Additional file 3: of IFNγ and TNFα synergistically induce apoptosis of mesenchymal stem/stromal cells via the induction of nitric oxide

Figure S3. ER stress was upregulated by blocking autophagy. MSCs were treated or not (mock) for 48 h with IFNγ/TNFα (10 ng/ml each), alone or in combination with chloroquine, rapamycin. ER stress-related gene transcripts were quantified by real-time PCR. (TIF 645 kb

Additional file 1: of IFNγ and TNFα synergistically induce apoptosis of mesenchymal stem/stromal cells via the induction of nitric oxide

Figure S1. Effect of a combination of IFNγ and TNFα on the inhibition of autophagy in MSCs. Representative Western blot of LC3I/LC3II and p62 from both wild-type and iNOS−/− BM-MSCs pretreated or not with IFNγ/TNFα (10 ng/ml each) for 16 h and treated with chloroquine (CQ) for the indicated time. (TIF 212 kb

Additional file 2: of IFNγ and TNFα synergistically induce apoptosis of mesenchymal stem/stromal cells via the induction of nitric oxide

Figure S2. IFNγ/TNFα induce ER stress in BM-MSCs dependently iNOS activity. (a and b) Both wide type and Fas−/−, BM-MSCs were treated with IFNγ/TNFα (10 ng/ml each) in the absence or presence (1 h pre-incubation) of L-NMMA (1 mM) for 24 h, ER stress-related transcripts were quantified by real-time PCR. (TIF 316 kb

TSCs display thymus identity.

<p>(a) RNAs were extracted from TSC2, 1307-6.1.7 and mTEC8 cells, and transcripts were detected by RT-PCR for the expression of indicated genes. (b) Immunoblot analysis of CBX4, delta Np63, TAp63 and DNMT3a in extracts of TSC2, mTEC1 and mTEC8 cells. GAPDH was used as a loading control.</p

Established TSC cells express markers of non-hematopoietic stem cells.

<p>(a) Representative spindle-like morphology of TSC clone 2 established from C57BL/6 E14.5 thymus repeated subculture and limiting dilution cloning.(b) Flow cytometric analysis of WT TSC with antibodies to Sca-1, CD29, CD44, CD45, CD73, CD105, CD133, CD80, MHC class I and II.</p

Established Thymic Epithelial Progenitor/Stem Cell-Like Cell Lines Differentiate into Mature Thymic Epithelial Cells and Support T Cell Development

<div><p>Common thymic epithelial progenitor/stem cells (TEPCs) differentiate into cortical and medullary thymic epithelial cells (TECs), which are required for the development and selection of thymocytes. Mature TEC lines have been widely established. However, the establishment of TEPC lines is rarely reported. Here we describe the establishment of thymic epithelial stomal cell lines, named TSCs, from fetal thymus. TSCs express some of the markers present on tissue progenitor/stem cells such as Sca-1. Gene expression profiling verifies the thymic identity of TSCs. RANK stimulation of these cells induces expression of autoimmune regulator (Aire) and Aire-dependent tissue-restricted antigens (TRAs) in TSCs <i>in vitro</i>. TSCs could be differentiated into medullary thymic epithelial cell-like cells with exogenously expressed NF-κB subunits RelB and p52. Importantly, upon transplantation under the kidney capsules of nude mice, TSCs are able to differentiate into mature TEC-like cells that can support some limited development of T cells <i>in vivo</i>. These findings suggest that the TSC lines we established bear some characteristics of TEPC cells and are able to differentiate into functional TEC-like cells <i>in vitro</i> and <i>in vivo</i>. The cloned TEPC-like cell lines may provide useful tools to study the differentiation of mature TEC cells from precursors.</p> </div

TSCs differentiate into Aire-expressing TECs <i>in vitro</i>.

<p>(a) Immunoblot analysis of Aire, delta Np63, DNMT3a, c-Myc, p52 and. RelB in extracts of TSCs stably overexpressed with p52 and RelB for 11 days. (b) Immunofluorescence analysis for UEA-1 and K8 in TSCs stably overexpressed with p52 and RelB for 11 days.</p

TSCs express cell surface markers of TEPCs.

<p>(a) Flow cytometry analysis of WT TSC line with anti-K5, anti-K8, anti-MTS24, anti-MTS10, anti-CDC205, anti-EpCAM1, 3T3 cells as a negative control for anti-CD205 and anti-EpCAM1. (b) Immunostaining of WT TSC line and 1307-6.1.7 cells with anti-K5 (green), anti-K8 (blue), anti-EpCAM1 (green), anti-Aire (red). (c) Immunostaining of WT TSC line with anti-K8 (blue) and anti-pan-cytokeratin (green).</p

TSCs express Aire and tissue-restricted antigens after stimulation.

<p>(a) RT-PCR analysis for the expression of <i>aire</i>, aire-dependent <i>i-fabp</i> and aire-independent <i>crp</i> and <i>col2</i> in non-cloned WT TSC cells and cloned TSC cells (TSC2) treated with agonistic antibody to RANK (50 ng/ml) for 4 days. <i>Tubulin</i> was used as loading control. The data represented three individual experiments with similar results. (b) Quantitative PCR of mRNA expression for <i>aire</i>, <i>spt1</i> and <i>crp</i> in TSC cells treated with agonistic antibody to RANK (50 ng/ml) for 4 days. <i>Tubulin</i> was used as a reference for data normalization. Bar graphs showed means ± standard deviations of at least three independent experiments. * p < 0.05. (c and d) Immunoblot analysis of Aire in extracts of 1307-6.1.7 cell line or TSCs treated with agonistic mAb to RANK and/or agonistic mAb to LTβ receptor, TSA (0.3 µM), AZA (0.3 µM) (LTβ represents mAb to LTβ receptor; RANK represents agonistic antibody to RANK). Tubulin was used as a loading control. Data represent three independent experiments with similar results.</p

TSCs can partially support the T lymphocytes differentiation in vivo.

<p>(a) Flow cytometric analysis of thymocyte subset distribution in thymus-like tumors and corresponding spleens of nude mice 7 weeks after engraftment with re-aggregates containing thymocytes with TSCs or MEFs as defined by CD4, CD8, B220, and CD3. (b) Gross anatomy of kidneys engrafted 7 weeks earlier with MEFs (as negative control), wild type E14.5 thymus (as positive control), re-aggregates of 1×10⁴ TSCs plus MEFs or 2×10⁵ TSCs plus MEFs. (c) Flow cytometric analysis of lymphocyte subset distribution in thymus-like tumors and corresponding spleens of nude mice grafted with re-aggregates containing 1×10⁴ TSCs plus MEFs or 2×10⁵ TSCs plus MEFs as defined by CD4, CD8, B220, and CD3. (d) Frequencies of T cell populations (D3⁺ cells, CD4⁺ cells and CD8⁺ cells) in spleens of nude mice engrafted with TSC cells or MEF cells. * p < 0.05. Data represented the means ± standard deviations of three independent experiments with at least three mice per group. (e) Immunostaining of the reconstituted thymus-like tumor with anti-K8, anti-K14 and UEA-1-biotin. All data represent three individual experiments with similar results.</p