Project description:To investigate how ex vivo culture affects chromatin accessibility in cultured HSC, we performed the Assay for Transposase Accessible Chromatin with high-throughput sequencing (ATAC-Seq) on cLT (CD34+CD90+CD45RA-) and cST populations purified from 8 day cultured lineage depleted cord blood (lin- CB) cells treated with 3-Factor (4HPR+UM171+SR1), U+S or 4HPR as well as untreated and vehicle-treated (DMSO) control populations. The subsequent ATAC-seq data was compared to chromatin accessibility signatures generated from uncultured hematopoietic stem and progenitor populations (Takayama, et al.). We found that ex vivo culture shifted cLT and cST cells isolated from control or untreated samples to a chromatin accessibility profiles not found in LT-HSC, suggesting some loss of a stem-cell associated chromatin state. By contrast, 4HPR-treated, to some extent, and 3-Factor-treated HSC maintained chromatin accessibility features of uncultured LT-HSC.
Project description:To investigate the efficacy of nicotinamide treatment using our ex-vivo primary lymphocyte model, we performed high-throughput RNA sequencing on libraries generated from untreated and nicotinamide treated samples. PBMC isolated from FRDA affected individuals were cultured to prepare the primary lymphocyte cell lines. The primary cultured cells were either treated with 10mM nicotinamide or without the addition of drug during the 3-days treatment. RNA was extracted after the treatment and then RNA-seq libraries were generated by standard protocols.
Project description:The CD4+ regulatory T (Treg) cell lineage comprises thymus-derived (t)Treg cells and peripherally induced (p)Treg cells. As a model for Treg cells, studies employ TGF-β-induced (i)Treg cells generated from CD4+ conventional T (Tconv) cells in vitro. Here, we describe the relationship of iTreg cells to tTreg and Tconv cells. Proteomic analysis revealed that iTreg, tTreg and Tconv cell populations each have a unique protein expression pattern. iTreg cells had very limited overlap in protein expression with tTreg cells, regardless of cell activation status and instead shared signaling and metabolic proteins with Tconv cells. tTreg cells had a uniquely modest response to CD3/CD28-mediated stimulation. As a benchmark, we used a previously defined proteomic signature that sets ex vivo naïve and effector phenotype Treg cells apart from Tconv cells and includes unique Treg cell properties (Cuadrado et al., Immunity, 2018). This Treg cell core signature was largely absent in iTreg cells. We also used a proteomic signature that distinguishes ex vivo effector Treg cells from Tconv cells and naïve Treg cells. This effector Treg cell signature was partially present in iTreg cells. In conclusion, iTreg cells are distinct from tTreg cells and share limited features with ex vivo Treg cells at the proteomic level.
Project description:To investigate the efficacy of nicotinamide treatment using our ex-vivo primary lymphocyte model, we performed high-throughput RNA sequencing on libraries generated from untreated and nicotinamide treated samples.
Project description:Lymph node stromal cells were isolated from young C57Bl6/J mice and ex-vivo expanded and purified for gene expression analysis. The aim was to assess gene expression patterns for cultured FRCs. Two replicate samples of untreated young (4-6 weeks old) C57BL6/J mice
Project description:RNA-seq analysis was performed to compare differentially expressed genes in freshly isolated and ex-vivo cultured human cord blood CD34+ cells. Mitochondrion related genes are upregulated in CD34+ hematopoietic stem and progenitor cells upon ex vivo culture. In vivo transplantation experiments demonstrate that stemness of CD34+ cells is significantly decreased due to oxidative stress induced by ex vivo culture.
Project description:Human naïve CD4+ T cells (CD4+ CD45RA+ CD25- CD45RO- CD8- CD14- CD15- CD16- CD19- CD34- CD36- CD56- CD123- TCRγ/δ- HLA-DR- and CD235a-) were magnetically negatively isolated from peripheral blood. Cells were stimulated with anti-CD3/anti-CD28 antibodies plus IL-2, and samples were taken at 6h, 24h, 48h and 6d of stimulation. Mock stimulation control cells (sample group G02) received no further compounds, whereas induced regulatory T cells (iTregs) were either differentiated under addition of TGF-b (sample group G03) or TGF-b + retinoic acid + rapamycin (sample group G05). As control, naïve CD4+ T cells were left unstimulated (0h; sample group G01). Ex vivo isolated CD25-high cells were included as positive control for the Treg signature (“nTreg”; sample group G07). Tregs were defined by expression of FOXP3, the “master” transcription factor of Tregs. Samples from 3 male healthy donors (age 34 to 38 years) were prepared with the Qiagen Allprep kit and protein precipitate was solubilized (5 min, 95°C) in freshly prepared buffer containing 4% (w/v) SDS, 25 mM HEPES pH 7.6, 1mM DTT. Samples were prepared using the FASP assay and peptides were labeled with TMT 10-plex reagents and MS data acquired on a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer.
Phenotyping, stability and functional analyses for iTregs induced under these conditions are available in Schmidt A et al., PLoSONE 2016, PMID: 26886923). In the publication associated to this dataset, the time-course proteomic profiling during human Treg differentiation is presented and integrated with RNA-Seq data from the same cells (including additional iTreg culture conditions and 2h time points for RNA-Seq). The data underwent clustering, network analysis and disease enrichment, which revealed many known regulators of Tregs along with novel candidate genes putatively involved in FOXP3 induction, the biological importance of which was validated with a targeted shRNA screen.