Journal: bioRxiv

Article Title: Computational Identification of Ligand-Receptor Pairs that Drive Human Astrocyte Development

doi: 10.1101/2022.05.31.491513

Figure Lengend Snippet: Ligand activity depends on hCO developmental stage. A . Following a 30-day exposure in hCOs, mouse-identified ligands 1-5 do not significantly modulate astrocyte or neuronal gene signatures (N.S. p = .781, Mann Whitney U) (n = 8 control organoids, 12 ligand-exposed organoids). B . Human-identified ligands 6-10 significantly increase expression of astrocyte gene signatures and decrease expression of neuronal genes (p = .002, Mann Whitney U). C . Heatmap of neuronal and astrocyte gene expression following ligand exposure. Classic signature genes for each population are highlighted. D . Schematic of proposed timeline of the gliogenic switch in organoid cultures. E . Variability of gliogenic switch in hCOs. 10 separate hCO differentiations (n = 5 hiPSC lines) were assayed for (1) % GFAP+ cells by IHC, (2) GFAP mRNA by qPCR, and (3) number of cells bound to HepaCAM+ immunopanning plate at 10-day intervals from days 70-110 in culture. Thresholds were set for each assay to determine that gliogenesis had begun. These include >5% GFAP/DAPI+ cells for IHC (top panel), a CT cutoff <30 for GFAP qPCR (middle panel), and >10,000 HepaCAM immunopanned astrocyte per organoid (lower panel). F . Representative GFAP and TUJ1 staining of day 40, day 100, day 200, and day 300 hCOs. Scale bar =100 µm, G-I . Result of ligand exposures (6-10) at different stages of hCO culture. Readouts are fold change of astrocyte and neuronal gene signatures compared to control hCOs using RNA-seq. (N.S. p = .643, ***p<.0001, Mann Whitney U) (n = 6-8 control organoids and 6-12 experimental organoids). J-K . Expression of target receptors for NicheNet-predicted ligands 6-10 throughout hCO development (***p<.0001, ***p<.0001, *p = .013, Mann Whitney U).

Article Snippet: Antibodies used were DAPI (in VECTASHEILD, Vector Laboratories, Cat. H-1500), GFAP (DAKO, Cat. Z0334, dilution 1:1500), and Ki67 (BD, Cat. b550609, dilution 1:50).

Techniques: Activity Assay, MANN-WHITNEY, Expressing, Staining, RNA Sequencing Assay

Journal: bioRxiv

Article Title: Computational Identification of Ligand-Receptor Pairs that Drive Human Astrocyte Development

doi: 10.1101/2022.05.31.491513

Figure Lengend Snippet: Impact of candidate ligand exposures on fetal astrocytes. A . GW 17-20 cortices were immunopanned for CD49f+ immature astrocytes, which were cultured for 10 days in the presence or absence of the ligand cocktail. B . Astrocyte and neuronal gene signatures assessed by RNA-seq of CD490f+ cells. Fold change represents expression in ligand conditions vs control media (p < .001, Mann Whitney U). C . GFAP+ cell process traces from ligand-exposed and control CD49f+ fetal cells. Scale bar = 50 µm. D . GFAP+ cell process quantification. Primary branches extend from the nucleus. Secondary branches extend from primary branches. Boundary size is the area (x 10 3 µm 2 ) of the image field that one cell occupies. (*p<.01, **p<.001, *** p<.0001, Mann Whitney U). E . Timeline of EdU exposure. Fetal astrocytes were cultured for 8 days after purification with ligand exposure from days 1-7. EdU added at day 1 until duration of experiment. F . No significant change in percent of EdU+ nuclei between control and ligand-exposed cells. G . Representative images of DAPI and EdU+ cells after 8 days in culture.

Article Snippet: Antibodies used were DAPI (in VECTASHEILD, Vector Laboratories, Cat. H-1500), GFAP (DAKO, Cat. Z0334, dilution 1:1500), and Ki67 (BD, Cat. b550609, dilution 1:50).

Techniques: Cell Culture, RNA Sequencing Assay, Expressing, MANN-WHITNEY, Purification

Journal: bioRxiv

Article Title: PKCδ splicing variant (PKC δ III) induces neurite outgrowth in PC12 cells in the absence of NGF

doi: 10.1101/2023.08.23.554440

Figure Lengend Snippet: PKC δ SV induces neurite outgrowth in the absence of NGF in an ERK-independent manner. (A) Representative fluorescent images of PC12 cells containing the indicated EGFP-tagged PKCδ proteins. Cells were cultured with or without 1ug/ml doxycycline for 48h. After fixation, DNA was stained with DAPI and actin filament was stained with TRITC conjugated phalloidin. Scale bar represents 50μm. (B) Boxplots of total neurite length per a GFP-positive cell among PC12 cells containing the indicated PKCδ constructs. **** p <0.001 (one-way ANOVA [Dunnett’s test]). (C) Quantification of cells with >5um neurites in GFP-positive cells. Graph represent mean ± SD of n =5. * p <0.05; ** p <0.01 (one-way ANOVA [Dunnett’s test]). (D) PC12 cells were cultured with or without 100ng/ml NGF. After 2, 4, and 7 days, whole cell lysates were harvested. The cell lysates were subjected to immunoblotting analysis using the indicated antibodies. n = 3. (E) PC12 cells containing PKCδ constructs were cultured with 1ug/ml doxycycline. PC12 cells were also cultured with or without 100ng/ml NGF. 48h later, cells were harvested and the whole cell lysate were subjected to immunoblotting analysis using the indicated antibodies. n = 3.

Article Snippet: After blocking with 3% BSA, cells were stained with TRITC conjugated phalloidin (H-1600, Vector laboratories) and mounted with HardSet Antifade Mounting Medium with DAPI (H-1500, Vector laboratories).

Techniques: Cell Culture, Staining, Construct, Western Blot