Gene expression profiling of skin adipocyte and adipocyte derived cells in mice 5 days after injury
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ABSTRACT: Cells were isolated from day 5wounds from dorsal skin wound beds of 7-9 weeks old using FACS. This experiment describes the gene expression profile associated with mature adipocytes, adipocyte-derived cells and myofibroblasts during tissue repair.
Project description:Lineage negative (CD45- & CD31-) cells were isolated from uninjured skin and day 5 dorsal skin wound beds of 7-week-old AdipoqCre; mT/mG mice using FACS. This experiment describes multiple subsets of wound bed myofibroblasts and identifies that Adipoq-traced cells contribute to the myofibroblast pool in wound beds.
Project description:APs were isolated from naïve skin and day 5wounds from dorsal skin wound beds of 7-9 weeks old using FACS. This experiment describes changes in AP gene expression associated with injury and subsequent tissue repair.
Project description:Mesenchymal cells were isolated from day 5 dorsal skin wound beds of 7-weeks old and >24-months old using FACS. This experiment describes multiple unique subsets of wound bed myofibrolbasts capable of contributing to tissue repair.
Project description:Cells were isolated from day 5wounds from dorsal skin wound beds of 7-9 weeks old using FACS. This experiment describes the gene expression profile associated with different immune cell subsets during tissue repair.
Project description:Cells were isolated from wounded skin tissue and administered into single cell RNA transcriptome sequencing to deconstruct cell hierarchy of dermal fibroblast population and underlying regulating mechanisms. In brief, 7~9 weeks old male C57BL/6 mice were anesthetized and one full-thickness wound (1.0 cm x 0.5 cm in size) was generated on shaved dorsal skin. Animals were sacrificed at indicated intervals after and wound bed tissues were collected for cell isolation and subsequent 10x Genomics single cell RNAseq. Sequencing was performed on Illumina Novaseq6000 platform (Illumina). The generated raw gene expression matrix was converted into Seurat objects using the R package Seurat v3.0. After low quality cells and outliers were discarded, 10233 cells from non-lesional skin, 7807 cells from wound beds at post-wounding day 2 (PWD2), and 4925 cells from wound tissue at PWD7 were used for downstream analysis. Next, unsupervised clustering and gene expression were visualized with the Seurat 3.0 on R studio, and assignment and identification of cell clusters was based on the expression of validated marker genes. Subsequent in-depth analysis was assisted by GENE DENOVO Inc (Guangzhou, China). Together, our study has identified a specific dermal fibroblast population with high DPP4, Sca1 and WNT2 expression, which is committed into adipocyte upon wounding, and define the differentiation trajectory of this adipocyte precursor during skin regeneration.
Project description:Adipocytes in dermis are considered to be important participants in skin repair and regeneration, but the role of subcutaneous white adipose tissue (sWAT) in skin repair is poorly understood. Here, we revealed the dynamic changes of sWAT during wound healing process. Lineage tracing mouse studies revealed that adipocytes from sWAT would migrate into the wound bed and participate in the formation of granulation tissue. Moreover, sWAT undergoes beiging after skin injury. Inhibition of sWAT beiging by genetically silencing PRDM16, a key regulator to beiging, hindered wound healing process. The transcriptomics results suggested beige adipocytes in sWAT abundantly express neuregulin 4 (Nrg4) which regulated macrophage polarization and the function of myofibroblasts. In diabetic wounds, the beiging of sWAT was significantly suppressed. Thus, adipocytes from sWAT regulate multiple aspects of repair and may be therapeutic for inflammatory diseases and defective wound healing associated with aging and diabetes.
Project description:Abstract Background In obesity, adipose tissue undergoes a remodeling process characterized by increased adipocyte size (hypertrophia) and number (hyperplasia). The individual ability to tip the balance toward the hyperplastic growth, with recruitment of new fat cells through adipogenesis, seems to be critical for a healthy adipose tissue expansion, as opposed to the development of inflammation and detrimental metabolic consequences. However, the molecular mechanisms underlying this fine-tuned regulation are far from being understood. Methods We analyzed by mass spectrometry-based proteomics visceral white adipose tissue (vWAT) samples collected from C57BL6 mice fed with a HFD for 8 weeks. A subset of these mice, called low responders (LowR HFD), showed a low susceptibility to the onset of adipose tissue inflammation, as opposed to their HFD counterpart. We identified the discriminants between LowR HFD and HFD vWAT samples and explored their function in Adipose Derived human Mesenchymal Stem Cells (AD-hMSCs) differentiated to adipocytes. Results We quantified 6051 proteins. Among the candidates that most differentiate LowR HFD from HFD vWAT, we found proteins involved in adipocyte function, including adiponectin and hormone sensitive lipase, suggesting that adipocyte differentiation is enhanced in LowR HFD, as compared to HFD. The chromatin modifier SET and MYND Domain Containing 3 (SMYD3), whose function in adipose tissue was totally unknown, was another top-scored hit. SMYD3 expression was significantly higher in LowR HFD vWAT, as confirmed by western blot analysis. In vitro, we found that SMYD3 mRNA and protein levels decrease rapidly along the differentiation process of AD-hMSCs. Moreover, SMYD3 knock-down at the beginning of adipocyte differentiation resulted in reduced cell proliferation and, at longer term, reduced lipid accumulation in adipocytes. Conclusions Our study describes for the first time the role of SMYD3 as a regulator of adipocyte proliferation during the early steps of adipogenesis.
Project description:FACS-purified adipocyte progenitors from murine subcutaneous adipose tissue were cultured under conditions promoting general adipogenic differentiation or beige/brite adipocyte differentiation (treatment with cPGI2). Time course expression profiling was performed during differentiation. In addition, some cultures of differentiated adipocytes were stimulated with norepinephrine for 3 hours. In parallel, differentiation and norepinephrine stimulation of progenitors from interscapular brown fat was performed and profiled.
Project description:The diverse transcriptional mechanisms governing cellular differentiation and development of mammalian tissue remains poorly understood. Here we report that TAF7L, a paralogue of TFIID subunit TAF7, is enriched in adipocytes and mouse white fat tissue (WAT). Depletion of TAF7L reduced adipocyte-specific gene expression and compromised adipocyte differentiation as well as WAT development. Ectopic expression of TAF7L in myoblasts reprograms these muscle precursors into adipocytes upon induction. Genome-wide mRNA-seq expression profiling and ChIP-seq binding studies confirmed that TAF7L is required for activating adipocyte-specific genes via a dual mechanism wherein it interacts with PPARM-NM-3 at enhancers and TBP/Pol II at core promoters. In vitro binding studies confirmed that TAF7L forms complexes with both TBP and PPARM-NM-3. These findings suggest that TAF7L plays an integral role in adipocyte gene expression by targeting enhancers as a cofactor for PPARM-NM-3 and promoters as a component of the core transcriptional machinery. Genome-wide mapping of TAF7L and additional factors, and mRNA-seq expression profiling prior to and following mouse adipocyte differentiation.