Journal: PLoS ONE

Article Title: Adipose Stromal Cells Contain Phenotypically Distinct Adipogenic Progenitors Derived from Neural Crest

doi: 10.1371/journal.pone.0084206

Figure Lengend Snippet: A . Quantification of surface marker expression in both GFP+ and GFP− ASCs of either P0-Cre/FR or Wnt1-Cre/FR mice with flow cytometric analysis. The majority of both GFP+ and GFP− cells expressed MSC markers including CD29, CD44, CD90, CD105, and Sca-1. B . The proportion of both CD24- and CD34-positive cells in the GFP+ population was also higher, and the proportion of double-positive cells in the GFP+ population was more than five-fold higher compared to those in GFP− cells. Representative flow cytometry profiles of CD24/CD34 double-positive cells from P0-Cre/FR mice are shown. C . The GFP+ population had a significantly higher proportion of p75NTR-positive cells. Data ( A–C ) are shown as the mean + SEM of 4 independent experiments for each condition. * p <0.05 versus GFP− cells, t test. D . Immunofluorescent staining for p75NTR, S100, Nestin, GFAP, and fibronectin ( red ) of either P0-Cre/FR ASCs ( upper five panels ) or Wnt1-Cre/FR ( lower two panels ) shows that the majority of GFP+ cells ( green ) co-expressed p75NTR, S100, and Nestin ( arrow ) but were negative for GFAP and fibronectin. A few GFP-negative but p75NTR- S100-, or Nestin-positive cells were also observed ( arrowhead ). Note that the majority of GFP− cells were positive for fibronectin. Scale bar = 50 µm. E . RT-qPCR analysis for pluripotent markers Sox2, Nanog , and Oct3/4 on FACS-purified GFP− and GFP+ cells from P0-Cre/FR mice. Results are normalized based on GAPDH expression and shown as relative changes to GFP− cells. Data are shown as the mean + SEM of 3–4 independent experiments for each condition.

Article Snippet: Flow cytometry using a FACSCalibur flow cytometer and CellQuest software (BD Biosciences, San Diego, CA) was performed to characterize ASCs using the following primary antibodies (applied in optimal amounts): PE-conjugated hamster anti-CD29 antibody (Biolegend, San Diego, CA), PE-Cy5-conjugated mouse anti-CD44 antibody (BD Biosciences), APC-conjugated rat anti- Sca-1 antibody (BD Biosciences), APC-conjugated rat anti- CD90 antibody (BD biosciences), and PE-conjugated rat anti-CD105 antibody (eBioscience, San Diego, CA) as mouse MSC markers, PE-Cy5-conjugated mouse anti-CD31 antibody (BD biosciences) as an endothelial marker, PE-Cy5-conjugated mouse anti-CD45 antibody (eBioscience) as a hematopoietic cell marker, and PE-conjugated mouse anti-CD24 antibody (eBioscience) and APC-conjugated mouse anti-CD34 antibody (eBioscience) as preadipocyte markers.

Techniques: Marker, Expressing, Flow Cytometry, Staining, Quantitative RT-PCR, Purification

Journal: Stem Cell Reports

Article Title: Troy/Tnfrsf19 marks epidermal cells that govern interfollicular epidermal renewal and cornification

doi: 10.1016/j.stemcr.2021.07.007

Figure Lengend Snippet: Characterization of Troy -expressing cells in adult murine telogen skin (A) Experimental setup. (B and C) Representative flow cytometry scatterplots of viable cells isolated from Troy -EGFP knockin mice stained for ITGA6 (CD49f) (B) and SCA1 (C). Histogram of Troy -EGFP expression normalized to mode (B). Dark green, ITGA6 bright cells; green, ITGA6 mid cells; gray, ITGA6 dim cells (B). Column chart indicating the percentage of SCA1 – and SCA1 + cells within the Troy -EGFP + population (C). The data are presented as mean ± SEM (n = 3 mice). Student's t test, ∗∗∗∗ p < 0.0001. (D) Heatmap of the bulk mRNA sequencing showing the differentially expressed genes comparing Troy -EGFP + and Troy -EGFP – ITGA6 bright cells. (E–H) Representative image of paraffin sections of telogen back skin stained RNAscope probes against Troy/Tnfrsf19 and Krt10 . Yellow arrows in (G) and (H) indicate basal layer cells positive for Krt10 mRNA and Troy mRNA. (I) Tail epidermal whole mount of Troy -EGFP mice stained for EGFP and KI67. (J) Schematic representation of the genetic constructs. (K) Representative flow cytometry scatterplot of viable SCA1 + ITGA6 + cells assessed for expression of Troy -EGFP and KI67-tagRFP. Column chart indicating the percentage of KI67-tagRFP – and KI67-tagRFP + cells within the Troy -EGFP + population. The data are presented as mean ± SEM (n = 3 mice). Student's t test, ∗∗∗ p < 0.001. See also Figure S3 and .

Article Snippet: Cells were stained on ice for 1 h with the following antibodies: rat anti-human/mouse ITGA6 (CD49f)-PE/Cy7 (555736, BD Biosciences, or 313621, BioLegend), rat anti-mouse Ly6A (SCA-1)-APC (17-5981-81, eBioscience) and rat anti-mouse CD34 (560230, BD Biosciences).

Techniques: Expressing, Flow Cytometry, Isolation, Knock-In, Staining, Sequencing, Construct

Journal: Stem Cell Reports

Article Title: Troy/Tnfrsf19 marks epidermal cells that govern interfollicular epidermal renewal and cornification

doi: 10.1016/j.stemcr.2021.07.007

Figure Lengend Snippet: Characterization of Troy -expressing cells using organoid technology (A) Experimental setup. (B) Representative flow cytometry scatterplots of viable cells isolated from Troy -EGFP knockin mice stained for ITGA6 and SCA1 (n = 4). (C) Representative bright-field images of sorted cell populations grown as organoids for 7 days. (D–F) Quantification of the cell viability per well (D), number of organoids formed per well (E), and size of organoids formed (F) after 7 days of culture. The data are presented as mean ± SEM (two to four replicates/wells per mouse, n = 4 mice). Student's t test, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. A.U., arbitrary units; L.U., light units.

Article Snippet: Cells were stained on ice for 1 h with the following antibodies: rat anti-human/mouse ITGA6 (CD49f)-PE/Cy7 (555736, BD Biosciences, or 313621, BioLegend), rat anti-mouse Ly6A (SCA-1)-APC (17-5981-81, eBioscience) and rat anti-mouse CD34 (560230, BD Biosciences).

Techniques: Expressing, Flow Cytometry, Isolation, Knock-In, Staining

Journal: Stem Cell Reports

Article Title: Troy/Tnfrsf19 marks epidermal cells that govern interfollicular epidermal renewal and cornification

doi: 10.1016/j.stemcr.2021.07.007

Figure Lengend Snippet: Single-cell transcriptomics of Troy -expressing cells and other epidermal cells (A) Experimental setup. (B) Representative flow cytometry scatterplots of viable cells isolated from Troy -EGFP knockin mice stained for ITGA6 (n = 4). Sorting gates are indicated in colors: ITGA6 – cells (red), ITGA6 bright Troy -EGFP – cells (yellow), and ITGA6 bright Troy -EGFP + cells (green). (C and D) t -SNE plot indicating the four different clusters identified within the epidermal cell populations (C) and the sorting gates (D). (E) Stacked column chart indicating cluster breakdown by gate. (F) Cell-cycle analysis. (G) Key marker gene expression per cluster. (H) Heatmap showing marker gene expression averaged per cell population for all identified epidermal clusters. (I–K) Violin plots comparing the EGFP and ITGA6 protein expression intensity (I), Itga6 expression levels (J), and the expression levels of Ly6a , Krt14 , and Krt10 (K) between the cells in clusters 0 and 2. Horizontal bars indicate the median and cross bars indicate the quartiles. See also Figure S4 .

Article Snippet: Cells were stained on ice for 1 h with the following antibodies: rat anti-human/mouse ITGA6 (CD49f)-PE/Cy7 (555736, BD Biosciences, or 313621, BioLegend), rat anti-mouse Ly6A (SCA-1)-APC (17-5981-81, eBioscience) and rat anti-mouse CD34 (560230, BD Biosciences).

Techniques: Single-cell Transcriptomics, Expressing, Flow Cytometry, Isolation, Knock-In, Staining, Cell Cycle Assay, Marker

Journal: Cell reports

Article Title: In Situ Modification of Tissue Stem and Progenitor Cell Genomes

doi: 10.1016/j.celrep.2019.03.105

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rat Anti-Mouse Ly-6A/E (Sca-1) APC , Thermo Fisher Scientific , Cat #17-5981-82; RRID: AB_469487.

Techniques: Blocking Assay, Plasmid Preparation, Staining, Flow Cytometry, Virus, Recombinant, Cell Culture, Saline, TaqMan Assay, Control, Software, Irradiation