Journal: Stem Cell Research & Therapy

Article Title: Investigation of de novo mutations in a schizophrenia case-parent trio by induced pluripotent stem cell-based in vitro disease modeling: convergence of schizophrenia- and autism-related cellular phenotypes

doi: 10.1186/s13287-020-01980-5

Figure Lengend Snippet: Establishment and molecular characterization of NPC lines and mature neuronal cultures. Investigation of target genes KHSRP and LRRC7. a Changes in gene expression patterns in NPCs and neurons derived from the case-parent trio. b , c NPCs and neurons derived from hiPSCs by the hippocampal neuronal differentiation protocol were investigated by immunofluorescence staining and visualized by confocal fluorescent microscopy. Immunocytochemical staining shows Nestin/Sox2 ( b ) and Map 2/Prox1 ( c ) positivity in these established neural cell types. Scale bars = 100 μm. d , e Immunofluorescence staining for KHSRP and LRRC7 in neurons. KHSRP ( d ) shows nuclear and cytoplasmic localization, while LRRC7 ( e ) localized postsynaptically in neurons. Scale bars = 50 μm

Article Snippet: The samples were then incubated for 1 h at room temperature with antibodies anti-SOX2 (monoclonal/mouse, 1:20 dilution; MAB2018, R&D Systems, Minneapolis, USA) and anti-Nestin (polyclonal/rabbit, 1:250 dilution; ab92391, Abcam, Cambridge, UK) or for overnight at 4 °C with antibodies anti-PROX1 (polyclonal/rabbit, 1:500 dilution; ab101851, Abcam, Cambridge, UK) and anti-MAP 2 (monoclonal/mouse, 1:500 dilution; M1406, Sigma/Merck, Darmstadt, Germany or polyclonal/rabbit, 1:1000 dilution; ab5622, Millipore, MA, USA).

Techniques: Gene Expression, Derivative Assay, Immunofluorescence, Staining, Microscopy

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Transdifferentiating Astrocytes Into Neurons Using ASCL1 Functionalized With a Novel Intracellular Protein Delivery Technology

doi: 10.3389/fbioe.2018.00173

Figure Lengend Snippet: Small molecule only group after 2 days of priming by LDN193189 and SB431542 followed by 10 days of treatment with DAPT. (A) Cells express the early neural marker TUJ1 and weakly express the neural stem cell marker SOX2. Scale bar is 100 μm. (B) High magnification of (A) . Scale bar is 30 μm. (C) Cells are negative for the mature neural marker MAP2. Scale bar is 100 μm. (D) High magnification of (C) . Scale bar is 10 μm (E) Cells are negative for the mature neural marker NEUN and the neurotransmitter glutamate expressed by excitatory interneurons GLUT. Scale bar is 100 μm. (F) High magnification of (E) . Scale bar is 20 μm. (G) Cells are negative for the neurotransmitter tyrosine hydroxylase expressed by dopaminergic neurons TH, the neurotransmitter glutamic acid decarboxylase expressed by inhibitory interneurons GAD65/67, and the neurotransmitter choline acetyltransferase expressed by motor neurons CHAT. Scale bar is 100 μm. (H) High magnification of (G) . Scale bar is 30 μm. Note that some fluorescence is picked up by all the cells producing background fluorescence which can be ignored.

Article Snippet: The cells were stained for SOX2-PE Mouse IgG2A (R&D Systems) with IgG2A-PE isotype control (R&D Systems), TUJ1-PerCP Mouse IgG2A (R&D Systems) with IgG2A-PerCP isotype control (R&D Systems), MAP2-PE Mouse IgG1 (Clone AP20, Milli-Mark) with Mouse IgG1 PE isotype control (R&D Systems), and NEUN-PE Mouse IgG1 (Clone A60) with Mouse IgG1 PE isotype control (R&D Systems).

Techniques: Marker, Fluorescence

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Transdifferentiating Astrocytes Into Neurons Using ASCL1 Functionalized With a Novel Intracellular Protein Delivery Technology

doi: 10.3389/fbioe.2018.00173

Figure Lengend Snippet: Plastic astrocytes generate neurospheres and mature neurons after 12 days of exposure to ASCL1-IPTD, including 2 days of priming by LDN193189 and SB431542 followed by 10 days of DAPT. (A) Cells express the early neural marker TUJ1 and the neural stem cell marker SOX2. Scale bar is 100 μm. (B) High magnification of (A) . Scale bar is 30 μm. (C) Cells express the mature neural marker MAP2. Scale bar is 100 μm. (D) High magnification of (C) . Scale bar is 30 μm (E) Cells express the mature neural marker NEUN and the neurotransmitter glutamate expressed by excitatory interneurons GLUT. Scale bar is 100 μm. (F) High magnification of (E) . Scale bar is 30 μm. (G) Cells are negative for the neurotransmitter tyrosine hydroxylase expressed by dopaminergic neurons TH, and negative for the neurotransmitter choline acetyltransferase expressed by motor neurons CHAT, but do express the neurotransmitter glutamic acid decarboxylase expressed by inhibitory interneurons GAD65/67. Scale bar is 100 μm. (H) High magnification of (G) . Scale bar is 30μm. Note that some fluorescence is picked up by all the cells producing background fluorescence which can be ignored.

Article Snippet: The cells were stained for SOX2-PE Mouse IgG2A (R&D Systems) with IgG2A-PE isotype control (R&D Systems), TUJ1-PerCP Mouse IgG2A (R&D Systems) with IgG2A-PerCP isotype control (R&D Systems), MAP2-PE Mouse IgG1 (Clone AP20, Milli-Mark) with Mouse IgG1 PE isotype control (R&D Systems), and NEUN-PE Mouse IgG1 (Clone A60) with Mouse IgG1 PE isotype control (R&D Systems).

Techniques: Marker, Fluorescence

Journal: Molecular oncology

Article Title: SETD2 loss in renal epithelial cells drives epithelial-to-mesenchymal transition in a TGF-β-independent manner.

doi: 10.1002/1878-0261.13487

Figure Lengend Snippet: Fig. 7. SOX2, OCT2, and PRRX1 are downstream effectors of the SETD2-regulated EMT program. (A) Expression of SOX2, OCT2, and PRRX1 in TGF-b-treated WT (72 h), SETD2 KO, and SETD2 rescue tested by RT-qPCR (run in triplicate). (B) Migration capacity by wound healing assay, (C) invasiveness by transwell assay, and (D) stemness by 3D spheroid formation assay in RPTEC WT GFP (control vector), SETD2 KO1 and KO2, and SOX2/OCT2/PRRX1-transduced WT RPTEC lines. Images are taken at 49 magnification, scale bar: 1000 lm for (B) and (D) and at 2.59 magnification, scale bar: 1200 lm for (C). Data are represented as mean SEM for triplicate reactions for B–D. P-value is calculated by one-way ANOVA in (A), (B), and (D). Two-way ANOVA is used for statistical test for (C). ****P < 0.0001; ***P < 0.001; **P < 0.01; *P < 0.05; ns, P ≥0.05. (E) Model of the SETD2 loss-driven EMT program through cell intrinsic (transcriptional) and cell extrinsic (paracrine) mechanisms.

Article Snippet: See the T erm s and C onditions (https://onlinelibrary.w iley.com /term s-and-conditions) on W iley O nline L ibrary for rules of use; O A articles are governed by the applicable C reative C om m ons L icense #ab1791), SOX2 (R&D Systems; #AF2018-SP), OCT2 (Thermo Fisher; #39-5400), PRRX1 (Novus Biologicals, St. Charles, MO, USA; #NBP1-06067), anti-FLAG (Sigma-Aldrich; #F1804), GAPDH (Cell Signaling Technology; #2118S), and lamin B1 (Proteintech; #12987-1-AP).

Techniques: Expressing, Quantitative RT-PCR, Migration, Wound Healing Assay, Transwell Assay, Tube Formation Assay, Control, Plasmid Preparation

Journal: Neural regeneration research

Article Title: Multiple factors to assist human-derived induced pluripotent stem cells to efficiently differentiate into midbrain dopaminergic neurons.

doi: 10.4103/1673-5374.378203

Figure Lengend Snippet: Figure 2 ｜ The modified dual SMAD pathway inhibition protocol yielded a low proportion of dopaminergic neurons. (A) Representative images of human induced pluripotent stem cells (hiPSC) with immunostaining of pluripotency markers, octamer-binding transcription factor 4 (OCT4) and stage-specific embryonic antigen-4 (SSEA4). Scale bars: 50 μm. (B) Representative images of neural precursor cells (NPC) induced from hiPSC with immunostaining of SRY-box transcription factor 2 (SOX2) and Nestin. Scale bars: 50 μm. (C) Representative images of hiPSC-derived neurons with immunostaining of microtubule- associated protein 2 (MAP2) (green) and human nuclei (HuNu) antibody (red). Scale bars: 50 μm. (D) Analysis of neuronal differentiation efficiency (number of cells expressing HuNu: n = 443 cells in dual SMAD pathway inhibition protocol). (E) Representative images of hiPSC-derived neurons with immunostaining of markers for different neuronal subtypes: tyrosine hydroxylase (TH), gamma-aminobutyric acid (GABA), vesicular glutamate transporter 1 (VGLUT1) (all in green), and MAP2 (red) grown on mouse astrocytes. Scale bars: 50 μm. (F) Differentiation ratio of dopaminergic neurons, GABAergic neurons, and glutamatergic neurons were assessed based on the proportion of TH, GABA, and VGLUT1 positive cells among MAP2 positive cells, respectively. At least three independent replicates were performed of each experiment. SMAD: Suppressor of mother against decapentaplegic.

Article Snippet: Primary antibodies included: octamer-binding transcription factor 4 (OCT4), a marker used for the identification of iPSC (rabbit, 1:100, BioVision, Milpitas, CA, USA, Cat# 6765-100, RRID: AB_2936816), stage-specific embryonic antigen-4 (SSEA4), a marker used for identification of iPSC (mouse, 1:100, Invitrogen, Rockford, AL, USA, Cat# 41-4000, RRID: AB_2533506), SRY-box transcription factor 2 (SOX2), a marker used for identification of NPC (mouse, 1:500, R&D Systems, Minneapolis, MN, USA, Cat# MAB2018, RRID: AB_358009), Nestin, a marker used for identification of NPC (rabbit, 1:1000, Millipore, Darmstadt, Germany, Cat# ABD69, RRID: AB_2744681), microtubule-associated protein 2 (MAP2), a marker of dendrites (rabbit, 1:1000, Millipore, Burlington, MA, USA, Cat# AB5622, RRID: AB_91939), MAP2 (mouse, 1:1000, Millipore, Cat# AMAb91375, RRID: AB_2716657), beta III tubulin (TUJ1), a neuronal marker (mouse, 1:1000, Sigma-Aldrich, Cat# T8660, RRID: AB_477590), anti-human nuclei (HuNu), a marker of nuclei in human cells (mouse, 1:500, Millipore, Cat# MAB1281, RRID: AB_94090), anti-vesicular glutamate transporter 1 (VGLUT1), a glutamatergic neuronal marker (mouse, 1:250, Millipore, Cat# MAB5502, RRID: AB_262185), gamma-aminobutyric acid (GABA), a GABAergic neuronal marker (rabbit, 1:1000, Sigma-Aldrich, Cat# A2052, RRID: AB_477652), tyrosine hydroxylase (TH), a dopaminergic neuronal marker (rabbit, 1:1000, Millipore, Cat# AB152, RRID: AB_390204), TH (chicken, 1:500, Millipore, Cat# AB9702, RRID: AB_570923), FOXA2, a midbrain neuronal marker (rabbit, 1:500, Cell Signaling Technology, Danvers, MA, USA, Cat# 8186, RRID: AB_10891055), and paired like homeodomain 3 (PITX3), another midbrain neuronal marker (rabbit, 1:500, Millipore, Cat# AB5722, RRID: AB_91997).

Techniques: Modification, Inhibition, Immunostaining, Binding Assay, Derivative Assay, Expressing

Journal: Neural regeneration research

Article Title: Multiple factors to assist human-derived induced pluripotent stem cells to efficiently differentiate into midbrain dopaminergic neurons.

doi: 10.4103/1673-5374.378203

Figure Lengend Snippet: Figure 4 ｜ The SHH + FGF8 differentiation protocol yielded a high proportion of dopaminergic neurons, with a small fraction of neurons that expressed FOXA2, a marker of midbrain dopaminergic neurons. (A) Representative images of neural precursor cells (NPC) induced from human induced pluripotent stem cells (hiPSC) with immunostaining of Forkhead box protein A2 (FOXA2) and SRY-box transcription factor 2 (SOX2). Scale bars: 50 μm. (B) Representative images of hiPSC-derived neurons in the SHH + FGF8 protocol with immunostaining of microtubule- associated protein 2 (MAP2) (green) and human nuclei (HuNu) antibody (red). Scale bars: 50 μm. (C) Comparison of the differentiation efficiency of neurons in the SHH + FGF8 protocol and modified dual SMAD pathway inhibition protocol (number of cells expressing HuNu: n = 607 cells in SHH+FGF8 protocol, n = 443 cells in dual SMAD pathway inhibition protocol). (D) Representative images of hiPSC-derived neurons in the SHH + FGF8 protocol with immunostaining of tyrosine hydroxylase (TH) (green) and MAP2 (red). Scale bars: 50 μm. (E) The proportion of dopaminergic neurons in the SHH + FGF8 protocol and modified dual SMAD pathway inhibition protocol was determined by calculating the percentage of TH-positive cells in MAP2-positive cells. (number of cells expressing MAP2: n = 581 cells in SHH + FGF8 protocol, n = 339 cells in dual SMAD pathway inhibition protocol). (F) The white box area in the image is enlarged and displayed on the right. NPC were infected with a DAT-Cherry lentivirus and then cultured on a glial cell feeder layer to label dopaminergic neurons in the later differentiation stage. Representative images of hiPSC-derived neurons in the SHH + FGF8 protocol with immunostaining of TH (green) and beta III tubulin (TUJ1) (white) on day 33. Scale bars: 50 μm (left), 10 μm (right). (G) The white box area in the image is enlarged and displayed at the bottom. Immunofluorescence staining of FOXA2 (red) and TH (green). Scale bars: 20 μm (upper panels), 5 μm (lower panels). At least three independent replicates were performed for each experiment. Data are presented as mean ± SEM, ****P < 0.0001. Statistical significance was evaluated by two-tailed unpaired Student’s t-test (C, E). FGF8: Fibroblast growth factor 8; ns: not significant; SHH: Sonic Hedgehog; TH: tyrosine hydroxylase.

Article Snippet: Primary antibodies included: octamer-binding transcription factor 4 (OCT4), a marker used for the identification of iPSC (rabbit, 1:100, BioVision, Milpitas, CA, USA, Cat# 6765-100, RRID: AB_2936816), stage-specific embryonic antigen-4 (SSEA4), a marker used for identification of iPSC (mouse, 1:100, Invitrogen, Rockford, AL, USA, Cat# 41-4000, RRID: AB_2533506), SRY-box transcription factor 2 (SOX2), a marker used for identification of NPC (mouse, 1:500, R&D Systems, Minneapolis, MN, USA, Cat# MAB2018, RRID: AB_358009), Nestin, a marker used for identification of NPC (rabbit, 1:1000, Millipore, Darmstadt, Germany, Cat# ABD69, RRID: AB_2744681), microtubule-associated protein 2 (MAP2), a marker of dendrites (rabbit, 1:1000, Millipore, Burlington, MA, USA, Cat# AB5622, RRID: AB_91939), MAP2 (mouse, 1:1000, Millipore, Cat# AMAb91375, RRID: AB_2716657), beta III tubulin (TUJ1), a neuronal marker (mouse, 1:1000, Sigma-Aldrich, Cat# T8660, RRID: AB_477590), anti-human nuclei (HuNu), a marker of nuclei in human cells (mouse, 1:500, Millipore, Cat# MAB1281, RRID: AB_94090), anti-vesicular glutamate transporter 1 (VGLUT1), a glutamatergic neuronal marker (mouse, 1:250, Millipore, Cat# MAB5502, RRID: AB_262185), gamma-aminobutyric acid (GABA), a GABAergic neuronal marker (rabbit, 1:1000, Sigma-Aldrich, Cat# A2052, RRID: AB_477652), tyrosine hydroxylase (TH), a dopaminergic neuronal marker (rabbit, 1:1000, Millipore, Cat# AB152, RRID: AB_390204), TH (chicken, 1:500, Millipore, Cat# AB9702, RRID: AB_570923), FOXA2, a midbrain neuronal marker (rabbit, 1:500, Cell Signaling Technology, Danvers, MA, USA, Cat# 8186, RRID: AB_10891055), and paired like homeodomain 3 (PITX3), another midbrain neuronal marker (rabbit, 1:500, Millipore, Cat# AB5722, RRID: AB_91997).

Techniques: Marker, Immunostaining, Derivative Assay, Comparison, Modification, Inhibition, Expressing, Infection, Cell Culture, Immunofluorescence, Staining, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Scalable purification enables high-quality virus-like particles for therapeutic translation

doi: 10.1016/j.jbc.2025.110946

Figure Lengend Snippet: Chromatographic purification of TIGER-converting PE-ENVLPEs+. A , anti-Cas9 ( top ) and anti-VSV-G ( bottom ) immunoblots of fractions of PE-ENVLPEs+ collected from a 20-ml CC700 column. B , DEAE ion-exchange chromatogram of PE-ENVLPEs+, prepurified on a 20-ml CC700 column; numbers denote absorbance peaks. C , anti-Cas9 (top) and anti-VSV-G (bottom) immunoblots of fractions of PE-ENVLPEs+ from experiment (B). D , E , DEAE ion-exchange chromatogram and immunoblots analogous to (B, C) for PE-ENVLPEs+, purified directly from the cell-culture medium. F , mass spectrometric classification of proteins detected in purified PE-ENVLPEs+. Three technical replicates, mean ± SD. G , fluorescence microscopic images of TIGER-reporter cells, 48 h after application of purified PE-ENVLPEs+; scale bar = 200 μm. H , two-photon fluorescence tomogram of posterior segment of the intact eye of a heterozygous TIGER-reporter mouse treated with PE-ENVLPEs+; scale is in μm. In, filtered production medium; MW, molecular weight; P, peak; CDS1, PE-ENVLPEs + purified by CC700 and DEAE and concentrated by ultracentrifugation; “

Article Snippet: For ENVLPEs+, pCMV-VSV-G plasmid (3200 ng), pCMV ENVLPE+ plasmid (3740 ng, Addgene 232427), pCMV iPE-C P2A Csy4 plasmid (2110 ng, Addgene 232428), and pegRNA PP7 plasmid (10,140 ng, Addgene 232435, modified to target the TIGER construct) were used for transfection with 40 μl of JetPrime in a total volume of 1 ml of JetPrime buffer per 150 mm dish.

Techniques: Purification, Western Blot, Cell Culture, Fluorescence, Molecular Weight, In Vivo, Virus