ABSTRACT: This study determined whether human adipose stem cells (hASCs) could be reprogrammed into induced pluripotent stem (iPS) cells using the 4 factors OCT4, SOX2, KLF4, and cMYC. We showed successfull reprogramming could be achieved in which fat/adipose cells were able to assume embryonic-like phenotypes after lentiviral transduction with the 4 factors. Further, we believe these fat cells are intrinsically better-suited for reprogramming compared to fibroblasts, and result in higher yields of iPS cells. Microarray study was performed in duplicates (hASCs, iPS cells derived from hASCs, and hESCs). Reference control was pooled RNA taken from H9 hESCs, embryoid bodies, and some differentiated cell types such as endothelial cells.
Project description:This study determined whether human adipose stem cells (hASCs) could be reprogrammed into induced pluripotent stem (iPS) cells using the 4 factors OCT4, SOX2, KLF4, and cMYC. We showed successfull reprogramming could be achieved in which fat/adipose cells were able to assume embryonic-like phenotypes after lentiviral transduction with the 4 factors. Further, we believe these fat cells are intrinsically better-suited for reprogramming compared to fibroblasts, and result in higher yields of iPS cells. Overall design: Microarray study was performed in duplicates (hASCs, iPS cells derived from hASCs, and hESCs). Reference control was pooled RNA taken from H9 hESCs, embryoid bodies, and some differentiated cell types such as endothelial cells.
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 ‘minicircle’ 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 GSM374067 and GSM374068 in conjunction with this series). Overall design: Total RNA from H7 hESCs (n = 2) and minicircle-derived human iPS cells (n = 2 subclones from adipose tissue of two individual patients) was hybridized to four Agilent Whole Human Genome microarrays. A pooled reference sample was used that contained total RNA from H9 hESCs, H9 embryoid bodies, cardiac and endothelial cells derived from H9 hESCs, cardiac fetal tissue, and human umbilical vein endothelial cells (HUVECs).
Project description:Assays to assess the quality of the reprogramming of human Induced pluripotent stem cells are needed. We have previously shown that hESC can be differentiated into embryonic and fetal type of red blood cells that sequentially express three types of hemoglobins recapitulating early human erythropoiesis. We report here that we have produced iPS from three somatic cell types: adult skin fibroblasts as well as embryonic and fetal mesenchymal stem cells. We show that some of these iPS are fully reprogrammed into a pluripotent state that is undistinguishable from that of hESCs based and low and high-throughput expression analysis and detailed expression of globin expression patterns suggesting that reprogramming with the four original Yamanaka pluripotency factors leads to complete erasure of all functionally important epigenetic marks associated with hematopoietic differentiation regardless of the age or the tissue type of the donor cells. We also report that reprogramming can also lead to abnormal iPS that are undistinguishable from hES cells by morphology, and by the expression of their endogenous genes but that are grossly abnormal in their differentiation potential. The most likely cause of abnormal reprogramming is failure to silence the virally transduced reprogramming factors. The ability to produce large number of erythroid cells with embryonic and fetal-like characteristics is likely to have many translational applications the gene expression in different source of iPS and standard ES cells H1
Project description:In this study, we aimed to gain further insight on the role of glucocorticoids (GCs) in adipocyte differentiation. For the future drugability of candidate targets, it is of utmost importance to find factors relevant to human biology. Thus, we analyzed the transcriptome of GC-induced primary human adipose stem cells (hASCs) isolated from paired subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) to identify novel factors downstream of GC action. We used microarrays to detail the global program of gene expression following GC treatment and identified distinct classes of up- and down-regulated genes during this process. Human preadipocytes (human adipose stem cells) were obtained from lipoaspirates or visceral fat biopsies by enzymatic digestions, followed by several steps of centrifugations (Mikkelsen et. al Cell. 2010 Oct 1;143(1):156-69.). Following isolation, human adipose stem cells were transfected with either siCtrl or siLMO3 oligonucleotides, followed by treatment with hydrocortisone.
Project description:In the current study, we have performed a gene expression analysis of well characterized and defined populations of human adipose-derived stem cells (hASCs) before and after in vitro induction of osteogenic and myogenic differentiation that allows identifying DNA methylation- regulated differentiation genes. We have also address the extent of the epigenetic programming of hASCs- derived differentiated cells by comparing the expression profiling of these cells with their somatic counterparts from primary tissues. Finally, we also compared the patterns of expression of hASCs (and their derivatives) Overall design: Identify all genes or probe-sets whose expression was significantly different between control hASCs and each one of the two differentiated cell types (DIF.M and DIF.O),
Project description:Human adipose stem cells (hASCs) play a crucial role in the fields of regenerative medicine and tissue engineering for different reasons: the abundance of adipose tissue, their easy harvesting, the ability to multipotent differentiation and the fact that they do not trigger allogeneic blood response or secrete cytokines that act as immunosuppressants. The vast majority of protocols use animal origin reagents, with the underlying risk of transmitting infections by non-human pathogens. We have designed a protocol to isolate and maintain the properties of hASCs avoiding xenogeneic reagents. These changes not only preserve hASCs morphology, but also increase cell proliferation and maintain their stem cell marker profile. On the other hand, human serum albumin (HSA), Tryple® and human Serum (HS), do not affect hASCs multipotent differentiation ability. The amendments introduced do not trigger modifications in the transcriptional profile of hASCs, alterations in key biochemical pathways or malignization. Thus, we have proven that it is possible to isolate and maintain hASCs avoiding animal reagents and, at the same time, preserving crucial culture parameters during long term culture. Thereby we have revealed a novel and effective tool for the improvement of clinical, cell-based therapies. Gene expression in human adipose stem cells isolated and maintained with human reagents were measured immediately after their isolation, and at passages 1, 3 and 5. Pools from the same 8 healthy donors were used to measure the 4 points of growth.
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:Both ovarian and pituitary hormones are required for the pubertal development of the mouse mammary gland. Estradiol directs ductal elongation and branching within the adipose stroma of the adolescent mouse mammary gland, while progesterone leads to tertiary branching and alveolar development. The purpose of this investigation was to identify the estrogen-responsive genes that are associated with estrogen-stimulated ductal elongation and branching in the mouse mammary gland in the absence of other ovarian hormones. We also wanted to determine if estrogen-responsive gene regulation at early stages of ductal elongation (ie. when ductal growth was minimal) was similar to those regulated after significant ductal elongation had occurred. To identify estrogen-regulated genes, ovariectomized prepubertal mice were exposed to 17beta-estradiol for four weeks, and mammary gland global gene expression analyzed by microarray analysis at various points during this time course. We determined that while many genes are regulated in all weeks of treatment, there remained a subset of genes that was uniquely regulated at each time-point. This observation was reflected in the biological functions of these genes; some categories were represented in all weeks of treatment while others were specific to only certain time-points. We have also identified estradiol-responsive genes in the mouse mammary gland that co-express with Estrogen Receptor alpha in human breast cancer, which may represent novel effectors of estrogen action and/or biomarkers for the progression of estrogen-dependent cancers and other estrogen-driven diseases. Experiment Overall Design: For each time-course experiment, one frozen #4 mammary gland was individually pulverized from four to five animals per treatment group, then homogenized in 3mL Trizol (Invitrogen, Carlsbad, CA) and RNA was prepared according to the manufacturer’s protocol. The RNA from individual animals was then pooled for each treatment group (four to five animals per group) and further purified using the QIAGEN (Valencia, CA) RNeasy Mini kit (Cat. No. 74104) clean-up protocol. Experiment Overall Design: Gene expression analysis was conducted using Agilent Mouse Oligo arrays (pattern number 011978) (Agilent Technologies, Palo Alto, CA). Total RNA was amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol. Starting with 500ng of total RNA, Cy3 or Cy5 labeled cRNA was produced according to manufacturer’s protocol. For each two color comparison, 750ng of each Cy3 and Cy5 labeled cRNAs were mixed and fragmented using the Agilent In Situ Hybridization Kit protocol. In each case, samples from estradiol-treated animals were co-hybridized with the day 7 placebo sample. Due to the rapid increase in adiposity in the mammary fat pad in the ovariectomized placebo-treated mice in days 14 and 28, the day 7 placebo sample was used as a control for all estradiol treated samples to avoid any variation due to this biological difference. Hybridizations were performed for 17 hours in a rotating hybridization oven using the Agilent 60-mer oligo microarray processing protocol. Slides were washed as indicated in this protocol and then scanned with an Agilent Scanner. Data was obtained using the Agilent Feature Extraction software (v7.5), using defaults for all parameters. Experiment Overall Design: The Agilent Feature Extraction Software performed error modeling, adjusting for additive and multiplicative noise. The resulting data were processed using the Rosetta Resolver® system (version 7.1) (Rosetta Biosoftware, Kirkland, WA).
Project description:Human somatic fibroblasts can be reprogrammed to induced pluripotent stem (iPS) cells by exogenic expression of the Yamanaka factors (OCT4, SOX2, KLF4 and MYC) after about 1 month. To gain some insight into the early processes operative in fibroblast reprogramming, we profiled genome-wide transcription levels using Illumina microarrays in the starting donor cells-human foreskin fibroblast (HFF1) cells and at three time points after OSKM transduction (24 h, 48 h, 72 h), as well as two iPS cell lines (iPS2, iPS4) and hES cell lines (H1, H9). We show that within the context of the viral transduction reprogramming protocol, the donor cell response to viral transfection perturbs redox homeostasis, which induces oxidative damage on the donor cells' protein and DNA. This leads to activation of p53, senescence, and apoptosis, greatly reducing the efficiency of reprogramming. Total RNA obtained from HFF1 (human foreskin fibroblast) cells, OSKM-transduced HFF1 cells after 24h, 48h, 72h, undifferentiated hESCs, iPSCs.