Project description:human Multipotent Adipose-Derived Stem (hMADS) cells constitute a highly interesting in vitro model to study adipocyte development in vitro. Using a chemically defined medium with several hormonal inducers, these cells can be differentiated into mature adipocytes within ~2 weeks. To gain a global view on transcripts which are induced or downregulated at various stages of adipocyte differentiation, we performed a microarray analysis with samples harvested at the stem cell stage, the terminally differentiated stage, as well as various time points in between.
Project description:The Mesoderm-specific transcript homolog protein MEST is a member of the alpha/beta hydrolase fold family that is expressed in adipose tissue, adipocytes as well as their precursor cells. To get an idea about potential functions of MEST in adipocyte development, we knocked down MEST in human Multipotent Adipose-Derived Stem (hMADS) cells, induced adipocyte differentiation by a chemically defined medium and performed gene expression profiling at the early stage of adipocyte differentiation.
Project description:Human multipotent adipose-derived stem (hMADS) cells were transfected at confluence with miR-26a or miR-C (non-targeting control microRNA). Two days later (=d0), adipocyte differentiation was induced. At d9 after start of differentiation, cells were harvested to identify mRNAs that are down- and upregulated by miR-26a versus miR-C.
Project description:Directed differentiation of cells in vitro is a powerful approach for dissection of developmental pathways, disease modeling and regenerative medicine, but analysis of such systems are complicated by heterogeneous and asynchronous cellular responses to differentiation-inducing stimuli. To enable deep characterization of heterogeneous cell populations, we developed an efficient digital gene expression profiling protocol that enables surveying of mRNA in thousands of single cells at a time. We then applied this protocol to profile 11,116 cells collected during directed differentiation of human adipose-derived stem/stromal cells. The resulting data reveals the major axes of cell-to-cell variation within and between time points and suggests a link between incomplete adipogenesis in vitro and adipocyte dysfunction in vivo. High-throughput single cell RNA-seq method applied to human adipose tissue-derived stromal/stem cells during differentiation towards an adipogenic fate
Project description:The Mesoderm-specific transcript homolog protein MEST is a member of the alpha/beta hydrolase fold family that is expressed in adipose tissue, adipocytes as well as their precursor cells. To get an idea about potential functions of MEST in adipocyte development, we knocked down MEST in human Multipotent Adipose-Derived Stem (hMADS) cells, induced adipocyte differentiation by a chemically defined medium and performed gene expression profiling at the early stage of adipocyte differentiation. Two-condition experiment, hMADS cells at day 3 after induction of adipocyte differentiation, comparison of cells transfected with an siRNA targeting MEST (siMEST) to cells transfected with a control siRNA (siC). Biological replicates: 3, indepently grown and harvested. On each array, one biological replicate of siMEST-transfected cells was directly compared to one biological replicate os siC-transfected cells (serving as reference sample). All hybridizations were repeated with reversed dye assignment (dye-swap) as technical replicates.
Project description:Mechanisms involved in adipose tissue expansion is of great interest in development of obesity and associated metabolic complications. Mesenchymal stem cell (MSC) differentiation in to adipocytes provides a model system to understand various gene expression pattern involved at different stages of adipogenesis. In this project we used bulk RNASeq to identify the genes that are selectively expressed at 3 different time points of adipocyte differentaition from MSCs.
Project description:Changes of gene expression in primary cell cultures of salmon adipose-derived stromal-vascular fraction were studied at six time-points that covered the key differentiation events: confluence, clonal expansion, determination and establishment of the mature adipocyte phenotype.
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:Directed differentiation of cells in vitro is a powerful approach for dissection of developmental pathways, disease modeling and regenerative medicine, but analysis of such systems are complicated by heterogeneous and asynchronous cellular responses to differentiation-inducing stimuli. To enable deep characterization of heterogeneous cell populations, we developed an efficient digital gene expression profiling protocol that enables surveying of mRNA in thousands of single cells at a time. We then applied this protocol to profile 11,116 cells collected during directed differentiation of human adipose-derived stem/stromal cells. The resulting data reveals the major axes of cell-to-cell variation within and between time points and suggests a link between incomplete adipogenesis in vitro and adipocyte dysfunction in vivo.
Project description:Post-transcriptional regulation of gene expression accomplished by microRNAs (miRNAs) importantly affects the complex gene regulatory network. In particular, miRNAs are known to be involved in recurrent motifs named miRNA-mediated feed forward loops (FFLs) where a transcription factor (TF) regulates a miRNA and they both regulate the expression level of a target RNA. Here, we focus on the identification of active FFLs during adipogenic differentiation. A list of putative feed-forward loops was generated based on sequence analysis of conserved and overrepresented motifs in the regulatory regions. Since this approach is not specific for adipogenesis and is known to generate false positive feed-forward loops, an experiment was designed to select active ones based on their dynamics using a model-based approach. Microarray time series of gene and miRNA expression data were collected at seven time points on human multipotent adipose-derived stem (hMADS) cells upon adipogenic differentiation. Three different dynamic models, sharing the same FFL topology but incorporating descriptions of increasing complexity of miRNA and mRNA dynamics, are identified on miRNA and mRNA expression data and compared based on identification criteria, namely: goodness of fit, precision of the estimates and comparison with submodels. 24 FFLs, able to properly reproduce data, are selected as active out of the 329 putative ones. This method considerably reduces the search space for new interactions between TFs, miRNAs and mRNAs and provides interesting biological results identifying genes known from the literature to be regulators in adipogenesis and adipocyte-related functions that can be interpreted as positive control of the validity of the apporoach. Therefore, the genes in the selected FFLs that are not yet known to be involved in this context are potential novel players in this regulatory network of adipogenesis and adipocyte function. Two independent cell culture experiments were performed as biological replicates during adipogenic differentiation of human mesenchymal stem cell. Cells where harvested at the pre-confluent stage as reference (day -3) and at seven subsequent time points during human adipogenic differentiation: day -2 and 0 before, and 1, 2, 5, 10, 15 days after induction of differentiation. All hybridizations were repeated with reversed dye assignment (dye-swap).