Fig. 3: Derivation of pNSCs without low-pH treatment.

a, Schematic and morphology of embryonic limb pNSC derivation without low-pH treatment. b, Limb pNSCs stably maintained for 50 passages. c, Immunofluorescence of limb pNSCs. Scale bar, 50 μm. d, FACS strategy for deriving adult lung pNSCs without low-pH treatment from Nes-GFP mouse. e, Morphology of Nes-GFP⁺ cells derived primary lung pNSC cluster. BF, bright-field. f, NSC-like cluster forming efficiency from sorted Nes-GFP⁺ lung cells. The data represent mean ± s.d. (n = 3 biological replicates). g, FACS strategy for deriving postnatal tail pNSCs without low-pH treatment from Nes-GFP mouse. h, Morphology of Nes-GFP⁺ cells derived primary tail pNSC cluster. i, NSC-like cluster forming efficiency from sorted Nes-GFP⁺ tail cells. The data represent mean ± s.d. (n = 3 biological replicates). j, Lung and tail pNSCs stably maintained for 50 passages. k, Immunofluorescence of NSC markers in lung and tail pNSCs. l, RT–qPCR analysis across samples. All data are calibrated to brain NSCs, whose expression is considered to be 1 for all genes. The data represent mean ± s.d. (n = 3 biological replicates). m, Heatmap of global gene expression pattern of the samples. n = 2 biological replicates, all replicates are shown. n, Hierarchical clustering of the cell lines based on m. o, Pairwise scatter-plots of lung and tail pNSCs versus brain NSCs and tissue cells. n = 2 biological replicates; the depicted results are an integration of data derived from all different biological samples. p, Bisulfite sequencing PCR on Nestin (up) and Col1a1 (down) in samples.