mRNA-electroporated mature dendritic cells retain transgene expression, phenotypical properties and stimulatory capacity after cryopreservation

Ponsaerts, Peter; Tendeloo, Viggo Van; Cools, Nathalie; Driessche, Ann Van; Lardon, Filip; Nijs, Griet; Lenjou, Marc; Mertens, Gerhard; Broeckhoven, Christine Van; Bockstaele, Dirk R. Van; Berneman, Zwi

doi:10.1038/sj.leu.2402511

Cited by 51 publications

(48 citation statements)

References 13 publications

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“…6, cell viability was high, and Ag was still detectable in the DC 2 days after the initial electroporation in virtually all cells when using this strategy. Compared with our previous results (20,29), related to the electroporation of DC cultured in serum in the presence of GM-CSF and IL-4, the level of protein expression, e.g., EGFP, was much lower in monocytes after 24 h (data not shown) and 48 h (Fig. 6C).…”

Section: Discussioncontrasting

confidence: 88%

“…This transfection technology resulted in high level transgene expression in Mo-DC using an EGFP reporter gene. More than 70% of the transfected cells showed high level EGFP expression (relative fluorescence between 10 2 and 10 3 decade as measured by flow cytometry) and retained their phenotypical properties after transfection (20,29). However, the use of this technology for transfection of short term, serum-free cultured DC, as presented in this study, resulted in substantial cell mortality among transfected cells (data not shown).…”

Section: Discussionmentioning

confidence: 65%

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Messenger RNA Electroporation of Human Monocytes, Followed by Rapid In Vitro Differentiation, Leads to Highly Stimulatory Antigen-Loaded Mature Dendritic Cells

Ponsaerts

Bosch

Cools

et al. 2002

The Journal of Immunology

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Dendritic cells (DC) are professional Ag-capturing and -presenting cells of the immune system. Because of their exceptional capability of activating tumor-specific T cells, cancer vaccination research is now shifting toward the formulation of a clinical human DC vaccine. We developed a short term and serum-free culture protocol for rapid generation of fully mature, viable, and highly stimulatory CD83+ DC. Human monocytes were cultured for 24 h in serum-free AIM-V medium, followed by 24-h maturation by polyriboinosinic polyribocytidylic acid (polyI:C). Short term cultured, polyI:C-maturated DC, far more than immature DC, showed typical mature DC markers and high allogeneic stimulatory capacity and had high autologous stimulatory capacity in an influenza model system using peptide-pulsed DC. Electroporation of mRNA as an Ag-loading strategy in these cells was optimized using mRNA encoding the enhanced green fluorescent protein (EGFP). Monocytes electroporated with EGFP mRNA, followed by short term, serum-free differentiation to mature DC, had a phenotype of DC, and all showed positive EGFP fluorescence. Influenza matrix protein mRNA-electroporated monocytes cultured serum-free and maturated with polyI:C showed high stimulatory capacity in autologous T cell activation experiments. In conclusion, the present short term and serum-free ex vivo DC culture protocol in combination with mRNA electroporation at the monocyte stage imply an important reduction in time and consumables for preparation of Ag-loaded mature DC compared with classical DC culture protocols and might find application in clinical immunotherapy settings.

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Section: Discussioncontrasting

confidence: 88%

Section: Discussionmentioning

confidence: 65%

Messenger RNA Electroporation of Human Monocytes, Followed by Rapid In Vitro Differentiation, Leads to Highly Stimulatory Antigen-Loaded Mature Dendritic Cells

Ponsaerts

Bosch

Cools

et al. 2002

The Journal of Immunology

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“…[24][25][26][27] Recently, mRNA electroporation of human monocyte-derived DC was described as a very efficient tool, without the need for viral vectors. 34,[36][37][38] Here, we further expand on the method of mRNA electroporation of human monocyte-derived DC. This procedure is considered to be very safe, because the mRNA has a short half-life in the cells, and is not integrated into the genome of the cells.…”

Section: Discussionmentioning

confidence: 99%

“…Previous reports described that mRNA electroporation does not influence DC viability, DC phenotype or allo-stimulatory capacity. 34,[36][37][38] We show that the transgene expression level can be modulated by varying the amount of mRNA used for electroporation. mRNA-electroporated DC can respond to CD40 ligation by IL-12 secretion, albeit slightly reduced compared to non-or mock-electroporated DC.…”

mentioning

confidence: 92%

Induction of Influenza Matrix Protein 1 and MelanA-specific T lymphocytes in vitro using mRNA-electroporated dendritic cells

et al. 2003

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Genetically modified dendritic cells (DC) constitute a promising approach in cancer immunotherapy. Viral gene delivery systems have been shown to be very efficient strategies, but safety concerns for their clinical use in immunotherapy remain an important issue. Recently, the technique of mRNA electroporation was described as a very efficient tool for the genetic modification of human monocyte-derived DC. Here, we show that transgene expression can be modulated by varying the amount of mRNA used for electroporation. We document that CD40 ligation leads to a significant production of IL-12 by the electroporated DC, although the level of IL-12 production is somewhat lower than for non-or mock-electroporated DC. Furthermore, we show that the electroporated DC can be frozen and thawed without loss of viability or function and that Influenza virus Matrix Protein 1 mRNA electroporated DC are capable of inducing a memory cytotoxic T lymphocyte response and are more potent in doing so than mRNA-pulsed DC. Similar results were obtained with MelanA/MART-1 mRNA electroporated DC. These results clearly indicate that mRNA-electroporated DC represent powerful candidates for use as tumor vaccines and could constitute an improvement compared with vaccines using peptide-pulsed DC.

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“…2,3 Directly after electroporation, ES cells were further cultured in standard ES cell medium supplemented with leukemia inhibitory factor (LIF). After 6 h of culture, cells were analysed by fluorescence microscopy in order to detect EGFP-positive cells.…”

mentioning

confidence: 99%

Messenger RNA electroporation is highly efficient in mouse embryonic stem cells: successful FLPe- and Cre-mediated recombination

et al. 2004

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Development of efficient short-term gene transfer technologies for embryonic stem (ES) cells is urgently needed for various existing and new ES cell-based research strategies. In this study, we present a highly efficient, nonviral non-DNA technology for genetic loading of mouse ES cells based on electroporation of defined mRNA. Here, we show that mouse ES cells can be efficiently loaded with mRNA encoding a green fluorescent reporter protein, resulting in a level of at least 90% of transgene expression without loss of cell viability and phenotype. To show that transgenes, introduced by mRNA electroporation, exert a specific cellular function in Short-term gene transfer in mouse embryonic stem (ES) cells is a potentially interesting strategy for various applications, including experiments involving sitespecific target gene excision using FLPe and Cre recombinase technology. 1 For this purpose, many studies report the use of plasmid DNA or viral vectors to introduce FLPe or Cre recombinase proteins in whole cell populations. However, these gene transfer strategies have several disadvantages. Gene transfer by plasmid DNA generally does not result in high numbers of transfected cells and is often associated with high cell mortality among transfected cells. The use of viral vectors for gene transfer can circumvent these problems, but is associated with more complex and laborious manipulations and safety issues. Below, we describe a nonviral non-DNA technique for rapid gene expression in mouse ES cells, based on electroporation of in vitro transcribed mRNA encoding defined proteins.To show that mouse ES cells can be genetically loaded with mRNA, several mouse ES cell lines were electroporated with in vitro transcribed mRNA, encoding the enhanced green fluorescent protein (EGFP), following a previously described procedure. 2,3 Directly after electroporation, ES cells were further cultured in standard ES cell medium supplemented with leukemia inhibitory factor (LIF). After 6 h of culture, cells were analysed by fluorescence microscopy in order to detect EGFP-positive cells. The following ES cell lines were examined: HM1, 4 R1 5 and E14. 6 As shown in Figure 1a, virtually all mRNA-electroporated HM-1 ES cells are positive for EGFP fluorescence, indicating high transfection efficiency and rapid translation following mRNA electroporation. At 48 h after electroporation, cells were harvested and analysed by flow cytometry for EGFP expression and cell viability (Figure 1b). The upper dot plots in Figure 1b

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mRNA-electroporated mature dendritic cells retain transgene expression, phenotypical properties and stimulatory capacity after cryopreservation

Cited by 51 publications

References 13 publications

Messenger RNA Electroporation of Human Monocytes, Followed by Rapid In Vitro Differentiation, Leads to Highly Stimulatory Antigen-Loaded Mature Dendritic Cells

Messenger RNA Electroporation of Human Monocytes, Followed by Rapid In Vitro Differentiation, Leads to Highly Stimulatory Antigen-Loaded Mature Dendritic Cells

Induction of Influenza Matrix Protein 1 and MelanA-specific T lymphocytes in vitro using mRNA-electroporated dendritic cells

Messenger RNA electroporation is highly efficient in mouse embryonic stem cells: successful FLPe- and Cre-mediated recombination

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