human ipsc derived nscs  (Axol Bioscience)

Journal: Science Advances

Article Title: DKK3-LRP1 complex and a chemical inhibitor regulate Aβ clearance in models of Alzheimer’s disease

doi: 10.1126/sciadv.adz2099

Figure Lengend Snippet: ( A and B ) Subcellular colocalization of fluorescently labeled Aβ 42 (FAM-Aβ 42 ; green) with LysoTracker (red) in SH-SY5Y neuroblastoma cells and C8-D1A astrocytic cells following treatment with CM as indicated. Nuclei are counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue). Scale bars, 50 μm. ( C ) Schematic of the experimental protocol used for culturing and differentiating neural stem cells derived from a patient with AD (AD-NSCs). ( D ) Images showing internalization of FAM-Aβ 42 (green) in differentiated AD-NSCs, colabeled with Tuj1 for neurons (red; top) or glial fibrillary acidic protein (GFAP) for astrocytes (red; bottom) under various CM treatments. Scale bar, 50 μm. Tuj1, β-III tubulin. ( E ) ELISA quantification of Aβ 42 uptake in cells treated with CTL, DKK1, or DKK3 CM containing soluble Aβ 42 . ( F ) Similar setup as in (E) but with Aβ 42 preincubated for 2 hours to allow for internalization, followed by a wash and an additional 8-hour incubation in fresh medium. ELISA quantified the decrease in internalized Aβ 42 as an estimate of cellular Aβ clearance (see Materials and Methods for details). Data are presented as mean ± SD. Statistical significance determined by Student’s t test, with ** P < 0.01, *** P < 0.001, and n.s. (not significant).

Article Snippet: Human iPSC-derived NSCs (Axol Bioscience, ax0111) were maintained according to the manufacturer’s protocols on Laminin-coated chamber slides (Thermo Fisher Scientific, 154526).

Techniques: Labeling, Derivative Assay, Enzyme-linked Immunosorbent Assay, Incubation

Journal: Science Advances

Article Title: DKK3-LRP1 complex and a chemical inhibitor regulate Aβ clearance in models of Alzheimer’s disease

doi: 10.1126/sciadv.adz2099

Figure Lengend Snippet: ( A ) Differentiated AD-NSCs were incubated with 1 μM Aβ 42 and various concentrations of purified DKK1-Flag or DKK3-Flag for 30 min, followed by IP of LRP1 and immunoblotting using specific antibodies. Five percent of the total lysate was used as input, with IgG serving as a negative CTL. ( B ) SH-SY5Y cells stably expressing mLRPIV treated as in (A) but with an expanded range of DKK1 or DKK3 concentrations. Levels of Aβ 42 following HA-tag IP were quantified using ELISA. ( C ) SPR analysis depicting the competitive binding dynamics of Aβ 42 to mLRPIV in the presence of 35.5 nM DKK3. The assay was conducted with varying concentrations of Aβ 42 . ( D ) IF imaging demonstrating the localization of DKK3-Flag (red) and mLRPIV-HA (green) in SH-SY5Y cells at 0, 30, and 60 min posttreatment with DKK3-Flag CM. Arrow indicates cell membrane localization; arrowhead points to intracellular localization. Nuclei are counterstained with DAPI (blue). Scale bar, 50 μm. ( E ) Immunoblot analysis showing levels of Mem and Cyt mLRPIV in SH-SY5Y cells after treatment with 50 nM recombinant DKK1 or DKK3 at specified time points. Na + - and K + -dependent ATPase (Na + ,K + -ATPase) and ACTIN were used as loading controls for membrane and cytoplasmic proteins, respectively. ( F ) Quantification of band density for proteins shown in (E). Membrane and cytoplasmic proteins were normalized to Na + ,K + -ATPase or ACTIN, respectively. Error bars represent mean ± SD from biological triplicates.

Article Snippet: Human iPSC-derived NSCs (Axol Bioscience, ax0111) were maintained according to the manufacturer’s protocols on Laminin-coated chamber slides (Thermo Fisher Scientific, 154526).

Techniques: Incubation, Purification, Western Blot, Stable Transfection, Expressing, Enzyme-linked Immunosorbent Assay, Binding Assay, Imaging, Membrane, Recombinant

Journal: NPJ Parkinson's Disease

Article Title: Formation of seeding-competent α-synuclein aggregates in parkin-deficient iPSC-derived human neurons

doi: 10.1038/s41531-025-01038-4

Figure Lengend Snippet: A Graphical overview of the applied dopaminergic (DA) differentiation protocol for differentiation of iPSC-derived PARK2 −/− and isogenic control NSCs. B Immunofluorescence staining of day 45 differentiated cultures for tyrosine hydroxylase (TH, dopaminergic neuronal marker), microtubule-associated protein 2 (MAP2, mature neuronal marker), FOXA2 (midbrain dopaminergic neuronal marker), β-tubulin-III (BTUB, general neuronal marker), glial-acidic fibrillary protein (GFAP, astrocyte marker), α-Synuclein (α-Syn), and synaptophysin (SYN, presynaptic marker). The nuclei were stained with DAPI (blue). Scale bar = 50 µm. C – E Quantification of MAP2 + mature neurons, TH + DA neurons, and GFAP + astrocytes showed no difference in the differentiation capacity of PARK2 −/− and isogenic control cells. Mean ± SEM, n = 7-15 coverslips from 3-5 independent differentiations. Statistical analysis: Student’s t -test, NS not significant.

Article Snippet: PARK2 −/− and isogenic control iPSC-derived NSCs were obtained from XCell Science Inc. (Novato, CA, USA).

Techniques: Derivative Assay, Control, Immunofluorescence, Staining, Marker

Journal: NPJ Parkinson's Disease

Article Title: Formation of seeding-competent α-synuclein aggregates in parkin-deficient iPSC-derived human neurons

doi: 10.1038/s41531-025-01038-4

Figure Lengend Snippet: A Graphical overview of PFF treatment. PARK2 −/− and isogenic control iPSC-derived NSCs were differentiated for 38 days before treatment with human recombinant S129A-α-Syn PFF (14 µg/ml). Excess PFFs were washed out after 24 h, and cells were for 45 days before fixation. B Differentiated cultures were immunofluorescence-stained for pSer129 α-Syn (green) in combination with MAP2 (purple) to label neuronal cell bodies, and the nuclei were stained with DAPI (blue). Pictures were acquired randomly with a X60 objective on a Zeiss Observer Z1 microscope. Scale bar = 20 µm. Scalebar on zoomed images = 10 µm. C Quantification of total pSer129 staining per affected cell. D Affected cells were quantified as the percentage of cells with pSer129 α-Syn inclusion + . This analysis was performed on pictures taken with a X20 objective. E Number of pSer129 α-Syn + inclusions per affected cell. F – H Inclusion size distribution according to small (25-100 pixels), medium (101-300 pixels), and large (>300 pixels) inclusions. Mean ± SEM, n = 10 coverslips from 3 independent differentiations. Statistical analysis: Two-way ANOVA, NS = not significant, * p < 0.05, ** p < 0.01, *** p < 0.001. I I 3 neurons were transfected with lentivirus encoding sgRNA targeting either PARK2 , SNCA or a non-targeting guide (control). Following transfection, neurons were treated with PFFs for 14 days, and pSer129 α-Syn levels were quantified. Mean ± SEM, n = 3 independent experiments. Statistical analysis: paired t-test; dotted lines represent the experimental pairs across genotypes.

Article Snippet: PARK2 −/− and isogenic control iPSC-derived NSCs were obtained from XCell Science Inc. (Novato, CA, USA).

Techniques: Control, Derivative Assay, Recombinant, Immunofluorescence, Staining, Microscopy, Transfection