Fig 1: qRT-PCR analysis.Notes: Unlabeled and labeled iPSC-NPs with CZF and PLL-coated ?-Fe2O3 were analyzed 2 weeks after the onset of differentiation. (A) The expression of the neural genes NES, TUBB3, SYP, and GFAP. (B) The expression of the mDN genes FOXA2, EN1, NURR1 and TH (*P<0.05). The average expression of studied markers in the unlabeled undifferentiated control was set as zero.Abbreviations: qRT-PCR, quantitative real-time reverse transcription polymerase chain reaction; iPSC-NPs, induced pluripotent stem cell-derived neural precursors; CZF, silica-coated cobalt zinc ferrite nanoparticles; PLL-coated ?-Fe2O3, poly-l-lysine-coated iron oxide superparamagnetic nanoparticles; SYP, synaptophysin; GFAP, glial fibrillary acidic protein; mDN, midbrain dopaminergic neuron.
Fig 2: Abnormal epicardial thickening with dilated coronary vessels and large nerves in aortic stenosis cases. Representative images of the epicardium at two magnifications in: (a,b) normal hearts; (c,d) severe aortic stenosis; (e,f) critical aortic stenosis. Note the remarkable thickened epicardium containing dilated coronary vessels and large nerves (brown) in cases with aortic stenosis. TUBB3, ßIII-tubulin; CV, coronary vessel.
Fig 3: BCL7A regulates cell type specification via Notch and Wnt signaling AHeatmap of significantly dysregulated genes in BCL7A KO compared to wt eNPCs.B, CGO terms of the top 10 biological processes for up- (B) and down-regulated (C) genes.DList of the top 10 significantly enriched canonical pathways for up- and down-regulated genes.EHeatmap of significantly dysregulated genes encoding components of the Wnt and Notch signaling pathways.FImmunoblots of spontaneously differentiated wt and BCL7A KO eNPCs in presence of 1 µM of DAPT or 3 µM of CHIR99021.GImmunofluorescence staining of eNPCs undergoing spontaneous differentiation for 7 days. BCL7A KO eNPCs were incubated for 72 h (24 h in proliferation plus 48 h in differentiation medium) with DMSO, 1 µM of DAPT, 3 µM of CHIR99021 or a combination of 1 µM of DAPT +3 µM of CHIR99021. Quantification of the ß-III tubulin-positive or GFAP-positive cells is shown on the right (n = 3–6 technical replicates from three independent experiments). Scale bar = 50 µm.HSholl analysis of ß-III tubulin-positive immature neurons after 7 days of spontaneous differentiation (n = 4 biological replicates with > 30 cells per condition). Scale bar = 20 µm.ImRNA expression levels of TUBB3 and GFAP at 15 and 25 days of spontaneously differentiating BCL7A KO compared parental smNPCs. BCL7A KO smNPCs were treated with DAPT, CHIR99021, DAPT + CHIR99021, or DMSO (as control = ctr) during the initial 7 days of spontaneous differentiation (n = 3–7 biological replicates).JImmunoblots for non-phospho (active) ß-catenin and TCF4/TCF7L2 in proliferating and 48 h-differentiated wt and BCL7A KO eNPCs. Respective densitometries are shown on the right (n = 5–8 biological replicates per condition).KScheme depicts Notch activation and Wnt inhibition in BCL7A KO NPCs. As a consequence of BCL7A deficiency, cells exhibit a decreased expression of ß-catenin and TCF4/TCF7L2, which results in a negative feedback to Notch and Wnt pathways. Data information: Data in (G), (H), (I), and (J) are presented as mean ± SEM. In (G) and (I), one-way ANOVA with Bonferroni's post hoc test for multiple comparisons was used while in (H), two-way RM ANOVA followed by Tukey's multiple comparisons test was performed. Data in (J) were analyzed via unpaired two-tailed Student's t test. *P < 0.05, **P < 0.01, ****P < 0.0001, ns, not significant. Source data are available online for this figure.
Fig 4: Crosstalk between Notch/Wnt signaling and BCL7A-containing SWI/SNF/BAF complex delineates NPC differentiation (relative to Fig 5) A, BSchematic representation of (A) Notch and (B) Wnt signaling pathways overlaid with IPA Molecule Activity Predictor based on differentially expressed genes (DEGs, surrounded by pink lines) in wt versus BCL7A KO eNPCs. Color intensity corresponds to fold change (red and green shades) or prediction strength (orange and blue shades).CGenome browser screenshots of SMARCA4/BRG1 ChIP-seq signals from BCL7A KO and wt eNPCs (left panels) as well as BCL7A KO and parental smNPCs (right panels) showing reduced SMARCA4/BRG1 occupancy at genes influencing Notch (Hey1/HEYL1, NOTCH3) and Wnt (Sox8, APCDD1, DKK1) signaling pathway.DGenome browser screenshots of SMARCA4/BRG1 Chip-seq signals in BCL7A KO and wt eNPCs showing reduced SMARCA4/BRG1 enrichment at Wnt signaling targets upon BCL7A loss.EImmunofluorescence staining of wt eNPCs treated with DAPT (1 µM), CHIR99021 (3 µM) or DMSO (ctr) following 7 days of spontaneous differentiation. The percentage of cells positive for ß-III tubulin and GFAP is shown on the right (n = 2–4 technical replicates from three independent experiments). Scale bar = 50 µm.FSholl analysis of ß-III tubulin-positive immature wt neurons following 7 days of spontaneous eNPC differentiation upon DAPT (1 µM), CHIR99021 (3 µM) or DMSO treatment (n = 3 biological replicates, each experiment with 10–15 cells per condition). Scale bar = 20 µm.GmRNA expression levels of TUBB3 and GFAP of spontaneously differentiating parental smNPCs treated with DAPT, CHIR99021, DAPT + CHIR99021, or DMSO as control (ctr) (n = 4–6 biological replicates).HSimplified schematic representation of canonical Wnt/ß-catenin signaling. In the absence of Wnt, cytosolic ß-catenin is constantly degraded through the actions of a destruction complex (AXIN, APC, CK1, GSK3) that phosphorylates ß-catenin, thereby tagging it for proteasomal degradation. Upon Wnt signaling activation, the destruction complex is recruited to LRP6 receptors resulting in the inhibition of ß-catenin phosphorylation, thereby allowing ß-catenin stabilization. Following translocation to the nucleus, ß-catenin then binds to DNA-bound TCF/LEF complexes to initiate Wnt target gene expression.IImmunoblots for non-phospho (active) ß-catenin and TCF4/TCF7L2 in proliferating and 48 h-differentiated wt and BCL7A KO eNPCs following treatment with DMSO or CHIR99021.JImmunostaining for non-phospho (active) ß-catenin in BCL7A KO smNPCs showing increased levels of nuclear ß-catenin upon Wnt signaling stimulation via CHIR99021 treatment. Quantification of fluorescence intensities within the nucleus are shown on the right (n = 1 biological replicate with 20–30 cells per condition). Scale bar = 5 µm. Data information: Data are presented as mean ± SEM. In (E), (G), and (J), data were statistically analyzed via one-way ANOVA followed by Bonferroni's post hoc test for multiple comparisons, while in (F), two-way RM ANOVA followed by Tukey's multiple comparisons test was performed. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant. Source data are available online for this figure.
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