Nucleofection Is the Most Efficient Nonviral Transfection Method for Neuronal St - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-4 23:57 |只看该作者 |倒序浏览 |打印

作者：Konstantin Cesnuleviciusa, Marco Timmera, Maike Wesemanna, Tobias Thomasb, Tanja Barkhausenc, Claudia Grothea作者单位：aDepartment of Neuroanatomy and Center for Systems Neuroscience Hannover, 8 `) {7 N$ V' M9 D, I- e& F/ q" ]0 z


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      【摘要】
   Neuronal progenitor cells (NPCs) play an important role in potential regenerative therapeutic strategies for neurodegenerative diseases, such as Parkinson disease. However, survival of transplanted cells is, as yet, limited, and the identification of grafted cells in situ remains difficult. The use of NPCs could be more effective with regard to a better survival and maturation when transfected with one or more neurotrophic factors. Therefore, we investigated the possibility of transfecting mesencephalic neuronal progenitors with different constructs carrying neurotrophic factors or the expression reporters enhanced green fluorescence protein (EGFP) and red fluorescent protein (DsRed). Different techniques for transfection were compared, and the highest transfection rate of up to 47% was achieved by nucleofection. Mesencephalic neuronal progenitors survived the transfection procedure; 6 hours after transfection, viability was approximately 40%, and the transfected cells differentiated into, for example, tyrosine hydroxylase-positive neurons. Within the group of transfected cells, many progenitors and several neurons were found. To provide the progenitor cells with a neurotrophic factor, different isoforms of fibroblast growth factor-2 were introduced. To follow the behavior of the transfected cells in vitro, functional tests such as the cell viability assay (water-soluble tetrazolium salt assay ) and the cell proliferation assay (5-bromo-2'-deoxyuridine-enzyme-linked immunosorbent assay) were performed. In addition, these transfected NPCs were viable after transplantation, expressed tyrosine hydroxylase in vivo, and could easily be detected within the host striatum because of their EGFP expression. This study shows that genetic modification of neural progenitors could provide attractive perspectives for new therapeutic concepts in neurodegenerative diseases. 6 m1 o( {) R5 z& o6 R$ I9 h/ {
      【关键词】 Dopaminergic neurons Neural stem cells Nucleofection Transfection Transplantation
   INTRODUCTION; p& _3 a1 D5 M) Q; h( e, a

Transplantation of embryonic dopaminergic (DA) cells . The focus of this study was the two latter problems." s1 q$ l( E) |6 K$ P4 B

Transplantation of in vitro expanded and subsequent differentiated ventral mesencephalic progenitor (VMP) cells into DA neurons also resulted in behavioral recovery, but after grafting these proliferated cells failed to show a substantial gain in number compared with embryonic cells from later stages . However, cotransplantations of two or more different cell sources are only a makeshift; an optimized approach should provide the cells of interest directly with one or more neurotrophic factors by introducing foreign DNA for ectopic expression.

Viral techniques are the most efficient systems to deliver DNA into cells ; and lipofection, which is a liposome-based method that uses, for example, Lipofectamine (Invitrogen, Carlsbad, CA, http://www.invitrogen.com). All of these techniques have been applied successfully, although the efficiency of these methods is quite low and most protocols are designed for permanent cell lines.  k- y1 `; A2 q" m1 \, U9 x6 W

Two years ago, a new highly effective nonviral method for gene transfer into primary cells was developed by Amaxa Biosystems (Cologne, Germany, http://www.amaxa.com) . So far, there have been no reports about nucleofection of neuronal progenitors from the brain, including dopaminergic ones.
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With regard to a putative application of genetically modified neurons in brain restoration, the following objectives were investigated in the present study: first, to compare different nonviral transfection methods and to establish an efficient one for VMP cells; second, to transfect VMP cells with both a reporter gene (EGFP/DsRed) and with an efficient growth factor gene (FGF-2); third, to analyze whether transfection of the cells alters their composition or changes their morphology, their properties, or their expansion and differentiation potential; and fourth, to determine whether these manipulated cells can survive transplantation.
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Here we demonstrate that nucleofection shows transfection rates of nearly 50% of primary VMP cultures when using EGFP plasmid and, in addition, an efficient expression of FGF-2. Furthermore, these cells can be grafted efficiently and are therefore an attractive alternative cell source for the treatment of, for example, PD.

MATERIALS AND METHODS, Q2 I: H, ~- h- o9 {9 B

Preparation of Embryonic Tissue1 }4 f5 ^' H0 p! m3 h4 E

Ventral mesencephalic (VM) progenitors were obtained from fetuses of Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA, http://www.criver.com) at embryonic day 11.5 (E11.5). The VM tissue was dissected as described by Nikkhah et al. .( @+ q. e, l- X
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Cell Culture
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After dissociation of the tissue  and transplantation on DIV6), medium II was removed from the flasks, the surface was rinsed with phosphate-buffered saline once, and the cells were removed with trypsin/EDTA (PAA) incubation for 3¨C4 minutes. Trypsinization was stopped by adding medium I. Afterward, the cells were counted, the volumes were adjusted, and the transfection procedure was performed (described below). After transfection, the cells were seeded on 96-well plates with a density of 15,000; 30,000 or 60,000 cells in 100 µl/well and incubated for 24 hours with medium I for attachment. Then medium I was replaced by medium II for the next 4 days of proliferation (to expand transfected culture). For some experiments, medium II was changed to medium III (DMEM/F12, 0.25% BSA, B27, 1% FCS, 100 µM ascorbic acid, and 2 mM glutamine) at day 5 of proliferation for differentiation of transfected progenitor cultures. The cells were incubated with medium III for the next 6 days.) r6 ]( G! V3 u' E( ~
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Transfection' `) Q$ C+ u" x3 _' T$ B; h- g

Three different methods were analyzed using primary mesencephalic progenitor cultures: chemical transfection with Lipofectamine 2000 reagent (Invitrogen), electroporation using EasyjecT Optima electroporator (EquiBio, Kent, U.K., http://www.flowgen.co.uk), and nucleofection with Nucleofector device (Amaxa). DNA plasmid constructs used for transfection were as follows: pEGFP-N2 (Clontech, Palo Alto, CA, http://www.clontech.com), pDsRed-N2 (Clontech), pCIneo-FGF18kDa ; pCIneo was used as empty control vector (Promega, Madison, WI, http://www.promega.com).
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Lipofection. The procedure was performed according to the manufacturer's protocol. An optimization test was performed once to find the optimal ratio of DNA and Lipofectamine 2000 reagent. The "rapid" transfection protocol in 96-well plates was also used to have similar conditions compared with other methods. Shortly before the cell seeding, the DNA and reagent mixes were prepared in 96-well plates. Each well contained 50 µl of OptiMEM I medium (Gibco) solution with 0.5 µg of DNA mixed with 0.5 µl of Lipofectamine 2000 reagent. The detached cells (see above) were adjusted to the volume of 60,000 cells per 100 µl in medium I, and 100 µl of the cell suspension was added to each well on the day of transfection. Cells were incubated for 24 hours. After incubation the medium was replaced by medium II, and the cells were cultured as described above.# g* a2 D9 t4 `: L  ?8 t

Electroporation. To test the capability of physical transfection to efficiently deliver DNA to primary VMP cells, we used an electroporation technique under high-voltage and low-capacitance settings: EasyJecT electroporator was set to voltage of 350 V, and capacitance parameter varied from 75 to 1,500 µF to establish an optimal setup. The optimal parameters were as follows: 350 V, 75 µF, 100 µg of DNA/500,000 cells, DIV3 as the day for transfection, and 4 days of further proliferation. The detached cells (see above) were adjusted to the volume of 500,000 cells per milliliter in medium I. Afterward, the cells were centrifuged (600 rpm for 5 minutes), and the medium was removed. Four hundred µl of electroporation buffer (50 mM K2HPO4¡¤3H2O , pH = 7.35), 10 µl of magnesium sulfate (1 M MgSO4¡¤7H2O, pH = 6.7), and 100 µg of DNA were added, and the cells were resuspended. There was no incubation on ice before or after the electroporation. After the pulse, 500 µl of medium I were added to neutralize the electroporation buffer, and the cells were seeded on the 96-well plates with a density of 30,000 cells per well. After incubation for 24 hours the medium was replaced by medium II and the cells were cultured as described above. It is important not to vortex or mix the cells after the pulse, for it can cause decreased cell survival.1 o/ I/ v* L1 O2 @9 ~3 u

Nucleofection. For nucleofection of primary mesencephalic progenitor cultures, Basic Nucleofector Kit for primary mammalian neurons (Amaxa) was used. In the beginning, two programs were chosen: A-033 and O-005, both of which were designed for transfection of primary neurons. Program A-033 showed better results in transfection efficiency and was used for further experiments. The transfection was performed according to the manufacturer's protocol for adult rat neuronal stem cells. Detached cells (see above) were adjusted to a volume of 2,000,000 cells per milliliter in medium I. Afterward, the cells were centrifuged (600 rpm for 5 minutes), and medium was removed. The cells were resuspended in 100 µl of nucleofection solution with 5 µg of plasmid DNA. After the pulse, 500 µl of RPMI 1640 medium (Biochrom AG, Berlin, http://www.biochrom.de) containing 10% FCS was immediately added to neutralize the nucleofection solution. Cells were seeded on 96-well plates at a final volume of 15,000 or 30,000 cells per well and cultured further (see above).+ k6 h' ^; Y4 t5 v" Y
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Immunocytochemistry

The immunostaining was performed according to the previously described protocol . Detection of bound primary antibody was performed with either Cy2- (1:200) or Cy3- (1:600) conjugated secondary antibodies. Nuclei were visualized by 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) staining when required.8 u$ ]; L+ _* V$ u

Cell-Enzyme-Linked Immunosorbent Assay

Cell-enzyme-linked immunosorbent assay (cell-ELISA) was performed in 96-well microtiter plates as previously described .
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Cell Viability and Proliferation Assay
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Cell viability and proliferation were evaluated after nucleofection by WST-1 assay (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com)  according to the manufacturer's protocol. To each well of the 96-well plate, each containing 100 µl of medium, 10 µl of WST-1 reagent was added. The control well contained medium and WST-1 reagent but no cells. After incubation for 105 minutes in the incubator (37¡ãC), the relative absorbance was measured with ELISA reader (Mikrotek Laborsysteme GmbH, Overath, Germany, http://www.mikrotek.de) at 490 nm. To achieve viability curves, different time points were selected (6 hours, 48 hours, 5 days, 7 days, and 11 days after nucleofection). In some experiments, cell-ELISA for FGF-2 expression was performed with the same cells following WST-1 assay.
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Cell Proliferation Assay

Cell proliferation was evaluated after nucleofection by 5-bromo-2'-deoxyuridine (BrdU) incorporation ELISA (Roche) according to the manufacturer's protocol. Different time points (18 hours, 48 hours, 5 days, 7 days, and 11 days after nucleofection) were selected.
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Western Blotting; `5 o1 h& Y( q% [: ~
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Western blot analysis was performed as previously described . Protein was received from expanded cells after nucleofection. The same amounts of protein were loaded on each gel. FGF-2 was detected by using a monoclonal anti-FGF-2 antibody (1:250) (Transduction Laboratories, Lexington, KY, http://www.bdbiosciences.com/pharmingen) and the enhanced chemiluminescence system (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) followed by documentation in the LAS-3000 (Fuji, Duesseldorf, Germany, http://www.fujifilm.de).
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Cell Counting
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EGFP-positive or red fluorescent cells (DsRed-positive) were counted to measure the transfection rate 24¨C48 hours after transfection. Cells were counted at x10 magnification using an inverse Olympus fluorescent microscope (Tokyo, http://www.olympus-global.com). Five fields were randomly selected in every well, and at least four to six wells were counted for each sample. Transfection rate was calculated from the relationship between EGFP- and DAPI-positive, or DsRed- and DAPI-positive cells, respectively. The number of TH-positive cells was measured as a relative grade, since these cells gather into the clusters after differentiation and it is hard to obtain the exact number. The following grades were used: 5 , preferably large and small clusters of TH-positive cells in the culture; 4 , preferably large clusters; 3 , preferably small clusters and separate cells; 2 , preferably separate cells; 1 , only a few TH-positive cells in the culture. All experiments were performed at least in triplicate.
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FACS Analysis0 I; h% C* N; \

Primary VMP cells were collected as described above. Prior to nucleofection the cells were expanded in culture for 6 days under proliferation conditions. Afterward, the cells were transfected with EGFP plasmid and reseeded for the following proliferation or differentiation. Either proliferated or differentiated transfected cells were taken for further analysis. Analysis of EGFP expression was performed using the FL1 channel of a FACSCalibur flow cytometer (BD Biosciences, San Diego, http://www.bdbiosciences.com). Nontransfected cells served as negative controls to determine background fluorescence. Cell debris was excluded from analysis by forward- and side-scatter gating. Data were analyzed using the software Cell Quest Pro software (BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen).

Generation of Immortalized VMP Cells

The freshly prepared dissociated VMP cells (see above) were cultivated for 3 days under proliferation conditions and then nucleofected with the construct containing the T-large antigen of SV40 (pSV3neo). After transfection, the cells were grown in selection medium with G418 for 2 weeks and then cloned using limiting dilution. The fastest growing clones were selected and frozen for storage.: I1 z( d+ @: N1 y; u

Transplantation and Evaluation of the Graft
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The procedure was described in detail previously . The sections were also immunostained with monoclonal anti-green fluorescent protein (anti-GFP) antibody (1:200) (Roche) and visualized by Cy2 (1:200) secondary antibody.
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Statistical Analysis9 S% J! D6 j% H" r

Results are expressed as means ¡À SD. Statistical evaluation was performed using the SigmaStat program (Version 2.0; Jandel Corporation, San Rafael, CA, http://www.systat.com). Comparisons between the groups were performed using one-factor analysis of variance (ANOVA) followed by the Tukey's post hoc test if the data were normally distributed or by Kruskal-Wallis one-way analysis of variance on ranks followed by Dunn's post hoc test if the data were not normally distributed. If only two groups were compared, a two-tailed t test for normally distributed data or Mann-Whitney test for data not normally distributed was performed. p values of
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RESULTS! W1 A5 c% x9 v1 Q8 G5 Z
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Comparison of Different Nonviral Transfection Methods Showed Highest Efficiency for Nucleofection

To establish an efficient nonviral transfection protocol for primary VMP cells, electroporation, lipofection, and nucleofection were compared. DsRed- and EGFP-positive cells were counted 48 hours after transfection. All three methods resulted in the delivery of the reporter gene coding for DsRed or EGFP even if the transfection rates differed significantly (Fig. 1A, i). The highest transfection efficiency was reached by nucleofection: 47.6 ¡À 8.6% (p . }/ ~" Z1 Q; ^& E4 Q

Figure 1. Transfection of primary VMP cells. Comparison of different nonviral transfection methods showed highest efficiency for nucleofection. (A): Comparison of transfection efficiencies after electroporation, lipofection, and nucleofection of two reporter constructs, pDsRed-N2 and pEGFP-N2. i, electroporation reached 3.2% transfection rate with pDsRed-N2 and 8.1% with pEGFP-N2; lipofection: 1.1% (DsRed) and 10.7% (EGFP); nucleofection: 16.7% (DsRed) and 47.6% (EGFP). Red fluorescence represents DsRed-positive transfected cells (ii), green fluorescence represents EGFP-positive transfected cells (iii), and phase-contrast of these cells is shown (iv) 3 days after electroporation in proliferation phase. Scale bar = 100 µm. (B): Comparison of electroporated and lipofected cell cultures in proliferation phase. Shown are transfected cells visualized by DsRed (i), progenitor cells immunostained for nestin (ii), and nuclei visualized by DAPI (blue; ii). Merge picture (iii) represents colocalization of nestin and DsRed (arrowheads). Scale bars = 50 µm (electroporation), 100 µm (lipofection). (C): Transfection with pDsRed-23kDa-FGF-2. Red fluorescence represents DsRed expression (i, iii), blue represents nuclei visualized by DAPI (ii), and green fluorescence represents immunocytochemistry for nestin (ii, iv). Arrowheads show DsRed colocalization with nestin; arrows show DsRed-positive nuclei. Scale bars = 50 µm (electroporation), 20 µm (lipofection). (D): 18-kDa FGF-2 overexpression 2 days after electroporation, lipofection, and nucleofection with pCIneo-FGF18kDa. i, FGF cell-ELISA shows FGF-2 expression levels. ii, the representative Western blot shows FGF-2 expression after nucleofection. The plots represent the average of three independent experiments. Error bars indicate SD; *, p 6 q9 @9 K0 X' {; I

To confirm that transfected cells are neural progenitors, nestin immunocytochemistry was performed (Fig. 1B, i¨Cvi). After electroporation, 86.9 ¡À 7.2% of the cells were immunopositive for nestin, and after lipofection, 79.7 ¡À 1.07%, indicating high progenitor richness within the VMP cultures. There was a colocalization of DsRed and nestin immunoreactivity in both methods, with a more intense cytoplasmatic DsRed signal after electroporation (Fig. 1B, iii, vi).( S8 V0 }$ W; j; R1 H
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Cell counting 3 days (proliferation phase) and 11 days (differentiation phase) after transfection revealed that during proliferation lipofected cultures showed higher cell numbers than electroporated ones; 55.8 ¡À 16.3% of the electroporated cells and 228.2 ¡À 79.8% of lipofected cells were detected after the transfection compared with the control group (the cells of the control group were detached and reseeded but not transfected). This difference was not significant (p > .05). At the end of the differentiation phase (11 days after transfection, 6 days in differentiation medium), there was no more difference in cell numbers (data not shown).5 ~( V8 h. K; G

In addition to the transfection of reporter genes, we were interested in delivering a "gene of interest." Therefore, we expressed in the cells a 23-kDa isoform of FGF-2 protein (which is localized in the nucleus . An analysis 48 hours after transfection with pDsRed-23kDa-FGF-2 revealed that nestin-positive cells expressing DsRed colocalized with DAPI nuclear staining (Fig. 1C, i¨Civ).
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Evaluation of the FGF-2 expression levels 3 days after transfection by cell-ELISA showed that nucleofection resulted in an almost twofold increase in FGF-2 expression (181.5 ¡À 19.4%; Fig. 1A, i), and this was confirmed in Western blot (Fig. 1D, ii). Electroporation showed only a slight overexpression (122.8 ¡À 23.2%; p , g1 o; K2 W; a& ]& |+ D

Electroporation Did Not Alter the Cellular Composition and Differentiation Potential of Primary Mesencephalic Progenitors
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After the transfection and subsequent differentiation (described in Materials and Methods), cultures were evaluated with respect to culture composition. Immunocytochemistry showed the presence of differentiated populations of VMP cells: ß-III tubulin-positive neurons, including TH-positive ones, and GFAP-positive astrocytes (Fig. 2B, ii, iv, vi). We found transfected cells in each cell population (Fig. 2B, i¨Cvi); however, the majority of them were astrocytes, whereas only a few transfected TH-positive neurons were detected (Fig. 2B, i¨Cii).

Figure 2. Characterization of primary VMP cultures after electroporation. (A): Analysis performed 11 days after transfection with pDsRed-N2 (4 days in proliferation phase and 7 days in differentiation phase). i, cell-enzyme-linked immunosorbent assay analysis shows distribution of different cell types in VMP culture after electroporation, both after proliferation and differentiation: ß-III-tubulin-positive neurons, nestin-positive progenitors, TH-positive neurons, and GFAP-positive astrocytes. ii, the relative amount of dopaminergic phenotype in the neuronal population displayed no differences between transfected and nontransfected cultures. (B): Immunostaining of transfected culture after differentiation. The cells were transfected with pDsRed-N2 (i¨Civ) or with pEGFP-N2 (v, vi). i, transfected cells expressed DsRed. ii, TH identified dopaminergic neurons; arrowheads show colocalization of DsRed and TH. iii, transfected cells expressed DsRed. iv, ß-III-tubulin-positive neurons; arrowheads show colocalization of DsRed and ß-III-tubulin. v, transfected cells express EGFP; vi, GFAP identified astrocytes; arrowheads show colocalization of EGFP and GFAP. Scale bars = 50 µm. (C): The transfection rates of VMP cells after electroporation under different capacitance conditions. The cells were transfected with pDsRed-N2. i, increase of capacitance parameter elevated transfection efficiency under the high voltage conditions. It was possible to increase transfection rate up to 7.1%. ii, TH immunoreactivity of differentiated VMP cells after electroporation under different capacitance conditions. Increase of capacitance significantly reduced TH-positive neuron population. The plots represent the average of three independent experiments. Error bars indicate the standard deviation; *, p

To increase the number of transfected cells, the capacitance parameters from 75 to 1,500 µF were varied and resulted in an increase of the transfection efficiency from 3.2 ¡À 1.49% to 7.1 ¡À 4.51% (transfection with pDsRed-N2), but the increase was not significant (Fig. 2C, i; p > .05; F = 1.473). To analyze possible negative effects on survival of dopaminergic neurons after electroporation, TH immunocytochemistry was performed. Higher capacitance conditions resulted in a lower number of surviving cells (visualized by nuclear DAPI staining) and dramatically decreased the population of TH-positive neurons (data not shown). We analyzed the relationship between capacitance variables and TH positivity of electroporated culture and found that it showed a strong negative correlation (Fig. 2C, ii; Pearson correlation, r = ¨C.829, p

Using the established conditions, we analyzed the population composition of the electroporated VMP cultures during proliferation and after differentiation by cell-ELISA. TH-positive neurons and astrocytes increased in number after differentiation, but not ß-III-tubulin-positive neurons or nestin-positive progenitors (Fig. 2A, i). In nontransfected cultures, TH-positive neurons increased from 12.6 ¡À 3.4% to 29.4 ¡À 4.02%, and in electroporated cultures, they increased from 12.8 ¡À 3.36% to 27.3 ¡À 4.08% (p  .05), but more astrocytes were found in electroporated cultures compared with nontransfected ones (p  .05), and electroporated cultures showed a decrease from 60.7 ¡À 12.45% to 41.3 ¡À 4.28% (p  .05), whereas in electroporated cultures, nestin positivity was reduced from 44.0 ¡À 7.33% to 34.7 ¡À 7.93% after differentiation (p 9 f9 P  K  d' l+ m

Lipofection
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To compare the physical transfection method with the chemical liposome-based approach, Lipofectamine 2000 reagent (Invitrogen) and pEGFP-N2 were used for transfection of primary VMP cells. Culture composition of differentiated transfected cells (described in Materials and Methods) was evaluated by immunocytochemistry. Both populations of differentiated VMP cells (astrocytes ), including TH-positive ones, were present; however, no transfected TH-positive neurons were found, but some ß-III-tubulin-positive neurons were (Fig. 3, i¨Ciii, i'¨Ciii'), and astrocytes preferably expressed EGFP (Fig. 3, iv¨Cvi). The transfection efficiency was not improved by enhancing the concentration of plasmid DNA (data not shown), changes in plasmid DNA concentration did not correlated with survival of TH-positive cells, and the number of surviving TH-positive cells was greatly variable (data not shown).

Figure 3. Characterization of differentiated primary ventral mesencephalic progenitor cells after lipofection; analysis performed 11 days after transfection with pEGFP-N2 (4 days in proliferation phase and 7 days in differentiation phase). Immunostaining of transfected culture. i, transfected cells expressed enhanced green fluorescence protein (EGFP) (green). ii, ß-III-tubulin identified neurons (red). Arrowheads show ß-III-tubulin immunoreactive neuron expressing EGFP. iii, merge picture shows colocalization of EGFP and ß-III-tubulin. Note the arrows and stars following the fibers of transfected neuron (i¨Ciii). Insets (i'¨Ciii') represent another sample of ß-III-tubulin immunoreactive neuron expressing EGFP under higher magnification. iv, transfected cells expressed EGFP (green). v, GFAP identified astrocytes (red). vi, merge picture shows colocalization of EGFP and GFAP (arrowheads). Contrary to electroporation, no transfected TH-positive cells were found. Scale bars = 50 µm (i¨Cvi), 25 µm (i'¨Ciii'). Abbreviations: GFAP, glial fibrillary acidic protein; GFP, green fluorescent protein./ ?' |2 Z$ b' D, ]/ b% q5 j

Nucleofection

Neither electroporation nor lipofection resulted in efficient gene delivery to primary VMP cells despite a high number of transfected cells displayed by nucleofection. To evaluate the transfection efficiency in more detail, transfected cultures were stained with DAPI (for visualization of nuclei) and quantified (Fig. 4A, i¨Cii). The counting showed an approximately 48% transfection rate (Fig. 1A, i). Similar to the cell culture protocols after electroporation or lipofection, the nucleofected cultures were further expanded for 4 days and differentiated for 6 days, and then immunocytochemistry was performed. Mainly, three populations within the differentiated VMP cultures were obvious (Fig. 4B, i¨Cvi), and the amount of TH-positive neurons was similar to that in nontransfected cultures (data not shown). Among transfected cells, few TH-positive neurons were found (Fig. 4B, i¨Cii); other neurons were visualized by ß-III-tubulin (Fig. 4B, iii¨Civ) and astrocytes (GFAP-positive; Fig. 4B, v¨Cvi). With regard to the stability of transfection, 30.8 ¡À 9.6% of the previous fluorescence signal was still detected after 11 days in culture (Fig. 4C), and overexpression of FGF-2 was still present (153.4 ¡À 60.5%; p

Figure 4. Immunofluorescence analysis of transfected primary mesencephalic (VMP) cultures after nucleofection with pEGFP-N2. (A): Green fluorescence of VMP cells 24 hours after transfection. i, transfected VMP cells expressed EGFP. ii, nuclei (blue) were visualized by DAPI staining. Scale bars = 100 µm. (B): Immunostaining of transfected culture after differentiation: i, ii, TH; iii, iv, ß-III-tubulin-positive neurons; v, vi, GFAP. i, iii, v, transfected cells expressed EGFP; ii, iv, vi, arrowheads and arrows show colocalization of corresponding marker and EGFP. Scale bars = 50 µm. (C): EGFP fluorescence analysis of transfected VMP culture. EGFP expression levels were measured after 24 hours and after 11 days in cell culture to evaluate the durability of protein expression. At the end of differentiation phase, EGFP expression was reduced threefold. (D): 18-kDa FGF-2 expression at the end of differentiation phase. The cells were transfected with pCIneo-FGF18kDa. Overexpression of FGF-2 was still detectable 11 days after nucleofection. (E): Transfection with pCIneo-FGF18kDa and immunostaining for FGF-2. Immunofluorescence analysis did not show any differences between nontransfected (i) and transfected (ii) cultures either in proliferation phase (i, ii) or in differentiation phase (iii). Scale bars = 50 µm. (F): Immunostaining of VMP cells in proliferation phase for FGF-2 showed two nestin-positive (ii) cell populations with different FGF-2 localization (i), with preferably nuclear localization (arrowheads) or with preferably cytoplasmic localization (arrow). iii, merge picture depicts colocalization of FGF-2 and nestin. Scale bars = 50 µm. (G): VMP cells transfected with pEGFP-23kDa-FGF-2. i, the cells were immunostained for FGF-2. ii, EGFP was expressed in nuclei. iii, arrows show the colocalization of EGFP and FGF-2 in transfected cells. Scale bars = 50 µm. Error bars indicate SD; **, p

Nucleofection with FGF-22 J5 [; a( g: u* n4 V

Since we found that nucleofection produces a high transfection rate, transfection of VMP cells with pCIneo-FGF18kDa, leading to overexpression of the 18-kDa isoform of FGF-2, was performed and followed by immunocytochemistry. Immunofluorescence analysis showed no differences in FGF-2 immunoreactivity between nontransfected and nucleofected cells, either in proliferation phase (Fig. 4E, i¨Cii) or after differentiation (Fig. 4E, iii). However, in proliferation phase at least two different cell populations with regard to FGF-2 cellular localization could be distinguished: cells with predominantly nuclear FGF-2 localization, which did not colocalize with nestin (Fig. 4F, i¨Ciii, arrowheads), and cells with predominantly cytoplasmic FGF-2 localization, which colocalized with nestin (Fig. 4F, i¨Ciii, arrow). To confirm the synthesis of FGF-2 in the cells, we used the construct coding for the 23-kDa isoform of FGF-2, which has a nuclear localization, fused with EGFP (pEGFP-23kDa-FGF-2). Fluorescence analysis after transfection showed that a number of FGF-2-positive cells had a nuclear green fluorescence signal (Fig. 4G, i¨Ciii).0 }# |% S8 X& Q6 K6 @# q
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To investigate the possible effects of endogenous overexpression of FGF-2 on primary VMP cells, cells transfected for expression of the 18-kDa isoform of FGF-2 (pCIneo-FGF18kDa) were analyzed with regard to cell viability and proliferation (Fig. 5A, i). The toxicity of this method was more than 50%; 6 hours after nucleofection, 47.3 ¡À 12.9% of the cells transfected with empty control vector (pCIneo) survived, compared with nontransfected controls (p  .05; Fig. 5A, v). This situation did not change within the first 2 days; at the end of proliferation phase, however (on day 5 after transfection), the cells transfected with control vector (pCIneo) showed an increase in cell number up to 71.4 ¡À 30.5%; FGF-2 overexpressing cells increased only up to 45.7 ¡À 24.3% (p 7 t" }1 C& `, ]1 H& }) {3 e
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Figure 5. Functional analysis of VMP cultures after overexpression of FGF-2 18 kDa by nucleofection (part I). (A): Cell viability and cell number were evaluated by WST-1 assay. i, the actual reagent absorbance on different time points during the cell culture protocol. ii¨Civ, phase-contrast microscopy of the transfected and nontransfected differentiated cultures at 11 days after transfection; cell clustering and continuous cell loss were detected in non-tf cultures (11 days). v¨Cviii, the plots show in detail the cell number changes during the cell culture protocol on different time points; spontaneous proliferation of the cells was excluded, whereas the cell number of non-tf cultures was considered as 100% on every time point. (B): The proliferation rate was evaluated by BrdU incorporation-ELISA. i, actual reagent absorbance on different time points during the cell culture protocol. ii, iii the plots show in detail the proliferation rates of each transfected group in comparison with non-tf control; spontaneous proliferation of the cells was excluded, whereas the proliferation rate of non-tf cultures was considered as 100% on every time point. Scale bar = 100 µm. The plots represent the average of three independent experiments. Error bars indicate SD; *, p $ V7 |* o7 y, T6 k: J
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To investigate the proliferation rate of primary VMP cells after transfection, BrdU incorporation ELISA was performed (Fig. 5B, i). As in the previous assay, the VMP cells were transfected with pCIneo-FGF18kDa for overexpression of the 18-kDa isoform of FGF-2 (FGF-2 group) and with pCIneo as a control. For detailed analysis, the groups transfected with pEGFP-23kDa-FGF-2 and nucleofected without plasmid were added to this assay as additional controls. The transfected cells showed reduced proliferation compared with nontransfected controls 18 hours after transfection (64.2 ¡À 9.2% and 64.5 ¡À 10.2% in FGF-2 and tagged groups, respectively; p ' s! R, O% _- V4 u" x
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To analyze the 18-kDa FGF-2 overexpression profile during the cell culture protocol, we performed FGF-ELISA and adjusted the values to the corresponding values of the WST-1 assay to exclude the influence of cell number differences. Analysis showed that 6 hours after nucleofection, FGF-2 overexpression had already reached 427.8 ¡À 213.5% of the level of nontransfected controls (p
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Figure 6. Functional analysis of the primary VMP cultures after overexpression of 18-kDa FGF-2 by nucleofection (part II). (A): BrdU incorporation of the cells transfected with regressive concentrations of pCIneo-FGF18kDa. The concentration was reduced from 5.0 µg to 0.5 µg of plasmid DNA. There was no difference in proliferation rate between the transfected groups. (B): The number of TH-positive cells after differentiation of transfected cultures was analyzed by cell-ELISA. Two different conditions were evaluated: when the exogenous FGF-2 was added to proliferation medium (PM FGF-2; white bars), and proliferation phase without exogenous FGF-2 (PM ¨C FGF-2; gray bars). A slight rescue effect of exogenous FGF-2 was detected only in the group transfected with the highest concentration of plasmid DNA (5.0 µg). (C): FGF-2 expression pattern after the transfection with pCIneo-FGF18kDa during the cell culture protocol. i, already, 6 hours after transfection FGF-2 was overexpressed almost fivefold (FGF-2 group) in comparison with non-tf controls. ii, a comparison of FGF-2 expression in the group overexpressing 18-kDa FGF-2 on different time points in cell culture. The highest FGF-2 expression was detected in the proliferation phase. (D): The regressive overexpression of 18-kDa FGF-2 after transfection was confirmed by Western blotting; actin served as loading control. i, immortalized and physiological VMP cells were transfected with regressive concentrations of pCIneo-FGF18kDa. ii, quantitative densitometry of Western blotting. After transfection with 3.0 µg of plasmid 18-kDa FGF-2 overexpression was reduced to 30% in comparison with the transfection with 5.0 µg. The plots represent the average of three independent experiments. Error bars indicate SD; *, p & S9 i; {6 b3 j2 D4 B3 W% D

To investigate a possible toxic effect of overflowing the cells with 18-kDa FGF-2, we transfected the VMP cells with decreasing concentrations of plasmid DNA (pCIneo-FGF18kDa) from 5.0 µg (standard concentration) to 0.05 µg. (Fig. 6A). To confirm the regressing FGF-2 expression in transfected cells, we used a cell line of immortalized VMP cells for transfection and performed Western blotting 24 hours after transfection (Fig. 6D, i). Western blot analysis showed that after transfection with 3.0 µg of plasmid DNA, the FGF-2 expression was reduced to 29.7 ¡À 10.86% (Fig. 6D, ii; p * `% F$ J: [  o5 _- X$ B

To investigate the influence of different 18-kDa FGF-2 overexpression levels on the survival of TH-positive neurons, two different culture conditions were analyzed using TH cell-ELISA. First, when the transfected cells were cultivated without exogenous FGF-2 during the proliferation phase, and second, when the exogenous FGF-2 was added to proliferation medium to induce rescue effects (Fig. 6B). Analysis showed that TH positivity of transfected cultures varied from 90.8 ¡À 18.99% (0.5-µg group) to 79.9 ¡À 12.32% (5.0-µg group) in the presence of exogenous FGF-2 (PM FGF-2), in comparison with 82.5 ¡À 18.5% (0.5-µg group) to 69.1 ¡À 16.66% (5.0-µg group) without exogenous FGF-2 (PM ¨C FGF-2). A slight rescue effect of exogenous FGF-2 was found only in the 5.0-µg group (Fig. 6B; p 9 P* _& C. T; Q" N. c$ y2 O- {0 Z
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Flow Cytometry

We have further analyzed the VMP cells nucleofected with pEGFP-N2 by FACS. It revealed a transfection efficiency of 45.2 ¡À 13.9% (Fig. 7A, i¨Cii). Prior to FACS analysis, the transfected cells were cultured further in either proliferation or differentiation medium after nucleofection for an additional 4 days, and FACS analysis revealed that differentiated cells were¡ªas expected¡ªsmaller than proliferating cells, resulting in a cell size shift of 10 ¡À 2.5% (Fig. 7B, i¨Cii; p
+ I) x: X: `% T) Y/ q0 j
Figure 7. Fluorescence-activated cell sorting (FACS) analysis of ventral mesencephalic progenitor (VMP) cells nucleofected with pEGFP-N2 and transplantation of them into 6-hydroxydopamine lesioned rat brains. (A): Representative plots of FACS analysis of green fluorescent cells (green) shows a transfection rate of 45%. Autofluorescence of nontransfected cells (red) was excluded from the analysis. (B): FACS analysis of relative cell size shows the 10% cell size decrease after 4 days in differentiation phase compared with proliferation phase. (C): Transplantation of VMP cells transfected with pEGFP-N2. i, TH-positive neurons in the medial and lateral (arrows) deposits of the graft. Inset (i') represents migrated TH-positive cells, which do not show typical neuronal morphology and were smaller than the neurons at the graft site. ii, the picture shows the higher magnification of the grafted cells. ii', the EGFP-expressing cells (green) were visualized by anti-GFP staining. Scale bar = 250 µm. *, p , A- o! K8 B1 G+ z4 k$ e
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Transplantation of Nucleofected VMP Cells into 6-OHDA Lesioned Rat Brains. {8 ^& u& X6 g

We next examined whether transfected VMP cells (a) could survive transplantation and (b) express TH after grafting. TH immunohistochemistry showed that transfected VMP cells survive the transplantation (tx) procedure and are TH immunopositive in vivo, but compared with the number of grafted cells, only a small percentage survived (Fig. 7C, i¨Cii). In addition, the grafted cells were still EGFP-positive in vivo (Fig. 7C, ii'). We found some macrophages within the tx region (not shown). The degree of reinnervation is very limited, because we had already sacrificed the animals 1 week after the cell tx. Interestingly, some grafted cells migrated away from the tx site but nevertheless expressed some TH (Fig. 7C, i'). These migrated cells looked much smaller than the DA neurons, and the TH positivity was much lower.: _2 J0 x% @  u
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DISCUSSION
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In this study, we compare three different approaches for transfection of primary VMP cells: electroporation, lipofection, and nucleofection. Our results show significant improvement in transfection efficiency of primary VMP cells by using nucleofection. Using standard electroporation or lipofection, approximately 10% of transfection efficiency was reached, whereas nucleofection was nearly 5 times more efficient, allowing to high concentrations of a gene product to be reached. These findings show a higher efficiency compared with the results of Bauer et al., who report only 150 transfected primary VMP cells out of 100,000 cells by lipofection . However, we showed significant differences in transfection efficiency between EGFP and DsRed constructs.
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Previous reports show that nucleofection is a powerful tool for gene delivery . In the present study, the transfected VMP cells were grafted into their main target area, the striatum, where DA neurons are usually implanted in clinical applications. Furthermore, our grafted VMP cells differentiated into neuronal phenotype and expressed TH. Here, for the first time, we report on nucleofection as an efficient method for nonviral transfection of dopaminergic progenitors.

We demonstrate here that not only could the reporter gene be efficiently delivered to VMP cells by nucleofection, but the gene expressing a growth factor could as well. First we transfected the cells with a construct, coding for the 23-kDa isoform of FGF-2 gene tagged with EGFP (or DsRed) to evaluate the quality of the transfection. We found the typical restriction to a nuclear expression of FGF-2 for this isoform .).
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Nucleofection is an electroporation-based gene transfer .
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Our study further shows that after differentiation of transfected progenitors, the transfected cells were detected in both neuronal and glial populations, but only a few transfected TH-positive neurons were detected (after electroporation and nucleofection) within the dopaminergic neuron population. The fivefold enhancement of transfection efficiency could not increase the number of transfected dopaminergic neurons, whereas the overall amount of these neurons was not affected by transfection. These findings suggest that this type of the neuronal cells is extremely sensitive to any kind of genetic modification. Yet this fact does not interfere with the idea of transplanting genetically modified VMP cells into the host brain, because: (a) it could be favorable if the graft contains not the pure population of dopaminergic neurons but a mixed composition of other neuron types and glial cells to induce maximum effect (whereas recent studies suggest that astrocytes not only give a trophic support to the neurons but also play an important role for fate decision by progenitors before or after transplantation . But cotransplantation approaches are only a vehicle, and these results give evidence that the same results could be achieved directly without the necessity of a cograft.* f+ R0 ]# b( e8 ~7 G

However, the overexpression of the 18-kDa isoform of FGF-2 in VMP cells seems to reduce their viability and proliferation during the proliferation phase (under serum-free conditions) in vitro. We demonstrated that in the first days after transfection, the expression of the delivered gene was the strongest; FGF-2 overexpression was increased fourfold in transfected VMP cells, but FGF-2 was also overexpressed in cells transfected with empty control vector, indicating that nucleofection itself causes an increase in FGF-2 expression too, probably through interactions with heat shock proteins (HSPs) .). Therefore, we speculate that the negative effects of 18-kDa FGF-2 overexpression on VMP cell viability and proliferation could be caused by the domination of proapoptotic mechanisms over antiapoptotic ones induced by a bimodal action of FGF-2.
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The ventral midbrain-derived VMP cells (stage E12) were not (pre-)differentiated in vitro prior to transplantation, as done previously by Timmer et al.  Nevertheless, the cells were TH-immunoreactive in vivo, suggesting that the differentiation was initiated by local stimuli within the host striatum. These results indicate that an in vitro differentiation is not necessary before grafting in terms of TH expression; however, predifferentiation seems to be important for preventing cell migration and to get more surviving TH  cells in vivo. Altogether, our in vivo results show that (a) VMP cells survive transplantation after nucleofection; (b) the transfected cells remain EGFP-positive in vivo and can therefore be detected easily within the host brain after transplantation, which has so far been a major problem; and (c) nucleofection does not prevent VMP cells to differentiate into a DA phenotype in vivo.; z/ x" ~# I) m
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With regard to the implementation of novel cell replacement strategies for Parkinson disease, these results are promising for the detection of implanted cells and transfecting them with growth factor(s); nevertheless, cell survival has to be improved (e.g., by immunosuppression).5 j  w/ f: G& k0 w% g

Conclusions
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This study shows that nucleofection is the most effective nonviral gene delivery method for primary VMP cells, which are the important source of dopaminergic neurons for cell replacement strategies in Parkinson disease. It allows the transfection of neural progenitors with high efficiency and does not alter the population composition and differentiation potential. It also delivers a growth factor gene into the cells efficiently that allows the manipulation of cell properties in vitro. Unfortunately, the comparatively high toxicity of the method to the neuronal progenitors remains a limiting factor for the wide application in transplantation studies and needs to be improved. The effective nonviral transfection method could minimize the risks associated with viral transfection, which include strong immunoinflammatory response, possible protein processing interference, and abnormal protein targeting. These results could help to optimize current transplantation strategies by manipulating both primary neuronal cells and neuronal stem cells before using them for therapeutic applications.

DISCLOSURES% T3 [: T* |# H% L3 z4 O, b
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The authors indicate no potential conflicts of interest./ w4 v! |4 M- J/ h; p/ o- G

ACKNOWLEDGMENTS4 N* j: }/ I6 ~) x% n9 H4 ]4 J

This work was supported by the Center for Systems Neuroscience, Hannover Medical School, Hannover, Germany. We thank the Department of Experimental Nephrology (Hannover Medical School) for the permission to use the Amaxa Nucleofector device. We thank Anastassiia Vertii, Kerstin Kuhlemann, Hella Brinkmann, Natascha Heidrich, Hildegard Streich, Ursula Jensen, and J¨¹rgen Wittek for excellent technical support and Peter Claus, Julia Jungnickel, Kirsten Haastert, Alexander Bruns, Ester Lipocatic, and Fabian Himstedt for discussions. K.C. and M.T. contributed equally to this work.
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