fis toolbox  (MathWorks Inc)

Journal: Neural Regeneration Research

Article Title: R-28 cell-derived extracellular vesicles protect retinal ganglion cells in glaucoma

doi: 10.4103/NRR.NRR-D-24-00709

Figure Lengend Snippet: In vitro testing of R-28 cell-derived EVs on RGC survival and regeneration. Representative images of untreated control wells (A), R-28 cell-derived EVs treated wells (B), and CNTF-treated wells (C) are shown with the graphs showing the total surviving RGC number (D), the number of RGC with neurites (E), and the longest neurite length (F) in primary retinal cell culture after 3 days. Data are expressed as the mean ± SEM. Images were stained with a nuclear (DAPI, blue) and RGC marker (β-III tubulin, green). Scale bars: 50 µm. All experiments were performed in three independent biological replicates. CNTF: Ciliary neurotrophic factor; DAPI: 4′,6-diamidino-2-phenylindole; EV: extracellular vesicles; RGC: retinal ganglion cells.

Article Snippet: To test the therapeutic effect of R-28-derived EVs on human ESC-derived RGC (H7/H9 immortalized cell line; WiCell, Madison, WI, USA, #WA07, RRID: CVCL_S800) were differentiated from CRISPR-modified ESC generously donated from Prof Donald Zacks laboratory (Johns Hopkins University, Baltimore, MD, USA) and licensed for use from WiCell (Material Transfer Agreement issue-164634007).

Techniques: In Vitro, Derivative Assay, Control, Cell Culture, Staining, Marker

Journal: Neural Regeneration Research

Article Title: R-28 cell-derived extracellular vesicles protect retinal ganglion cells in glaucoma

doi: 10.4103/NRR.NRR-D-24-00709

Figure Lengend Snippet: R-28 cell-derived EVs promote human ESC-derived RGC survival in vitro . Images show untreated controls, R-28 cell-derived EV treated, and CNTF-treated hESC-derived RGCs (green, βIII-tubulin) after injury induced by the microtubule poison, colchicine. Scale bar: 50 µm. Data are presented as mean ± SEM. All experiments were performed in three independent biological replicates. CNTF: Ciliary neurotrophic factor; ESC: embryonic stem cells; EV: extracellular vesicles; RGC: retinal ganglion cells.

Article Snippet: To test the therapeutic effect of R-28-derived EVs on human ESC-derived RGC (H7/H9 immortalized cell line; WiCell, Madison, WI, USA, #WA07, RRID: CVCL_S800) were differentiated from CRISPR-modified ESC generously donated from Prof Donald Zacks laboratory (Johns Hopkins University, Baltimore, MD, USA) and licensed for use from WiCell (Material Transfer Agreement issue-164634007).

Techniques: Derivative Assay, In Vitro

Journal: Neural Regeneration Research

Article Title: R-28 cell-derived extracellular vesicles protect retinal ganglion cells in glaucoma

doi: 10.4103/NRR.NRR-D-24-00709

Figure Lengend Snippet: R-28 cell-derived EVs show protective trend for RGCs in a chronic glaucoma model. (A) Experimental design of the in vivo study. R-28 cell-derived EVs were intravitreally injected weekly beginning 1 week after microbead injection, and animals’ IOPs were measured twice a week. Four weeks after weekly EV injection, animals were sacrificed and histologically analyzed. After injection, microbeads localized (arrow) around the iridocorneal angle (B). IOP (mmHg) of healthy animals (blue) and animals receiving intracameral injection of microbeads with (green) or without (brown) intravitreal EV treatments is shown (C). (D, E) Representative images (D) and quantification (E) of Brn3a + (green) RGCs from the three groups on week 5. Scale bars: 50 µm. Data are presented as mean ± SEM. n = 3–5. EV: Extracellular vesicles; IOP: intraocular pressure; RGC: retinal ganglion cells; PBS: phosphate buffered saline.

Article Snippet: To test the therapeutic effect of R-28-derived EVs on human ESC-derived RGC (H7/H9 immortalized cell line; WiCell, Madison, WI, USA, #WA07, RRID: CVCL_S800) were differentiated from CRISPR-modified ESC generously donated from Prof Donald Zacks laboratory (Johns Hopkins University, Baltimore, MD, USA) and licensed for use from WiCell (Material Transfer Agreement issue-164634007).

Techniques: Derivative Assay, In Vivo, Injection, Saline

Journal: Neural Regeneration Research

Article Title: R-28 cell-derived extracellular vesicles protect retinal ganglion cells in glaucoma

doi: 10.4103/NRR.NRR-D-24-00709

Figure Lengend Snippet: Differentially expressed miRNA shown as abundance and fold change heat map profiles. Heatmaps show the upregulated and downregulated normalized counts of miRNA from injured RGCs treated with R-28 cell-derived EVs compared to injured untreated (A, B), injured RGCs treated with R-28 derived EVs compared to uninjured treated (D, E), and uninjured RGCs treated with R-28 cell-derived EVs compared to injured untreated (G, H), both statistically significant ( P < 0.05; A, D, G) and those trending towards significance ( P < 0.1; B, E, H) with abundance profiles shown in associated bar charts (C, F, I, respectively). * P < 0.05, ** P < 0.01. Data are presented as mean ± SEM. n = 3. EV: Extracellular vesicles; RGC: retinal ganglion cells.

Article Snippet: To test the therapeutic effect of R-28-derived EVs on human ESC-derived RGC (H7/H9 immortalized cell line; WiCell, Madison, WI, USA, #WA07, RRID: CVCL_S800) were differentiated from CRISPR-modified ESC generously donated from Prof Donald Zacks laboratory (Johns Hopkins University, Baltimore, MD, USA) and licensed for use from WiCell (Material Transfer Agreement issue-164634007).

Techniques: Derivative Assay

Journal: Stem cell research

Article Title: Generation and characterization of POMC-tdTomato reporter human pluripotent stem cell lines

doi: 10.1016/j.scr.2026.103905

Figure Lengend Snippet: (A) Schematic of CRISPR/Cas9-mediated knock-in strategy for insertion of a tdTomato reporter into the endogenous POMC locus. Primer sets 1–3 are positioned outside the RHA or LHA and within tdTomato where appropriate, as indicated. (B) Transfection of WA09 parental line followed by expansion, single-cell sorting and clonal expansion yielded five independent clonal lines (1G, 1B2, 2A, 8A, and 10E). (C) PCR genotyping using three primer sets confirmed locus-specific insertion and distinguished homozygous (1G, 1B2) from heterozygous (10E) knock-in clones. (D) RT-qPCR analysis of pluripotency markers OCT4, SOX2, and NANOG in validated clones, normalized to the parental WA09 line and compared against ectoderm-differentiated WA09 as a negative control. (E) Immunocytochemistry confirming OCT4, SOX2, and NANOG protein expression in homozygous (1G) clone. (F) Sanger sequencing verifying precise tdTomato reporter integration at the POMC locus. (G) Immunostaining of directly differentiated cultures of clone 1G into three germ layers showing expression of lineage-specific markers: PAX6 (ectoderm), TBXT (mesoderm), and SOX17 (endoderm). (H) Live-cell imaging (Incucyte) of 1G POMC-tdTomato clone at day 21 of hypothalamic neuronal differentiation showing robust tdTomato fluorescence. (I) Representative spontaneous action potential firing of POMC-tdTommato (W09 1G clone)-expressing hypothalamic neuron at −50 mV.

Article Snippet: Cell Source , WA09 (H9) (WiCell Research Institute, Inc.).

Techniques: CRISPR, Knock-In, Transfection, Single Cell, Clone Assay, Quantitative RT-PCR, Negative Control, Immunocytochemistry, Expressing, Sequencing, Immunostaining, Live Cell Imaging, Fluorescence