Journal: Molecular and Cellular Biology

Article Title: Sox17-Mediated Maintenance of Fetal Intra-Aortic Hematopoietic Cell Clusters

doi: 10.1128/MCB.01485-13

Figure Lengend Snippet: Overexpression of Sox17 in CD45low c-Kithigh cells from the E10.5 AGM region leads to the formation of ball-like cell clusters. (A) Tissue sections of E10.5 embryos were stained with anti-Sox17 (green) and anti-CD31 (red) antibodies. Sox17 expression was observed in the nucleus. DA, dorsal aorta. (B) Transduction of Sox17 into E10.5 CD45low c-Kithigh AGM cells induced ball-like cell clusters. In vehicle- and Sox17MA-transduced cultures, there were colonies of scattered hematopoietic cells on OP9 stromal cells. Bars, 100 μm. (C) Expression of hematopoietic transcription factors and markers of endothelial cells was observed in Sox17-transduced cells by RT-PCR. (D) The ability of Sox17-transduced cells to form multilineage colonies in a semisolid medium was greater than that of vehicle- or Sox17MA-transduced cells. Data are averages of results of 4 experiments. (E) Sox17-transduced cell clusters were cocultured with new OP9 stromal cells. After 3 days, large clusters were observed in the cocultures. Bar, 100 μm. (F) Sox17-transduced cells dispersed by pipetting were cocultured with new OP9 stromal cells. After 3 days, a large number of GFP+ cell clusters was observed in the cocultures. Bar, 100 μm. (G) Morphologies of vehicle-, Sox17-, and Sox17MA-transduced cells as shown by Giemsa staining. (H) The expression of markers for macrophages (Mac-1) and granulocytes (Gr1) was hardly detected in Sox17-transduced cells.

Article Snippet: In some experiments, the cells were also stained with PE-conjugated anti-Gr1 (RB6-8C5) and allophycocyanin (APC)-conjugated anti-Mac-1 (M1/70) antibodies (eBioscience, San Diego, CA).

Techniques: Over Expression, Staining, Expressing, Transduction, Reverse Transcription Polymerase Chain Reaction

Journal: Molecular and Cellular Biology

Article Title: Sox17-Mediated Maintenance of Fetal Intra-Aortic Hematopoietic Cell Clusters

doi: 10.1128/MCB.01485-13

Figure Lengend Snippet: Sox17-transduced cells reconstitute hematopoiesis in recipient mice. (A) Donor chimerism in competitive reconstitution assays using 1 × 105 or 2 × 106 cells transduced with Sox17-IRES-GFP (Sox17) or 1 × 105 cells transduced with IRES-GFP (Mock) (CD45.2+) and 2 × 105 BM competitor cells (CD45.1+). Because IRES-GFP-transduced CD45low c-Kithigh cells rapidly lost their proliferative activity (Fig. 8A), we could not transplant 2 × 106 Mock cells. The chimerism in CD45.2+ donor-derived PB cells from each recipient mouse was analyzed monthly (five independent analyses were conducted for 2 × 106 Sox17 cells and 1 × 105 Mock cells, while four independent analyses were conducted for 1 × 105 Sox17 cells). (B) Flow cytometric analysis to monitor donor-derived multilineage reconstitution in mice receiving transplants of Sox17-transduced cells. (Left) Representative dot plots show BM chimerism in mice into whom IRES-GFP- or Sox17-IRES-GFP-transduced cells (CD45.2+) and BM competitor cells (CD45.1+) had been transplanted. (Right) Representative dot plots of myeloid (Gr1 and Mac-1), B-lymphocyte (B220), and T-lymphocyte (CD4 and CD8) lineages in the PB, BM, SP, and thymuses of mice into whom Sox17-transduced cells had been transplanted (Sox17-CD45.2+ cells). (C) Chimerism in the PB, BM, SP, and thymuses of mice at 3 months after transplantation of Sox17-transduced cells (2 × 106 CD45.2+ cells) (n = 5). Shown are the contributions of Sox17-expressing donor cells to erythroid (Ter119), myeloid (Gr1 and Mac-1), B-lymphocyte (B220), and T-lymphocyte (CD4 and CD8) lineages in the PB, BM, SP, and thymus (n = 5).

Article Snippet: In some experiments, the cells were also stained with PE-conjugated anti-Gr1 (RB6-8C5) and allophycocyanin (APC)-conjugated anti-Mac-1 (M1/70) antibodies (eBioscience, San Diego, CA).

Techniques: Transduction, Activity Assay, Derivative Assay, Transplantation Assay, Expressing

Journal: bioRxiv

Article Title: Deciphering sources of PET signals in the tumor microenvironment of glioblastoma at cellular resolution

doi: 10.1101/2023.01.26.522174

Figure Lengend Snippet: (A) Schematic illustration of the in vitro scRadiotracing workflow in human glioma samples (n=20), leading to calculation of radioactivity per single tumor cell and TAM. Pseudocolor plots derived from flow cytometry show the applied gating strategy. The single cell suspension (left) was separated into TAM-enriched (CD11b(+), second from left) and tumor enriched (third from left) fractions. Tumor cells were defined via GFAP or 5-ALA after confirmation of 5-ALA-positivity during surgery or after confirmation of GFAP-positivity during neuropathological workup of the same samples. 5-ALA positive cells co-localized with GFAP-positive cells in samples where both markers allowed tumor cell identification (right). (B) Relative distribution of tumor cells and TAMs in the single cell suspension of human glioma samples revealed similar heterogeneity in human high-grade glioma (HGG, n=10) compared to low-grade glioma (LGG, n=2). The initial two patients did not receive an analysis of the single cell suspension. (C) Regression model indicated strong contribution of TAMs (CD11b(+) cells) but not of CD11b(−)/non-tumor cells to the measured activity in the investigated TAM-enriched samples of patients that underwent biopsy (n=10). Linear regression, β = standardized regression coefficient, error bands represent 95% confidence interval. (D) Comparison of single cell tracer uptake (scTSPO) of tumor cells and TAMs in samples of human HGG (n=11) and LGG (n=9). Tumor cells of HGG indicated higher scTSPO than tumor cells of LGG, whereas there was no significant difference for TAMs (unpaired t-test). Tumor cells of HGG showed higher scTSPO than TAMs of HGG (paired t-test). Mean±SEM. (E) Correlation of TSPO-PET signals with scTSPO elucidated a strong correlation between PET signals and tumor cell TSPO enrichment and no correlation between PET signals and TAM TSPO enrichment. N=13, R = Pearson’s coefficient of correlation. Error bands represent 95% confidence interval. (F) Example of three patients with HGG investigated via in vitro scRadiotracing. All three patients showed similar signals in amino acid PET (FET-PET) and only little contrast enhancement in MRI. The patient with high tumoral TSPO-PET signal (upper row, glioblastoma, WHO grade IV, high-affinity binding status (HAB)) showed distinctly more TSPO tracer uptake in both tumor cells and TAMs when compared to the patients with only faint (middle row, astrocytoma, WHO grade III, high-affinity binding status) or low (bottom row, glioblastoma, WHO grade IV, low-affinity binding status (LAB)) tumoral signal in TSPO-PET. The patient with glioblastoma and low-affinity binding status (bottom row) showed nearly absent single cell tracer uptake of TAMs but notable TSPO tracer uptake of tumor cells. T1w = T1-weighted, FLAIR = Fluid-attenuated inversion recovery.

Article Snippet: Cell pellets were resuspended in 100 μl cold D-PBS and stained with fluorochrome-conjugated antibodies recognizing human CD11b and GFAP (Miltenyi Biotec, 130-113-810 and 130-123-846) as described above.

Techniques: In Vitro, Radioactivity, Derivative Assay, Flow Cytometry, Suspension, Activity Assay, Comparison, Binding Assay

Journal: bioRxiv

Article Title: PU.1 expression defines distinct functional activities in the phenotypic HSC compartment in a mouse model of inflammatory stress

doi: 10.1101/2021.10.25.465758

Figure Lengend Snippet: A) Experimental design for analysis of SLAM cells from PU . 1-EYFP mice treated for 20d ± IL-1β. B) Representative FACS plot showing phenotypic HSC LT frequencies within the SLAM gate in PU . 1-EYFP mice treated for 20d ± IL-1β. C) Summary data showing phenotypic HSC LT frequency within the SLAM gate of PU . 1-EYFP mice treated for 20d ± IL-1β (n = 6/grp). Individual values are shown, bars represent mean values. Data are compiled from two independent experiments. D) EPCR expression in phenotypic HSC LT within the SLAM gate of PU . 1-EYFP mice treated for 20d ± IL-1β (n = 6/grp). Data are expressed as geometric mean fluorescence intensity (MFI) Individual values are shown, bars represent mean values. Data are compiled from two independent experiments. E) Representative FACS plot showing frequencies of CD41 + and Mac-1 + cells within the SLAM gate in PU . 1-EYFP mice treated for 20d ± IL-1β. F) Frequency of CD41+ cells within the SLAM gate of PU . 1-EYFP mice treated for 20d ± IL-1β (n = 3-5/grp). Individual values are shown, bars represent mean values. Data are representative of two independent experiments. G) Frequency of Mac-1+ cells within the SLAM gate of PU . 1-EYFP mice treated for 20d ± IL-1β (n = 3-5/grp). Individual values are shown, bars represent mean values. Data are representative of two independent experiments. H) Experimental design for Fluidigm qRT-PCR array analysis of PU.1 lo and PU.1 hi SLAM cells from PU . 1-EYFP mice treated for 20d ± IL-1β (n = 8/grp). I) Fluidigm qRT-PCR analysis of self-renewal and metabolism genes in PU.1 lo and PU.1 hi SLAM cells from PU . 1-EYFP mice treated for 20d ± IL-1β. Data are expressed as log 10 fold change versus PU.1 lo -IL-1β. Box represents upper and lower quartiles with line representing median value. Whiskers represent minimum and maximum values. Data are representative of two independent experiments. J) Fluidigm qRT-PCR analysis of myeloid and megakaryocyte/erythroid genes in PU.1 lo and PU.1 hi SLAM cells from PU . 1-EYFP mice treated for 20d ± IL-1β. Data are expressed as log 10 fold change versus PU.1 lo -IL-1β. Box represents upper and lower quartiles with line representing median value. Whiskers represent minimum and maximum values. Data are representative of two independent experiments. Data are shown as means ± SD. Significance was determined by ANOVA with Tukey’s post-test. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: For mature BM cell analyses to monitor BM chimerism, peripheral blood cells were RBC depleted using ACK as above and stained with an antibody cocktail of CD8-PE (Biolegend, 100708), Mac-1 PE/Cy7, IgM APC (eBioscience, 17-1590-82), CD3 AF700 (Biolegend, 100216), CD19 APC/Cy7 (Biolegend, 115530), Gr1 PB (Biolegend, 108430), CD4 BV510 (Biolegend, 100449), Ly6C BV605 (BD Biosciences, 563011), B220 BV875 (Biolegend, 103246), and incubated for 30 minutes on ice.

Techniques: Expressing, Fluorescence, Quantitative RT-PCR

Journal: bioRxiv

Article Title: PU.1 expression defines distinct functional activities in the phenotypic HSC compartment in a mouse model of inflammatory stress

doi: 10.1101/2021.10.25.465758

Figure Lengend Snippet: A) Experimental design for analysis of PU . 1-EYFP mice treated for 1d ± IL-1 (n = 3/grp). B) Representative FACS plot showing fractionation of SLAM gate by EPCR and CD34 levels. C) Analysis of PU . 1-EYFP reporter expression in SLAM fractions. Data are expressed as geometric mean fluorescence intensity (MFI). Individual values are shown, bars represent mean values. Data are representative of two independent experiments. D) Experimental design and representative FACS plots showing frequency of PU.1 hi and PU.1 lo SLAM cells, for analysis of PU.1 hi and PU.1 lo SLAM cells isolated from PU . 1-EYFP mice treated for 1d ± IL-1 (n = 4-6/grp). E) Representative FACS plot showing phenotypic HSC LT frequencies within the SLAM gate in PU . 1-EYFP mice treated for 20d ± IL-1β. F) Summary data showing phenotypic HSC LT frequency within the SLAM gate of PU . 1-EYFP mice treated for 20d ± IL-1β (n = 4-6/grp). Individual values are shown, bars represent mean values. Data are compiled from two independent experiments. G) EPCR expression in phenotypic HSC LT within the SLAM gate of PU . 1-EYFP mice treated for 1d ± IL-1β (n = 4-6/grp). Individual values are shown, bars represent mean values. Data are compiled from two independent experiments. H) Representative FACS plot showing frequencies of CD41 + and Mac-1 + cells within the SLAM gate in PU . 1-EYFP mice treated for 1d ± IL-1β. I) Frequency of CD41+ cells within the SLAM gate of PU . 1-EYFP mice treated for 1d ± IL-1β (n = 5/grp). Individual values are shown, bars represent mean values. Data are representative of two independent experiments. J) Frequency of Mac-1+ cells within the SLAM gate of PU . 1-EYFP mice treated for 1d ± IL-1β (n = 5/grp). Individual values are shown, bars represent mean values. Data are representative of two independent experiments. K) Experimental design for Fluidigm qRT-PCR array analysis of PU.1 lo and PU.1 hi SLAM cells from PU . 1-EYFP mice treated for 1d ± IL-1β (n = 8/grp). L) Fluidigm qRT-PCR analysis of genes in PU.1 lo and PU.1 hi SLAM cells from PU . 1-EYFP mice treated for 1d ± IL-1β. Data are expressed as log 10 fold change versus PU.1 lo -IL-1β. Box represents upper and lower quartiles with line representing median value. Whiskers represent minimum and maximum values. Data are representative of two independent experiments. Data are shown as means ± SD. Significance was determined by ANOVA with Tukey’s post-test. *p<0.05; **p<0.01; ***p<0.001.

Article Snippet: For mature BM cell analyses to monitor BM chimerism, peripheral blood cells were RBC depleted using ACK as above and stained with an antibody cocktail of CD8-PE (Biolegend, 100708), Mac-1 PE/Cy7, IgM APC (eBioscience, 17-1590-82), CD3 AF700 (Biolegend, 100216), CD19 APC/Cy7 (Biolegend, 115530), Gr1 PB (Biolegend, 108430), CD4 BV510 (Biolegend, 100449), Ly6C BV605 (BD Biosciences, 563011), B220 BV875 (Biolegend, 103246), and incubated for 30 minutes on ice.

Techniques: Fractionation, Expressing, Fluorescence, Isolation, Quantitative RT-PCR