DNA methylation and transcriptional trajectories during human development and reprogramming of isogenic pluripotent stem cells

Abstract

Determining cell identity and maturation status of differentiated pluripotent stem cells (PSCs) requires knowledge of the transcriptional and epigenetic trajectory of organs during development. Here, we generate a transcriptional and DNA methylation atlas covering 21 organs during human fetal development. Analysis of multiple isogenic organ sets shows that organ-specific DNA methylation patterns are highly dynamic between week 9 (W9) and W22 of gestation. We investigate the impact of reprogramming on organ-specific DNA methylation by generating human induced pluripotent stem cell (hiPSC) lines from six isogenic organs. All isogenic hiPSCs acquire DNA methylation patterns comparable to existing hPSCs. However, hiPSCs derived from fetal brain retain brain-specific DNA methylation marks that seem sufficient to confer higher propensity to differentiate to neural derivatives. This systematic analysis of human fetal organs during development and associated isogenic hiPSC lines provides insights in the role of DNA methylation in lineage commitment and epigenetic reprogramming in humans.While DNA methylation and gene expression data are widely available for animal models, comprehensive data from human development is rarer. Here, the authors generated transcriptional and DNA methylation data from 21 organs during human development and 6 isogenic induced pluripotent stem cell lines.

Fig. 1

Transcriptional and DNA methylation of a collection of organs of first and second trimester. a The collection of 111 samples analysed for gene expression (by next generation sequencing (NGS) DeepSAGE) and 105 samples analysed for DNA methylation (by Illumina 450 K array). See also Supplementary Data File 1. Mat, maternal. b Multidimensional scaling (Euclidian distance) of the transcriptional profiles of the 111 samples. The colors represent the different germ layers whereas the shapes indicate the gestational age. See also Supplementary Fig. 2a. c Multidimensional scaling (Euclidian distance) of the DNA methylation profiles of the 105 samples. The colors represent the different germ layers whereas the shapes indicate the gestational age. See also Supplementary Fig. 2b. d Hierarchical clustering (Euclidian distance) of the 105 DNA methylation samples. e Multidimensional scaling (Euclidian distance) of the DNA methylation profiles excluding the extraembryonic samples. The colors represent the different germ layers whereas the shapes indicate the gestational age. Dashed arrows represent the trend of DNA methylation dynamics during development in similar organs

Fig. 2

Organ-specific upregulated and downregulated genes and associated changes in DNA methylation. a Numbers of differentially expressed genes (FDR < 0.01, general linearized model) between W8-12 and W20-22 in different organs. Heart V, heart ventricle; Mother, maternal endometrium. b Heatmap of the upregulated genes that were uniquely assigned to one organ. See also Supplementary Data File 2. c Heatmap of the downregulated genes that were uniquely assigned to one organ. See also Supplementary Data File 2. d Boxplots illustrating the organ-specific methylation changes (delta beta) of the nearest proximal promoter (PP) and the gene body (GB) of the loci identified in b, c, excluding the maternal endometrium. The red line indicates a delta beta of −0.3, 0 and 0.3. See also Supplementary Data File 3

Fig. 3

DNA methylation signatures of isogenic sets of organs. a Violin plots showing the distribution of the DNA methylation (beta values) in each of the 14 isogenic organs at W9, W18 and W21. Heart A, heart atrium; Heart V, heart ventricle; Sp. cord, spinal cord. b–d Heatmaps illustrating hyper- (top) and hypomethylated (bottom) CpGs per organ with a beta value difference of > 0.2 or < 0.2, respectively, at W9 b, W18 c and W21 d. Due to the quantity of hypermethylated and hypomethylated CpGs in the placenta at W9 and W18, those were removed but are given in Supplementary Fig. 3b, c. e Venn diagram illustrating the overlap of the hypermethylated (bottom) and hypomethylated (top) CpGs of the organs at W9 and W21. The numbers in brackets represent the CpGs including the placenta. The overlapping CpGs per organ are given in Supplementary Fig. 3d. f Genic and CGI-centric annotation of the identified hyper- (left) and hypomethylated (right) CpGs given as odds ratio (OR). CGI, CpG island; DP, distal promoter; GB, gene body; IG, intergenic; NC, non-CGI; PP, proximal promoter; SHE, shelf; SHO, shore. g Number of organ-specific hypermethylated and hypomethylated differentially methylated regions (DMRs) per isogenic organ. Tongue and muscle were pooled. For the nearest associated loci see Supplementary Data File 4.The number of organ-specific hypermethylated and hypomethylated individual CpGs are given in brackets

Fig. 4

Epigenetic memory of isogenic human iPSCs from six organs a Illustration describing the experimental setup for reprogramming. OSKM, reprogramming factors POU5F1, SOX2, KLF4, MYC. b Bright-field pictures of the primary cells (first row, scale bar 50 µm) and the iPSC colonies of clone #1 (second row, scale bar 1 mm) from the six organs. The iPSCs (clones #1) were immunostained for POU5F1 and TRA-1-81 (third row, scale bar 100 µm) and NANOG and SSEA4 (fourth row, scale bar 100 µm). For the single channels and the immunostaining of clones #2 see Supplementary Fig. 4a. c Real-time quantitative PCR analysis of the 12 iPSCs for endogenous SOX2, endogenous OCT4 and NANOG. NKX2.5^eGFP/w hESCs were used as positive and fetal skin as negative control. Each bar represents the mean of three technical replicates ± standard deviation. d Violin plots depicting the distribution of the DNA methylation (beta values) of the 12 iPSCs and their isogenic organ of origin. e Hierarchical clustering of the 12 iPSCs and their isogenic organ of origin based on the DNA methylation profiles. f Heatmap of uniquely hypermethylated (top) and hypomethylated (bottom) CpGs in the 12 iPSCs compared to the six organs of origin with a beta value difference of > 0.2 or < 0.2, respectively. See also Supplementary Data File 5. g Heatmap depicting uniquely hypermethylated (top) and hypomethylated (bottom) CpGs in the brain triplet (two iPSC clones and brain) compared to the five triplets with a beta value difference of > 0.2 or < 0.2, respectively. The genic location and gene identity are provided