Project description:Background: Obesity is characterized as a disease that directly affects the whole-body metabolism and is associated with excess fat mass and several related comorbidities. Dynamics of the adipocyte hypertrophy and hyperplasia play an important role in health and disease, especially in obesity. Human adipose-derived stem cells (hASC) represent an important source for understanding the entire adipogenic differentiation process. However, little is known about the triggering step of adipogenesis in hASC. Here, we performed a proteogenomic approach for understanding the protein abundance alterations expression changes during the initiation of the adipogenic differentiation process. Methods: hASC were isolated from adipose tissue from three donors, characterized and expanded. Cells were cultured for 24 hours in adipogenic differentiation medium followed to protein extraction. We used shotgun proteomics to compare the proteomic profile of 24h-adipogenic differentiated and undifferentiated hASC. Besides, we used our previously next-generation sequencing data (RNA-seq) from the total and polysomal mRNA fractions of hASC to study post-transcriptional regulation during the initial steps of adipogenesis. Results: We identified a total of 3,.420 proteins out of and 48,.336 peptides, being 92 exclusively identified proteins in the undifferentiated hASC and 53 exclusive proteins in 24h-differentiated cells. Using a stringent criterion, we identified 33 differentially abundant proteins when compare 24h-differentiated versus undifferentiated hASC (14 upregulated and 19 downregulated, respectively). Among the upregulated proteins, we shortlist identified several adipogenic-related proteins. Combined analysis of the proteome and the transcriptome allowed the identification of positive correlation coefficients between proteins and mRNAs. Conclusions: These results demonstrate a specific proteome profile related to adipogenesis at the very beginning (24h) of the differentiation process in hASC, which represents an important piece for a better understanding of human adipogenesis and obesity. In addition, the adipogenic differentiation is fine-tuning regulated at transcriptional, post-transcriptional and post-translational levels.
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:Examination of EBf1 binding by ChIP-seq in differentiated human adipose stromal cell (hASC) pre-adipocyte Pre-Adipocytes differentiated in-vitro were fixed in 1% formaldehyde for 15 min at room temperature and quenched for 5 min by adding glycine to a final concentration of 0.125 M. ChIP assays were then performed with custom-made EBF1 antibody or rabbit IgG . ChIP-sequencing libraries were prepared using NEBnext chip-seq library Prep master mix set from 5 ng of anti-EBF1 and anti-IgG ChIP DNA, respectively. Sequence data were generated with Illumina HiSeq 2000 single-read sequencing and aligned against the human genome (hg19, NCBI).
Project description:Human abdominal adipose tissue was obtained with informed consent from a 33-year old Caucasian female (BMI = 32.96 Kg/m2) undergoing lipoaspiration. Adipose stromal cells (hASCs) were isolated and differentiated into adipocytes in vitro. Two technical replicates from 9 time points relative to induction of adipogenesis (day 0). Also, one sample from pre-adipocytes (day -2) grown without FGF.
Project description:Owing to the risk of insertional mutagenesis, viral transduction has been increasingly replaced by nonviral methods to generate induced pluripotent stem (iPS) cells. We report the use of âminicircleâ DNA, a vector type that is free of bacterial DNA and capable of high expression in cells. Here we use a single minicircle vector to generate transgene-free iPSCs from adult human adipose stem cells. (Note: Our Nature Methods publication included analysis of array data from GSM378832 (Foreskin), GSM378833-GSM378838 (JT-iPSC), and GSM378817-GSM378820 (H1, H7, H9, H13, H14) in conjunction with this series). Total RNA from human adipose stem cells (hASC, n = 3 replicate samples), hASC-derived iPS cells using lentiviral factors (lenti-iPSC, n = 3 replicate samples), and minicircle-derived human iPS cells (mc-iPSC, n = 3 subclones from adipose tissue of three individual patients) was hybridized to nine Affymetrix GeneChip Human Genome U133 Plus 2.0 Arrays.
Project description:Human abdominal adipose tissue was obtained with informed consent from a 33-year old Caucasian female (BMI = 32.96 Kg/m2) undergoing lipoaspiration. Adipose stromal cells (hASCs) were isolated and differentiated into adipocytes in vitro. Two technical replicates from 9 time points realtive to induction of adipogenesis (day 0). Also, two technical replicated from pre-adipocytes (day -2) grown without FGF. Two individual replicates (day 1 and day 7) were removed due to failed hybridizations.