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: International Journal of Bioprinting

Article Title: 344 Laser bioprinting of human iPSC-derived neural stem cells and neurons: Effect on cell survival, multipotency, differentiation, and neuronal activity

doi: 10.18063/ijb.v9i2.672

Figure Lengend Snippet: Printing process and cell culture homogeneity. (A) Schematic sketch of the laser-assisted bioprinting technique. (B) Example of bioprinted neuronal stem cells (NSCs): Fluorescence microscopic image of NSCs printed in a dot-pattern with a dot-to-dot distance of 600 µm, immunostained with nestin antibody (green fluorescence) for neuronal stem cells 2 days after printing. (C) Quantitative investigation of NSC’s differentiation toward neurons and astrocytes (without printing); counting of positive cells for mature neuronal (MAP2), proliferation (Ki67) and astrocyte (GFAP) markers among NSCs, d5, d10, d20 neurons and astrocytes from fluorescent microscopic images; representative images are depicted in Figure S2 (in Supplementary File). (D) Western blot (left) analysis of neuronal proteins and its quantitative representation (right) of the expression level of β3-tubulin, doublecortin (DCX), MAP2, synapto-physin (SYN), synapsin-1 (SYP1), PSD95, and GFAP of cells in NSC, d5, d10, d20 neuron and astrocyte culture. Data are normalized relative to internal standard β-actin band density.

Article Snippet: hiPSC-derived NSCs (ax0011, Axol Biosciences Ltd., Cambridge, UK) were used in this study.

Techniques: Cell Culture, Fluorescence, Western Blot, Expressing

Journal: International Journal of Bioprinting

Article Title: 344 Laser bioprinting of human iPSC-derived neural stem cells and neurons: Effect on cell survival, multipotency, differentiation, and neuronal activity

doi: 10.18063/ijb.v9i2.672

Figure Lengend Snippet: Cell viability, colony morphology, development of dendrites. (A and B) Fluorescent microscopic images of live/dead-staining 24 hours after printing for NSCs, d5, d10, and d20 neurons with calcein AM (green, viable cells) and ethidium homodimer-1 (red, dead cells). Live/dead staining revealed agglomeration of printed cells, increasing with duration of pre-differentiation. Dead cells could be seen within these agglomerations in high-magnification images (B). (C) Left: Viability 24 hours post-printing of printed, donor, and control cells (NSCs, d5, d10, and d20 neurons), given in percent. Control cells were not in contact to bioink or substrate, but stored in a vial, while printed and donor cells were suspended in bioink for printing. Obviously, viability decreases with duration of pre-differentiation period and viability of printed cells is mostly higher than that of donor and control cells. Right: Statistical analysis, P values for unpaired two-sample t-test. Statistical significance (highlighted in red) was determined for higher viability of printed cells when compared to control and donor cells (five out of eight cases) and for decreased viability with increasing pre-differentiation period (15 out of 18 cases). (D and E) Formation of neuronal dendrites in smaller (D) and larger (E) droplets of bioink with lower and higher number of NSCs printed on MatrigelTM substrate is depicted in phase contrast imaging 1 day post-printing and in fluorescence imaging with marker β3-tubulin and Hoechst 33342 at day 7 of post-printing differentiation. The number of neuronal dendrites relative to the number of neuronal cells is lower in smaller droplets and higher in larger droplets. Scale bars are 1 mm (D and E, except for right column), 500 μm (A) and 100 μm (all others).

Article Snippet: hiPSC-derived NSCs (ax0011, Axol Biosciences Ltd., Cambridge, UK) were used in this study.

Techniques: Staining, Control, Imaging, Fluorescence, Marker

Journal: International Journal of Bioprinting

Article Title: 344 Laser bioprinting of human iPSC-derived neural stem cells and neurons: Effect on cell survival, multipotency, differentiation, and neuronal activity

doi: 10.18063/ijb.v9i2.672

Figure Lengend Snippet: Proliferation and differentiation toward neurons and glial cells in printed patterns. All blue staining are general cell nuclei staining with Hoechst 33342. Scale bars: 100 μm. (A–C) Proliferation of NSCs (A and B) and d20 neurons (C) 2 days (A) and 12 days (B and C) post-printing. Ki67 staining marked nuclei of proliferating cells in red, while green staining of NSCs (β3-tubulin (A); MAP2 (B)) and d20 neurons (MAP2 (C)) stains whole cells. While both NSCs and d20 neurons migrated post-printing and proliferation of NSCs was observed everywhere, d20 neurons proliferated only at the positions of printed droplets with high cell density. (D) TBR1 and MAP2 staining of printed NSCs (left) and d5 neurons (right) 22 days after printing. The position of four printed droplets can still be seen in TBR1 panels, while there is only one droplet position depicted in MAP2 panels due to higher resolution microscopy. (E and F) NSC spontaneous differentiation to glial cells like astrocytes and oligodendrocytes under neuronal differentiation conditions. Staining with markers GFAP (red, for astrocytes), MAP2 (green), and S100B (green, for mature astrocytes) at days 2, 23, and 37 post-printing demonstrates differentiation to astrocytes under neural differentiation conditions. Staining with oligodendrocytic marker O4 (red) at days 35 and 67 of neuronal differentiation post-printing demonstrated many O4-positive cells already present on day 35, mostly pre-oligodendrocytes with simple dendritic extensions (without bifurcations) and a few immature oligodendrocytes (with a few bifurcations). On day 67, mature oligodendrocytes (with long extensions and many complex bifurcations) were also observed. (G) vGLUT1 staining (green) of glutamatergic neurons and GABA staining (red, same image section) of GABAergic neurons at day 30 of post-differentiation of printed NSCs. Most of the NSCs differentiated to glutamatergic neurons and only a smaller proportion became GABAergic neurons.

Article Snippet: hiPSC-derived NSCs (ax0011, Axol Biosciences Ltd., Cambridge, UK) were used in this study.

Techniques: Staining, Microscopy, Marker

Journal: International Journal of Bioprinting

Article Title: 344 Laser bioprinting of human iPSC-derived neural stem cells and neurons: Effect on cell survival, multipotency, differentiation, and neuronal activity

doi: 10.18063/ijb.v9i2.672

Figure Lengend Snippet: Printed NSCs, neurons and astrocytes 23 days post-printing. Neuronal differentiation of printed cells, compared among NSCs, NSCs with 20% of astrocytes, NSCs with 50% of astrocytes, astrocytes only, d20 neurons, d20 neurons with 20% of astrocytes, and d20 neurons with 20% of NSCs. MAP2 (green), GFAP (red), and Hoechst 33342 (blue) staining show neurons, astrocytes, and nuclei of all cells, respectively. Images with two different magnifications are depicted. Distance between droplets in printed patterns is 1 mm; scale bar: 500 μm.

Article Snippet: hiPSC-derived NSCs (ax0011, Axol Biosciences Ltd., Cambridge, UK) were used in this study.

Techniques: Staining

Journal: International Journal of Bioprinting

Article Title: 344 Laser bioprinting of human iPSC-derived neural stem cells and neurons: Effect on cell survival, multipotency, differentiation, and neuronal activity

doi: 10.18063/ijb.v9i2.672

Figure Lengend Snippet: Formation of neuronal networks and synapses. Development of pre-and post-synaptic compartments by printed NSCs (A and B) and d20 neurons (C and D), both neuronal post-differentiated for 20 days and stained with pre-synaptic synaptophysin (red) and post-synaptic density protein PSD95 (green) together with Hoechst 33342 cell nuclei staining (blue). (B and D) Details of (A) and (C), respectively. For each panel (A–D), the right image is a merger of the others; the yellow color in these merged images is an overlap of red and green color. Both synaptic markers were found in abundance after 20 days of neuronal differentiation, and synaptic compartments could be seen in juxtaposition (red and green spot next to each other) and in colocalization (yellow spot as overlap of red and green spot). This demonstrates neuronal maturation, extensive formation of synapses, and connection of neurons via synapses. Scale bar: 50 μm.

Article Snippet: hiPSC-derived NSCs (ax0011, Axol Biosciences Ltd., Cambridge, UK) were used in this study.

Techniques: Staining

Journal: International Journal of Bioprinting

Article Title: 344 Laser bioprinting of human iPSC-derived neural stem cells and neurons: Effect on cell survival, multipotency, differentiation, and neuronal activity

doi: 10.18063/ijb.v9i2.672

Figure Lengend Snippet: Functionality of neuronal network dependence on cell type. (A) Rating of the neuronal activity visualized by calcium imaging applying a grading system ranging from 0 (no activity) to 24 (abundant and intensive activity with bursting events). Different printed cell types and cell compositions were differentiated post-printing with different durations of neuronal differentiation period. Mean and standard error of mean (SEM) of the ratings for the activity of NSCs (8.9 ± 0.3), NSCs mixed with 20% (8.6 ± 0.5) or 50% (9.3 ± 1.0) of astrocytes, astrocytes (4.2 ± 0.9) alone, d20 neurons (7.7 ± 0.5), and d20 neurons mixed with 20% of astrocytes (6.2 ± 0.6) or NSCs (6.4 ± 0.8) are depicted, and also averaged over different durations of neuronal post-differentiation period. (B) Statistical analysis of P values for unpaired two-sample t -test, which are highlighted in red if less than the significance level of 0.05.

Article Snippet: hiPSC-derived NSCs (ax0011, Axol Biosciences Ltd., Cambridge, UK) were used in this study.

Techniques: Activity Assay, Imaging

Journal: International Journal of Bioprinting

Article Title: 344 Laser bioprinting of human iPSC-derived neural stem cells and neurons: Effect on cell survival, multipotency, differentiation, and neuronal activity

doi: 10.18063/ijb.v9i2.672

Figure Lengend Snippet: Data acquisition and analysis. (A) Highly contrasted fluorescence image of NSCs at Diff 0/67. Bright spots are neural cells (neurons and glia). The enlarged area shows detail of the field of view, with the brightest spots (yellow outlines) automatically ascribed as regions of interest (ROIs). (B) Fluorescence traces of spontaneous activity for 10 randomly selected ROIs. Sharp increases in fluorescence reveal neuronal activity and are associated to elicited action potentials. The simultaneous activation of a group of neurons shapes a network burst (yellow rectangle). (C) Top: Raster plot of spontaneous activity extended to 1000 neurons. Black dots are neuronal activations. Network bursts appear as vertical bands of coordinated activity. Bottom: Corresponding “global network activity” revealing a persistent background activity of about 10% of the network and 5 significantly strong peaks that correspond to network bursts (blue dots and yellow rectangle). (D) Sketch of functional connectivity and network measures for two contrasting situations of a highly segregated network (left) and a highly integrated one (right). The top matrices show the functional communities as colored boxes along the diagonal. Dots are functional connections, either intra-modular (black) or inter-modular (blue). The bottom graphs show the corresponding network maps. (E) Functional connectivity matrix of the experiment shown in panels A–C, highlighting the functional communities. The combination of moderate Q and low G eff values indicates a network that exhibits clear functional communities that are well linked among themselves, suggesting a balanced network.

Article Snippet: hiPSC-derived NSCs (ax0011, Axol Biosciences Ltd., Cambridge, UK) were used in this study.

Techniques: Fluorescence, Activity Assay, Activation Assay, Functional Assay

Journal: International Journal of Bioprinting

Article Title: 344 Laser bioprinting of human iPSC-derived neural stem cells and neurons: Effect on cell survival, multipotency, differentiation, and neuronal activity

doi: 10.18063/ijb.v9i2.672

Figure Lengend Snippet: Contrasting activity between NSCs and printed neurons. (A) Representative raster plots (top) and global network activity (bottom) of NSC cultures along development, showing the emergence of collective activity in the form of network bursts and the progressive increase of burst sizes (blue dots). (B) Corresponding data plot for printed neurons. Bursts emerge since early days in culture but remain small. Spontaneous activity is overall weaker than NSCs at late developmental times. (C) Comparison of average neuronal activity for NSCs and printed neurons along development. Activity in NSCs gradually increases along time, while printed neurons maintain a low activity. Data are shown as mean ± standard error of mean. Each data point is an average over four cultures, and lines are linear fits. (D) Comparison of burst sizes between NSCs and d5-printed neurons. Burst sizes for NSCs significantly increase along development and are much higher than for printed neurons. Color boxes show the extent of the distribution; dots are all the burst sizes observed in four realizations of each condition, and black crosses indicate mean ± standard deviation. Significance analyzed by a multiple comparison test in ANOVA. *** P < 0.001.

Article Snippet: hiPSC-derived NSCs (ax0011, Axol Biosciences Ltd., Cambridge, UK) were used in this study.

Techniques: Activity Assay, Comparison, Standard Deviation