BioProject

Human cells produce thousands of lipids that impact biological processes in ways we are only starting to characterize. More...

Human cells produce thousands of lipids that impact biological processes in ways we are only starting to characterize. The cellular composition in lipids changes during differentiation and also varies across individual cells of the same type. Yet, whether and how cell-to-cell differences in lipid composition affect cell phenotypes remain unknown. Here we have measured the lipidomes and transcriptomes of individual human dermal fibroblasts by coupling high-resolution mass spectrometry imaging to single-cell transcriptomics. We find that the cell-to-cell variation of specific lipid metabolic pathways contributes to the establishment of cell states involved in the organization of skin architecture. In fact, sphingolipid composition defines fibroblast subpopulations and its metabolic rewiring drives cell state transitions. These data uncover a role for cell-to-cell lipid heterogeneity in the determination of cell states and reveal a new regulatory component to the self-organization of multicellular systems. Overall design: Dermal human fibroblasts were grown in DMEM supplemented with 10% (v/v) fetal bovine serum (FBS), 4.5 g/l glucose, 2 mM L-glutamine, 1 U/ml penicillin/streptomycin. The cell lines were grown under a controlled atmosphere in the presence of 5% CO2 at 37 °C. Cells were grown in a flask until 90% confluence. The medium was removed and trypsin-EDTA solution (0.05% trypsin, 0.02% EDTA) was added for 3-5 min. Then, the medium was added back to block the protease action, and the cells were collected into a plastic tube. After centrifugation for 5 min at 1200 rpm, the cell pellet was resuspended in fresh medium, gently mixed, and placed in a new plastic flask with fresh growth medium. Bulk RNA sequencing was performed on the following FACS sorted populations of dHFs: Cholera Toxin positive (n=2), ChTxB/ShTxB1a/ShTxB2e positive (n=2), ShTxB1a/ShTxB2e positive (n=3) and ShTxB2e positive (n=1) and to a control unsorted population (n=2). Total RNA was isolated from FACS sorted dHFs populations using Rneasy Mini kits (Qiagen, Germany) according to the manufacturer instructions. The yield and the integrity of the RNA were determined using a spectrophotometer (NanoDrop ND-1000; Thermo Scientific, USA). Total RNA (10ng-1mg, depending on the different population) were submitted for RNA-seq with GENEWIZ, NJ. Libraries were prepared using Illumina HiSeq platform with ultra-low input configuration and sequenced with 2× 150 bp sequencing configuration to a depth of 350 million reads (GENEWIZ, NJ). Sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36. The trimmed reads were mapped to the Homo sapiens GRCh38 reference genome available on ENSEMBL using the STAR aligner v.2.5.2b. scRNA-seq experiments were performed using 10X Genomics Chromium cRNA-seq kit v3.1. following provider instructions to obtain 3500 cells per replicate. Libraries were sequenced at the depth of 300 million reads corresponding to an average of 80k reads per cell. Data was pre-processed using cellranger and velocyto v0.17 and then analyzed using scanpy. Less...