Project description:To identify the functional non-coding RNAs in human DC development, we explored the gene expression profiles during DC development from peripheral monocytes. Using RNA-seq, we identified many annotated lncRNAs with markedly altered expressions during human DC differentiation and maturation. RNA samples from human peripheral blood monocytes and monocyte-derived DCs were collected. RNAs from 3 donors were pooled together to form monocyte sample or DC sample and then these two samples were subjected to RNA-seq and analysis.
Project description:Transcriptome analysis of monocytes directly exposed to cell-to-cell contact with Natural Killer (NK) cells or separated by a transwell membrane and their subsequent monocyte-derived dendritic cells. The role of Natural Killer (NK) cells in the early differentiation of monocytes into dendritic cells (DCs) is poorly understood. Their interaction is thought to be restricted to a bilateral cross talk of soluble cytokines. However, many authors have shown that NK cells effect over myeloid cells is dependent on direct cell-to-cell contact. In order to understand what determines a major effect of NK cells over monocytes and their derived DCs, total RNA samples from purified monocytes and monocyte-derived DCs, exposed to direct contact with NK cells or separated by a transwell membrane were amplified and hybridized to the Affymetrix Human GeneChip 2.0 ST array. Analyses were performed using BRB-ArrayTools version 4.5.0, developed by Dr. Richard Simon and the BRB-ArrayTools Development Team. We found that compared to untouched monocytes, cell-to-cell contact with NK cells modified the expression of 283 genes whereas contact through a transwell membrane modified the expression of 35 genes. After differentiation and compared to DCs derived from untouched monocytes, DCs derived from monocytes allowed to direct cell-to-cell contact with NK cells had the expression of 128 genes modified whereas DCs derived from monocytes primed through a transwell membrane had only 2 genes modified. Our results suggested that cell-to-cell interactions with NK cells are important to imprint a stable transcriptional program in monocytes lasting even after their further differentiation into DCs.
Project description:We report the application of single-molecule-based sequencing technology for high-throughput profiling of histone modifications in human monocyte devried DCs. By obtaining about 15 million mapped reads for each sample from chromatin immunoprecipitated DNA, we generated genome-wide chromatin-state maps of human monocytes and monocyte-derived DCs. This study provides a framework for the application of comprehensive chromatin profiling towards characterization of diverse mammalian cell populations.
Project description:In mice, two restricted DC progenitors, macrophage-dendritic progenitor (MDP) and common dendritic cell progenitor (CDP) demonstrate increasing commitment of DC lineage as they sequentially lose granulocyte and monocyte potential respectively. Identifying these progenitors has enabled understanding of the role of DCs and monocytes in immunity and tolerance in mice. In humans, however, restricted monocyte and DC progenitors remain unknown. Progress in studying human DC development has been hampered by lack of an in vitro culture system that recapitulates in vivo DC hematopoiesis. Here we report a culture system that supports development of CD34+ hematopoietic stem cell progenitors into the three major human DC subsets, monocytes, granulocytes, NK and B cells. Using this culture system we defined the pathway for human DC development, and revealed the sequential origin of human DCs from increasingly restricted progenitors: a granulocyte-monocyte-DC progenitor (hGMDP) that develops into a monocyte-DC progenitor (hMDP) that develops into monocytes and a common DC progenitor (hCDP) that is restricted to produce the three major DC subsets. The phenotype of the DC progenitors partially overlaps with granulocyte monocyte progenitors (GMPs). These progenitors reside in human cord blood and bone marrow but not in the blood or lymphoid tissues in the steady state. We performed whole transcriptome expression analysis on monocytes and subsets of dendritic cells i.e. CD1c+ DCs, CD141+ DCs and CD303+ pDCs isolated from blood or differentiated in culture from cord blood CD34+ cells in presence of MS5 stromal cells and Flt3l, GM-CSF and SCF cytokines.