Journal: Journal of Extracellular Vesicles

Article Title: Extracellular vesicle‐mediated delivery of CRISPR/Cas9 ribonucleoprotein complex targeting proprotein convertase subtilisin‐kexin type 9 (Pcsk9) in primary mouse hepatocytes

doi: 10.1002/jev2.12389

Figure Lengend Snippet: Schematic illustration of EV‐mediated CRISPR/Cas9 RNP delivery knocking out Pcsk9 leads to increased LDLR recycling and LDLR expression on the cell surface. (a) PCSK9 binds the LDLR/LDL‐C complex and marks it for degradation by the lysosome. In the absence of PCSK9, the LDLR is free to bind and process plasma LDL‐C, whereafter, it is recycled back to the membrane surface to clear more LDL‐C from circulation. (b) Upon extracellular vesicle‐mediated CRISPR/Cas9 delivery, knock‐out of the Pcsk9 gene would reduce the LDLR/LDL‐C complex's degradation and increase LDLR recycling.

Article Snippet: Membranes were blocked for 1 h in 5% Bovine Serum Albumin (BSA) (w/v) in Tris buffered saline (TBS).Primary antibodies included anti‐rabbit DmrA (635089, Clontech), anti‐mouse Cas9, (NBP2‐36440, Novus Biologicals, clone 7A9‐3A3), anti‐rabbit TSG101 (30871, Abcam), anti‐mouse CD63 (8219, Abcam), mouse anti‐syntenin (TA504796, Origene), mouse anti‐CD81 (SC‐166029, Santa Cruz), anti‐mouse PCSK9 (#MA5‐32843, ThermoFisher, clone 2F1), anti‐rabbit LDLR (#MA5‐32075, ThermoFisher, clone SJ0197) and mouse anti‐β‐actin (Cell Signaling Technology, clone 8H10D10).

Techniques: CRISPR, Expressing, Clinical Proteomics, Membrane, Knock-Out

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular vesicle‐mediated delivery of CRISPR/Cas9 ribonucleoprotein complex targeting proprotein convertase subtilisin‐kexin type 9 (Pcsk9) in primary mouse hepatocytes

doi: 10.1002/jev2.12389

Figure Lengend Snippet: EVs mediate CRISPR/Cas9 RNP delivery in vitro (a) Schematic illustration of N‐Myr‐2xFKBP12/Cas9‐FRB construct and (b) Cas9‐EV production by transiently transfecting HEK293FT cells with N‐Myr‐FKBP12/FRB‐Cas9, VSV‐G and gRNA plasmids followed by differential ultracentrifugation. (c) NTA showed Cas9 EVs had a size distribution between 50‐250 nm with a mean peak size of 113,2 ± 2,3 nm. (d) TEM images showed Cas9 EVs had an average size around 100 nm. Scale bar = 100 nm (E) Western blot analysis demonstrated Cas9 EVs are enriched with Cas9 protein, FKBP12, VSV‐G and are positive for EV marker TSG101, CD63, CD81, synthenin‐1 and β‐actin. (f) Schematic illustration of CRISPR/Cas9‐mediated NHEJ leading to eGFP+ expression in Cas9 stoplight reporter cells. Administration of Cas9 EVs demonstrated successful CRISPR/Cas9‐mediated genome editing in Cas9 stoplight reporter cells visualized with (g) fluorescence microscopy analysis and (H) quantified by flow cytometry analysis with an efficiency of ±28,1%. Representative images of four individual experiments. Data presented as Mean ± SEM, analyzed using t‐test with * p < 0,05 and ** p < 0,01. Scale bar = 200 μm.

Article Snippet: Membranes were blocked for 1 h in 5% Bovine Serum Albumin (BSA) (w/v) in Tris buffered saline (TBS).Primary antibodies included anti‐rabbit DmrA (635089, Clontech), anti‐mouse Cas9, (NBP2‐36440, Novus Biologicals, clone 7A9‐3A3), anti‐rabbit TSG101 (30871, Abcam), anti‐mouse CD63 (8219, Abcam), mouse anti‐syntenin (TA504796, Origene), mouse anti‐CD81 (SC‐166029, Santa Cruz), anti‐mouse PCSK9 (#MA5‐32843, ThermoFisher, clone 2F1), anti‐rabbit LDLR (#MA5‐32075, ThermoFisher, clone SJ0197) and mouse anti‐β‐actin (Cell Signaling Technology, clone 8H10D10).

Techniques: CRISPR, In Vitro, Construct, Western Blot, Marker, Expressing, Fluorescence, Microscopy, Flow Cytometry

Journal: Journal of Extracellular Vesicles

Article Title: Extracellular vesicle‐mediated delivery of CRISPR/Cas9 ribonucleoprotein complex targeting proprotein convertase subtilisin‐kexin type 9 (Pcsk9) in primary mouse hepatocytes

doi: 10.1002/jev2.12389

Figure Lengend Snippet: Cas9 EV mediated delivery of CRISPR/Cas9 successfully targets Pcsk9 in mouse hepatocytes leading to increased LDLR levels and LDL‐C uptake (a) Schematic illustration of MMgRNA targeting the Pcsk9 gene (b) Agarose gel of T7E1‐treated PCR products amplified from lentivirus treated NIH3T3 cells show NHEJ frequencies for the different gRNA's (c) Quantification of on‐target indel mutation frequency by respective MgRNA's from lentivirus transduced cells (d) Agarose gel of T7E1‐treated PCR products amplified from mouse hepatocytes treated with EVs carrying either NTgRNa, MMgRNA 5, 8 or 9 (D) Quantification of NHEJ activity of Cas9 EV MMgRNA treated mouse hepatocytes (e) RT‐qPCR of relative Pcsk9 mRNA levels showed MMgRNA 9 successfully targets the exon splicing donor slice thereby interfering with Pcsk9 pre‐mRNA processing. (f) Western blot analysis was conducted on Cas9 EV‐treated mouse hepatocytes using antibodies against LDLR, PCSK9, and β‐actin. (g) Quantification of LDLR levels, normalized to β‐actin, demonstrated that administration of Cas9 EVs loaded with gRNA promoted the expression of the mature form of LDLR. (h) Quantification of intracellular PCSK9 levels, also normalized to β‐actin, suggested that PCSK9 protein levels remained relatively unchanged 3–4 days post‐administration of the Cas9 EVs. (H) Microscopic analysis and (i) flow cytometry analysis of pHrodo uptake of TOP‐EV treated mouse hepatocytes shows mouse hepatocytes treated with Cas9 TOP‐EV carrying MMgRNA 8 or 9 results in a higher uptake of LDL‐C compared to Cas9 EV NTgRNA treated hepatocytes. Data expressed as mean ± SEM, analyzed using with ordinary one‐way ANOVA with * p < 0.05 and ** p < 0.01. Scale bar = 200 μm.

Article Snippet: Membranes were blocked for 1 h in 5% Bovine Serum Albumin (BSA) (w/v) in Tris buffered saline (TBS).Primary antibodies included anti‐rabbit DmrA (635089, Clontech), anti‐mouse Cas9, (NBP2‐36440, Novus Biologicals, clone 7A9‐3A3), anti‐rabbit TSG101 (30871, Abcam), anti‐mouse CD63 (8219, Abcam), mouse anti‐syntenin (TA504796, Origene), mouse anti‐CD81 (SC‐166029, Santa Cruz), anti‐mouse PCSK9 (#MA5‐32843, ThermoFisher, clone 2F1), anti‐rabbit LDLR (#MA5‐32075, ThermoFisher, clone SJ0197) and mouse anti‐β‐actin (Cell Signaling Technology, clone 8H10D10).

Techniques: CRISPR, Agarose Gel Electrophoresis, Amplification, Mutagenesis, Activity Assay, Quantitative RT-PCR, Western Blot, Expressing, Flow Cytometry

Journal: Stem cells (Dayton, Ohio)

Article Title: Conversion of human umbilical cord mesenchymal stem cells in Wharton's jelly to dopaminergic neurons in vitro: potential therapeutic application for Parkinsonism.

doi: 10.1634/stemcells.2005-0053

Figure Lengend Snippet: Figure 1. The grouping and scheme of in vitro incubation of HUMSCs. Tyrosine hydroxylase–positive populations were gener- ated from undifferentiated HUMSCs by a three-step in vitro differ- entiation method. Abbreviations: DMEM, Dulbecco’s modified Ea- gle’s medium; FBS, fetal bovine serum; FGF, fibroblast growth factor; HUMSC, human umbilical mesenchymal stem cell; NCM, neuronal-conditioned medium; Shh, sonic hedgehog.

Article Snippet: In stage 3, cells were supplemented with NCM or 10% FBS-DMEM in the presence of the murine N-terminal fragment of sonic hedgehog (SHH) (500 ng/ml, 461-SH, R&D Systems Inc., Minneapolis, http://www.rndsystems.com) and murine FGF8 isoform b (FGF8) (100 ng/ml, 423-F8, R&D Systems Inc.) for 3, 6, 9, or 12 days.

Techniques: In Vitro, Incubation, Modification

Journal: Stem cells (Dayton, Ohio)

Article Title: Conversion of human umbilical cord mesenchymal stem cells in Wharton's jelly to dopaminergic neurons in vitro: potential therapeutic application for Parkinsonism.

doi: 10.1634/stemcells.2005-0053

Figure Lengend Snippet: Figure 2. HUMSC differentiation into dopaminergic, norepinephrine, and GABAergic neurons in vitro. (A): Photomicrographs showing TH immunocytochemistry of cultured HUMSCs. The cells expressed TH after incubation with NCM for 6 days and then SHH and FGF8 in DMEM for 3 days. In addition to TH-positive neurons, DBH-positive (B) and GAD-positive (C) neurons were detected. Human-specific nuclear antigen are in green, and DBH and GAD are in red. Arrows indicate cells stained positively for TH, DBH, or GAD. Scale bar 100 m. (D): Histograms showing the percentage of TH-positive cells after incubation with NCM, SHH, and FGF8. (Results represent the mean standard error from three different experiments. At least 200 cells were counted from 10 randomly selected microscopic fields in each experiment. Statistics consisted of one-way ANOVA followed by the LSD test; *statistical difference at p .05 compared with NCM-only group.) (E): TH expression in cultured cells by Western blotting. The molecular weight of rat and human TH were 60 and 68 kDa, respectively. Rat SN served as positive control. (F): Dopamine concentration in culture medium after HUMSCs were treated with NCM, SHH, and FGF8. (Results represent the mean standard error from three different experiments. Statistics consisted of one-way ANOVA followed by the LSD test; *statistical significance at p .05 compared with DMEM and NCM-only groups.) Abbreviations: ANOVA, analysis of variance; DBH, dopamine--hydroxylase; DMEM, Dulbecco’s modified Eagle’s medium; FGF, fibroblast growth factor; LSD, least-significant difference; HUMSC, human umbilical mesenchymal stem cell; NCM, neuronal-conditioned medium; Shh, sonic hedgehog; SN, substantia nigra; TH, tyrosine hydroxylase.

Article Snippet: In stage 3, cells were supplemented with NCM or 10% FBS-DMEM in the presence of the murine N-terminal fragment of sonic hedgehog (SHH) (500 ng/ml, 461-SH, R&D Systems Inc., Minneapolis, http://www.rndsystems.com) and murine FGF8 isoform b (FGF8) (100 ng/ml, 423-F8, R&D Systems Inc.) for 3, 6, 9, or 12 days.

Techniques: In Vitro, Immunocytochemistry, Cell Culture, Incubation, Staining, Expressing, Western Blot, Molecular Weight, Positive Control, Concentration Assay, Modification

Journal: Stem cells (Dayton, Ohio)

Article Title: Conversion of human umbilical cord mesenchymal stem cells in Wharton's jelly to dopaminergic neurons in vitro: potential therapeutic application for Parkinsonism.

doi: 10.1634/stemcells.2005-0053

Figure Lengend Snippet: Figure 5. Rotation behavior in response to amphetamine tested at 1, 2, 3, 4, and 5 months after lesion. A significant decrease in the number of amphetamine-induced turning was seen in animals with grafted cells treated with NCM SHH FGF8 (, n 6) compared with control (lesion-only) animals (F, n 12) and lesioned animals that received grafted cells treated with NCM (E, n 12). Statistics consisted of two-way ANOVA followed by the LSD test. (* Significant difference at p .05 between NCM SHH FGF8-treated group compared with the control and NCM groups at the same time point. # Significant difference at p 0.05 between the control and NCM groups over 1-month intervals.) Abbreviations: ANOVA, analysis of variance; FGF, fibroblast growth factor; LSD, least-significant difference; NCM, neuronal- conditioned medium; Shh, sonic hedgehog.

Article Snippet: In stage 3, cells were supplemented with NCM or 10% FBS-DMEM in the presence of the murine N-terminal fragment of sonic hedgehog (SHH) (500 ng/ml, 461-SH, R&D Systems Inc., Minneapolis, http://www.rndsystems.com) and murine FGF8 isoform b (FGF8) (100 ng/ml, 423-F8, R&D Systems Inc.) for 3, 6, 9, or 12 days.

Techniques: Control

Journal: iScience

Article Title: Cocaine and habit training cause dendritic spine rearrangement in the prelimbic cortex

doi: 10.1016/j.isci.2023.106240

Figure Lengend Snippet: Antibodies used in this report

Article Snippet: anti-Cofilin , N-terminus domain , Rabbit , ECM Biosciences , CP1131 , 1:1000.

Techniques: Concentration Assay, Sequencing

Journal: iScience

Article Title: Cocaine and habit training cause dendritic spine rearrangement in the prelimbic cortex

doi: 10.1016/j.isci.2023.106240

Figure Lengend Snippet:

Article Snippet: anti-Cofilin , N-terminus domain , Rabbit , ECM Biosciences , CP1131 , 1:1000.

Techniques: Virus, Plasmid Preparation, Recombinant, Software