Fig 2: Similarities in neuronal differentiation. LUHMES cells from two different sources [University of Konstanz (UKN); American Type Culture Collection (ATCC)] were cultured and differentiated according to the published standard protocol. a Morphology and structural properties of undifferentiated LUHMES (d0) cells (of both SP) were assessed by immunocytochemistry staining for ß-III tubulin, Nestin and H-33342. b Morphology and structural properties of differentiated LUHMES (d6) cells (of both SP) seeded at a density of 0.5 × 105 cells/cm2 were assessed by immunostaining for ß-III tubulin (red), MAP2 (green) and H-33342 (blue). c LUHMES cells were differentiated for the indicated time points (0; 2; 4; 6; 8 days) and mRNA samples were prepared Changes in gene expression of the neurodevelopmental marker genes DRD2, GFRA1, v-MYC, TUBB3; ACHE, SNAP25, GRIN1, SYP, HES5 and VMAT-2 of LUHMES cells from both SP were monitored by qPCR. Values are expressed relative to expression levels in undifferentiated LUHMES cells. Data are means ± SEM for three different experiments. (Color figure online)

Fig 3: Amino acid changing SNVs. SNVs affecting proteins in LUHMES from two different sources [University of Konstanz (UKN); American Type Culture Collection (ATCC)] were filtered from the total SNVs called as described in the material and methods section in detail. a Genes affected by common LUHMES SP and “UKN” or “ATCC” specific amino acid changing SNV. The genes marked in red were further assessed on protein level for differences between the two SP. b Undifferentiated (d0) and mature (d6) LUHMES cells were lysed and analysed by Western blot analysis using anti- NRXN3, anti-SIRT6 and anti-GAPDH antibodies. c, d Densitometric quantification of band intensities of NRXN3 in d6 cells (c) and SIRT6 in d6 cells (d). e Densitometric quantification of band intensities of HSF1 in d6 cells. Intensities are depicted relative to the intensity in the “UKN” SP. f LUHMES (d6) cells of both SP were treated with indicated concentrations of Geldanamycin for 24 h. After incubation cells were lysed and analysed by Western blot using anti-HSF1, anti-HSP70 and anti-GAPDH antibodies. g Densitometric quantification of band intensities of HSP70 induction by Geldanamycin. Intensities are depicted relative to the intensity in the untreated “UKN” SP. Band intensities of 5 and 50 nM Geldanamycin were combined. h LUHMES (d6) cells of both SP untreated (c) and with indicated time after start of heat shock (43° C/1 h). After incubation, cells were lysed and analysed by Western blot using anti-HSF1 and anti-GAPDH antibodies

Fig 4: Similarities and differences in the reaction to different toxicants. LUHMES cells from two different sources [University of Konstanz (UKN); American Type Culture Collection (ATCC)] were cultured according to a standard protocol. LUHMES cells were seeded at a density of 1.5 × 105 cells/cm2 at d2. Medium was exchanged at d4 and exposed to MPP+ starting at d6. a Viability was assessed at d9 by measuring resazurin reduction and LDH release after cells have been exposed to MPP+ [5 µM] for times as indicated. Data are means ± SD of three independent experiments.***p = 0.001 b UKN and ATCC LUHMES were incubated with indicated concentrations of MPP+. After 48 h viability was assessed measuring resazurin reduction and LDH release, Data are means ± SD of three independent experiments. c Intracellular levels of total glutathione (GSH + GSSG) were measured in cells treated with MPP+ [5 µM] for indicated time periods and represent means ± SD of three independent experiments. d Intracellular levels of total ATP were measured in cells treated with MPP+ [5 µM] for indicated time periods. Data are means ± SD of three independent experiments.***p = 0.001

Fig 5: Evidence for in vitro endocytosis of colonic EVs and neuronal cells.(A) Coincubation of human neuronal cells (LUHMES, ATCC CRL-2927) with colonic EVs from patients with PI-IBS-D revealed increased NMDA NR2B expression starting at 24 hours and reaching significance at 48 hours. (B and C) Coincubation of human neuronal cells with colonic EVs from patients with PI-IBS-D lead to significantly diminished miR-23a (B) and miR-23b (C) after 24, 48, and 72 hours versus control participants. (D) Coincubation of mouse DRG cells with colonic EVs from patients with PI-IBS-D resulted in significantly increased NMDA NR2B expression at 48 and 72 hours. (E and F) Coincubation of mouse DRGs with colonic EVs from patients with PI-IBS-D lead to decreased miR-23a expression (E) at 24, 48, and 72 hours and decreased miR-23b expression (F) at 48 and 72 hours. (G) IHC showing endocytosis of human colonic EVs (red), neuronal cells (green), and DAPI (blue) after 8 hours of incubation. Scale bars: 10 μm. (H–M) Relative GAS5 (H and I), miR-23a (J and K), and miR-23b (L and M) expression in human colonoids incubated for 24 and 48 hours with colonic EVs from patients with PI-IBS-D or control participants. At 48 hours, GAS5 was significantly increased, and miR-23a and miR-23b were decreased. (N) Endocytosis of human colonic EVs (red) into the colonoids after 48 hours of incubation. Epithelial cell marker (EpCam, green) and EVs labeled with PKH26 (red). Scale bars: 10 μm. *P = 0.05, **P = 0.01 by unpaired t test.