Fig. 4: Molecular roadmap of ciRPE chemical reprogramming.

a Schematic illustrating the multi-omics sequencing strategy employed to analyze the reprogramming process from MEFs to ciRPE cells. b PCA of RNA seq and CUT&Tag data (H3K4me3, H3K27ac, and H3K27me3) were performed on samples collected at days 0, 7, 18, and 32 (ciRPE) of reprogramming, along with pRPE cells serving as the control. c Heatmap showing differentially expressed genes in MEF to ciRPE cell reprogramming samples at indicated time points. The number above heatmap indicates independent biological replicates. Representative genes (left side of the heatmap) and associated gene ontology (GO; right side of the heatmap) for each block are shown. Red and blue indicate upregulated and downregulated genes, respectively. Differential expression was analyzed using the R package limma (v3.58.1) following normalization with edgeR (v4.0.16). Significantly changed genes were defined by |log₂ fold change| > 1.5 and adjusted p < 0.01 (Benjamini-Hochberg correction). d Dynamics of CUT&Tag peaks (H3K4me3, H3K27ac, H3K27me3) associated with cluster-specific genes (C). The red line indicates the median CUT&Tag peaks over time, the blue line represents the median RNA expression levels, and the gray background lines depict individual peak values across time points. e Uniform manifold approximation and projection (UMAP) analysis of integrated scRNA-seq data from cells collected at the indicated time points during the reprogramming process of MEFs to ciRPE cells. f UMAP plot showing identified cell types in samples collected at the indicated time points during the reprogramming process. g Dot plot illustrating the expression of representative marker genes across different cell types during the reprogramming process. h RNA velocity streamline plot illustrating the transitions of cell populations during the reprogramming process using the scVelo method. The arrows represent the flow determined by the ratio of unspliced to spliced transcripts, predicting dynamic changes in cell identity. Black arrows indicate RNA velocity flow based on the unspliced-to-spliced transcript ratio, while the gray to teal arrows are used for visual enhancement to highlight specific trajectories. i Heatmap showing the gene expression similarity between cell types during the reprogramming process and those reported in previous studies. pEF cells represent the data used for small molecule prediction as described above, while pRPE data were obtained from the GSE183572 dataset.