Nucleofection Is a Valuable Transfection Method for Transient and Stable Transgene Expression in Adipose Tissue-Derived Stem Cells

Zaragosi, Laure‐Emmanuelle; Billon, Noëlle; Ailhaud, Gérard; Dani, Christian

doi:10.1634/stemcells.2006-0235

Cited by 44 publications

(49 citation statements)

References 31 publications

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“…hMADS cell culturing and differentiation hMADS-3 cells (Rodriguez et al 2005;Zaragosi et al 2007) were cultured and differentiated into mature adipocytes as described in the Supplemental Material. From day 13 to day 16 of differentiation, hMADS adipocytes were treated with rosiglitazone to form brite adipocytes or with DMSO to maintain white adipocytes.…”

Section: Methodsmentioning

confidence: 99%

Browning of human adipocytes requires KLF11 and reprogramming of PPARγ superenhancers

et al. 2014

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Long-term exposure to peroxisome proliferator-activated receptor g (PPARg) agonists such as rosiglitazone induces browning of rodent and human adipocytes; however, the transcriptional mechanisms governing this phenotypic switch in adipocytes are largely unknown. Here we show that rosiglitazone-induced browning of human adipocytes activates a comprehensive gene program that leads to increased mitochondrial oxidative capacity. Once induced, this gene program and oxidative capacity are maintained independently of rosiglitazone, suggesting that additional browning factors are activated. Browning triggers reprogramming of PPARg binding, leading to the formation of PPARg ''superenhancers'' that are selective for brown-in-white (brite) adipocytes. These are highly associated with key brite-selective genes. Based on such an association, we identified an evolutionarily conserved metabolic regulator, Kruppel-like factor 11 (KLF11), as a novel browning transcription factor in human adipocytes that is required for rosiglitazone-induced browning, including the increase in mitochondrial oxidative capacity. KLF11 is directly induced by PPARg and appears to cooperate with PPARg in a feed-forward manner to activate and maintain the brite-selective gene program.

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Section: Methodsmentioning

confidence: 99%

Browning of human adipocytes requires KLF11 and reprogramming of PPARγ superenhancers

et al. 2014

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“…While others showed the percentage of cells exhibiting green fluorescent protein reporter gene (Aluigi et al 2006;Zarogasi et al 2007), we measured the mean intensity of luciferase reporter gene in viable cells using the pRL-CMV plasmid DNA. Our results showed that the mean expression in viable hMSC was four times higher (21.79%) when the U-23 pulsing program was used compared with C-17 (5.62%).…”

Section: Discussionmentioning

confidence: 99%

Extended and stable gene expression via nucleofection of MIDGE construct into adult human marrow mesenchymal stromal cells

et al. 2011

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Human mesenchymal stromal cell (hMSC) is a potential target for cell and gene therapy-based approaches against a variety of different diseases. Whilst cationic lipofection has been widely experimented, the Nucleofector technology is a relatively new non-viral transfection method designed for primary cells and hard-to-transfect cell lines. Herein, we compared the efficiency and viability of nucleofection with cationic lipofection, and used the more efficient transfection method, nucleofection, to deliver a construct of minimalistic, immunologically defined gene expression encoding the erythropoietin (MIDGE-EPO) into hMSC. MIDGE construct is relatively safer than the viral and plasmid expression systems as the detrimental eukaryotic and prokaryotic gene and sequences have been eliminated. Using a plasmid encoding the luciferase gene, we demonstrated a high transfection efficiency using the U-23 (21.79 ± 1.09%) and C-17 (5.62 ± 1.09%) pulsing program in nucleofection. The cell viabilities were (44.93 ± 10.10)% and (21.93 ± 5.72)%, respectively 24 h post-nucleofection. On the other hand, lipofection treatment only yielded less than 0.6% efficiencies despite showing higher viabilities. Nucleofection did not affect hMSC renewability, immunophenotype and differentiation potentials. Subsequently, we nucleofected MIDGE-EPO using the U-23 pulsing program into hMSC. The results showed that, despite a low nucleofection efficiency with this construct, the EPO protein was stably expressed in the nucleofected cells up to 55 days when determined by ELISA or immunocytochemical staining. In conclusion, nucleofection is an efficient non-viral transfection approach for hMSC, which when used in conjunction with a MIDGE construct, could result in extended and stable transgene expression in hMSC.

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“…Due to the apoptosis and the low cell density, mesenchymal cells lost their chondrifying characteristics, therefore detection of metachromasial extracellular matrix was almost impossible on the last day of culturing (day 6). Thus, although electroporation gives high transfection efficiency on different cell lines [39,40] it does not seem applicable to deliver vectors into primary mesenchymal chondrifying cell cultures.…”

Section: Discussionmentioning

confidence: 99%

Optimalized transient transfection of chondrogenic primary cell cultures

et al. 2010

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Abstract:We aimed to find a transfection method which provides high efficiency with minimal cytotoxic and/or apoptotic effects for gene transfer into multilayer primary chondrogenic cell cultures. The pEGFP-C1 plasmid was introduced into the cell culture and the efficiency of transformation quantified by GFP fluorescence; the resulting nucleofection was effective but resulted in severe apoptosis. Two liposomal reagents designed to allow transfection into adherent cells did not deliver the plasmids sufficiently and cartilage formation did not occur. In addition, a third liposomal compound, recommended for transfection into either adherent or suspension cell cultures, lead to acceptable transfection efficiency but no cartilage formation. When an amphiphilic reagent was used however, there was acceptable transfection efficiency as well as cartilage formation. The viability of the cells which were transfected using the amphiphilic reagent remained unaffected but proliferation was severely diminished, particularly in the presence of GFP. In addition, the amount of cartilage decreased when GFP was expressed, despite unchanged levels of mRNAs of sox9 and aggrecan core protein, factors reflecting on the efficiency of chondrogenesis. Overexpression of both the constitutively active delta and gamma isoforms of catalytic subunit of calcineurin, a protein phosphatase described as a positive regulator of chondrogenesis, decreased protein level of Sox9 and subsequent cartilage formation. In conclusion, we found that amphiphilic reagent applied prior to the adhesion of cells provides a useful means to transfer plasmids to primary differentiating chondrogenic cells.

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Nucleofection Is a Valuable Transfection Method for Transient and Stable Transgene Expression in Adipose Tissue-Derived Stem Cells

Abstract: STEM CELLS 2007;25:790 -797

Cited by 44 publications

References 31 publications

Browning of human adipocytes requires KLF11 and reprogramming of PPARγ superenhancers

Browning of human adipocytes requires KLF11 and reprogramming of PPARγ superenhancers

Extended and stable gene expression via nucleofection of MIDGE construct into adult human marrow mesenchymal stromal cells

Optimalized transient transfection of chondrogenic primary cell cultures

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