ABSTRACT: Comparson of Biphenotypic hepatocytes with Mature Hepatocytes Biphenotypic hepatocytes were isolated from DDC-injured liver as Sox9+EpCAM- cells. Gene expression profile of biphenotypic hepatocytes were compared with that of Mature hepatocytes.
Project description:Transcriptional profiling of human preadipocytes comparing preadipocytes cultured in control media vs co-culture with PBMC's after 3 days. Goal was to elucidate novel expression patterns in preadipocytes during exposure to human immune cells. 2-condition experiment, Preadipocytes+Media vs Preadipocytes+PBMC. Biological replicates: 4 experimental replicates.
Project description:During a T cell response, naïve CD8 T cells differentiate into effector cells. Subsequently, a subset of effector cells termed memory precursor effector cells (MPECs) further differentiates into functionally mature memory CD8 T cells. The transcriptional network underlying this carefully scripted process is not well understood. Here, we report that the transcription factor FoxO1 plays an integral role in facilitating effector to memory transition and functional maturation of memory CD4 and CD8 T cells. We find that FoxO1 is not required for differentiation of effector cells, but in the absence of FoxO1, memory CD8 T cells displayed features of scenescence and progressive attrition in polyfunctionality, which in turn led to impared recall responses and poor protective immunity. These data suggest that FoxO1 is essential for active maintenance of functional CD8 T cell memory and protective immunity. Under competing conditions in bone marrow Single-cell suspensions from splenocytes of eight samples WT (control) and FoxO1-/- (experimental) LCMV-immune mice were prepared using standard procedures. CD8 T cells were then isoloated using Thy1.2 (CD90.2) (30-H12) microbeads (Miltenyi Biotec). Cells were then stained with anti-CD8, anti-CD44 and Db/NP396 MHC class I tetramer. Activated (CD8+CD44hi), naive (CD8+CD44lo), and virus-specific CD8 T cells were sorted using FACSAria II instrument (BD Biosciences). The purity of the cells was >95%. Total RNA was extracted from the sorted cells by Trizol Reagent. RNA samples were reverse transcribed and Cy3-labeled cDNAs were hyrbidized to Agilent whole Mouse Genome Oligo Microarrays. Fluorscence signals were detected using Agilent's Microarray Scanner system, data was analyzed using the Rosetta Resolver gene expression data analysis system and genes with a fold change < and p-values <0.01 were identified. Microarray data discussed in the paper is focused on virus-specific memory CD8 T cells from samples WT_Tet_2 vs KO_Tet_2.
Project description:The role of B cells after transplant regarding allograft rejection or tolerance has become a topic of major interest. Recently, in renal transplant recipients, a B cell signature characterized by the overexpression of CD19+CD38hiCD24hi transitional B cells has been observed in operationally tolerant patients and in Belatacept treated patients with significant lower incidence of donor specific antibodies. The phenotypic and functional characterization of these transitional B cells is far to be exhaustive. We present the first transcriptomic and phenotypic analysis associated with this phenotype. Three populations were studied and compared: (i) transitional CD24hiCD38hi (ii) CD24+CD38- and (iii) CD24intCD38int populations. Peripheral blood mononuclear cells were isolated from 5 healthy donors and CD20+ B cells were isolated by magnetic beads. Three B cells populations were then sorted by flow cytometry: CD19+CD24hiCD38hi (test), CD19+CD24+CD38- (control 1) and CD19+CD24intCD38int (control 2)
Project description:Much remains unknown about the signals that induce early mesoderm to initiate hematopoietic differentiation. Here we show that endoglin (Eng), a receptor for the TGFβ superfamily, identifies all cells with hematopoietic fate in the early embryo. These arise in an Eng+Flk1+ mesodermal precursor population at E7.5, a cell fraction also endowed with endothelial potential. In Eng knockout embryos, hematopoietic colony activity and numbers of CD71+Ter119+ erythroid progenitors were severely reduced. This coincided with severely reduced expression of embryonic globin and key BMP target genes including the hematopoietic regulators Scl, Gata1, Gata2 and Msx-1. To interrogate molecular pathways active in the earliest hematopoietic progenitors, we applied transcriptional profiling to sorted cells from E7.5 embryos. Eng+Flk-1+ progenitors co-expressed TGFβ and BMP receptors and target genes. Furthermore, Eng+Flk-1+ cells presented high levels of phospho-SMAD1/5, indicating active TGFβ and/or BMP signaling. Remarkably, under hematopoietic serum-free culture conditions, hematopoietic outgrowth of endoglin-expressing cells was dependent on TGFβ superfamily ligands: BMP4, BMP2, or TGF-β1. These data demonstrate that the E+F+ fraction at E7.5 represents mesodermal cells competent to respond to TGFb1, BMP4, or BMP2, shaping their hematopoietic development, and that endoglin is a critical regulator in this process by modulating TGF/BMP signaling. E7.5 pooled embryos (25 litters; 300 embryos approximately) were dissected and 3,000 cells were sorted in triplicate for Eng-Flk1-, Eng-Flk1+, Eng+Flk1+, and Eng+Flk1- fractions. Microarray results were analyzed with GeneSpring GX software.
Project description:Expression profiling of hepatocytes-derived ductal cells with properties intermediate between mature hepatocytes and cholangiocytes. Chimeric adult mice were generated where mature hepatocytes were marked with a fluorescent red marker. Chronic injury was induced for ~6weeks and three cell types were isolated by FACS (Influx, BD) for expression analysis by RNAseq based on cell surface phenotype and origin: hepatocytes (n=3), hepatocyte-derived oval cells (1c3+, n=5), and cholangiocyte-derived oval cells (1c3+, n=5).
Project description:Analysis of Hoechst dye 33342-effluxing side population (SP) cells from B-CLL peripheral blood mononuclear cells. 9 biological replicates from B-CLL patients sorted into CD5+CD19+ SP and non-SP subsets. Two color comparative gene expression using Agilent microarrays.
Project description:By employing single-cell transcriptional profiling, we uncovered that mature hepatocytes re-activate reprogramming/progenitor-related genes (RRG) and dedifferentiate to liver progenitor-like cells (LPLC). Because we found macrophages regulated hepatocyte dedifferentiation, We also dissected the transcriptional profiling of hepatocytes in single-cell level during DDC injury after macrophage-depletion via clondronate liposomes. Besides, we uncovered the heterogeneity of liver macrophages in normal and DDC-injured liver via scRNA-seq as well.
Project description:In this experiment, dendritic cells and monocytes obtained from melanoma patients who underwent immunotherapy were stimulated with a maturation cocktail then either treated or not treated with denlieukin diftitox (DD; ONTAK) and then subjected to transcription profiling to investigate the effects of DD on the maturation and activation of dendritic cells and monocytes.