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IntDCs versus LCs

ABSTRACT: miRNA profiling of CD34+ derived, in vitro-generated Langerhans cells (LCs) and interstitial-type dendritic cells (intDCs). Epidermal Langerhans cells (LCs) form a network of dendritic cells (DCs) in basal/suprabasal layers of the skin and are characterized by a unique phenotype (CD207+ CD1abright CD324+ CD11b-). Due to their specific location at body surfaces, LCs are constantly exposed to environmental stimuli. Conversely, interstitial-type DCs (intDCs) are located in adjacent tissues and can be discriminated from LCs phenotypically (CD207- CD1adim CD324- CD11b+). These DCs constitute a second line of defense against pathogens that have crossed the epithelial barrier. During development both DC subsets originally arise from myeloid progenitor cells via monocyte-committed intermediates in response to specific microenvironmental signals. Additionally certain pools of DCs can be replenished by tissue resident cells or monocytes in response to specific microenvironmental signals (2, 4, 5). In vitro generated GM-CSF/IL-4-dependent DCs derived either from CD14+ monocytes or via CD14+CD11b+ monocyte intermediates from CD34+ progenitor cells share many characteristics with intDCs in vivo. On the other hand, LCs generated from CD34+ cells under serum-free TGF-beta1-dependent conditions phenotypically resemble epidermal-resident LCs. Since these two DC subsets are of considerable interest for clinical cell therapy studies, an improved understanding of their development and function is of substantial relevance. To identify differentially expressed miRNAs in DC subsets, we performed a miRNA screen. Therefore, we generated LCs or intDCs from human CD34+ cord blood haematopoietic progenitor cells as described. For intDCs, CD34+ cells (1 x 104 to 2 x 104/ml per well) were cultured in 24-well tissue culture plates in serum free CellGro® DC medium (CellGenix, Freiburg, Germany) supplemented with GM-CSF (100 ng/ml), SCF (20 ng/ml), FL (50 ng/ml) and TNFalpha (2.5 ng/ml) for 3-5 days and subsequently with GM-CSF (100 ng/ml) and IL-4 (2.5 ng/ml) for 4-5 days. For LCs, CD34+ cells (1 x 104 to 2 x 104/ml per well) were cultured in 24-well tissue culture plates in serum free CellGro® DC medium (CellGenix, Freiburg, Germany) supplemented with GM-CSF (100 ng/ml), SCF (20 ng/ml), FL (50 ng/ml), TNFalpha (2.5 ng/ml) and TGF-beta1 (0.5 ng/ml). The well-established immunophenotype of LCs (CD1ahi CD11b- CD207+ CD324+) and intDCs (CD1a+ CD11b+ CD207- CD324-) was confirmed by FACS. These two DC subsets were purified and submitted to differential miRNA profiling.

ORGANISM(S): Homo sapiens

PROVIDER: GSE20215 | GEO | 2010/02/12

SECONDARY ACCESSION(S): PRJNA124139

REPOSITORIES: GEO

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Project description:miRNA profiling of CD34+ derived, in vitro-generated Langerhans cells (LCs) and interstitial-type dendritic cells (intDCs). Epidermal Langerhans cells (LCs) form a network of dendritic cells (DCs) in basal/suprabasal layers of the skin and are characterized by a unique phenotype (CD207+ CD1abright CD324+ CD11b-). Due to their specific location at body surfaces, LCs are constantly exposed to environmental stimuli. Conversely, interstitial-type DCs (intDCs) are located in adjacent tissues and can be discriminated from LCs phenotypically (CD207- CD1adim CD324- CD11b+). These DCs constitute a second line of defense against pathogens that have crossed the epithelial barrier. During development both DC subsets originally arise from myeloid progenitor cells via monocyte-committed intermediates in response to specific microenvironmental signals. Additionally certain pools of DCs can be replenished by tissue resident cells or monocytes in response to specific microenvironmental signals (2, 4, 5). In vitro generated GM-CSF/IL-4-dependent DCs derived either from CD14+ monocytes or via CD14+CD11b+ monocyte intermediates from CD34+ progenitor cells share many characteristics with intDCs in vivo. On the other hand, LCs generated from CD34+ cells under serum-free TGF-beta1-dependent conditions phenotypically resemble epidermal-resident LCs. Since these two DC subsets are of considerable interest for clinical cell therapy studies, an improved understanding of their development and function is of substantial relevance. To identify differentially expressed miRNAs in DC subsets, we performed a miRNA screen. Therefore, we generated LCs or intDCs from human CD34+ cord blood haematopoietic progenitor cells as described. For intDCs, CD34+ cells (1 x 104 to 2 x 104/ml per well) were cultured in 24-well tissue culture plates in serum free CellGroÂ® DC medium (CellGenix, Freiburg, Germany) supplemented with GM-CSF (100 ng/ml), SCF (20 ng/ml), FL (50 ng/ml) and TNFalpha (2.5 ng/ml) for 3-5 days and subsequently with GM-CSF (100 ng/ml) and IL-4 (2.5 ng/ml) for 4-5 days. For LCs, CD34+ cells (1 x 104 to 2 x 104/ml per well) were cultured in 24-well tissue culture plates in serum free CellGroÂ® DC medium (CellGenix, Freiburg, Germany) supplemented with GM-CSF (100 ng/ml), SCF (20 ng/ml), FL (50 ng/ml), TNFalpha (2.5 ng/ml) and TGF-beta1 (0.5 ng/ml). The well-established immunophenotype of LCs (CD1ahi CD11b- CD207+ CD324+) and intDCs (CD1a+ CD11b+ CD207- CD324-) was confirmed by FACS. These two DC subsets were purified and submitted to differential miRNA profiling. Two conditioned experiment LCs vs. intDCs. Biological replicates: 3 LCs, 3 intDCs, independently differentiated, harvested and purified. 3 replicates each were pooled and hybridized on one array. Supplementary files linked below.

Project description:The ontogeny of human Langerhans cells (LCs) remains poorly characterized, in particular the nature of LC precursors and the factors that may drive LC differentiation. Through a systematic transcriptomic analysis of TSLP-activated dendritic cells (DCs), we unexpectedly identified markers that have been associated with a skin-homing potential as well as with a LC phenotype. We performed transcriptomic analysis of TSLP-activated blood DCs, as compared to freshly purified, Medium-, and TNF-activated DCs. Among TSLP up-regulated genes, we identified molecules associated with skin homing, LC phenotype, and LC function, as determined by a literature-based survey. Conversely, genes not expressed in LCs were not found among TSLP-induced genes. Further experiments showed that TGF-? synergized with TSLP leading to the differentiation of blood BDCA-1+ DCs into bona fide Birbeck granule-positive LCs. The ontogeny of human Langerhans cells (LCs) remains poorly characterized, in particular the nature of LC precursors and the factors that may drive LC differentiation. Through a systematic transcriptomic analysis of TSLP-activated dendritic cells (DCs), we unexpectedly identified markers that have been associated with a skin-homing potential as well as with a LC phenotype. We performed transcriptomic analysis of TSLP-activated blood DCs, as compared to freshly purified, Medium-, and TNF-activated DCs. Among TSLP up-regulated genes, we identified molecules associated with skin homing, LC phenotype, and LC function, as determined by a literature-based survey. Conversely, genes not expressed in LCs were not found among TSLP-induced genes. Further experiments showed that TGF-? synergized with TSLP leading to the differentiation of blood BDCA-1+ DCs into bona fide Birbeck granule-positive LCs. Dendritic cells (DC) were purified from the blood of healthy human donors. Each replicate comes from a different donor. There are 6 freshly purified samples (Control_0h), 4 DC samples cultured during 6h with no additional stimulus than the medium (Control_6h), 5 samples cultured during 6h with recombinant TSLP (Thymic Stromal Lymphopoietin at 20 ng/uL) (TSLP_6h) and 3 samples cultured during 6h with recombinant TNF (Tumor necrosis factor at 20 ng/mL) (TNF_6h).

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IntDCs versus LCs

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