Journal: Journal of Translational Medicine

Article Title: Identifying the functions of two biomarkers in human oligodendrocyte progenitor cell development

doi: 10.1186/s12967-021-02857-8

Figure Lengend Snippet: Identification of hOPC markers. a Three markers of hOPCs were identified using cell immunofluorescence staining: PDGFR-α, A2B5, and NG2. Scale bar is 200 μm. b Images of single cells detached in the presence of PDGFR-α+, A2B5+, and NG2+ cells, as observed using flow cytometry. Ch01: Bright field, Ch03: A2B5-PE, Ch07: PDGFR-α-BV421, Ch11: NG2-APC. Scale bar is 20 μm. c Gating strategy to define PDGFR-α/A2B5/NG2 cells. R4: PDGFR-α+ cells, R5: A2B5+ cells, R2: NG2+ cells. d scRNA-seq of hOPCs. Maps of t-SNE of 7885 cells from high-dimensional images of hOPCs coloured according to the cell type. hOPC human oligodendrocyte progenitor cells, PDGFR-α platelet-derived growth factor receptor alpha, scRNA-seq single-cell RNA sequencing

Article Snippet: The cells were surface-stained with PDGFR-α BV421 mouse anti-human (Cat. #562799, BD Biosciences, Franklin Lake, NJ, USA), NG2 APC mouse anti-human (Cat. #FAB2585A, R&D Systems, MN, USA), or A2B5 PE mouse anti-human (Cat. #130-093-581, Miltenyi Biotec, Bergisch-Gladbach, Germany) antibodies for 30 min at 4 °C.

Techniques: Immunofluorescence, Staining, Flow Cytometry, Derivative Assay, RNA Sequencing

Journal: Journal of Translational Medicine

Article Title: Identifying the functions of two biomarkers in human oligodendrocyte progenitor cell development

doi: 10.1186/s12967-021-02857-8

Figure Lengend Snippet: The expression of PDGFR-α in positive and negative cells. a Gating strategy to define PDGFR-α+ cells in A2B5+/− and NG2+/− populations. b Images of single cells detached in the presence of A + P +, A − P +, N + P +, and N − P + cells. c , d Cell counts of different cell populations. Bars represent the mean. Error bars show the standard error of the mean. A + P + , PDGFR-α+ cells in A2B5+ cells; A − P + , PDGFR-α+ cells in A2B5− cells; N + P + , PDGFR-α+ cells in NG2+ cells; N − P + , PDGFR-α+ cells in NG2− cells

Article Snippet: The cells were surface-stained with PDGFR-α BV421 mouse anti-human (Cat. #562799, BD Biosciences, Franklin Lake, NJ, USA), NG2 APC mouse anti-human (Cat. #FAB2585A, R&D Systems, MN, USA), or A2B5 PE mouse anti-human (Cat. #130-093-581, Miltenyi Biotec, Bergisch-Gladbach, Germany) antibodies for 30 min at 4 °C.

Techniques: Expressing

Journal: Journal of Translational Medicine

Article Title: Identifying the functions of two biomarkers in human oligodendrocyte progenitor cell development

doi: 10.1186/s12967-021-02857-8

Figure Lengend Snippet: Differential expression analysis. a Volcano plot illustrating differentially regulated gene expression between the four groups of cells. Red dots indicate upregulated genes, and blue dots indicate downregulated genes. Values are presented as the log2 of tag counts. b Heat map showing the relative gene expression in NG2+/− and A2B5+/− cells. Red and green indicate upregulated and downregulated genes, respectively. c , d Graph showing the FPKM value of markers in different cell populations. Bars represent the mean. Error bars show the standard error of the mean. FPKM fragments per kilobase of exon model per million mapped fragments

Article Snippet: The cells were surface-stained with PDGFR-α BV421 mouse anti-human (Cat. #562799, BD Biosciences, Franklin Lake, NJ, USA), NG2 APC mouse anti-human (Cat. #FAB2585A, R&D Systems, MN, USA), or A2B5 PE mouse anti-human (Cat. #130-093-581, Miltenyi Biotec, Bergisch-Gladbach, Germany) antibodies for 30 min at 4 °C.

Techniques: Quantitative Proteomics, Gene Expression

Journal: The Journal of Neuroscience

Article Title: Embryonic Pericytes Promote Microglial Homeostasis and Their Effects on Neural Progenitors in the Developing Cerebral Cortex

doi: 10.1523/jneurosci.1201-21.2021

Figure Lengend Snippet: Figure 2. Administration of APB5 induced pericytes to undergo apoptosis. A, A schematic showing the strategy of in vivo adminis- tration of anti-PDGFRb antibodies (APB5). B, Representative immunostaining for CD31 (green), NG2 (magenta), and PDGFRb (top) or PDGFRa (bottom; cyan) of the E14 cerebral wall. In top figures, white arrowhead indicates NG21PDGFRb 1 pericytes and yellow arrowhead indicates NG21PDGFRb – OPCs. In bottom figures, white arrowhead indicates NG21PDGFRa– pericytes and yellow arrowhead indicates NG21PDGFRa1 OPCs. C, Immunostaining for NG2 (green), CD31 (magenta), and DAPI (blue) in the E14 cerebral wall. D, A graph comparing the proportion of pericyte coverage, which was calculated by dividing the merged area of NG2 and CD31 by the total CD311 area in the pallium (not including the meninges), between the saline-treated, isotype control antibody-treated, and APB5-treated brains (two-sided Steel–Dwass test; the average value of four sections from each animal is plotted; N = 6 mice; p= 1.000 and p=0.011; left to right). Data are presented as the mean value 6 SD. E, Immunostaining for Cl-Casp3 (green), CD31 (magenta), and DAPI (blue) in the cerebral wall of the mice treated with antibodies or saline. The white broken line indicates the ap- ical surface. Scale bar: 50mm (B) or 100mm (C, E).

Article Snippet: Single cells were stained with rat FITC rat anti-mouse CD31 mAb (1:200, catalog #102405, BioLegend, RRID: AB_312900) or rat FITC IgG2a isotype control Ab (1:200, catalog #400505, BioLegend, RRID: AB_2736919), rat anti-NG2 mAb (1:200, catalog #MAB6689, R&D Systems, RRID: AB_10890940), which was labeled by PE using an Ab-10 Rapid R-Phycoerythrin Labeling kit (LK34, Dojindo), or rat PE IgG1 isotype control Ab (1:200, catalog #400408, BioLegend, RRID: AB_ 326514), and rat BV421 anti-PDGFRa mAb (1:200, catalog #135923, BioLegend, RRID: AB_2814036) or rat BV421 IgG2a isotype control antibody (1:200, catalog #407117, BioLegend, RRID: AB_2687343) for 1 h on ice.

Techniques: In Vivo, Immunostaining, Saline, Control

Journal: The Journal of Neuroscience

Article Title: Embryonic Pericytes Promote Microglial Homeostasis and Their Effects on Neural Progenitors in the Developing Cerebral Cortex

doi: 10.1523/jneurosci.1201-21.2021

Figure Lengend Snippet: Figure 6. An in vitro coculture showed that pericytes promoted microglial proliferation. A, FACS analysis of pallial cells collected from E14 mice to detect PDGFRb , NG2, and PDGFRa expres- sion. B, Pericyte sorting strategy by FACS. NG21PDGFRa– cells were collected from E14 cerebral wall cells. C, A schematic showing the bilayer culture of CD11b1 microglia with or without NG21PDGFRa– cells (pericytes) using the Transwell insert. D, Immunostaining for CX3CR1 and Ki67 in microglia cultured with or without NG21PDGFRa cells (pericytes). E, A bar graph com- paring the density of CX3CR11 cells (two-sided Mann–Whitney U test; the average value of six fields per independent culture is plotted; N = 6 independent cultures; p = 0.002). F, A bar graph comparing the proportion of Ki671 cells among CX3CR11 cells (two-sided Mann–Whitney U test; the average value of six fields per independent culture is plotted; N = 6 independent cultures; p = 0.002). G, Immunostaining for CX3CR1 and BrdU in microglia cultured with or without NG21PDGFRa cells (pericytes). Scale bar: 20mm. H, A bar graph comparing the proportion of BrdU1 cells among CX3CR11 cells (two-sided Mann–Whitney U test; the average value of six fields per independent culture is plotted; N = 6 independent cultures; p = 0.002). Data are pre- sented as the mean value 6 SD. Scale bar: 50 mm (D) or 20mm (G).

Article Snippet: Single cells were stained with rat FITC rat anti-mouse CD31 mAb (1:200, catalog #102405, BioLegend, RRID: AB_312900) or rat FITC IgG2a isotype control Ab (1:200, catalog #400505, BioLegend, RRID: AB_2736919), rat anti-NG2 mAb (1:200, catalog #MAB6689, R&D Systems, RRID: AB_10890940), which was labeled by PE using an Ab-10 Rapid R-Phycoerythrin Labeling kit (LK34, Dojindo), or rat PE IgG1 isotype control Ab (1:200, catalog #400408, BioLegend, RRID: AB_ 326514), and rat BV421 anti-PDGFRa mAb (1:200, catalog #135923, BioLegend, RRID: AB_2814036) or rat BV421 IgG2a isotype control antibody (1:200, catalog #407117, BioLegend, RRID: AB_2687343) for 1 h on ice.

Techniques: In Vitro, Immunostaining, Cell Culture, MANN-WHITNEY

Journal: Frontiers in Physiology

Article Title: Human Induced Pluripotent Stem Cell-Derived Brain Endothelial Cells: Current Controversies

doi: 10.3389/fphys.2021.642812

Figure Lengend Snippet: Major iterations of hPSC-derived iBMEC protocols.

Article Snippet: 2017 , . Fluids barriers CNS , Modified 2014 protocol , E8 and E6 media, E6 for 4 days then continued as protocol. , MR90-4 iPSCs CC3 iPSCs, CD12 iPSCs, and SM14 iPSCs in growth factor-reduced Matrigel (VWR) in E8 medium , E8 medium was prepared by adding 100 μL of human insulin solution (Sigma-Aldrich), 500 μL of 10 mg/mL of human holo-transferrin (R&D Systems), 500 μL of 100 μg/mL human basic fibroblast growth factor (bFGF; PeproTech), and 500 μL of 2 μg/mL TGFβ1 (PeproTech) to 500 mL of E4. The final concentrations are 2.14 mg/L insulin, 100 μg/L bFGF, 2 μg/L TGFβ1, and 10.7 mg/L holo-transferrin E6 medium was prepared by adding 100 μL of human insulin solution and 500 μL of 10 mg/mL of human holo-transferrin to 500 mL of E4. The final concentrations are 2.14 mg/L insulin and 10.7 mg/L holo-transferrin UM and EC same as 2–14 , , PECAM-1 (Rabbit, Thermo Scientific) GLUT-1 (Mouse, Thermo Scientific) OCCLUDIN (Mouse, Thermo Scientific) CLAUDIN-5 (Mouse, Thermo Scientific) VE-Cadherin (Goat, R&D Systems) GFAP (Rabbit, Dako) PDGFR-B (Rabbit, SCBT) NG2 (Mouse, SCBT) αSMA (Mouse, SCBT) , TEER , Intracellular accumulation of rhodamine 123 (a Pgp substrate) was evaluated in the absence of bFGF and RA. Cells were incubated with 10 μM PSC833 or 10 μM MK-571 for 1 h at 37°C. They were then incubated for an additional house with 10 μM rhodamine 123 or 10 μM H2DCFDA..

Techniques: Clinical Proteomics, Derivative Assay, Irradiation, Control, Modification, Incubation