Journal: Stem Cell Research & Therapy

Article Title: TNFα/TNFR2 signaling pathway: an active immune checkpoint for mesenchymal stem cell immunoregulatory function

doi: 10.1186/s13287-020-01740-5

Figure Lengend Snippet: MSC WT and TNFR2 KO characterization. a MSCs WT showed normal spindle-shaped fibroblast-like appearance (passage 3) (× 4) while MSCs TNFR2 KO exhibited a more heterogeneous morphology (passage 3) (× 4). b Flow cytometry analyses of the surface expression of CD45, CD34, CD44, CD105, CD73, CD90, and SCA1 in MSCs WT and TNFR2 KO (passage 3). Both MSC populations were negative for CD45 and CD34 and positive for the rest of the markers studied. The dark gray histograms represent isotype controls. Data are representative of n = 6 in 3 independent experiments. c Osteogenic differentiation: both MSCs WT and TNFR KO (passage 3) were incubated in osteogenic differentiation medium for 17 days followed by Alizarin Red S staining (× 4). d Adipogenic differentiation: both MSCs WT and TNFR2 KO (passage 3) were incubated in adipogenic differentiation medium for 21 days followed by Oil Red O staining (× 4)

Article Snippet: Cells were then stained with Oil Red for 5 min. For osteocyte differentiation, MSCs were cultured in differentiation medium (StemXVIVO Osteogenic/Adipogenic Base Media supplemented with P/S/N at 1:100 dilution + StemXVIVO Mouse/Rat Osteogenic Supplement 20×) (R&D Systems) for 17 days and stained 3 min with 2% Alizarin Red.

Techniques: Flow Cytometry, Expressing, Incubation, Staining

Journal: Stem Cell Research & Therapy

Article Title: TNFα/TNFR2 signaling pathway: an active immune checkpoint for mesenchymal stem cell immunoregulatory function

doi: 10.1186/s13287-020-01740-5

Figure Lengend Snippet: MSCs’ suppressive effect is diminished in the absence of the TNFα/TNFR2 signaling pathway. Activated CFSE + CD4 + or CD8 + effector T cells were co-cultured with MSCs WT or TNFR2 KO in different MSC/T cell ratios ( n = 6). Proliferation of CD4 + T cells ( a ) and CD8 + T cells ( b ) was measured by flow cytometry. The first bar represents the unstimulated T cells alone ( n = 6), the second bar represents the bead-stimulated T cells alone in RPMI ( n = 8), and the third bar is for the stimulated T cells alone in 50% RPMI+50% MEMα ( n = 6). All data are collected from 3 different experiments. c A flow cytometry representative of proliferation assay at a 1:4 MSC/T cell ratio. Non-stimulated T cells have a single peak represented in blue. Upon stimulation, T cells alone or in co-culture with MSCs start to proliferate, and one can see less intensity of CFSE fluorescence. Data are represented as mean value ± SEM. One-way ANOVA analysis was performed to generate P values. ns, non-significant; * P < .05, ** P < .01, *** P < .001, **** P < .0001. b Anti-CD3 and anti-CD28 activation beads

Article Snippet: Cells were then stained with Oil Red for 5 min. For osteocyte differentiation, MSCs were cultured in differentiation medium (StemXVIVO Osteogenic/Adipogenic Base Media supplemented with P/S/N at 1:100 dilution + StemXVIVO Mouse/Rat Osteogenic Supplement 20×) (R&D Systems) for 17 days and stained 3 min with 2% Alizarin Red.

Techniques: Cell Culture, Flow Cytometry, Proliferation Assay, Co-Culture Assay, Fluorescence, Activation Assay

Journal: Stem Cell Research & Therapy

Article Title: TNFα/TNFR2 signaling pathway: an active immune checkpoint for mesenchymal stem cell immunoregulatory function

doi: 10.1186/s13287-020-01740-5

Figure Lengend Snippet: The expression of TNFR2 on MSCs is directly correlated to their higher immunomodulation of CD4 + T convs. Activated CD3 + effector T cells were co-cultured with MSCs WT or TNFR2 KO in a fixed 1/5 ratio. After 3 days, T cells were collected and different activation markers were studied. Cells were gated on CD4 + Foxp3 − T conventional cells. For each marker, the strategy of gating is indicated on the left ( y -axis) and below ( x -axis) the figure. The x -axis represents each activation marker, and the y -axis represents the CD4 population. Left dot plots represent the Fluorescence Minus One (FMO) controls, and right dot plots represent the main samples. Each dot represents a measured value ( n = 6) collected from 2 different experiments. For each group of values, horizontal lines represent the mean value and standard error of the mean. MFI values have been normalized with the T cells alone control group. One-way ANOVA analysis was performed to generate P values. ns, non-significant; * P < .05, ** P < .01, *** P < .001, **** P < .0001

Article Snippet: Cells were then stained with Oil Red for 5 min. For osteocyte differentiation, MSCs were cultured in differentiation medium (StemXVIVO Osteogenic/Adipogenic Base Media supplemented with P/S/N at 1:100 dilution + StemXVIVO Mouse/Rat Osteogenic Supplement 20×) (R&D Systems) for 17 days and stained 3 min with 2% Alizarin Red.

Techniques: Expressing, Cell Culture, Activation Assay, Marker, Fluorescence, Control

Journal: Stem Cell Research & Therapy

Article Title: TNFα/TNFR2 signaling pathway: an active immune checkpoint for mesenchymal stem cell immunoregulatory function

doi: 10.1186/s13287-020-01740-5

Figure Lengend Snippet: The expression of TNFR2 on MSCs is directly correlated to their higher immunomodulation of CD8 + T convs. Activated CD3 + effector T cells were co-cultured with MSCs WT or TNFR2 KO in a fixed 1/5 ratio. After 3 days, T cells were collected and different activation markers were studied. Cells were gated on CD8 + Foxp3 − T conventional cells. For each marker, the strategy of gating is indicated on the left ( y -axis) and below ( x -axis) the figure. The x -axis represents each activation marker, and the y -axis represents the CD8 population. Left dot plots represent the Fluorescence Minus One (FMO) controls, and right dot plots represent the main samples. Each dot represents a measured value ( n = 6) collected from 2 different experiments. For each group of values, horizontal lines represent the mean value and standard error of the mean. MFI values have been normalized with the T cells alone control group. One-way ANOVA analysis was performed to generate P values. ns, non-significant; * P < .05, ** P < .01, *** P < .001, **** P < .0001

Article Snippet: Cells were then stained with Oil Red for 5 min. For osteocyte differentiation, MSCs were cultured in differentiation medium (StemXVIVO Osteogenic/Adipogenic Base Media supplemented with P/S/N at 1:100 dilution + StemXVIVO Mouse/Rat Osteogenic Supplement 20×) (R&D Systems) for 17 days and stained 3 min with 2% Alizarin Red.

Techniques: Expressing, Cell Culture, Activation Assay, Marker, Fluorescence, Control

Journal: Stem Cell Research & Therapy

Article Title: TNFα/TNFR2 signaling pathway: an active immune checkpoint for mesenchymal stem cell immunoregulatory function

doi: 10.1186/s13287-020-01740-5

Figure Lengend Snippet: MSCs can modulate T cell capacity to produce pro or anti-inflammatory cytokines via the TNFα/TNFR2 signaling pathway. Effector T cells were co-cultured with MSCs WT or TNFR2 KO in a fixed 1/5 ratio. After 3 days, T cells were collected, activated with PMA/ionomycin, and then blocked with GolgiPlug (a protein transport inhibitor). Intracellular pro-inflammatory cytokine ( a , b ) and anti-inflammatory cytokine ( c , d ) production was determined in both CD4 + and CD8 + T cells. Cells were first gated on CD4 + Foxp3 − or CD8 + Foxp3 − T cells to precisely analyze the conventional T cell population. For each marker, the strategy of gating is indicated on the left ( y -axis) and below ( x -axis) the figure. The x -axis represents each cytokine, and the y -axis represents the CD4 or CD8 populations. Each dot represents a measured value ( n = 12) collected from 2 different experiments. For each group of values, horizontal lines represent the mean value and standard error of the mean. One-way ANOVA analysis was performed to generate P values. ns, non-significant; ** P < .01, *** P < .001

Article Snippet: Cells were then stained with Oil Red for 5 min. For osteocyte differentiation, MSCs were cultured in differentiation medium (StemXVIVO Osteogenic/Adipogenic Base Media supplemented with P/S/N at 1:100 dilution + StemXVIVO Mouse/Rat Osteogenic Supplement 20×) (R&D Systems) for 17 days and stained 3 min with 2% Alizarin Red.

Techniques: Cell Culture, Marker

Journal: Stem Cell Research & Therapy

Article Title: TNFα/TNFR2 signaling pathway: an active immune checkpoint for mesenchymal stem cell immunoregulatory function

doi: 10.1186/s13287-020-01740-5

Figure Lengend Snippet: Expression of TNFR2 by MSCs is directly correlated to their higher Foxp3 T reg induction capacity. Activated CD4 + or CD8 + effector T cells were co-cultured with MSCs WT or TNFR2 KO in different ratios ( n = 9). After 3 days, the expression of Foxp3 among CD4 + T cells ( a ) and CD8 + T cells ( b ) was measured by flow cytometry. Cells were gated on CD4 + CD25 + Foxp3 + or CD8 + CD25 + Foxp3 + T cells. The gating strategy is indicated on the left and under the figures. The first black bar represents the percentage of Foxp3-expressing cells among total effector T cells used in co-cultures at day 0 ( n = 9) while the second bar represents the percentage of Foxp3-expressing T cells alone after 3 days of culture ( n = 9). ( c ) CD4 + T cells collected from the spleen of WT or TNFα deficient mice were co-cultured with MSCs WT. Cells were gated on CD4 + CD25 + Foxp3 + . Data are represented as the mean value ± SEM collected from 3 different experiments. One-way ANOVA or unpaired Student t test analysis was performed to generate P values; *** P < .001. D0, day 0

Article Snippet: Cells were then stained with Oil Red for 5 min. For osteocyte differentiation, MSCs were cultured in differentiation medium (StemXVIVO Osteogenic/Adipogenic Base Media supplemented with P/S/N at 1:100 dilution + StemXVIVO Mouse/Rat Osteogenic Supplement 20×) (R&D Systems) for 17 days and stained 3 min with 2% Alizarin Red.

Techniques: Expressing, Cell Culture, Flow Cytometry