Fig. 1: Generation of self-organizing neural and mesodermal progenitors from human PSCs in adherent culture.

a Schematic representation illustrating the strategy employed to generate the soNMJ model and NPs from hPSCs. b qPCR analysis on day 6 demonstrated that administration of dual SMADi (2SMADi) from day 0 – day 6 resulted in the differentiation of hPSCs to NPs expressing high levels of SOX2, SOX1, NKX1.2 and PAX6. Administration of 2SMADi from day 3 − day 6 was sufficient to generate both NP cells and PSM cells expressing MEOX1, FOXC1, FOXC2 and MYF5. The statistical tests employed included an unpaired t-test with Welch’s correction. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. Each dot with the same colour/shape represents different wells from a single differentiation experiment of a specific cell line. The dashed line is the median value. XM001: N = 3, n = 9; H1: N = 2, n = 6; H9: N = 1, n = 3. Source data are provided as a Source Data file. c Representative brightfield images of NP and NP + PSM cultures at day 6. Circles mark PSM colonies. Scale bars: 100 µm. d Immunofluorescence analysis at day 6 of differentiation showed that exposure to 2SMADi since day 0 resulted in the generation of SOX2⁺ NPs in the absence of mesodermal cells. Co-expression of PAX3 and SOX2 corresponded to dorsal NPs. Conversely, exposure to 2SMADi after day 3 resulted in the segregation of NMP cells to SOX2⁺ NPs and PAX3⁺ PSM cells. e We quantified the number of PAX3⁺ cells in H1 (37.5% ± 6.6%), H9 (30.6% ± 6.5%) and XM001 (30.6% ± 7.6) lines, and the number of SOX2⁺ cells in H1 (60.4% ±6.4%), H9 (64% ± 6.7%) and XM001 (71.8% ± 5.6%). The statistical tests employed included one-way ANOVA with Bonferroni’s multiple comparison test. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001. Each dot with the same colour/shape represents a sample from a single differentiation experiment of a specific cell line. The dashed line is the median value. XM001: N = 3, n = 9; H1: N = 3, n = 9; H9: N = 3, n = 9. Scale bars: 50 µm. Source data are provided as a Source Data file. f Immunofluorescence analysis at day 20 of differentiation showed that early exposure to 2SMADi from day 0 - day 6 resulted in the generation of TUBB3⁺ neurons. Exposure to 2SMADi after day 3 resulted in the segregation of NMPs into DESMIN⁺ myoblast cells and TUBB3⁺ neurons. The immunofluorescence analysis was performed in the H1, H9 and XM001 cell lines (also see Table S1). Scale bars 75 µm. NPs Neural progenitors, PSM Pre-somitic mesoderm, 2SMADi dual-SMAD-inhibition, soNMJ model: self-organizing neuromuscular junction model.