Nucleofection Is an Efficient Nonviral Transfection Technique for Human Bone Mar - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:10 |只看该作者 |倒序浏览 |打印

作者：Michela Aluigia, Miriam Foglia, Antonio Curtia, Alessandro Isidoria, Elisa Gruppionib, Claudia Chiodonid, Mario P. Colombod, Piera Versurac, Antonia DErrico-Grigionib, Elisa Ferria, Michele Baccarania, Roberto M. Lemolia作者单位：a Institute of Hematology and Medical Oncology L. e A. Sergnoli and Stem Cells Research Center, S. Orsola-Malpighi Hospital, Bologna, Italy;b Molecular and Transplantation Pathology Laboratory Felice Addarii andc Department of Surgery and Transplant, Section of Ophthalmology, University of Bologna,  . \  q6 s+ t+ O) ]2 e( Z
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      【摘要】
   Viral-based techniques are the most efficient systems to deliver DNA into stem cells because they show high gene transduction and transgene expression in many cellular models. However, the use of viral vectors has several disadvantages mainly involving safety risks. Conversely, nonviral methods are rather inefficient for most primary cells. The Nucleofector technology, a new nonviral electroporation-based gene transfer technique, has proved to be an efficient tool for transfecting hard-to-transfect cell lines and primary cells. However, little is known about the capacity of this technique to transfect adult stem cells. In this study, we applied the Nucleofector technology to engineer human bone marrow¨C derived mesenchymal stem cells (hMSCs). Using a green fluorescent protein reporter vector, we demonstrated a high transgene expression level using U-23 and C-17 pulsing programs: 73.7% ¡À 2.9% and 42.5% ¡À 3.4%, respectively. Cell recoveries and viabilities were 38.7% ¡À 2.9%, 44.5% ¡À 3.9% and 91.4% ¡À 1.3%, 94.31% ¡À 0.9% for U-23 and C-17, respectively. Overall, the transfection efficiencies were 27.4% ¡À 2.9% (U-23) and 16.6% ¡À 1.4% (C-17) compared with 3.6% ¡À 2.4% and 5.4% ¡À 3.4% of other nonviral transfection systems, such as FUGENE6 and DOTAP, respectively (p < .005 for all comparisons). Nucleofection did not affect the immunophenotype of hM-SCs, their normal differentiation potential, or ability to inhibit T-cell alloreactivity. Moreover, the interleukin-12 gene could be successfully transfected into hMSCs, and the immunomodulatory cytokine was produced in great amount for at least 3 weeks without impairment of its biological activity. In conclusion, nucleofection is an efficient nonviral transfection technique for hMSCs, which then may be used as cellular vehicles for the delivery of biological agents. 5 H" C  l* w1 D
      【关键词】 Nucleofection Mesenchymal stem cells Interleukin- Gene therapy
   INTRODUCTION

Gene transfer technology has many potential applications in medicine. The delivery of genes into cells, in vitro and in vivo, has not only become an important tool for research but has also progressed in numerous clinical studies. Phase I and phase II gene-based clinical trials have been conducted for the treatment of cancer, neurodegenerative disorders, cardiomyopathies, and infectious diseases.
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To date, the most commonly used systems to deliver DNA into primary cells are viral-based techniques. Adenovirus, retrovirus, and lentivirus are probably the most effective gene transfer vehicles that can provide high transduction efficiency, also in nondividing cells, integration into the host cell genome, and high level of gene expression . Therefore, nonviral transfection methods have been developed. Commonly used nonviral transfection systems, including lipofection/polyfection reagents, microinjection, gene-gun, and electroporation, are much less efficient compared with viral transduction and often present high cell mortality due to toxicity.6 s  J  X; Y5 l

The Nucleofector technology is a new nonviral transfection method especially designed for primary cells and hard-to-transfect cell lines. Nucleofection combines electrical parameters and cell-type solutions to drive plasmid DNA, oligonucleotides, as well as siRNA directly into the cell nucleus. Consequently, transfection of cells is no longer dependent on cell division, and high transfection efficiencies can be obtained in fresh or immortalized primary cells .4 d  {# B8 v( `# ], O
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Human bone marrow (BM) includes at least two populations of hematopoietic and nonhematopoietic stem cells, the so-called mesenchymal stem cells (hMSCs) .
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Both animal mesenchymal stem cells and hMSCs have been successfully transduced in vitro with exogenous genes without impairment of their functional capacity .
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In the present study, we evaluated the efficacy of the Nucleofector technology as a gene delivery vehicle into hMSCs. Our data demonstrate that nucleofection is an efficient nonviral transfection technique for hMSCs, which can serve as cellular vehicles for the delivery and local production of biological agents, such as interleukin-12 (IL-12).

MATERIALS AND METHODS

Cell Isolation and Culture/ K- H) H7 G+ s7 p; G' L
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hMSCs were isolated from BM aspirates of normal donors after obtaining informed consent. The mononuclear cell fraction was separated by centrifugation over a Ficoll-Paque gradient (Amersham Bioscience, Piscataway, NJ, http://www.amersham.com), resuspended in proliferation medium consisting of low-glucose Dulbecco¡¯s modified Eagle¡¯s medium (BioWhittaker, Cambrex Bio Science, Caravaggio, Italy, http://www.cambrex.com), 10% fetal bovine serum (Gibco-Invitrogen, Carlsbad, CA, http://www.invitrogen.com), 2 mM L-glutamine, and 1% antibiotics (MP Biomedicals, Verona, Italy, http://www.mpbio.com), and plated at an initial seeding density of 5 x 105 cells/ml. After 3 days, the nonadherent cell fraction was removed by washing with phosphate-buffered saline (PBS), and monolayers of adherent cells were cultured until they reached 70%¨C90% confluence. Cells were then trypsinized (0.25% trypsin with 0.1% EDTA) (EuroClone, West York, UK, http://www.euroclone.net), subcultured at a density of 5 x 103 cells/cm2, and used for further experiments within passages 2 to 4.+ u- e5 x! J- L( D6 v! k1 E
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Immunophenotype Studies
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Dual-color immunofluorescence was performed using the following panel of phycoerythrin-conjugated or fluorescein isothiocyanate¨Cconjugated monoclonal antibodies (mAbs): anti-human CD14, anti-human CD13 (Becton, Dickinson and Company, San Jose, CA, http://www.bd.com); anti-human CD105, anti-human CD106, anti-human CD44, anti-human CD45, anti-human CD90, anti-human CD29 (Chemicon, Te-mecula, CA, http://www.chemicon.com); anti-human HLA-DR, anti-human CD34 (BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen); and anti-human HLA-I (Euro-Clone). Negative controls were isotype-matched irrelevant mAbs (BD Pharmingen). For cell-surface staining, cells (5 x 105) were incubated in the dark for 30 minutes at 4¡ãC in PBS-1% bovine serum albumin (BSA). After washing, cells were resuspended in PBS and analyzed using FACScan equipment (Becton, Dickinson and Company, Mountain View, CA, http://www.bd.com). A minimum of 10,000 events was collected in list mode on FACScan software.! q9 x6 b3 I2 S& u
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Osteogenic and Adipogenic Differentiation of hMSCs7 @# I% Z' o' r* J$ d" Z

The capacity of hMSCs to differentiate along the osteogenic and adipogenic lineages was assessed as described elsewhere . Briefly, to induce osteogenesis, hMSCs were cultured at 3.1 x 103 cells/cm2 either in six-well plates or in Lab-Tek II Chamber slide (Nunc, Rochester, NY, http://www.nuncbrand.com) for 24 hours in proliferation medium. Cells were subsequently cultured for 3 weeks in the presence of 10 mM ß-glycerophosphate, 0.1 µM dexamethasone, and 50 µM ascorbic acid (all from Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) with medium change every 3 to 4 days. Control cells were grown in proliferation medium. Differentiation was assessed by nitroblue tetrazolium/X-phosphate staining, which reveals alkaline phosphatase.; _) i2 r8 u5 S9 f* {. e+ g

To induce adipogenesis, hMSCs were seeded at a concentration of 2.1 x 104 either in six-well plates or in Lab-Tek II Chamber slide and induced by adding 0.5 mM isobuthyl-1-methylxanthine, 0.1 µM dexamethasone, 10 µg/ml insulin, and 0.2 µM indomethacin (Sigma-Aldrich). The medium was replaced every 3 to 4 days for 21 days. At the end of the culture, the adipocytes were visualized by hematoxylin-eosin staining, which evidences lipid droplet in the cytoplasm.% j/ ], p, M# A0 T

Reverse Transcription¨CPolymerase Chain Reaction
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Total RNA from both untreated and differentiated hMSCs was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA; www.invitrogen.com). RNA concentration and purity was assessed spectrophometrically by UV reading. For reverse transcription¨Cpolymerase chain reaction (RT-PCR), cDNA was synthesized in a 20-µl reaction containing 2 µg of total RNA using SuperScript II System (Invitrogen) according to the manufacturer¡¯s instructions. PCR was performed with Taq Polymerase (Roche Applied Science, Mannheim, Germany, https://www.roche-applied-science.com) using the following gene-specific primers: osteopontin (264 bp) forward: 5'-ATGGCCGAGGTGATAGTGTGGT-3' and reverse: 5'-ATCTGGACTGCTTGTGGCTGTG-3'; peroxisome proliferator¨Cactivated receptor gamma 2 (PPAR2) (264 bp) forward: 5'-GGAGCCCAAGTTTGAGTTTGCTGT-3' and reverse: 5'-TAGCTGCACGTGTTCCGTGACAAT-3'; ß2-microglobulin (162bp): forward: 5'-CTCGCGCTACCTTCTCTTTCT-3' and reverse: 5'-TCCATTCTTCAGTAAGTCAACT-3'. PCR products were analyzed by 2% agarose gel electrophoresis and visualized by ethidium bromide staining.

Plasmids, e6 r  Q# b$ Z: K) c- X

Both human IL-12 (hIL-12) subunits, p35 and p40, were cloned, separated by an IRES sequence, into pcDNA3. Briefly, cDNA for p35 and p40 fragments of IL-12 were cloned using RT-PCR poly(A)  RNA obtained from a lymphoblastoid cell line, obtained by mononuclear cells treated with mitogenic stimuli. The p35 product was ligated into the EcoRV/XhoI sites of pcDNA3 to obtain pcDNAhp35. The p40 amplified product was cloned into the NcoI/BamHI sites of plasmid vector pLTM1 containing the IRES sequence to give pLTMp40. To excise the IRES/p40 fusion product, pLTMp40 was linearized by ClaI digestion, blunt-ended with T4 DNA polymerase, digested with BamHI, and ligated into pcDNAhp35 digested with XhoI and blunt-ended. The cytomegalovirus (CMV) promoter pcDNA3/NT-GFP was used to gauge transfection efficiency and pcDNA3 as a mock vector (Invitrogen).) t) c, `9 Q" u7 B7 Y' l/ o
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Cell Transfection
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Nucleofection of hMSCs was performed according to the optimized protocols provided by the manufacturer (Amaxa Biosystem, Cologne, Germany, www.amaxa.com). Briefly, cells were gently resuspended in 100 µl of Human Mesenchymal Nucleofector Solution (Amaxa Biosystem), mixed with cDNA, and pulsed with either the program U-23 (high-transfection efficiency) or C-17 (high cell survival). Immediately after, cells were transferred into prewarmed fresh medium in six-well plates. Cells were analyzed 24, 48, and 72 hours after Nucleofection for both viability and green fluorescent protein (GFP) expression.
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Transfection with two lipid-based systems, FuGENE6 and DOTAP, was performed following the producer guidelines (Roche Applied Science). In brief, cells were plated in six-well plates at a cell density of 105 cells/well and allowed to grow overnight to achieve 70%¨C80% confluence. Transfection complex, consisting of different DNA/transfection reagent ratios (wt/vol), was directly added to the wells in the presence of serum-containing medium, and cells were assayed 24 to 72 hours later for the GFP.; ~3 O5 {7 ^, i8 |9 p. D

Analysis of GFP Expression! L+ `8 S; ^  q2 K
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Analysis of GFP fluorescence in hMSCs was performed by flow cytometry. Briefly, cells were detached from culture flasks by incubation with 0.25% Trypsin-EDTA for 5 minutes, recovered by centrifugation, and washed in flow cytometry buffer consisting of 2% BSA (Sigma-Aldrich) and 0.1% sodium azide (Sigma-Aldrich) in PBS. Cells were collected by centrifugation and resuspended in flow cytometer buffer containing 2% paraformaldeyde immediately before analysis. Nonspecific fluorescence was determined using hMSCs not GFP-transfected. Samples were analyzed counting 10,000 events on a Becton Dickinson instrument using Cell-Quest Software (Becton, Dickinson and Company, Mountain View, CA, http://www.bd.com). GFP  cells cultured in six-well plates or Lab-Tek II chamber slides were observed at x20 to x40 original magnification with a Zeiss Axiovert 10 inverted microscope equipped with an epifluorescent condenser HBO 100W/2 (lamp excitation range, 450 to 490 nm ); micrographs were recorded with a Nikon Coolpix digital image system./ ]+ U+ F) m( d& K* z: v9 E
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Enzyme-Linked Immunosorbent Assay  A3 f! \1 w* d8 H; _% d- _( M+ e

To assess IL-12 production, supernatants were collected at different times after nucleofection and assayed in duplicate for IL-12 (p70) concentration by using an enzyme-linked immunosorbent assay (ELISA) kit (R&D Systems Inc., Minneapolis, http://www.rndsystems.com) according to the manufacturer¡¯s instructions.
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T-Cell Proliferation Assay7 s' G  ]! a" ], |" E/ d, U8 ~2 u1 F
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A previously reported method was used thymidine incorporation was measured as counts per minute (cpm) on a TopCount NxT ß-counter (PerkinElmer). Results were expressed as mean cpm ¡À standard deviation.. D) d, F. e9 K
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Cytotoxicity Assay4 C. o6 t( V3 |, e. ^- E
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Highly purified natural killer (NK) cells were suspended at 105/ml in culture medium supplemented with recombinant human IL-12 (1 ng/ml) (R&D Systems) or the same amount of IL-12 produced by hMSCs nucleofected with IL-12 plasmid. Cultures were incubated for 24 hours in a humidified atmosphere of 5% CO2 in air. At the end of culture period, viable and activated cells were counted using the trypan blue exclusion method and used as effectors in a standard 4-hour 51Cr release assay against K562 target cells. Percentage of specific lysis was calculated as .
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Statistical Analysis3 r7 \  Z5 |  o8 O

Results are expressed as mean ¡À standard deviation. The results were analyzed with the paired nonparametric Wilcoxon rank-sum test. Two-sided p values " d7 A! Y+ C8 H, A. I  n
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RESULTS
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MSC Culture# I$ S; Y$ Z$ `; p: C2 b
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BM hMSCs were successfully culture-expanded from all donors (n = 11). A morphologically homogeneous population of fibroblast-like cells was seen at a median of 13 days. After the second or the third passage, growing cells were uniformly positive for CD13, CD90, CD105, CD106, CD44, CD29, and HLA-I antigens and negative for hematopoietic lineage markers such as CD14, CD34, CD45, and HLA-II (Fig. 1).
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Figure 1. Representative flow cytometric analysis of cultured hMSCs. Filled histograms indicate isotype-matched mouse immunoglobulin G antibody control staining. hMSCs were negative for CD45, CD34, CD14, and class II MHC and positive for CD13, CD90, CD44, CD105, CD106, CD29, and class I MHC. Abbreviations: hMSC, human mesenchymal stem cell; MHC, myosin heavy chain.
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Nucleofection of hMSCs Induced High Transient Transfection Efficiency Compared with Other Nonviral Systems$ p2 W  {/ x7 O- E3 B, q9 p

hMSCs were transfected by the Nucleofector technology following the optimized protocol provided by the manufacturer. To this end, we used 5 x 105 cells, 2 µg of pcDNA3.1/NT-GFP, and two different pulsing programs, U-23 and C-17, specifically indicated for high transfection efficiency and high cell survival, respectively. After nucleofection, cells were allowed to adhere overnight at 37¡ãC in a humidified atmosphere of 5% CO2 in air, and then dead cells and debris were removed by medium exchange and transgene expression evaluated by fluorescence-activated cell sorter (FACS) analysis. GFP levels in hMSCs were evaluated at different time points, 24, 48, and 72 hours after nucleofection, to look for the highest transfection efficiency. No significant differences in GFP expression were observed at all times analyzed and with both pulsing programs (data not shown). As shown in Figure 2A, 72 hours after nucleofection, GFP  cells represented more than 40% of transfected hMSCs with both pulsing programs. Specifically, application of U-23 program, developed to induce a higher transgene expression, resulted in 73.7% ¡À 2.9% GFP  cells with a mean viability of 91.4% ¡À 1.3%. The overall recovery of viable GFP  hMSCs was 38.8% ¡À 2.9% (n = 5) (Fig. 2A). Conversely, C-17 program, designed to improve cell viability, resulted in 42.5% ¡À 3.4% GFP  cells with a mean cell viability of 94.3% ¡À 0.9% and an overall recovery of 44.5% ¡À 3.9% (n = 5). Only the transfection efficiency between the two pulsing programs was statistically significant (p = .01). Both programs showed a significantly higher transfection efficiency compared with two different lipid-based nonviral systems, FuGENE6 and DOTAP, which had been successfully used in many different primary cell models  (Fig. 2B). The percentage of GFP expression in viable MSCs 72 hours after transfection was 4.4% ¡À 2.2% for FuGENE6 and 6.8% ¡À 4.1% for DOTAP (n = 3), with a mean cell viability greater than 80%, 94% ¡À 6.7%, and 85.5% ¡À 9.3%, respectively. Cell recovery was 80.6% ¡À 33.1% for FuGENE6 (n = 3) and 98.5% ¡À 48.1% for DOTAP (n = 3) due to the less-invasive nature of these reagents.6 E2 o; g2 Y$ M; a5 j" I7 [

Figure 2. Transfection efficiency of expanded human MSCs. (A): MSCs were pulsed with U-23 and C-17 programs in the presence of 2 µg of pcDNA3/NT-GFP. After overnight incubation, cell debris and dead cells were removed by changing culture medium and cells were cultured for additional 48 hours before analysis. (B): MSCs were transfected with lipid-based reagents FuGENE6 and DOTAP with 2 µg of pcDNA3/NT-GFP. For DOTAP transfection, only medium was replaced after 5 to 6 hours. Cells were recovered after 72 hours and analyzed. Transfection efficiency was scored by fluorescence-activated cell sorter analysis and reported as percentage of GFP  cells; the percentage of viable cells was estimated by trypan blue exclusion count; the recovery is the percentage of collected cells after nucleofection compared with plated cells; yield is the percentage of viable GFP-expressing hMSCs compared with plated cells. *p - e+ R9 M# G! o8 k4 A. ~0 d& |8 j

Overall, the yield of GFP  hMSCs after transfection with the two lipid-based systems was 3.6% ¡À 2.4% (FuGENE6) and 5.4% ¡À 3.4% (DOTAP). By comparison, nucleofection induced significantly higher numbers of transfected cell, as the total yield of GFP-expressing hMSCs was 27.4% ¡À 2.9% and 16.6%¡À 1.4% for U-23 and C-17 programs, respectively (p 2 V* _( `4 U. t
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To assess the stability of the Nucleofection-based transfection, the time course and the level of GFP expression were determined over a period of 3 weeks. In accordance with the transient nature of plasmid-based transfection, GFP levels in hMSCs declined over time because cells were not cultured in the presence of selective antibiotic. The protein production was sustained at high levels (>60%) for 7 days (Fig. 3). Afterward, although GFP expression decreased due to the dilution of the plasmid in culture, the percentage of GFP cells was still as high as 32.8% ¡À 6.3% after 3 weeks.( {) ]# p; L1 c# y1 s% ?

Figure 3. Time-course analysis of GFP expression in nucleofected human mesenchymal stem cell. Cells nucleofected with U-23 pulsing program and GFP reporter vector were expanded in culture, and GFP expression was evaluated by fluorescence-activated cell sorter at different time points after nucleofection. Abbreviation: GFP, green fluorescent protein.
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Nucleofection Does Not Impair Phenotypical and Functional Properties of hMSCs In Vitro) q. B; ?6 w' _4 X
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Taken together, these experiments demonstrated that Nucleofector technology is an efficient method to induce high transgene expression in BM-derived hMSCs and may be superior to other transfection systems. We then evaluated whether nucleofected hMSCs have altered phenotypic and/or functional properties. FACS analysis of GFP-nucleofected cells 72 hours after transfection showed the positivity of CD13, CD29, CD105, and class I MHC antigens and the lack of expression of the hematopoietic stem cell marker CD34 and class II MHC (Fig. 4).7 J" s. u8 K; V2 q! b

Figure 4. Nucleofection-based transfection did not alter the immuno-phenotype of human MSCs. One million cells were pulsed with program U-23 in the presence of 4 µg of pcDNA3/NT-GFP. After 72 hours, cells were stained with antibodies and fluorescence-activated cell sorter analyzed as reported in Material and Methods. The representative scatters of GFP  MSCs counterstained with phycoerythrin-conjugated CD-34, HLA-II, HLA-I, CD13, CD105, and CD29 demonstrated the MSC phenotype of transfected cells. Abbreviations: GFP, green fluorescent protein; MSC, mesenchymal stem cell.

To test the differentiation potential of hMSCs, GFP  cells were induced to differentiate toward the osteogenic and adipogenic lineages in vitro (Fig. 5). When cultured in the presence of ß-glycero-phosphate, ascorbic acid and dexamethasone cells underwent a change in their morphology from spindle shape to cuboidal and formed large nodules by day 18 of induction. Upregulation of alkaline phosphatase activity and deposition of a hydroxyapatite mineralized extracellular matrix was evidenced by NBT/X phosphate staining (Fig. 5A, panels d¨Cf). GFP-nucleofected hMSCs cultured in monolayer in the presence of isobutylmethylxanthine turned into adipocytes with the production of large lipid-filled vacuoles within the cytoplasm (Fig. 5A, panels a¨Cc). Additionally, bone differentiation was confirmed by molecular analysis of osteopontin mRNA, whose expression is restricted to the osteogenic lineage (Fig. 5B). Adipogenic differentiation is induced by the nuclear receptor and transcription factor PPAR2, which was detected in the cultured cells by RT-PCR (Fig. 5B). Interestingly, hMSCs still expressed GFP after differentiation (Fig. 5A).8 B& U; `9 x* U

Figure 5. Nucleofected human mesenchymal stem cells retain their differentiation potential. GFP  cells were induced to differentiate toward adipocytes and osteoblasts for 25 and 21 days, respectively. (A): Cells were observed under phase contrast (a, d) and by florescence microscopy (b, e). Cells were also fixed and stained with hematoxylineosin and nitroblue tetrazolium /X-phosphate to reveal adipocytic (c, f) and ostoblastic differentiation, respectively. Original magnification x 20. (B): Molecular analysis of mRNA transcript upregulated during differentiation. Osteoponin (upper panel), PPAR2 (middle panel), and beta2-microglobulin (lower panel). Abbreviations: B2M, beta 2-microglobulin; GFP, green fluorescent protein; OPN, osteopontin; PPAR2, peroxisome proliferator¨Cactivated receptor gamma-2.
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Several reports have demonstrated that MSCs are able to suppress immune responses by inhibiting T-cell proliferation . Therefore, we analyzed the effect of the addition of nucleofected hMSCs on CD3  T cells stimulated with the mitogen PHA. As shown in Figure 6, hMSCs nucleofected with either mock or GFP-encoding plasmid caused the significant inhibition of T-cell proliferation (p = .013 for GFP  hMSCs).
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Figure 6. Effect of human MSCs on T-cell proliferation induced by mitogens. In each experiment, triplicates of peripheral blood lymphocytes were stimulated with phytohemagglutinin and incubated with different cultured MSC populations. The data shown were obtained from three experiments and are expressed as mean counts per minute ¡À standard deviation.*p . y+ L8 U+ ]3 \) F6 r% n) O# V

IL-12 gene can be nucleofected into hMSCs and the cytokine produced at high levels without impairment of its biological activity.# m6 \4 w3 W# g" N7 [' j

Our preliminary results prompted us to transfect hMSCs using the Nucleofector technology to induce the expression of the immunomodulatory cytokine IL-12. A CMV promoter plasmid encoding for both hIL-12 subunits, p35 and p40, was used in combination with transfection program U-23. After 48 hours, supernatants from untransfected and IL-12¨Ctransfected cells were analyzed to measure the production of the cytokine by an ELISA test. As shown in Figure 7A, IL-12 produced after nucleofection was as high as 2,146 ¡À 59 pg/ml. IL-12¨Ctransfected hMSCs were then expanded for several days and supernatants were collected 48 hours after each replating. As reported for GFP expression (Fig. 2), IL-12 levels declined with time, but significant production of IL-12 was seen after 20 days with an average of 290.5 ¡À 97.6 pg/ml (n = 3). Additionally, we investigated whether IL-12 secreted by nucleofected hMSCs retained its biological activity. To this end, NK cells were used as effector cells in a cytotoxicity test following activation by a 24-hour incubation with 1 ng/ml exogenous recombinant human IL-12 (rhIL-12) or with the same amount of cytokine from the medium of IL-12¨Cnucleofected MSCs. Supernatants from untransfected cells were used as control. As presented in Figure 7B, medium from control cells induced very little lysis of K562 cells, whereas the addition of exogenous rhIL-12 as well as medium from IL-12¨Ctransfected cells caused high NK-mediated target cell killing. Thus, nucleofection with IL-12 coding genes in hMSCs can achieve high production of transgene protein without modifying its biological properties.
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Figure 7. Nucleofection induces high amounts of biological active IL-12. hMSCs were transfected with pCDNA3 pIL-12 using pulsing program U-23. After 48 hours, supernatants were collected, cells were tripsinized, and cultures were expanded for an additional 21 days. Supernatants were then collected and analyzed for IL-12 detection by an ELISA test. CTR indicates untreated hMSCs (A). (B): NK cells were incubated in the presence of exogenous recombinant human IL-12 (1 ng/ml) and the same amount of cytokine present in postnucleofection supernatants for 24 hours. Then activated NK cells (effectors) were coincubated with K562 cells (targets) in a standard cytotoxicity test. Abbreviations: hMSC, human mesenchymal stem cell; IL, interleukin; NK, natural killer.

DISCUSSION2 m% a( F7 G! r( Y9 u0 v8 L0 x0 G
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hMSCs are multipotential cells capable of differentiation into bone, endothelium, adipose tissue, cartilage, muscle, and brain .
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As for most plasmid-based transfection techniques, nucleofection induced only a transient transgene expression in hMSCs. Culture-expanded GFP  nucleofected cells slowly lost the GFP expression as a consequence of dilution. However, even after 3 weeks in culture, a significant percentage of GFP  hMSCs could still be detected. In this study, we did not investigate whether the Nucleofector technology can induce gene integration in hMSCs after culture in selective medium. To this end, further studies using linearized plasmids are in progress. Of note, GFP-nucleofected hMSCs did not show impairment of their immunophenotype, differentiation potential, or immuno-regulatory capacity.
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To further assess the functional integrity of transgenic protein, IL-12 was transiently expressed by nucleofection in hMSCs. A high amount of the cytokine was produced within 48 hours and up to 3 weeks in culture. Most importantly, the cytokine retained its biological potential to activate NKs killing toward MHC class I cells. IL-12 was selected because it provided one of the most significant antitumor activities to stimulate effective cellular immune responses against tumor cells in cytokine gene-based cancer immunotherapy . Thus, the administration of IL-12 in the early phase after BM transplantation might have the potential to overcome GVHD and thereby facilitate HLA-mismatched allogenic BM transplantation in leukemic patients.
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In summary, our data clearly showed that nucleofection is an efficient method to induce high transgene expression in hMSCs without affecting pivotal properties of this versatile adult stem cell population. Nucleofected hMSCs could be used in several types of studies where a high expression of transgenic protein is required.0 v; }4 h, U# Y5 o+ @9 p8 K
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ACKNOWLEDGMENTS
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This work was supported by grants from the Italian Minister of Education, University and Scientific Research, the National Research Council of Italy, the Italian Association for Cancer Research, the University of Bologna (funds for selected topics), and Italian Association Against Leukemia Section of Bologna, Bologna, Italy./ I/ |9 d1 U* d8 v

DISCLOSURES; [2 M+ f9 ~3 L
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The authors indicate no potential conflicts of interest.6 q' b- b& _  N0 K1 V" \
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