neural progenitor cells ipsc derived npcs  (ATCC)

Journal: Cancers

Article Title: Glioblastoma Cells Induce Neuron Loss In Vivo and In Vitro

doi: 10.3390/cancers17172817

Figure Lengend Snippet: Establishment of the GSC–neuron coculture. ( a ) Neuron differentiation: iPSC-derived NPCs were induced to differentiate into neurons for three weeks and were characterized for the neuronal markers Tuj1 (green) and NeuN (red). Cell nuclei were stained with DAPI (blue). ( b ) Establishment of the GSC–neuron coculture: NeuN + neurons were used for the cocultures. GFP + GSCs (NSC11 and NSC20) were added directly to the neurons in the GSC media for 48 h to establish the coculture. The figure shows representative images of the GSCs (GFP: green)–neurons (NeuN: red) in the direct coculture. The images were captured with a confocal microscope using a 40× lens, and images of the neurons are presented as orthogonal projections.

Article Snippet: iPSC-derived NPCs (Catalog #ACS5004) were procured from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Derivative Assay, Staining, Microscopy

Journal: bioRxiv

Article Title: The YTHDF Proteins Shape the Brain Gene Signatures of Alzheimer’s Disease

doi: 10.1101/2024.10.23.619425

Figure Lengend Snippet: (A) Brightfield image of Cas9-expressing NPCs used for CRISPR experiments. (B) MAP2 and DAPI staining of NPCs. NPC-Cas9hy p7 was seeded as single cells in a 24-well plate pre-coated with GFR-Matrigel, at 200,000 cells per well. Cell culture medium NPC + Hygromycin (100 ug/ml) was changed every other day. Cells were fixed at d9 with 4% PFA for 15 min and stained for MAP2. (C) Gel image of T7 Endonuclease I assay result. Multiple cleavage products in the KO cells indicate genome editing at the intended site. Band intensities of full-length and cleavage products were quantified using Fiji, and gene editing efficiency was calculated using the following equation: 100 x (1 – (1-fraction cleaved)1/2) (see Methods). (D) Western blot results confirm the perturbation effects for m6A-associated RBPs. Whole-cell lysates from KO or NT control cells were subjected to western blot analysis using the Jess Simple Western platform. Digital gel images are shown for the 6 designated targets, probed for both Actin (red) and antibodies for the corresponding KO target (black). Actin-normalized intensities of KO targets are shown at the bottom.

Article Snippet: Neural progenitor cells (NPCs) derived from XCL-1 iPSCs were obtained from Stem Cell Technologies.

Techniques: Expressing, CRISPR, Staining, Cell Culture, T7EI Assay, Western Blot, Control, Simple Western

Journal: Scientific Reports

Article Title: Zika virus vertical transmission induces neuroinflammation and synapse impairment in brain cells derived from children born with Congenital Zika Syndrome

doi: 10.1038/s41598-024-65392-8

Figure Lengend Snippet: Reprogramming of CZS fibroblasts from skin biopsies into iPSC and differentiation into NPC. ( A ) Representative image of fibroblasts derived from skin explants from patients with CZS. ( B ) Representative image of the establishment of iPSC colonies from CZS patients. ( C ) Gene expression of for Nanog and SOX2 in IPSC by qRT-PCR. ( D ) Representative image of IPSC colonies shown in C, and LIN28, NANOG, OCT4, and SOX2 gene expression for all samples used in this study. ( E ) Representative images of morphological changes during the NPC production steps. ( F ) NPC displaying expression of the Nestin and SOX2 NPC markers. ( G ) Representative image of NPC by immunofluorescence. The NPC obtained displayed SOX2, Nestin, and Musashi 1 expression in all samples used in this study. MAP2, known as a neuron marker, was not expressed in NPC. ( A , B , D , E ) Objective magnification: 4x. 200 µm scale bars. ( G ) Objective magnification: 20x. 50 µm scale bars.

Article Snippet: Neural Progenitor Cells (NPC) derived from iPSC from CTRL and CZS were produced using the protocol described elsewhere , , illustrated in Fig. E. NPC revealed expression of Nestin, Sox2, and Musashi 1 (MS-1) and did not express MAP-2, confirming the NPC differentiation success (Fig. F,G).

Techniques: Derivative Assay, Gene Expression, Quantitative RT-PCR, Expressing, Immunofluorescence, Marker

Journal: Scientific Reports

Article Title: Zika virus vertical transmission induces neuroinflammation and synapse impairment in brain cells derived from children born with Congenital Zika Syndrome

doi: 10.1038/s41598-024-65392-8

Figure Lengend Snippet: CZS-derived neurons display reduced synaptogenesis, less glutamate production and differential astrocyte quantification. ( A ) Representative images depicting morphological changes during neuronal differentiation. ( B ) Gene expression upon neuronal differentiation. Cell culture showed expression of MAP2, a neuronal marker and low expression of Musashi1, an NPC marker. ( C ) Representative image of mixed culture upon neuronal differentiation, characterization by immunofluorescence. Cells revealed expression of MAP2, a neuronal marker, and low staining for GFAP, a glial marker, in both CTRL and CZS/A groups, and even less GFAP in the CZS/B group. None of the groups showed expression for Musashi1, a progenitor cell marker. ( D ) Correlation between CZS groups and CTRL neuronal cell lines (% of MAP2 and GFAP positive cells). It is possible to observe that the CZS/A group has similar proportions of cells when compared to the CTRL ( p = 0.3131), while the CZS/B group showed a significant reduction of GFAP compared to the CTRL ( p = 0.0065). ( E ) Synapsin 1 (pre-synaptic) and PSD-95 (postsynaptic) gene expression in neurons upon 4 weeks of differentiation, by qRT-PCR/ΔΔCt. A significant reduction in the expression levels of both genes was found in the neurons of patients with CZS (both CZS/A and CZS/B), when compared to the CTRL (Synapsin 1: CZS/A: p = 0.0363; CZS/B: p = 0.0264 / PSD95: CZS/A: p = 0.0130; CZS/B: p = 0.0118). (F) Representative image of puncta co-localization, showing the expression of MAP2 (white), Synapsin 1 (green) and PSD-95 (red). (G) Analysis of synaptic puncta counting. Synapsin1 was reduced in CZS groups (CZS/A p < 0.0001, CZS/B p < 0.0001). PSD95 was reduced in CZS groups ( p < 0.0001) and even more reduced in CZS/B, when compared to the CZS/A ( p = 0.0488). Both functional puncta and co-localization were reduced in both CZS groups, when compared to the CTRL (CZS/A, p < 0.0001/ CZS/B, p < 0.0001). Lastly, CZS/B showed even less functional puncta quantification than CZS/A ( p = 0.1750). ( H ) Analysis of glutamate production in the supernatant of neuronal culture. CZS groups presented a reduction in glutamate production, when compared to the CTRL group (CTRL vs. CZS1- 1 h: p = 0.0891; 24 h: p = 0.0522; 48 h: p = 0.0655; 72 h: p = 0.0584; 96 h: p = 0.0530 / CTRL vs. CZS2- 1 h: p = 0.1079; 24 h: p = 0.1120; 48 h: p = 0.3017; 72 h: p = 0.1841; 96 h: p = 0.1645 / CTRL vs. CZS3- 1 h: p = 0.0401; 24 h: p = 0.1211; 48 h: p = 0.9999; 72 h: p = 0.7336; 96 h: p = 0.5877 / CTRL vs. CZS4- 1 h: p = 0.0011; 24 h: p = 0.2016; 48 h: p = 0.9913; 72 h: p > 0.9999; 96 h: p = 0.9956). Glutamate levels were normalized relative to the total number of cells. ( A ) Objective magnification: 10x. 100 µm scale bars. ( C ) Objective magnification: 40x. 20 µm magnification bars. ( F ) Objective magnification: 63x. 10 µm scale bars. The statistical study was analyzed through Two-way ANOVA ( D , H ) and One-way ANOVA ( E , G ).

Article Snippet: Neural Progenitor Cells (NPC) derived from iPSC from CTRL and CZS were produced using the protocol described elsewhere , , illustrated in Fig. E. NPC revealed expression of Nestin, Sox2, and Musashi 1 (MS-1) and did not express MAP-2, confirming the NPC differentiation success (Fig. F,G).

Techniques: Derivative Assay, Gene Expression, Cell Culture, Expressing, Marker, Immunofluorescence, Staining, Quantitative RT-PCR, Functional Assay

Journal: Scientific Reports

Article Title: Zika virus vertical transmission induces neuroinflammation and synapse impairment in brain cells derived from children born with Congenital Zika Syndrome

doi: 10.1038/s41598-024-65392-8

Figure Lengend Snippet: CZS-derived Astrocytes display less glutamate and higher cytokine levels. ( A ) Representative images depicting morphological changes during the astrocyte differentiation protocol. ( B ) Representative images of astrocytes characterized by immunofluorescence. Astrocytes revealed GFAP expression in CTRL and CZS patients but not of Nestin, an NPC marker. ( C ) Gene expression upon astrocyte differentiation. Cell cultures displayed expression of GFAP ( p = 0.4541), a glial marker; and low expression of Nestin ( p = 0.3265), an NPC marker. ( D ) Analysis of glutamate levels in the supernatant of astrocytes culture. Compared to the CTRL, a reduction in glutamate levels was found at all time points investigated (CTRL vs. CZS1- 1 h: p = 0.0077; 24 h: p = 0.0739; 48 h: p = 0.0668; 72 h: p = 0.0536; 96 h: p = 0.1580 / CTRL vs. CZS2- 1 h: p = 0.0535; 24 h: p = 0.1220; 48 h: p = 0.5048; 72 h: p = 0.2292; 96 h: p = 0.9711 / CTRL vs. CZS3- 1 h: p = 0.0443; 24 h: p = 0.1301; 48 h: p = 0.9777; 72 h: p = 0.9250; 96 h: p = 0.6692 / CTRL vs. CZS4- 1 h: p = 0.9984; 24 h: p = 0.2498; 48 h: p = 0.9991; 72 h: p = 0.6305; 96 h: p = 0.2060). Glutamate levels were normalized relative to the total number of cells. ( E ) Quantification of cytokines in the supernatants of astrocytes. Compared to the CTRL, significantly higher levels of cytokines were found in CZS patients (IL-6—CZS1: p = 0.8759; CZS2: p = 0.8596; CZS3: p < 0.0001; CZS4: p = 0.0522) (IFNy – CZS1: p > 0.9999; CZS2: p < 0.0001; CZS3: p < 0.0001; CZS4: p > 0.9999) (IL-1ra—CZS1: p > 0.9999; CZS2: p < 0.0001; CZS3: p < 0.0001; CZS4: p > 0.9999) (GM-CSF—CZS1: p < 0.0001; CZS2: p < 0.0001; CZS3: p < 0.0001; CZS4: p < 0.0001) (TNFa—CZS1: p < 0.0001; CZS2: p < 0.0001; CZS3: p < 0.0001; CZS4: p < 0.0001) (RANTES—CZS1: p < 0.0001; CZS2: p < 0.0001; CZS3: p < 0.0001; CZS4: p < 0.0001) (Eoxatin—CZS1: p < 0.0001; CZS2: p < 0.0001; CZS3: p < 0.0001; CZS4: p > 0.9999) (VEGF—CZS1: p = 0,9998; CZS2: p = 0.0007; CZS3: p = 0.0001; CZS4: p = 0.1228) (IP-10—CZS1: p < 0.0001; CZS2: p < 0.0001; CZS3: p = 0,0125; CZS4: p < 0.0001) (MCP-1—CZS1: p = 0.5950; CZS2: p = 0.4712; CZS3: p < 0.0001; CZS4: p = 0.9999) (MIP-1a—CZS1: p = 0.0273; CZS2: p < 0.0001; CZS3: p < 0.0001; CZS4: p = 0.8552) (IL-10—CZS1: p = 0.6818; CZS2: p = 0.0051; CZS3: p < 0.0001; CZS4: p = 0.9851) (IL-15 – CZS1: p = 0.0021; CZS2: p = 0.0091; CZS3: p < 0.0001; CZS4: p = 0.1851) (IL-12p10—CZS1: p = 0.9474; CZS2: p = 0.0013; CZS3: p = 0.0002; CZS4: p = 0.9406). ( B ) Objective magnification: 40x. 20 µm scale bars. The data were analyzed by Two-way ANOVA ( D ) and One-way ANOVA ( E ).

Article Snippet: Neural Progenitor Cells (NPC) derived from iPSC from CTRL and CZS were produced using the protocol described elsewhere , , illustrated in Fig. E. NPC revealed expression of Nestin, Sox2, and Musashi 1 (MS-1) and did not express MAP-2, confirming the NPC differentiation success (Fig. F,G).

Techniques: Derivative Assay, Immunofluorescence, Expressing, Marker, Gene Expression