Fig 2: qPCR analysis of pluripotency and neuronal markersIn order to characterize the derivative cell populations, hESCs collected at the onset of the protocol (Day 0), and cells derived at all 3 time points of the differentiation protocol (i.e., at Day 7, Day 13 and Day 20) were analyzed for specific pluripotency markers (POU5F1, NANOG, SOX2, NES), major neuronal development transcription factors (SOX2, OTX2, FOXG1, NEUROD1) and genes related to cytoskeletal rearrangement during differentiation towards neuronal maturation (NES, VIM, TUBB3 and MAP2) by qRT-PCR. The expression of the pluripotency transcription factors POU5F1 and NANOG decreases to zero already at Day 7, while SOX2 and NES expression increases as hESCs commit to neuronal fate. NES expression decreases at Day 20, consistent with an arising neuronal, non-proliferative cell population of Day 20. Similar expression pattern was observed with FOXG1, one of the earliest telencephalic specific transcription factors. The expression of the transcription factor OTX2, which is known as a regulator of neurogenesis, increases as cells differentiate, promoting their commitment. This increasing pattern of expression from Day 0 to Day 20 is also seen in the expression of the key bHLH enhancer of transcriptional regulators of neurogenesis NEUROD1, the mature dendritic marker MAP2, the neuronal specific TUBB3, and the intermediate filament VIM. For the qPCR analysis we used samples collected from 3 separate experiments (cells collected from one well were considered one sample, 3 samples were collected per experiment, and each sample collected for analysis was run in technical triplicate.) Each value is plotted as a point (average value of technical triplicates), on top of the box plot. The box represents 25th to the 75th percentile, and the midline represents the median value, and the line goes from minimum to maximum.

Fig 3: The development of a malignant peripheral nerve sheath tumors (MPNST) model. (A) Representative immunocytochemical images of induced tumor Schwann cells (iSCs) either deprived of growth factors (-NRG) or treated with neuregulin and forskolin (+NRG, 50 ng/ml neuregulin, 5 mM Forskolin). Note the similar numbers of Nestin+ (green, NES), Hoechst+ (blue) iSCs that express Ki67 (red) across both conditions. (B) Quantification of percentage of Ki67+ cells revealed no difference between groups (Student’s t-test, n = 4, P = 0.3). (C) Note the presence of sphere formation when iSCs are grown in growth factor deprived conditions. (D) Karyotyping analysis indicates multiple chromosomal abnormalities, including polyploidy and monosomy cells. (E) Intradermal iSC injections into back skin resulted in the development of tumors within 16 weeks. (F–M) Representative histological images of H&E (F,G,J,K), s100 (H,L) and Desmin (I,M) from a biopsied sample of human MPNSTs (F–I) and from a sample from iSCs (J–M). Note the presence of necrosis, mitosis and spindle-shaped cells in both samples (F,G and J,K). Also note the presence of S100 (H,L) immunoreactivity, as well as the lack of Desmin immunoreactivity (I,M) in both samples. Scale bars = A (50 µm), C (200 µm), G–I (20 µm).

Fig 4: Mutations in PSEN1 Reduce Notch Signaling and Lead to Premature Terminal Differentiation(A) Representative images of day 27 iPSC-derived neural progenitors labeled for dividing cells (Ki67) and postmitotic neurons (TUJ1).(B) High-content analysis and quantification of the relative proportion of proliferative (Ki67 positive) and neuronal (TUJ1 positive) cells.(C and D) Representative western blot and quantification for the cleaved, active Notch intracellular domain (NICD), PAX6 neural progenitor marker, and TUJ1 neuronal marker. Note, due to variability in Ctrl1, PAX6 data were normalized to Ctrl2. Due to similar molecular weights, the ratio of PAX6 to TUJ1 intensity represents a measure of terminal differentiation. na, not available.(E) qPCR analysis of the expression of the Notch readout gene FABP7 (alias BLBP) and TUBB3 (neuronal tubulin).(F) RNA sequencing (RNA-seq) expression data from Kwart et al. (2019), demonstrating expression of neural stem cell (NSC) markers (PAX6, NES, and SOX2), Notch signaling readout genes (FABP7, HES1, and HES5), and adult NSC markers (HOPX, LGALS1, and ANXA2) (Berg et al., 2019; Edri et al., 2015) in 80 days in vitro (DIV) iPSC-derived neurons from wild type (WT) compared with isogenic PSEN1 or APP mutant lines. Adjusted p values are represented within the histogram.Blue, control; purple, APP mutant cells; pink, mutations in the PSEN1 extracellular loop; green, mutations in PSEN1 transmembrane and intracellular domains. The number of independent neural inductions is shown within the histograms. Data from different iPSC clones are depicted by gray data points (APP V717I, PSEN1 int4del, and PSEN1 R278I). *p < 0.05, **p < 0.01, ***p < 0.001 via ANOVA with post hoc Tukey’s analysis (normal distribution tested via the Shapiro-Wilk test), as indicated in Table S1. Scale bar represents 50 µm. Error bars represent standard error of the mean.

Fig 5: Immunofluorescence and Functional Comparison of DesLO, FeLO, iHEP, and PHH Cultures(A) Phase imaging of day 12 DesLO cultures shows the clear formation of tight junctions (indicated by the yellow arrow) characteristic of hepatic cells in culture and visible vascular structure (indicated by the red arrow) (D12: day 12). Scale bars, 100 µm.(B–H) Immunofluorescence staining for DAPI with CD34 and AAT (B), CK18 and CEBPA (C), CD31 and HNF4A (D), CD31 and ERG (E), CD34 and NES (F), and DES and HNR4A (H) of day 17 DesLO cultures confirming the presence of HLC, SLC, and ELC populations through staining of cell-type indicative markers (D17: day 17). Scale bars, 100 µm.(I) PAS, Oil Red O, and indocyanine green (ICG) stains indicating glycogen storage, lipid production, and solute transport activity, respectively, in iHEPs, FeLO, DesLO, and PHH cultures. Scale bar, 50 µm.(J) ELISAs of liver-specific proteins, CYP3A4 enzymatic activity, and urea production measured in iHEP (2 lots, n = 3 each), FeLO (n = 3), DesLO, (n = 3), and PHH (2 lots, n = 3 except ANGPTL3 first lot is n = 2, and CYP3A4 second lot is n = 4). For albumin day <2 PHH samples (left PHH bar) is 2 lots, n = 3 each (n = 6 total) and day 3 PHH samples (right PHH bar) is 2 lots, n = 3 each (n = 6 total). All other PHH samples are <2 days in culture. Dotted line indicates DesLO average.(K) Schematic of FXR regulation of CYP7A1 expression and bile acid synthesis.(L–N) qPCR showing expression of CYP7A1 (L), NR0B2 (M), and FGF19 (N) in iHEP (n = 2), FeLO (n = 3), DesLO (n = 3), and PHH (2 lots, n = 2 each) vehicle controls and with addition of GW4064. Fold change is over iHEP vehicle.Data are represented as mean ± SEM for (J) and (L–N).See also Figure S3.