Extended Data Fig. 5: scRNA-seq analysis of day-6 and day-29 skin organoids derived from WA25 and DSP-GFP cells.

a, g, Separate UMAP plots for WA25 and DSP-GFP cell clusters at day 6 (a) and day 29 (g). The major cell cluster groupings of surface epithelia, epidermis and mesenchyme are noted. Colours indicate cell state. The presumptive cell identities (based on a priori knowledge of marker genes) are listed. Cell clusters with no discernible identity had low expression of mitochondrial genes (‘low mito cells’) or high expression of long noncoding RNA genes (‘high lncRNA’). Day 6 (a): n = 11,785 WA25 cells, n = 11,544 DSP-GFP cells; day 29 (g): n = 9,268 WA25 cells, n = 9,013 DSP-GFP cells. Ten day-6 organoids from one experiment and five day-29 organoids from one experiment were randomly pooled for scRNA-seq analysis (per cell line). b, Dot plot for surface ectoderm and non-neural ectoderm (NNE) markers on the basis of RNA-seq data from a previous study¹¹. c, UMAP plot for HAND1, a key marker for NNE cells derived from human pluripotent stem cells. d, Dot plot for markers of surface ectoderm and anterior–posterior placodes. Gene expression frequency is indicated by spot size and expression level is indicated by colour intensity. e, UMAP overlay plot showing the distribution of OTX1 (marker of anterior placode and neuroectoderm) and GBX2 (marker of posterior placode). f, UMAP plots for key markers of epidermal progenitors, neuroectoderm, general placode and cycling cells. h, UMAP plots for specific marker genes that define epidermal, cycling, CNC and dermal cell subtypes. i, Magnified view of clusters 9 (CNC cell and Schwann cell precursors), 15 (neuroectoderm cells), 18 (PNS- or CNS-like neurons), 19 (myocytes) and 22 (melanocytes). Key marker genes are shown to label each cell cluster. SOX2 has broad expression across neuroectoderm and CNC cells. j, Dot plot for general mesenchymal markers (PRRX1 and PDGFRA), markers of all pharyngeal arches, markers of PA1 and markers of PA2–PA4. Gene expression frequency is indicated by spot size and expression level is indicated by colour intensity. The expression of HOX genes appears to be limited to a subset of cells with low mitochondrial gene expression. This figure corresponds with the data in Fig. 2.