Primary Adipocyte Culture: Adipocyte Purification Methods May Lead to a New Understanding of Adipose Tissue Growth and Development

Fernyhough, Melinda E.; Vierck, Janet L.; Hausman, Gary; Mir, P. S.; Okine, E. K.; Dodson, M. V.

doi:10.1007/s10616-005-2602-0

Cited by 82 publications

(105 citation statements)

References 5 publications

(4 reference statements)

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“…Primary mature s.c. adipocytes were isolated from 8-to 16-wk-old PDGFRA-nGFP mice adipose tissue using a previously described method (45). Primary adipocytes were cultured in adipocyte medium (DMEM-HG + 10% FCS + P/S/G) at 37°C and 5% CO 2 in the incubator for 8-10 d before exposing to reprogramming agents (see SI Appendix).…”

Section: Methodsmentioning

confidence: 99%

“…S.c. fat was harvested with consent from patients undergoing surgery for degenerative disk disease with approval from the Prince of Wales Hospital human research ethics committee. The s.c. adipocytes were harvested using a previously described method (45) with modifications (see SI Appendix). Harvested primary human adipocytes were exposed to reprogramming media containing 10 μM AZA + 200 ng/mL human recombinant PDGF-AB + 20% autologous serum + P/S/G for 2 d and then maintained in 200 ng/mL human recombinant PDGF-AB + 20% autologous serum + P/S/G for a further 23 d with every 3-4 d media change.…”

Section: Cellular Reprogrammingmentioning

confidence: 99%

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PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells

Chandrakanthan

Yeola

Kwan

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor–AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration.

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Section: Methodsmentioning

confidence: 99%

Section: Cellular Reprogrammingmentioning

confidence: 99%

PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells

Chandrakanthan

Yeola

Kwan

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…Briefly, collected fat pads were minced and digested with type I collagenase before centrifugation using similar procedures as described (53). The pellet is the stromal vascular fraction (SVF), and the floating cells were collected as the mature adipocyte fraction.…”

Section: Methodsmentioning

confidence: 99%

Ablation of XP-V gene causes adipose tissue senescence and metabolic abnormalities

Chen

Harris

Hatahet

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Obesity and the metabolic syndrome have evolved to be major health issues throughout the world. Whether loss of genome integrity contributes to this epidemic is an open question. DNA polymerase η (pol η), encoded by the xeroderma pigmentosum (XP-V) gene, plays an essential role in preventing cutaneous cancer caused by UV radiation-induced DNA damage. Herein, we demonstrate that pol η deficiency in mice (pol η −/− ) causes obesity with visceral fat accumulation, hepatic steatosis, hyperleptinemia, hyperinsulinemia, and glucose intolerance. In comparison to WT mice, adipose tissue from pol η −/− mice exhibits increased DNA damage and a greater DNA damage response, indicated by upregulation and/or phosphorylation of ataxia telangiectasia mutated (ATM), phosphorylated H 2 AX (γH 2 AX), and poly[ADP-ribose] polymerase 1 (PARP-1). Concomitantly, increased cellular senescence in the adipose tissue from pol η −/− mice was observed and measured by up-regulation of senescence markers, including p53, p16 Ink4a , p21, senescence-associated (SA) β-gal activity, and SA secretion of proinflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) as early as 4 wk of age. Treatment of pol η −/− mice with a p53 inhibitor, pifithrin-α, reduced adipocyte senescence and attenuated the metabolic abnormalities. Furthermore, elevation of adipocyte DNA damage with a high-fat diet or sodium arsenite exacerbated adipocyte senescence and metabolic abnormalities in pol η −/− mice. In contrast, reduction of adipose DNA damage with N-acetylcysteine or metformin ameliorated cellular senescence and metabolic abnormalities. These studies indicate that elevated DNA damage is a root cause of adipocyte senescence, which plays a determining role in the development of obesity and insulin resistance.T he human genome is constantly challenged by exogenous and endogenous DNA damaging agents. To ensure genome integrity, human cells have faithful DNA replication machinery and DNA repair systems that are coordinated by DNA damage response networks. In response to different extents or types of DNA lesions, the DNA damage response activates appropriate cellular responses, including transient or permanent (senescence) cell cycle arrest, or apoptosis, to minimize the detrimental effects of DNA lesions (1). Reduction or deficiency in DNA repair/replication enzyme activity is well documented to increase vulnerability for the development of cancer, neurodegenerative diseases, and aging (2). In addition, defective DNA repair enzymes are associated with the metabolic symptom; for example, DNA glycosylase (Neil1)-and OGG1-deficient mice are obese (3-5), and nucleotide excision repair protein ERCC1-XPF deficiency causes lipodystrophy (6). Furthermore, DNA damage response protein ataxia telangiectasia mutated (ATM) suppresses JNK activity through p53 signaling and mediates an antioxidant action that has been suggested to be relevant to the metabolic syndrome (7). Nucleotide excision repair XP-A protein may affect metabolism by altering mitochondrial...

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“…Visible connective and muscle tissue were carefully dissected away from the intramuscular adipose tissue. Adipocytes were isolated using an adaptation of the methods described by Fernyhough et al (2005). Briefly, adipose tissue was washed four times in sterile PBS-PS and was cut into approximately 1 cm 3 pieces.…”

Section: Methodsmentioning

confidence: 99%

Effect of level of eicosapentaenoic acid on the transcriptional regulation of Δ-9 desaturase using a novel in vitro bovine intramuscular adipocyte cell culture model

Waters

Kenny²,

Killeen³

et al. 2009

Animal

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Ruminant fat is often perceived as having a negative impact on human health; however, the composition of the fat is under complex biochemical control and can be improved through strategic manipulation of the animal's diet. There were two major objectives of this study, namely (i) to develop and validate a primary bovine intramuscular adipocyte cell line and (ii) to examine the effect of eicosapentaenoic acid (EPA) on the transcriptional regulation of D-9 desaturase in vitro using the novel cell line. Intramuscular adipose tissue was obtained from the Musculus longissimus thoracis of a beef heifer. Mature adipocytes were isolated and cultured, and subsequently harvested and evaluated for lipid accumulation and the expression of genes regulating key functional adipocyte protein markers at passages 10, 20 and 30. Isolated cells were shown to accumulate lipid in culture over time. Fatty acid analysis by gas chromatography was carried out at passage 30. Thirteen fatty acids ranging from tetradecanoic acid (C14:0) to the polyunsaturated fatty acid, docosahexaenoic acid (C22:6), were easily detected and measured. High-quality total RNA was isolated from adipocytes and the expression of peroxisome proliferator-activated receptor-g, fatty acid synthase, fatty acid-binding protein-4, adipocyte lipid-binding protein, CD36, D-9 desaturase, sterol regulatory element-binding protein (SREBP), microsomal triglyceride transfer protein and leptin genes were identified by reverse transcriptase-PCR and sequence analysis. Expression of the negative control, liver-specific hepatocyte nuclear factor-1alpha, was not detected. Adipocytes were subsequently incubated in medium containing 0, 50 or 100 mM EPA for 24 h. Increasing the EPA concentration of the culture media led to a linear increase in adipocyte EPA concentration ( P , 0.01). Expression of D-9 desaturase mRNA was decreased five-and seven-fold, respectively, following 50 and 100 mM EPA incubation compared to the control. Gene expression of SREBP-1c was decreased by 6-and 18-fold in cells supplemented with 50 and 100 mM EPA, respectively, compared to the control. Regression analysis showed a negative linear relationship between EPA concentration and the gene expression of both D-9 desaturase ( P , 0.001) and SREBP-1c ( P , 0.001), while a significant positive relationship was observed between D-9 desaturase and SREBP-1c gene expression ( P , 0.001). This is the first report demonstrating that EPA treatment of bovine intramuscular adipocyte cells decreased gene expression of both D-9 desaturase and SREBP-1c in vitro. The bovine adipocyte cell line developed here is an important resource for future studies facilitating less-expensive, rapid screening of research hypotheses and circumventing the limitations associated with the use of experimental animals including cost, inter-animal variation, pre-experimental management and ethics.

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Primary Adipocyte Culture: Adipocyte Purification Methods May Lead to a New Understanding of Adipose Tissue Growth and Development

Cited by 82 publications

References 5 publications

PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells

PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells

Ablation of XP-V gene causes adipose tissue senescence and metabolic abnormalities

Effect of level of eicosapentaenoic acid on the transcriptional regulation of Δ-9 desaturase using a novel in vitro bovine intramuscular adipocyte cell culture model

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