gene clusters with specific expression patterns. Line plot and heatmap (left panels) showing expression patterns of each cluster during naïve reprograming. Grey shades represent a 95% confidence interval around the mean value; Bar plot (middle panels) showing enrichment of p-value of representative GO term of each cluster; Bar plot (right panels) showing expression dynamics of selected genes across naïve reprogramming time course.

https://doi.org/10.7554/eLife.29518.006
Figure 3 with 2 supplements
Transient activation of transcripts with 8C-stage-like signatures during naïve reprogramming.

(A) Line plot showing expression dynamics of 8C-stage-specific genes during human embryonic development. Gray shades represent a 95% confidence interval around the mean value. (B) Line plot showing transcriptional dynamics of 8C-specific genes across naïve and primed reprogramming. (C–D) Bar plot showing the absolute expression values of ZSCAN4 (C) and MBD3L2 genes (D) in the naïve and primed reprogramming processes. Error bars represent a 95% confidence interval around the mean value. (E) Immunostaining images of ZSCAN4, MBD3L2-5 and DPPA3 expression in cells during naïve reprogramming. Scale bar, 50 μm. (F) Western blot results of MBD3L2-5 expression in naïve and primed reprogramming cells, niPSC-Ts and piPSC-Ts. β-ACTIN was used as endogenous control. (G) Line plot showing the expression patterns of 8C-specific TEs during human embryonic development. Gray shades represent a 95% confidence interval around the mean value. (H) Line plot showing expression dynamics of 8C-specific TEs during naïve reprogramming. (I–J) Heatmap of expression patterns of 8C-specific HERVK integrants across naïve reprogramming (I) and pre-implantation development (J). (K) Heatmaps showing different expression patterns of KRAB-ZNF genes in the naïve reprogramming process. K-means clustering was performed on KRAB-ZNF genes with k = 8 using R library ‘amap’. Distance between genes was measured based on their correlation.

https://doi.org/10.7554/eLife.29518.007
Figure 3—figure supplement 1
Dynamics of 8C-genes during naïve reprogramming.

(A) Bar plot showing the p-value of 8C-gene enrichments in each cluster measuring with Fisher’ exact test. (B) Line plot showing expression patterns of selected genes during human pre-implantation development. (C) Bar plot showing the absolute expression values of several 8C-genes in the naïve and primed reprogramming processes. Error bars represent a 95% confidence interval around the mean value. (D) Bar plot showing the absolute expression values of MBD3L3/4/5 genes in the naïve and primed reprogramming processes. Error bars represent a 95% confidence interval around the mean value. (E) Immunostaining images of ZSCAN4, MBD3L2-5 and DPPA3 expression in cells during primed reprogramming. Scale bar, 50 μm. (F) Immunostaining for OCT4, ZSCAN4 and MBD3L2-5 in niPSC-Ts and piPSCTs respectively. Scale bar, 20 μm.

https://doi.org/10.7554/eLife.29518.008
Figure 3—figure supplement 2
Dynamics of TEs during naïve reprogramming.

(A) Pie charts of genomic distribution of top 100 8C-TEs distribution (upper panel) and all TEs (lower panel), showing significant enrichment (Fisher’s exact test p-value=0.00408) in LTRs. (B) Heatmaps of expression patterns of SVA integrants during naïve reprogramming (upper panel) and embryonic development (lower panel). (C) Expression dynamics of LTR7-HERVH during embryonic development. (D) Bar plot showing expression dynamics of LTR7-HERVH during naïve and primed reprogramming. (E) Transcriptional dynamics of ZNF534 in naïve and primed reprogramming. Error bars represent a 95% confidence interval around the mean value.

https://doi.org/10.7554/eLife.29518.009
Figure 4 with 1 supplement
Changes in DNA methylation during naïve reprogramming.

(A) Bar plot showing changes in average DNA methylation ratios of all covered C sites during naïve and primed reprogramming. Error bars represent a 95% confidence interval around the mean value. (B) Box plot showing DNA methylation ratio dynamics of naïve specific DMRs during naïve and primed reprogramming. The middle lines of the boxes indicate the median, the outer edges represent the first and the third quartiles, and the whiskers indicate the 1.5 × interquartile range below the lower quartile and above the upper quartile. (C) Dynamics in the DNA methylation levels of naïve-specific DMR-related genes during naïve reprogramming. (D) DNA methylation over stable primary imprints during naïve and primed reprogramming. The middle lines of the boxes indicate the median, the outer edges represent the first and the third quartiles, and the whiskers indicate the 1.5 × interquartile range below the lower quartile and above the upper quartile.

https://doi.org/10.7554/eLife.29518.010
Figure 4—figure supplement 1
Dynamics of DNA methylation in naïve reprogramming.

(A) Histograms showing the distribution of methylation levels (%) across all CpGs at indicated time points during naïve reprogramming. a, average methylation level. (B) 5mC dynamics across naïve and primed reprogramming by HPLC-MS analysis. (C) Bar plot showing DMC dynamics during naïve reprograming. The ratio of hyper-DMCs divided by the number of all C sites (red bars) and the ratio of hypo-DMCs divided by the number of all C sites (blue bars) were plotted separately. (D) Heatmap showing dynamics of DNA methylation on hiF-T with niPSC-T and piPSC-T. Each line is a DMR called using mcomp from MOABS. Naïve specific DMRs were defined as those only detected in niPSC-Ts compared with hiF-Ts and piPSC-Ts. (E) Line plot showing the expression dynamics of selected genes related to DNA methylation (upper panel) and TET family genes (lower panel) in naïve reprogramming (solid line) and primed reprogramming (dash line) process.

https://doi.org/10.7554/eLife.29518.011
Figure 5 with 1 supplement
Histone modification profiles during naïve reprogramming.

(A) Alluvial plots showing the global dynamics of genes covered by different chromatin states during naïve reprogramming. Each line represents a gene. Red bar represents genes with promoter that covered only by H3K4me3. Yellow bar represents genes with promoter that covered only by H3K27me3. Blue bar represents genes with promoter that covered by both H3K4me3 and H3K27me3. Grey bar represents genes with promoter that covered by neither H3K4me3 nor H3K27me3. (B) Line plot showing dynamics of different histone modification signals across naïve and primed reprogramming. (C) Heatmap showing clusters of genes with different bivalency patterns across naïve and primed reprogramming. (D) Average profiles of H3K4me3 and H3K27me3 signals surrounding the TSS of genes characteristic for early embryogenesis and pre-implantation during naïve reprogramming. (E) Heatmap showing six clusters of H3K9me3 peaks with different patterns (left panel) and average H3K9me3 profiles around integrants of SVA family in cluster vi of the heatmap (right panel) during naïve reprogramming. (F) Average H3K9me3 profiling around MER9a3-HERVK-9 TE during naïve reprogramming.

https://doi.org/10.7554/eLife.29518.012
Figure 5—figure supplement 1
Dynamics of histone modifications in naïve reprogramming.

(A) Alluvial plots showing the global dynamics of genes covered by different chromatin states during naïve reprogramming. Each line represents a gene. Red bar represents genes with promoter that covered only by H3K4me2. Yellow bar represents genes with promoter that covered only by H3K4me3. Blue bar represents genes with promoter that covered by both H3K4me2 and H3K4me3. Grey bar represents genes with promoter that covered by neither H3K4me2 nor H3K4me3. (B) Enrichment of p-value of representative GO term of the selected clusters in Figure 5C. (C) Average H3K9me3 profiles around integrants of SVA family during naïve reprogramming.

https://doi.org/10.7554/eLife.29518.013
Figure 6 with 1 supplement
Integrative analysis of transcriptional and epigenetic dynamics during naïve reprogramming.

(A) Transcriptional dynamics of genes with different patterns are closely correlated with epigenetic modifications at promoter regions during naïve reprogramming. Black lines in the heatmaps separate genes regulated by histone modifications (upper panel, left part), DNA methylation (upper panel, right part) or both (upper panel, middle part). Line plots (lower panels) show the representative genes in each category that are regulated by H3K4me3 modification or DNA methylation. (B) Up-regulated genes with different CG ratios in their promoters exhibit different kinetics with regard to transcription, DNA methylation and H3K4me2/H3K4me3/H3K27me3 coverage during naïve reprogramming.

https://doi.org/10.7554/eLife.29518.014
Figure 6—figure supplement 1
Epigenetic changes of representative genes during naïve reprogramming path.

(A) Base-level visualization of DNA methylation and histone modifications in the promoter regions of representative genes. (B) Classification of genes by the CG ratio in their promoter. (C) Enrichment of p-value of representative GO term of clusters based on different CG ratios in promoters as shown in Figure 6B.

https://doi.org/10.7554/eLife.29518.015

Tables

Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional
information
gene ()NANA
strain, strain background ()NANA
genetic reagent ()NANA
cell line ()Human embryonic fibroblasts (HEFs);
Primary primed iPSC lines;
hiF-T cell lines; Secondary primed iPSC
lines; Secondary naïve iPSC lines
This paper;
Cacchiarelli, D., Trapnell, C.,
Ziller, M.J., Soumillon, M.,
Cesana, M., Karnik, R.,
Donaghey, J., Smith, Z.D.,
Ratanasirintrawoot, S.,
Zhang, X., et al. (2015).
Cell. 2015 Jul
16;162(2):412–424. doi:
10.1016/j.cell.2015.06.016.
Yan, L., Yang, M., Guo, H.,
Yang, L., Wu, J., Li, R., Liu, P.,
Lian, Y., Zheng, X., Yan, J.,
et al. (2013). Nat Struct
Mol Biol. 2013
Sep;20(9):1131–9. doi:
10.1038/nsmb.2660.
Epub 2013 Aug 11.
transfected construct ()dox-inducible, polycistronic
OKMS lentiviral vector
Addgene 51543.
Cacchiarelli, D., Trapnell, C.,
Ziller, M.J., Soumillon, M.,
Cesana, M., Karnik, R.,
Donaghey, J., Smith, Z.D.,
Ratanasirintrawoot, S.,
Zhang, X., et al.
(2015).Cell. 2015 Jul
16;162(2):412–424. doi:
10.1016/j.cell.2015.06.016.
biological sample ()hiF-T/0d/2d/6d/8d/12d/14d/20d/
24d+dox/24d-dox/niPSC-T;
Oocyte/Zygote/2 cell/4 cell/8 cell/
Morula/MTE/PTE/EPI/PE/hESC0/hESC10;
hiF-T/2d/6d/8d/14d/20d/24d+
dox/24d-dox/piPSC-T;
this paper;
antibodyanti-SSEA3, SSEA4, TRA-1–60, UTF1,
DPPA3, ZSCNA4, MBD3L2
Millipore MAB4304,
Millipore MAB4360, Abcam
ab24273, Santa Cruz sc-
67249, Millipore AB4340,
Abcam ab174802
recombinant DNA reagentNANA
sequence-based reagentKAPA Stranded mRNA-Seq Kit;
KAPA DNA Library Preparation Kits
KAPA KK8401;
KAPA KK8234
peptide, recombinant proteinhuman LIF recombinant protein;
bFGF recombinant protein
Peprotech 300–05;
Peprotech 450–33
commercial assay or kitbowtie; TopHat; Cufflinks;
edgeR; MACS2;
KAPA KK8401;
KAPA KK8234
chemical compound, drugActivin A; PD0325901; IM-12;
SB590885; WH-4–023; Y-27632;
Peprotech 120–14;
Stemgent 04–0012; Enzo
BML-WN102; R and D
systems 2650; A Chemtek
0104–002013; Stemgent
04–0012;
software, algorithmbowtie; TopHat; Cufflinks;
edgeR; MACS2;
PMID: 22388286;
PMID: 19289445;
PMID: 22383036;
PMID: 24743990;
PMID: 18798982;
RRID:SCR_005476;
RRID:SCR_013035;
RRID:SCR_014597;
RRID:SCR_012802;
RRID:SCR_013291
NA
other

Data availability

The following data sets were generated
  1. 1
    Integrative analysis of reprogramming human fibroblast cells to naïve pluripotency
    1. Wang Y
    2. Zhao C
    3. Hou Z
    4. Yang Y
    5. Bi Y
    6. Wang H
    7. Zhang Y
    8. Gao S
    (2017)
    Publicly available at the NCBI Gene Expression Omnibus (accession no: GSE89072).
The following previously published data sets were used
  1. 1
  2. 2
    Tracing pluripotency of human early embryos and embryonic stem cells by single cell RNA-seq
    1. Tang F
    2. Qiao J
    3. Li R
    (2013)
    Publicly available at the NCBI Gene Expression Omnibus (accession no: GSE36552).
  3. 3
    The DNA methylation landscape of human early embryos
    1. Guo H
    2. Zhu P
    3. Yan L
    4. Qiao J
    5. Tang F
    (2014)
    Publicly available at the NCBI Gene Expression Omnibus (accession no: GSE49828).

Additional files

Source data 1

Source Data for Figures 26 and Figure Supplements.

The Source Data files include the source R code for Figures 26. The R scripts use the data in the ‘Expression Data’ folder with the relative path. The ‘Expression Data’ folder contains the non-redundant related gene expression, methylation and histone modification tables for the main figures and figure supplements.

https://doi.org/10.7554/eLife.29518.016
Transparent reporting form
https://doi.org/10.7554/eLife.29518.017

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)