Fig. 6: Maintenance of stem cell characteristics and genomic stability across aESCs, iPSCs, and ntESCs.

a Schematic diagram of experiment. b UMAP visualization showing seven different cell types in PSCs cultured on a feeder layer, each represented by a distinct color. Each stem cell type is shown separately on the right. c UMAP visualization showing two cell types in PSCs feeder-free PSC, cultured on the feeder-free layer, each represented by a distinct color. Each stem cell type is shown separately on the right. d, e Violin plots depicting expression levels of key lineage-associated genes across seven cell types in the feeder layer and two cell types in the feeder-free layer, respectively. Endo differentiation propensity (DPR) expressed genes associated with endoderm development, including FOXA2, SOX17, and CER1^44,45. Meso DPR exhibited elevated expression of genes related to mesoderm formation, such as RSPO3, HAND1, and TBX3^44,45. NE DPR expressed early neural cell lineage markers FEZF1^77,78 and genes promoting stem cell differentiation into neural cells, such as SOX21 and SOX9^79,80,81,82. NNE DPR upregulated non-neuroectoderm genes TFAP2C, GATA3, DLX5, and CLDN10⁴⁵. ExE mech DPR upregulated ExE mech genes TIMP1, FN1, and COL1A2^37,83,84. d seven cell types are represented: Epi1 (n  =  14864 cells), Epi2 (n  =  650 cells), Endo DPR (n  =  949 cells), Meso DPR (n  =  567 cells), NE DPR (n  =  1162 cells), NNE DPR (n  =  1607 cells), and ExE mech DPR (n  =  616 cells). These data are from one biological replicate of iPSCs, aESCs, and ntESCs. e two cell types are shown: Epi (n  =  19669 cells) and ExE mech DPR (n  =  57 cells), derived from one biological replicate of iPSCs, aESCs, and ntESCs. For the box plots, the central line represents the median, the box boundaries correspond to the 25th and 75th percentiles. The length of the whiskers indicates 1.5 times the interquartile range from the first and third quartiles. f, g Comparison of heterogeneity in constituent cell types among aESCs, iPSCs, and ntESCs cultured on feeder layers and feeder-free layers, respectively. h The number of up- and down-regulated DEGs with FC > 1 or <1 and p-value < 0.05, as well as FC ≥ 1.5 or ≤ 0.67 and p-value < 0.05, in iPSCs and ntESCs cultured with and without feeder layers, compared to aESCs. Red and blue values on the bars represent the number of up- and down-regulated DEGs. Gene expression differences were compared using two-sided Wilcoxon Rank-Sum test. Due to the small number of DEGs, p values were not adjusted using the Benjamini-Hochberg method. i The top 10 Gene Ontology (GO) terms and KEGG pathways were identified through functional enrichment analysis, based on DEGs (FC > 1 or <1 and p-value < 0.05). For GO terms, p-values were adjusted using the Benjamini-Hochberg method. For KEGG pathways, due to the small number of DEGs, p-values were not adjusted using the Benjamini-Hochberg method. j, k Violin plots showing the differences in CNV level and transcriptional noise in Epi cells derived from the three PSC types (aESCs, iPSCs, and ntESCs). For feeder layer, n  =  5301 cells from one biological replicate of aESCs, n  =  3038 cells from one biological replicate of iPSCs, n  =  6525 cells from one biological replicate of ntESCs. For feeder-free, n  =  5929 cells from one biological replicate of aESCs, n  =  8733 cells from one biological replicate of iPSCs, n  =  5007cells from one biological replicate of ntESCs. The differences in CNV level and transcriptional noise were compared by two-sided Wilcoxon Rank-Sum test, with p < 0.0001 indicated by ****. For the box plots, the central line represents the median, the box boundaries correspond to the 25th and 75th percentiles. The length of the whiskers indicates 1.5 times the interquartile range from the first and third quartiles.