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Bio-Technical Methods Section

RNA-based gene transfer for adult stem cells and T cells

Abstract

Electroporation of mRNA has become an established method for gene transfer into dendritic cells for immunotherapeutic purposes. However, many more cell types and applications might benefit from an efficient mRNA-based gene transfer method. In this study, we investigated the potential of mRNA-based gene transfer to induce short-term transgene expression in adult stem cells and activated T cells, based on electroporation with mRNA encoding the enhanced green fluorescent protein. The results show efficient transgene expression in CD34-positive hematopoietic progenitor cells (35%), in in vitro cultured mesenchymal cells (90%) and in PHA-stimulated T cells (50%). Next to presentation of gene transfer results, potential applications of mRNA-based gene transfer in stem cells and T cells are discussed.

Introduction

Messenger (m)RNA-based gene transfer, as compared to plasmid DNA and viral vectors, has several advantages when thinking in terms of clinical applications.1 Since there is no danger of insertional mutagenesis and/or vector-induced immunogenicity,2, 3 in vitro/ex vivo manipulation of cells with mRNA is considered to be a valuable and safe alternative when cells need to be administered to patients. One drawback of mRNA-based gene transfer is that transgene expression is only transient. However, this should not be a problem because several applications require only a short term expression of a desired transgene. This has previously been demonstrated in many studies on the use of mRNA-loaded dendritic cells for immunotherapeutic purposes.4, 5, 6 However, many more cell types and applications might benefit from an efficient mRNA-based gene transfer method.5 We therefore examined the potential of mRNA electroporation as a nonviral non-DNA gene transfer tool for gene expression in adult stem cells, both hematopoietic and mesenchymal, and in activated T cells. Based on the expression of an enhanced green fluorescent reporter protein (EGFP), gene transfer efficiencies are described and potential applications discussed.

Materials and methods

Source of peripheral blood and bone marrow samples

Peripheral blood was obtained from hemochromatosis patients after informed consent or from buffy coats provided by the Antwerp Blood Transfusion Center. Bone marrow samples were provided by the Hematology Clinic of the Antwerp University Hospital after informed consent. Mononuclear cells from peripheral blood and bone marrow samples were isolated by Ficoll-Hypaque gradient separation (LSM, ICN Biomedicals). Isolated cells were directly used for electroporation experiments or cultured in vitro to obtain the desired cell type before electroporation experiments.

In vitro culture of human mesenchymal cells

Mononuclear bone marrow cells were plated in T75 culture flasks in complete medium consisting of Iscove's modified Dulbecco's medium supplemented with 2 mM L-glutamine (IMDM; Invitrogen), 100 U/ml penicillin (Invitrogen), 100 μg/ml streptomycin (Invitrogen), 1.25 μg/ml amphotericin B (Invitrogen) and 10% fetal calf serum (FCS; Sera Lab). After 48 h of culture, nonadherent cells were removed, and cells were further cultured in complete medium with 10% FCS until confluent. Flow cytometric phenotyping of in vitro cultured mesenchymal cells was performed using monoclonal antibodies directed against CD45 (in PerCP) and CD13 (in PE) (both from Becton Dickinson) on a FACScan analytical flow cytometer (Becton Dickinson). In vitro cultured mesenchymal cells were characterized as CD45 negative and CD13 positive.7

Phytohemagglutinin stimulation of T cells

For the generation of phytohemagglutinin (PHA)-stimulated T-cell cultures, 20 × 106 nonadherent PBMC were cultured in T25 culture flasks in AIM-V medium (Invitrogen) supplemented with 10% human AB serum (ICN Biomedicals) and 1 μg/ml PHA (Sigma). Cells were used for electroporation experiments after 72 h of stimulation. During flow cytometry analysis, T cells were stained with the monoclonal antibodies directed against CD2 (in PE), CD3 (in PerCP), CD4 (in PE) or CD8 (in PE) (all from Becton Dickinson).

Generation of Epstein–Barr virus-transformed B lymphoblastoid cell lines and Epstein–Barr virus-specific autologous T-cell lines

B lymphoblastoid cell lines (B-LCL) were generated by infection of PBMC with the supernatant of the Epstein–Barr virus (EBV)-producing B95-8 monkey cell line8 (kindly provided by Professor Christine Van Broeckhoven, University of Antwerp, Belgium). Cell lines were cultured in IMDM supplemented with 10% FCS. Polyclonal EBV-specific autologous T-cell lines were generated as reported by Savoldo et al.9 Briefly, PBMC were stimulated with irradiated autologous B-LCL at an effector/stimulator (E:S) ratio of 40:1 in IMDM supplemented with 10% FCS. Stimulated T-cells were used for electroporation experiments after 3 days of coculture or further expanded to culture EBV-specific T-cell lines. For the latter, cells were restimulated at day 10 with irradiated B-LCL (E:S ratio 4:1). From this point on, 40 U/ml recombinant human interleukin (IL)-2 (Biosource) was added to the cultures biweekly. On day 20, cultured EBV-specific T-cells were restimulated again with irradiated B-LCL (E:S ratio 4:1) and used 3 days thereafter for electroporation experiments. During flow cytometry analysis, T cells were stained with monoclonal antibodies directed against CD4 (in PE) or CD8 (in PE) (Becton Dickinson).

Electroporation of EGFP mRNA

mRNA encoding the EGFP was prepared from the pGEM4Z/EGFP/A64 vector10 (kindly provided by Dr Eli Gilboa, Duke University Medical Center, Durham, NC, USA) as previously described.11 Prior to electroporation, cells were washed twice with Optimix Washing Solution (EquiBio) and resuspended to a final concentration of 50 × 106 cells/ml in Optimix electroporation buffer (EquiBio). Subsequently, 0.2 ml of the cell suspension was mixed with 20 μg of in vitro transcribed EGFP mRNA and electroporated in a 0.4-cm cuvette (EquiBio) at 300 V and 150 μF using an Easyject Plus device (EquiBio). After electroporation, fresh complete medium was added to the cell suspension and cells were further cultured until flow cytometry analysis.

EGFP and cell viability analysis by flow cytometry

For flow cytometry analysis, cells were harvested, stained with appropriate monoclonal antibodies and resuspended in 500 μl of PBS (Invitrogen). To assess cell viability, ethidiumbromide (Sigma) was added to the cells (10 μl of a 20 μg/ml stock solution per 500 μl of cell suspension) prior to flow cytometry analysis. In order to exclude dead cells from the flow cytometry analysis, gating was performed on ethidiumbromide negative cells. Non- or mock-electroporated cells were used as control cells for EGFP analysis.

Results

Efficiency of mRNA electroporation in adult stem cells

Two adult bone marrow samples were electroporated with EGFP mRNA, as described in Materials and methods, and analyzed for EGFP expression after 24 h of culture. Flow cytometry analysis showed that gene transfer efficiency in the total population of fresh bone marrow cells is 25–30% (Figure 1a, histogram overlay). Moreover, the electroporation procedure did not induce significant T-cell mortality among electroporated cells (Figure 1a, dot plot). Electroporated bone marrow samples were also stained for CD34 (Figure 1b, dot plot), and further analysis revealed that 35% of the hematopoietic progenitor cell fraction (CD34+ cells, n=2) within adult bone marrow was efficiently electroporated with EGFP mRNA (Figure 1b, histogram overlay).

Figure 1
figure1

Efficiency of mRNA electroporation in adult stem cells. (a) Left side: dot plot showing side scatter (X-axis) vs ethidiumbromide staining (Y-axis) of EGFP mRNA-electroporated bone marrow mononuclear cells. The drawn gate shows the ethidiumbromide-negative viable cell population. Right side: histogram overlay showing EGFP fluorescence of EGFP mRNA-electroporated (filled histogram) vs mock-electroporated (open histogram) viable bone marrow mononuclear cells. Results are representative for two independent experiments. (b) Left side: dot plot showing side scatter (X-axis) vs CD34 staining (Y-axis) of viable EGFP mRNA-electroporated bone marrow mononuclear cells (gated on dot plot in (a)). The drawn gate shows the CD34+ progenitor cell population. Right side: histogram overlay showing EGFP fluorescence of EGFP mRNA-electroporated (filled histogram) vs mock-electroporated (open histogram) viable CD34+ progenitor cells. Results are representative for two independent experiments. (c) Left side: dot plot showing CD45 staining (X-axis) vs CD13 staining (Y-axis) of viable EGFP mRNA-electroporated mesenchymal cells. Right side: histogram overlay showing EGFP fluorescence of EGFP mRNA-electroporated (filled histogram) vs nonelectroporated (open histogram) viable mesenchymal cells. Results are representative for three independent experiments.

We also tried EGFP mRNA electroporation as a non-viral method to express a transgene in mesenchymal (stem) cells. For this, bone marrow-derived mesenchymal cells from three different donors were cultured as described in Materials and methods. Expanded mesenchymal cells were characterized by flow cytometry analysis based on negative staining for CD45 and positive staining for CD13 (Figure 1c, dot plot, 86±6%). At 24 h after electroporation, cells were analyzed by flow cytometry for EGFP fluorescence and cell viability. Results show EGFP fluorescence in 89±2% of viable EGFP mRNA-electroporated mesenchymal cells (Figure 1c, histogram overlay), combined with acceptable cell viability (67±16% for nonelectroporated cells vs 63±17% for EGFP mRNA-electroporated cells).

Efficiency of mRNA electroporation in activated T cells

To investigate the efficiency of mRNA-electroporation in T cells, nonstimulated and PHA-stimulated PBMC were electroporated with EGFP mRNA as described in Materials and methods, and analyzed for T-cell markers (CD3 vs CD4 or CD8) and EGFP expression after 24 h of culture (Figure 2a). Flow cytometry analysis showed that RNA-based gene transfer in nonstimulated T cells is very inefficient (Figure 2a: NO STIMULATION; 0.6±0.3% (n=3) EGFP+ cells for CD3+/CD4+ T cells; 0.8±0.6% (n=3) EGFP+ cells for CD3+/CD8+ T cells). In contrast, efficiency of mRNA electroporation was very high in PHA-stimulated T cells (Figure 2a: PHA STIMULATION; 46±14% (n=3) EGFP+ cells for CD3+/CD4+ T cells; 47±17% (n=3) EGFP+ cells for CD3+/CD8+ T cells). Furthermore, kinetics of EGFP expression in the CD2-positive viable PHA-stimulated T-cell population was followed by flow cytometry during a period of seven days (Figure 2b). Although the mean fluorescence intensity (MFI) of the EGFP+ cells started to decrease at day 2 after electroporation, EGFP expression could still be detected in 57±19% (n=4) of viable CD2-positive T cells at 4 days after electroporation. Considering cell viability, no significant difference was observed at 24 h after electroporation in nonelectroporated (87±4%, n=4) vs EGFP mRNA-electroporated T cells (87±2%, n=4). Next, we investigated the efficiency of mRNA electroporation in PBMC stimulated with an EBV-transformed autologous B-cell line (B-LCL). After 3 days of in vitro coculture, as described in Materials and methods, stimulated PBMC were electroporated with EGFP mRNA and analyzed for T-cell markers (CD4 or CD8) and EGFP expression after 24 h of culture (Figure 2c). While EGFP expression was very low in nonstimulated T-cells (Figure 2c: NO STIMULATION), a small number of EGFP-positive T cells could be detected in T-cell cultures stimulated with an autologous B-LCL (Figure 2c: B-LCL STIMULATION; 4.4±1.1% (n=3) EGFP+ cells for CD4+ T cells; 5.4±1.1% (n=3) EGFP+ cells for CD8+ T cells). In order to increase the number of EBV-stimulated T cells, PBMC were restimulated twice with autologous B-LCL as described in Materials and methods. At 3 days after the second restimulation, cultured cells were electroporated with EGFP mRNA followed by flow cytometry analysis 24 h after electroporation. Figure 2d shows an increased number of EGFP-positive T cells within the CD8+ T-cell population after multiple B-LCL stimulations (14% EGFP+ cells after three B-LCL stimulations vs 5.4% after 1 B-LCL stimulation vs 0.8% in nonstimulated PBMC).

Figure 2
figure2

Efficiency of mRNA electroporation in activated T cells. (a) Number of EGFP expressing CD4+ and CD8+ T cells in nonstimulated PBMC (NO STIMULATION) and in PHA-stimulated PBMC (PHA STIMULATION). Data are shown as mean+standard deviation of three independent experiments. (b) Percentage of EGFP+ cells (left Y-axis; EGFP, filled squares) among viable PHA-stimulated T cells and MFI of EGFP+ viable PHA-stimulated T-cells (right Y-axis; MFI, open squares) vs time after mRNA electroporation (X-axis). Representative example for four independent experiments. (c) Number of EGFP expressing CD4+ and CD8+ T cells in nonstimulated PBMC (NO STIMULATION) and in B-LCL-stimulated PBMC (B-LCL STIMULATION). Data are shown as mean+standard deviation of three independent experiments. (d) Histogram overlay shows the level of EGFP fluorescence within the CD8+ T-cell population of non-electroporated EBV-activated T cells (open histogram) and EGFP mRNA-electroporated EBV-activated T cells (filled histogram) after two restimulations with irradiated autologous B-LCL. Gating was carried out based on the CD8+ staining (dot plot).

Discussion

Gene therapy strategies used in clinical applications should be carefully developed in order to avoid serious adverse effects possibly caused by the use of plasmid DNA and viral vectors.2, 3 Gene transfer by plasmid DNA generally does not result in high numbers of transfected cells12 and is often associated with high cell mortality among transfected cells. The use of viral vectors for gene transfer can circumvent these problems, but their potential immunogenicity might cause serious problems.13, 14 Moreover, for both plasmid DNA transfer and viral gene transfer, integration of DNA fragments into or next to certain genes might occur, possibly resulting in undesired gene expression following use in vivo. In contrast, the use of mRNA for transfection of cell lines and primary cells is likely to be a safe, highly efficient and clinically applicable alternative to virus and/or plasmid DNA-mediated gene transfer.1 However, since transgene expression is only transient, the potential applications are limited to gene therapy strategies where no long-term gene-expression is required. Although most of the currently ongoing gene therapy research requires stable transgene expression,13, 15 several applications are arising where the use of mRNA will be preferable over the use of plasmid DNA and viral vectors. This has previously been demonstrated in many studies on the use of mRNA-loaded dendritic cells for immunotherapeutic purposes,6, 11, 16 where gene transfer by means of mRNA has become one of the most promising candidates for use in clinical trials.1, 5 Based on this success of mRNA-mediated gene transfer, novel applications are being explored.

Recently, both hematopoietic and mesenchymal stem cells have become of major interest for the development of cell replacement therapies.17, 18 Efficient short-term transgene expression of specific genes, growth factors or transcription factors in CD34+ progenitor cells might be interesting to promote growth of undifferentiated progenitor cells or to (re)direct their differentiation towards a specific lineage.19, 20 Multiple combinations of growth factors and chemicals have been described to differentiate mesenchymal cells into several different lineages of mesodermal origin, such as cartilage, bone, fat, tendon, muscle, myocardium and marrow stroma.18 More recently, strategies using gene-modified mesenchymal cells, by means of viral transduction, have been developed for differentiation of osteogenic cells.21, 22 Using the presented mRNA electroporation method, adult stem cells can be loaded with mRNA encoding a specific transcription factor, combinations of several transcription factors or even total mRNA fractions derived from specific cell types. It will be interesting to see whether these stem cells, after one or multiple mRNA electroporations, will be directed towards a specific lineage.

Previously published manuscripts already reported the inefficiency of plasmid DNA-based gene transfer in nonstimulated T cells, while enhanced efficiency of plasmid DNA transfection was shown in PHA-stimulated human T cells and activated CD4+ murine T cells (32 and 34% respectively).23, 24 Our results also show inefficient gene transfer by means of mRNA electroporation in nonstimulated T cells, while PHA-stimulated T cells can be efficiently loaded with mRNA (Figure 2a, around 50% for both CD4+ and CD8+ T cells). Moreover, transgene expression could also be detected in EGFP mRNA-electroporated EBV-stimulated T cells. Thinking in terms of clinical applications, and if short-term transgene expression is sufficient, the use of mRNA instead of plasmid DNA or viral vectors would be preferable. Applications for mRNA-based gene transfer in T cells can be found in the field of adoptive T-cell immunotherapy. Introduction of telomerase in order to increase the replicative lifespan of cloned antigen-specific T cells has been reported with the use of viral vectors.25, 26 Safety issues associated with the use of viral vectors can be circumvented by using mRNA for gene transfer. Also, directing or redirecting T-cell subtype differentiation by short-term introduction of mRNA encoding specific activating/inhibitory master regulatory genes27 might become an interesting research direction in fundamental research towards adoptive T-cell immunotherapy, both for immune stimulation and immune tolerance.

Based on the presented results and the potential applications, we conclude that, for several applications in both fundamental research and the treatment of hematologic and other diseases, mRNA electroporation might become an important tool for gene transfer in adult stem cells and activated T cells. Moreover, using the same electroporation procedure, it can be further investigated whether a combined electroporation of mRNA, in order to express specific genes, and siRNA, in order to shut down specific genes, might be beneficial for directing (stem) cell fate in vitro and in vivo.

References

  1. 1

    Sullenger BA, Gilboa E . Emerging clinical applications of RNA. Nature 2002; 418: 252–258.

    CAS  Article  Google Scholar 

  2. 2

    Hacein-Bey-Abina S, von Kalle C, Schmidt M, Le Deist F, Wulffraat N, McIntyre E et al. A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 2003; 348: 255–256.

    Article  Google Scholar 

  3. 3

    Marshall E . Gene therapy death prompts review of adenovirus vector. Science 1999; 286: 2244–2245.

    CAS  Article  Google Scholar 

  4. 4

    Van Tendeloo VF, Ponsaerts P, Lardon F, Nijs G, Lenjou M, Van Broeckhoven C et al. Highly efficient gene delivery by mRNA electroporation in human hematopoietic cells: superiority to lipofection and passive pulsing of mRNA and to electroporation of plasmid cDNA for tumor antigen loading of dendritic cells. Blood 2001; 98: 49–56.

    CAS  Article  Google Scholar 

  5. 5

    Ponsaerts P, Van Tendeloo VF, Berneman ZN . Cancer immunotherapy using RNA-loaded dendritic cells. Clin Exp Immunol 2003; 134: 378–384.

    CAS  Article  Google Scholar 

  6. 6

    Saeboe-Larssen S, Fossberg E, Gaudernack G . mRNA-based electrotransfection of human dendritic cells and induction of cytotoxic T lymphocyte responses against the telomerase catalytic subunit (hTERT). J Immunol Methods 2002; 259: 191–203.

    CAS  Article  Google Scholar 

  7. 7

    Chen ZZ, Van Bockstaele DR, Buyssens N, Hendrics D, De MI, Vanhoof G et al. Stromal populations and fibrosis in human long-term bone marrow cultures. Leukemia 1991; 5: 772–781.

    CAS  PubMed  Google Scholar 

  8. 8

    Miller G, Lipman M . Release of infectious Epstein–Barr virus by transformed marmoset leukocytes. Proc Natl Acad Sci USA 1973; 70: 190–194.

    CAS  Article  Google Scholar 

  9. 9

    Savoldo B, Huls MH, Liu Z, Okamura T, Volk HD, Reinke P et al. Autologous Epstein–Barr virus (EBV)-specific cytotoxic T cells for the treatment of persistent active EBV infection. Blood 2002; 100: 4059–4066.

    CAS  Article  Google Scholar 

  10. 10

    Nair SK, Boczkowski D, Morse M, Cumming RI, Lyerly HK, Gilboa E . Induction of primary carcinoembryonic antigen (CEA)-specific cytotoxic T lymphocytes in vitro using human dendritic cells transfected with RNA. Nat Biotechnol 1998; 16: 364–369.

    CAS  Article  Google Scholar 

  11. 11

    Ponsaerts P, Van Tendeloo VF, Cools N, Van Driessche A, Lardon F, Nijs G et al. mRNA-electroporated mature dendritic cells retain transgene expression, phenotypical properties and stimulatory capacity after cryopreservation. Leukemia 2002; 16: 1324–1330.

    CAS  Article  Google Scholar 

  12. 12

    Van Tendeloo VF, Willems R, Ponsaerts P, Lenjou M, Nijs G, Vanhove M et al. High-level transgene expression in primary human T lymphocytes and adult bone marrow CD34+ cells via electroporation-mediated gene delivery. Gene Therapy 2000; 7: 1431–1437.

    CAS  Article  Google Scholar 

  13. 13

    Van Tendeloo VF, Van Broeckhoven C, Berneman ZN . Gene therapy: principles and applications to hematopoietic cells. Leukemia 2001; 15: 523–544.

    CAS  Article  Google Scholar 

  14. 14

    Pfeifer A, Verma IM . Gene therapy: promises and problems. Annu Rev Genomics Hum Genet 2001; 2: 177–211.

    CAS  Article  Google Scholar 

  15. 15

    Van Tendeloo VF, Van Broeckhoven C, Berneman ZN . Gene-based cancer vaccines: an ex vivo approach. Leukemia 2001; 15: 545–558.

    CAS  Article  Google Scholar 

  16. 16

    Muller MR, Tsakou G, Grunebach F, Schmidt SM, Brossart P . Induction of chronic lymphocytic leukemia (CLL)-specific CD4- and CD8-mediated T-cell responses using RNA-transfected dendritic cells. Blood 2004; 103: 1763–1769.

    Article  Google Scholar 

  17. 17

    Srour EF, Jetmore A, Wolber FM, Plett PA, Abonour R, Yoder MC et al. Homing, cell cycle kinetics and fate of transplanted hematopoietic stem cells. Leukemia 2001; 15: 1681–1684.

    CAS  Article  Google Scholar 

  18. 18

    Tocci A, Forte L . Mesenchymal stem cell: use and perspectives. Hematol J 2003; 4: 92–96.

    Article  Google Scholar 

  19. 19

    Brun AC, Fan X, Bjornsson JM, Humphries RK, Karlsson S . Enforced adenoviral vector-mediated expression of HOXB4 in human umbilical cord blood CD34+ cells promotes myeloid differentiation but not proliferation. Mol Ther 2003; 8: 618–628.

    CAS  Article  Google Scholar 

  20. 20

    Svedberg H, Richter J, Gullberg U . Forced expression of the Wilms tumor 1 (WT1) gene inhibits proliferation of human hematopoietic CD34(+) progenitor cells. Leukemia 2001; 15: 1914–1922.

    CAS  Article  Google Scholar 

  21. 21

    Lou J, Xu F, Merkel K, Manske P . Gene therapy: adenovirus-mediated human bone morphogenetic protein-2 gene transfer induces mesenchymal progenitor cell proliferation and differentiation in vitro and bone formation in vivo. J Orthop Res 1999; 17: 43–50.

    CAS  Article  Google Scholar 

  22. 22

    Tsuda H, Wada T, Ito Y, Uchida H, Dehari H, Nakamura K et al. Efficient BMP2 gene transfer and bone formation of mesenchymal stem cells by a fiber-mutant adenoviral vector. Mol Ther 2003; 7: 354–365.

    CAS  Article  Google Scholar 

  23. 23

    Herndon TM, Juang YT, Solomou EE, Rothwell SW, Gourley MF, Tsokos GC . Direct transfer of p65 into T lymphocytes from systemic lupus erythematosus patients leads to increased levels of interleukin-2 promoter activity. Clin Immunol 2002; 103: 145–153.

    CAS  Article  Google Scholar 

  24. 24

    Lai W, Chang CH, Farber DL . Gene transfection and expression in resting and activated murine CD4 T cell subsets. J Immunol Methods 2003; 282: 93–102.

    CAS  Article  Google Scholar 

  25. 25

    Rufer N, Migliaccio M, Antonchuk J, Humphries RK, Roosnek E, Lansdorp PM . Transfer of the human telomerase reverse transcriptase (TERT) gene into T lymphocytes results in extension of replicative potential. Blood 2001; 98: 597–603.

    CAS  Article  Google Scholar 

  26. 26

    Schreurs MW, Scholten KB, Kueter EW, Ruizendaal JJ, Meijer CJ, Hooijberg E . In vitro generation and life span extension of human papillomavirus type 16-specific, healthy donor-derived CTL clones. J Immunol 2003; 171: 2912–2921.

    CAS  Article  Google Scholar 

  27. 27

    Zheng WP, Zhao Q, Zhao X, Li B, Hubank M, Schatz DG et al. Up-regulation of Hlx in immature Th cells induces IFN-gamma expression. J Immunol 2004; 172: 114–122.

    CAS  Article  Google Scholar 

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Acknowledgements

This work was supported by Grant No. G.0313.01 of the Fund for Scientific Research – Flanders, Belgium (FWO-Vlaanderen), by a grant of the Scientific Committee of the Fortis Bank (FB) Verzekeringen-financed Cancer Research and by a grant of the Belgian Federation against Cancer (BFK). We also acknowledge support from Grant No. 7.0004.03 (Levenslijn) of the FWO-Vlaanderen. VFIVT is a postdoctoral fellow of the FWO-Vlaanderen.

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Smits, E., Ponsaerts, P., Lenjou, M. et al. RNA-based gene transfer for adult stem cells and T cells. Leukemia 18, 1898–1902 (2004). https://doi.org/10.1038/sj.leu.2403463

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Keywords

  • stem cells
  • T cells
  • electroporation
  • mRNA
  • EGFP

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