Expression analysis of human adipose tissue stem cells (ADSCs) transduced with homeobox C8 (Hoxc8) or control vector, followed by treatment with adipogenic-induction media for 14 days
ABSTRACT: Adipogenesis occurs through a specific gene program in undifferentiated fat progenitors. We hypothesized that the properties of the fat progenitors are regulated by hox genes, the developmental genes essential in different tissue stem cells. Their biased expression in white and brown fat implies roles in distinguishing the two fat types. Among 39 Hox genes, Hoxc8 is highly enriched in undifferentiated adipose tissue stem cells (ADSCs) and down-regulated in differentiated adipocytes. Forced expression of Hoxc8 suppressed adipocyte differentiation of ADSCs. Using microarrays, we investigated the effect of Hoxc8 overexpression on global transcripts in ADSCs. We compared among four groups: untreated ADSCs, adipogenic induction media (MDI)-treated ADSCs, MDI-treated ADSC-vector and MDI-treated ADSC-Hoxc8. A number of, but not all, adipogenesis-related genes are suppressed by Hoxc8. This dataset illustrates the global effect of Hoxc8, a developmental transcription factor, on the expression of adipogenesis-related genes. Overall design: Gene expression was compared among untreated ADSCs (control), adipogenic induction media-treated ADSCs, adipogenic induction media-treated ADSC-vector (ADSCs transduced with control vector), and adipogenic induction media-treated ADSC-Hoxc8 (ADSCs transduced with human Hoxc8). Total RNA was isolated from ADSCs using the Qiagen RNeasy kit (Qiagen). At NimbleGen, quality and yield were verified before cDNA synthesis and Cy3-end labeling. The labeled cDNA samples were hybridized to Homo sapiens 4-Plex arrays (Roche NimbleGen, A4487001-00-01) that represent 24,000 human genes. Raw data files for each sample were normalized and background-corrected using a Robust Multi-Array Analysis as implemented by NimbleScan software. Students’ two-tail t-tests were conducted among the samples for each transcript and fold-change was determined. Transcripts whose abundance was significantly altered (P < 0.05) and an absolute fold change greater than 2 were defined as differentially regulated.
INSTRUMENT(S): NimbleGen Homo sapiens HG18 60mer expr 4plex (4x72k)
Project description:Adipogenesis occurs through a specific gene program in undifferentiated fat progenitors. We hypothesized that the properties of the fat progenitors are regulated by hox genes, the developmental genes essential in different tissue stem cells. Their biased expression in white and brown fat implies roles in distinguishing the two fat types. Among 39 Hox genes, Hoxc8 is highly enriched in undifferentiated adipose tissue stem cells (ADSCs) and down-regulated in differentiated adipocytes. Forced expression of Hoxc8 suppressed adipocyte differentiation of ADSCs. Using microarrays, we investigated the effect of Hoxc8 overexpression on global transcripts in ADSCs. We compared among four groups: untreated ADSCs, adipogenic induction media (MDI)-treated ADSCs, MDI-treated ADSC-vector and MDI-treated ADSC-Hoxc8. A number of, but not all, adipogenesis-related genes are suppressed by Hoxc8. This dataset illustrates the global effect of Hoxc8, a developmental transcription factor, on the expression of adipogenesis-related genes. Gene expression was compared among untreated ADSCs (control), adipogenic induction media-treated ADSCs, adipogenic induction media-treated ADSC-vector (ADSCs transduced with control vector), and adipogenic induction media-treated ADSC-Hoxc8 (ADSCs transduced with human Hoxc8). Total RNA was isolated from ADSCs using the Qiagen RNeasy kit (Qiagen). At NimbleGen, quality and yield were verified before cDNA synthesis and Cy3-end labeling. The labeled cDNA samples were hybridized to Homo sapiens 4-Plex arrays (Roche NimbleGen, A4487001-00-01) that represent 24,000 human genes. Raw data files for each sample were normalized and background-corrected using a Robust Multi-Array Analysis as implemented by NimbleScan software. Students’ two-tail t-tests were conducted among the samples for each transcript and fold-change was determined. Transcripts whose abundance was significantly altered (P < 0.05) and an absolute fold change greater than 2 were defined as differentially regulated.
Project description:Mesenchymal stromal cells (MSCs) can self-renew, differentiate into specialised cells and have different embryonic origins-ectodermal for dental pulp-derived MSCs (DPSCs) and mesodermal for adipose tissue-derived MSCs (ADSCs). Data on DPSCs adipogenic differentiation potential and timing vary, and the lack of molecular and genetic information prompted us to gain a better understanding of DPSCs adipogenic differentiation potential and gene expression profile. While DPSCs differentiated readily along osteogenic and chondrogenic pathways, after 21 days in two different types of adipogenic induction media, DPSCs cultures did not contain lipid vacuoles and had low expression levels of the adipogenic genes proliferator-activated receptor gamma (PPARG), lipoprotein lipase (LPL) and CCAAT/enhancer-binding protein alpha (CEBPA). To better understand this limitation in adipogenesis, transcriptome analysis in undifferentiated DPSCs was carried out, with the ADSC transcriptome used as a positive control. In total, 14,871 transcripts were common to DPSCs and ADSCs, some were unique (DPSCs: 471, ADSCs: 1032), and 510 were differentially expressed genes. Detailed analyses of overrepresented transcripts showed that DPSCs express genes that inhibit adipogenic differentiation, revealing the possible mechanism for their limited adipogenesis.
Project description:OBJECTIVES:In human post-menopausal osteoporosis, enhanced adipogenesis in bone marrow and enhanced formation of adipose tissue in vivo are observed. These changes correlate with reduced trabecular bone volume and increased adipocyte cell size as well as cell number. However, cellular and molecular mechanisms underlying osteoporosis-related changes in adipocyte cell volume are not known. This study was designed to compare adipogenic potential of adipose tissue-derived stem cells (ADSCs) obtained from ovariectomized mice with that of control ADSCs, and to analyse pathological mechanisms from the point of functional changes of ADSCs. MATERIALS AND METHODS:Healthy female C57BL/6J mice were randomly divided into ovariectomy and sham-surgery groups. Mouse ADSCs were isolated and cultured in vitro up to passage 3. After adipogenic induction, oil red O staining of lipid droplets was used to detect adipogenic ability of ADSCs; real-time PCR and immunofluorescence staining were used to detect expression of adipogenesis-related genes and proteins. RESULTS:As indicated by increased expression of adipogenic and lipogenic genes and proteins, and lipid droplets accumulation shown by oil red-O staining, adipogenic differentiation of ADSCs was significantly enhanced in the ovariectomy group compared to the sham-surgery group (P < 0.05). CONCLUSION:These findings suggest that enhanced adipogenic differentiation of ADSCs is likely to be the important cause for increased adipogenesis in vivo and subsequent obesity-like changes in body mass, in mice, after ovariectomy.
Project description:BACKGROUND: Previous studies on the effects of aging in human and mouse mesenchymal stem cells suggest that a decline in the number and differentiation potential of stem cells may contribute to aging and aging-related diseases. In this report, we used stromal cells isolated from adipose tissue (ADSCs) of young (8-10 weeks), adult (5 months), and old (21 months) mice to test the hypothesis that mechanical loading modifies aging-related changes in the self-renewal and osteogenic and adipogenic differentiation potential of these cells. RESULTS: We show that aging significantly reduced the proliferation and increased the adipogenesis of ADSCs, while the osteogenic potential is not significantly reduced by aging. Mechanical loading (10% cyclic stretching, 0.5 Hz, 48 h) increased the subsequent proliferation of ADSCs from mice of all ages. Although the number of osteogenic colonies with calcium deposition was increased in ADSCs subjected to pre-strain, it resulted from an increase in colony number rather than from an increase in osteogenic potential after strain. Pre-strain significantly reduced the number of oil droplets and the expression of adipogenic marker genes in adult and old ADSCs. Simultaneously subjecting ADSCs to mechanical loading and adipogenic induction resulted in a stronger inhibition of adipogenesis than that caused by pre-strain. The reduction of adipogenesis by mechanical strain was loading-magnitude dependent: loading with 2% strain only resulted in a partial inhibition, and loading with 0.5% strain could not inhibit adipogenesis in ADSCs. CONCLUSIONS: We demonstrate that mechanical stretching counteracts the loss of self-renewal in aging ADSCs by enhancing their proliferation and, at the same time, reduces the heightened adipogenesis of old cells. These findings are important for the further study of stem cell control and treatment for a variety of aging related diseases.
Project description:The environmental obesogen hypothesis proposes that pre- and postnatal exposure to environmental chemicals contributes to adipogenesis and the development of obesity. Tributyltin (TBT) is an agonist of both retinoid X receptor (RXR) and peroxisome proliferator-activated receptor gamma (PPARgamma). Activation of these receptors can elevate adipose mass in adult mice exposed to the chemical in utero. Here we show that TBT sensitizes human and mouse multipotent stromal stem cells derived from white adipose tissue [adipose-derived stromal stem cells (ADSCs)] to undergo adipogenesis. In vitro exposure to TBT, or the PPARgamma activator rosiglitazone increases adipogenesis, cellular lipid content, and expression of adipogenic genes. The adipogenic effects of TBT and rosiglitazone were blocked by the addition of PPARgamma antagonists, suggesting that activation of PPARgamma mediates the effect of both compounds on adipogenesis. ADSCs from mice exposed to TBT in utero showed increased adipogenic capacity and reduced osteogenic capacity with enhanced lipid accumulation in response to adipogenic induction. ADSCs retrieved from animals exposed to TBT in utero showed increased expression of PPARgamma target genes such as the early adipogenic differentiation gene marker fatty acid-binding protein 4 and hypomethylation of the promoter/enhancer region of the fatty acid-binding protein 4 locus. Hence, TBT alters the stem cell compartment by sensitizing multipotent stromal stem cells to differentiate into adipocytes, an effect that could likely increase adipose mass over time.
Project description:Adipose tissue hyperplasia with increased number of adipocytes is implicated in a protective rather than deleterious effect on obesity-associated metabolic disorder. It is poorly understood how the adipose tissue cellularity is regulated. Tc1 is a gene of vertebrates that regulates diverse downstream genes. Young Tc1-deleted mice fed on standard chow diet show expanded adipose tissue with smaller adipocytes in size compared to wild type controls, representing adipose tissue hyperplasia. Tc1(-/-) mice show enhanced glucose tolerance and reduced serum lipids. Adipocyte-derived stem cells (ADSCs) from Tc1(-/-) mice show enhanced proliferative and adipogenic capacity compared to wild type controls, suggesting that the adipose hyperplasia is regulated at the stem cell level. PPAR? and CEBP? are up-regulated robustly in Tc1(-/-) ADSCs upon induction for adipogenesis. Wisp2 and Dlk1, inhibitors of adipogenesis, are down-regulated in Tc1(-/-) ADSCs compared to controls. Tc1-transfected NIH3T3 cells show higher ?-catenin reporter signals than vector transfected controls, suggesting a role of canonical Wnt signaling in the Tc1-dependent adipose regulation. Our data support that Tc1 is a novel regulator for adipose stem cells. Adipose tissue hyperplasia may be implicated in the metabolic regulation of Tc1(-/-) mice.
Project description:Autologous fat grafting represents an attractive source for tissue engineering applications in the field of reconstructive medicine. However, in adipogenic differentiation protocols for human adipose-derived stem cells, the concentration of glucose and insulin varies considerably. With the intent to gain maximum tissue augmentation, we focused on the late phase of adipogenesis. In this study, we modified the differentiation protocol for adipose-derived stem cells by prolongation of the induction period and the application highly concentrated glucose and insulin. Human adipose-derived stem cells were isolated from subcutaneous depots and differentiated in a standard induction medium for the first two weeks, followed by two weeks with varying glucose and insulin concentrations. Morphological changes assessed using Oil-Red-O staining were examined for corresponding alterations in the expression of the adipogenic markers peroxisome proliferator-activated receptor gamma (PPAR?) and lipoprotein lipase (LPL). Furthermore, glucose and lactate levels in conditioned media were monitored over the period of differentiation. We found high-glucose media increasing the level of lipid accumulation and the size of single droplets whereas insulin significantly showed a dose-dependent negative effect on fat storage. However, whereas high glucose stimulated PPAR? transcription, expression levels in insulin-treated cells remained constant. Results permit assumptions that a high-glucose medium intensifies the degree of differentiation in mature adipocytes providing conditions to promote graft volume while we have identified highly concentrated insulin treatment as an inhibitor of lipid storage in the late adipogenic differentiation.
Project description:There is growing evidence that chronic exposure to inorganic arsenic (iAs) is associated with an increased prevalence of type 2 diabetes (T2D). However, the mechanisms for the diabetogenic effect of iAs are still largely unknown. White adipose tissue (WAT) actively stores and releases energy and maintains lipid and glucose homeostasis.We sought to determine the mechanisms of arsenic suppression of adipogenesis.The effects and associated mechanisms of iAs and its major metabolites on adipogenesis were determined in 3T3-L1 preadipocytes, mouse adipose-derived stromal-vascular fraction cells (ADSVFCs), and human adipose tissue-derived stem cells (ADSCs).Exposure of 3T3-L1 preadipocytes to noncytotoxic levels of arsenic, including inorganic arsenite (iAs3+, ? 5 ?M), inorganic arsenate (? 20 ?M), trivalent monomethylated arsenic (MMA3+, ? 1 ?M), and trivalent dimethylated arsenic (DMA3+, ? 2 ?M) decreased adipogenic hormone-induced adipogenesis in a concentration-dependent manner. In addition, iAs3+, MMA3+, and DMA3+ exhibited a strong inhibitory effect on adipogenesis in primary cultured mouse ADSVFCs and human ADSCs. Time-course studies in 3T3-L1 cells revealed that inhibition of adipogenesis by arsenic occurred in the early stage of terminal adipogenic differentiation and was highly correlated with the induction of C/EBP homologous protein (CHOP10), an endoplasmic reticulum (ER) stress response protein. Induction of CHOP10 by arsenic is associated with reduced DNA-binding activity of CCAAT/enhancer-binding protein ? (C/EBP?), which regulates the transcription of peroxisome proliferator-activated receptor ? and C/EBP?.Low-level iAs and MMA3+ trigger the ER stress response and up-regulate CHOP10, which inhibits C/EBP? transcriptional activity, thus suppressing adipogenesis. Arsenic-induced dysfunctional adipogenesis may be associated with a reduced capacity of WAT to store lipids and with insulin resistance.
Project description:The recent discovery of functional brown adipocytes in adult humans illuminates the potential of these cells in the treatment of obesity and its associated diseases. In rodents, brown adipocyte-like cells are known to be recruited in white adipose tissue (WAT) by cold exposure or ?-adrenergic stimulation, but the molecular machinery underlying this phenomenon is not fully understood. Here, we show that inducible brown adipogenesis is mediated by the microRNA miR-196a. We found that miR-196a suppresses the expression of the white-fat gene Hoxc8 post-transcriptionally during the brown adipogenesis of white fat progenitor cells. In mice, miR-196a is induced in the WAT-progenitor cells after cold exposure or ?-adrenergic stimulation. The fat-specific forced expression of miR-196a in mice induces the recruitment of brown adipocyte-like cells in WAT. The miR-196a transgenic mice exhibit enhanced energy expenditure and resistance to obesity, indicating the induced brown adipocyte-like cells are metabolically functional. Mechanistically, Hoxc8 targets and represses C/EBP?, a master switch of brown-fat gene program, in cooperation with histone deacetylase 3 (HDAC3) through the C/EBP? 3' regulatory sequence. Thus, miR-196a induces functional brown adipocytes in WAT through the suppression of Hoxc8, which functions as a gatekeeper of the inducible brown adipogenesis. The miR-196a-Hoxc8-C/EBP? signaling pathway may be a therapeutic target for inducing brown adipogenesis to combat obesity and type 2 diabetes.
Project description:Macrodactyly is a congenital malformation characterized by enlargement of bone and soft tissues in limbs, typically with excessive accumulation of adipose tissues. Although gain-of-function mutation of PIK3CA has been identified in macrodactyly, the mechanism of PIK3CA mutation in adipose accumulation is poorly understood. In this study, we found that adipocytes from macrodactyly were more hypertrophic than those observed in polydactyly. PIK3CA (H1047R) activating mutation and enhanced activity of PI3K/AKT pathway were detected in macrodactylous adipose-derived stem cells (Mac-ADSCs). Compared to polydactyly-derived ADSCs (Pol-ADSCs), Mac-ADSCs had higher potential in adipogenic differentiation. Knockdown of PIK3CA or inhibition by BYL-719, a potent inhibitor of PIK3CA, impaired adipogenesis of Mac-ADSCs in vitro. In vivo study, either transient treatment of ADSCs or intragastrical gavage with BYL-719 inhibited the adipose formation in patient-derived xenograft (PDX). Furthermore, RNA-seq revealed that E2F1 was up-regulated in Mac-ADSCs and its knockdown blocked the PIK3CA-promoted adipogenesis. Our findings demonstrated that PIK3CA activating mutation promoted adipogenesis of ADSCs in macrodactyly, and that this effect was exerted by the up-regulation of E2F1. This study revealed a possible mechanism for adipose accumulation in macrodactyly and suggested BYL-719 as a potential therapeutic agent for macrodactyly treatment.