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TISSUE-SPECIFIC STEM CELLS |
aDepartment of Neuroanatomy and Center for Systems Neuroscience Hannover,
bInstitute of Biochemistry, and
cDepartment of Trauma Surgery, Hannover Medical School, Hannover, Germany
Key Words. Dopaminergic neurons • Neural stem cells • Nucleofection • Transfection • Transplantation
Correspondence: Claudia Grothe, Dr. rer. nat., Department of Neuroanatomy, Center of Anatomy, OE 4140 Hannover Medical School, Carl-Neuberg-Strasse 1, D-30623 Hannover, Germany. Telephone: 49-511-532-2896; Fax: 49-511-532-2880; e-mail: grothe.claudia{at}mh-hannover.de
Received on March 24, 2006;
accepted for publication on August 4, 2006.
First published online in STEM CELLS EXPRESS August 10, 2006.
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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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 [22–24]. To overcome the above-mentioned limitations, the delivery of DNA encoding additional growth factors and/or reporter genes such as enhanced green fluorescence protein (EGFP) is necessary to promote the expansion and differentiation capacity and the survival potential, as well as to identify the donor cells within the host brain. It has been shown previously that basic fibroblast growth factor (FGF-2), which is a physiological neurotrophic factor in the nigrostriatal system, stimulates neurite formation, protects DA cells from 6-hydroxydopamine (6-OHDA) toxicity in vitro [25–31], and displays beneficial effects in vivo [32–34]. Our group has shown that cotransplantation of FGF-2 overexpressing Schwann cells with fetal mesencephalic cells increased survival, reinnervation, and functional recovery in Parkinsonian rats [35]. 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 [36–39], but viral vectors have many disadvantages, mainly involving safety risks (e.g., infections). In addition, the use of viral vectors such as adenoassociated virus, lentivirus, or retrovirus requires each cDNA to be cloned into specific vectors, cDNAs must not be too long, and these techniques are very demanding in technical skills, time, and laboratory safety precautions [40, 41]. Several nonviral methods are available for generating transiently and stably transfected cells, including calcium phosphate precipitation; the use of particles; electroporation, which is the most commonly used [42]; 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.
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) [41]. This is an electroporation-based method that uses the combination of a specific nucleofector solution and specific electrical parameters to achieve the delivery of plasmid DNA into the cell nucleus. It was successfully applied to hematological and immunological cells [43–45] and also to embryonic and adult stem cells [46–49]. To date, there have been only a few reports of gene delivery to neurons by using the recently developed nucleofector technology [40, 41, 50, 51], and there is only one report about nucleofection of NSCs, which was done with spinal cord progenitors [52]. So far, there have been no reports about nucleofection of neuronal progenitors from the brain, including dopaminergic ones.
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.
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.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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Cell Culture
After dissociation of the tissue [24] the cells were counted using a cell-counting chamber (hemocytometer) and adjusted to the final number of 2,000,000–3,000,000 cells per milliliter. One ml of the suspension was seeded per 25-mm2 cell culture dish (Nunc GmbH, Wiesbaden, Germany, http://www.nunc.de) with 5 ml of medium I (Dulbecco's modified Eagle's medium/Ham's F-12 medium [DMEM/F12], 3% fetal calf serum [FCS] [PAA Laboratories, Linz, Austria, http://www.paa.at], 20 ng/ml FGF-2 [18 kDa] [Peprotech, Rocky Hill, NJ, http://www.peprotech.com], B27, N2 [1 ml/100 ml of a 100x stock solution] [Gibco, Grand Island, NY, http://www.invitrogen.com], 1 mM sodium pyruvate, 0.25% bovine serum albumin [BSA] [Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com], and 2 mM glutamine). The culture dishes were precoated with polyornithine (Sigma-Aldrich, 0.1 mg/ml in 15 mM boric acid buffer, pH 8.4) and laminin (Sigma-Aldrich, 6 µg/ml) in distilled water for 24 hours at room temperature then washed twice with distilled water before cell seeding. After 24 hours of cell culture, medium I was removed and replaced by medium II (a modification of medium I, without FCS and B27 supplement). Then the cells were cultivated for 3 days. The day with medium I was considered day in vitro 0 (DIV0). On DIV3 (for fluorescence-activated cell sorting [FACS] 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 3–4 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.
Transfection
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 [56], pDsRed-23kDa-FGF-2, and pEGFP-23kDa-FGF-2 [57, 58]; pCIneo was used as empty control vector (Promega, Madison, WI, http://www.promega.com).
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.
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 [11.41 g], 20 mM potassium acetate [1.96 g], 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.
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).
Immunocytochemistry
The immunostaining was performed according to the previously described protocol [24]. The following primary antibodies were used: anti-tyrosine hydroxylase (anti-TH), 1:200 (Chemicon, Temecula, CA, http://www.chemicon.com); anti-nestin, 1:550 (Chemicon); anti-glial fibrillary acidic protein (anti-
-GFAP), 1:400 (Sigma-Aldrich); anti-ß-tubulin type III, 1:140 (Upstate, Charlottesville, VA, http://www.upstate.com); anti-fibroblast growth factor-2 (anti--FGF-2), 1:500 [59]. 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.
Cell-Enzyme-Linked Immunosorbent Assay
Cell-enzyme-linked immunosorbent assay (cell-ELISA) was performed in 96-well microtiter plates as previously described [60]. The following dilutions of primary antibodies were used: anti-TH, 1:200; anti-ß-III-tubulin, 1:140; anti-GFAP, 1:600, and anti-FGF-2, 1:300 (Transduction Laboratories). The relative absorbance was measured at 405 nm (Mikrotek Laborsysteme GmbH, Germany). Data were corrected for blank values obtained in the absence of the first antibody [29].
Cell Viability and Proliferation Assay
Cell viability and proliferation were evaluated after nucleofection by WST-1 assay (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com) [61] 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°C), 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.
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.
Western Blotting
Western blot analysis was performed as previously described [24, 62]. 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).
Cell Counting
EGFP-positive or red fluorescent cells (DsRed-positive) were counted to measure the transfection rate 24–48 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.
FACS Analysis
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.
Transplantation and Evaluation of the Graft
The procedure was described in detail previously [24]. Transplantation of progenitor cells transfected with EGFP construct was performed during the proliferation phase, one day after transfection into the striatum of 6-OHDA-lesioned adult Sprague-Dawley rats (for anesthesia, 6-OHDA lesion, and animal handling, see Timmer et al. [35]). The animals received eight 1-µl deposits containing 100,000 cells each. Intrastriatal stereotaxic injections according to the modified microtransplantation approach [54] were done at the following coordinates: tooth bar, 0; anterior posterior, 0, +1; lateral, –2.3, –3.3; vertical, –4, –5. Experimental protocols were approved by the Bezirksregierung Hannover, Germany, and meet the guidelines of the Tierschutzgesetz (under the law 25.05.1998). One week after grafting, the rats were perfused with 4% paraformaldehyde, coronal sections were cut on a freezing microtome at 30-µm thickness, and TH immunohistochemistry was performed [24]. 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.
Statistical Analysis
Results are expressed as means ± 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 <0.05 were considered significant.
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RESULTS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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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 ± 16.3% of the electroporated cells and 228.2 ± 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).
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 [57, 58]) fused with DsRed using a pDsRed-23kDa-FGF-2 construct. The 23-kDa FGF-2 is known to stimulate the survival and neurite formation of dopaminergic neurons both in vitro and in vivo [29, 35, 63]. 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–iv).
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 ± 19.4%; Fig. 1A, i), and this was confirmed in Western blot (Fig. 1D, ii). Electroporation showed only a slight overexpression (122.8 ± 23.2%; p < .05; F = 35.258), whereas lipofection failed to produce overexpression in the primary VMP cells (Fig. 1A, i).
Electroporation Did Not Alter the Cellular Composition and Differentiation Potential of Primary Mesencephalic Progenitors
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–vi); however, the majority of them were astrocytes, whereas only a few transfected TH-positive neurons were detected (Fig. 2B, i–ii).
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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 ± 3.4% to 29.4 ± 4.02%, and in electroporated cultures, they increased from 12.8 ± 3.36% to 27.3 ± 4.08% (p < .001; F = 89.369). Similarly, the astrocyte population increased from 12.2 ± 3.73% to 37.7 ± 5.88% in nontransfected cultures and from 10.9 ± 4.67% to 42.8 ± 3.87% in electroporated cultures (p < .001; F = 196.403). There was no difference between nontransfected and electroporated cells in the TH-positive population after differentiation (p > .05), but more astrocytes were found in electroporated cultures compared with nontransfected ones (p < .05). We found fewer ß-III-tubulin-positive neurons after differentiation; nontransfected cultures showed a ß-III-tubulin positivity decrease from 48.9 ± 9.96% to 42.0 ± 3.09% (p > .05), and electroporated cultures showed a decrease from 60.7 ± 12.45% to 41.3 ± 4.28% (p < .001, Fig. 2A, i). There was no reduction of nestin-positive progenitors in nontransfected cultures (38.9 ± 11.5% during proliferation and 38.6 ± 5.49% after differentiation; p > .05), whereas in electroporated cultures, nestin positivity was reduced from 44.0 ± 7.33% to 34.7 ± 7.93% after differentiation (p < .05; F = 3.169; Fig. 2A, i). The differentiation potential of primary VMP cells was not altered by electroporation, and the relative amount of TH-positive neurons in neuronal population increased from 25.7% (proliferation phase) to 70.1% (differentiation phase) in nontransfected controls, whereas in electroporated cultures, it increased from 21.1% to 66.2% (Fig. 2A, ii).
Lipofection
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 [GFAP-positive] and neurons [ß-III-tubulin-positive]), 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–iii, i'–iii'), and astrocytes preferably expressed EGFP (Fig. 3, iv–vi). 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).
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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 ± 12.9% of the cells transfected with empty control vector (pCIneo) survived, compared with nontransfected controls (p < .05; Fig. 5A, v). The cells overexpressing FGF-2 showed even lower viability (35.6 ± 18.3% survived); there was no significant difference between the transfected groups (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 ± 30.5%; FGF-2 overexpressing cells increased only up to 45.7 ± 24.3% (p < .05; Fig. 5A, vi). On day 7 after transfection, when the cells were exposed to differentiation medium, this significant difference disappeared (Fig. 5A, vii). At the end of the differentiation phase, the cell number in the FGF-2 overexpressing group reached the level of the nontransfected group (107.4 ± 26.3%; Fig. 5A, viii). Nevertheless, when nontransfected cells were analyzed under the phase-contrast microscope, cell clustering and continuous cell loss were detected, which caused a relative increase in cell numbers of the transfected groups (Fig. 5A, ii–iv).
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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 ± 213.5% of the level of nontransfected controls (p < .001; Fig. 6C, i). Admittedly, the cells transfected with empty control vector (pCIneo) also showed an increase in FGF-2 expression, up to 268.8 ± 66.15% of the level of nontransfected controls (p < .001; Fig. 6C, i), indicating that nucleofection itself causes an increase in FGF-2 expression. FGF-2 overexpression continuously decreased during the cell culture protocol, and in differentiation phase, it reached the level of nontransfected cells (Fig. 6C, i). In FGF-2-overexpressing cultures, the FGF-2 expression levels during the proliferation phase were significantly higher than in the differentiation phase (Fig. 6C, ii; p < .05).
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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 ± 18.99% (0.5-µg group) to 79.9 ± 12.32% (5.0-µg group) in the presence of exogenous FGF-2 (PM + FGF-2), in comparison with 82.5 ± 18.5% (0.5-µg group) to 69.1 ± 16.66% (5.0-µg group) without exogenous FGF-2 (PM – FGF-2). A slight rescue effect of exogenous FGF-2 was found only in the 5.0-µg group (Fig. 6B; p < .05) indicating a possible negative effect of excessive overexpression of 18-kDa FGF-2 on survival of TH-positive neurons.
Flow Cytometry
We have further analyzed the VMP cells nucleofected with pEGFP-N2 by FACS. It revealed a transfection efficiency of 45.2 ± 13.9% (Fig. 7A, i–ii). 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—as expected—smaller than proliferating cells, resulting in a cell size shift of 10 ± 2.5% (Fig. 7B, i–ii; p < .05), indicating an increase of smaller cell populations, which are more differentiated cells. These data are congruent with qualitative morphological observations of the cell culture.
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DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresAcknowledgmentsReferences |
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Previous reports show that nucleofection is a powerful tool for gene delivery [40, 50, 51] and gene silencing [41, 51] in postmitotic neurons. Transplantation of postmitotic neurons has so far been used for brain restoration, even though it has several limitations [1, 16]. NSCs seem to be more promising for brain repair approaches in terms of both genetic manipulations and transplantation [7, 9, 10, 66]. The only work of nucleofection of NSCs with GFP was done on spinal cord progenitors by Richard et al. [52], but they could not show the differentiation of transfected spinal progenitors toward neuronal fate either in vitro or in vivo. Richard et al. transplanted spinal cord progenitors ectopically into retina, where they found only astrocytes [52]. 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 [57, 58], independent of the transfection method used (nucleofection, standard electroporation, or lipofection). Second, in the same manner, cells were transfected with a construct coding for the untagged 18-kDa isoform of FGF-2, which is localized in both cytoplasm and nucleus, and we analyzed the overexpression level after the transfection. The FGF-2 overexpression was stronger after nucleofection than after electroporation, whereas lipofection failed to show significant overexpression of FGF-2. These findings suggest that nucleofection does not affect the quality of FGF-2 expression but results in higher amounts of growth factor, which could potentiate its effect on the cells. This approach could therefore be useful for transplantation purposes. We could also show that the fluorescence intensity of EGFP was reduced by threefold 2 weeks after transfection compared with the intensity 24 hours after transfection. These results suggest that nucleofection produces a transient rather than stable transfection, and this correlates with the findings of Lakshmipathy et al., which show a sixfold lower stable transfection rate in comparison with a transient one [48]. However, this finding could be rather an advantage than a disadvantage for transplantation purpose, because the crucial time for the survival of transplanted cells is the first 6 days after transplantation, which involves implantation procedure, the immediate period (first 1–3 days) after graft injection into new adult host environment, and the following phase of graft maturation when transplanted cells might die because of a lack of appropriate neurotrophic support. Therefore the highest concentration of the growth factor in the graft is essential during the first week after transplantation, whereas it is not necessary in later stages, when it could interfere with the physiological functioning of already integrated transplanted cells [26, 36] (for details, see the review of Brundin et al. [19].).
Nucleofection is an electroporation-based gene transfer [40, 52], but its predefined settings do not allow us to analyze which physical parameters could influence the functionality of transfected primary neuronal progenitors. For this purpose, we used a standard electroporation procedure, which allows us to manipulate voltage and capacitance parameters [67]. In our results, we could show that optimal conditions for transfection of primary VMP cells with standard electroporation are 350 V and 75 µF, and although increase of capacitance setting (and therefore increase of the pulse time) slightly elevates the transfection efficiency, at the same time, it greatly affects the survival of the VMP cells in general and especially of the TH-positive neurons after differentiation. However, under the optimal conditions, we found populations of differentiated cells in the same proportions as in nontransfected cultures, under both proliferating and differentiating conditions. These results suggest that electroporation under these optimal conditions does not alter the population composition and differentiation potential of primary VMP cells. In addition, it allows gene delivery into postmitotic cells such as neurons [42, 68, 69]. Since nucleofection is an electroporation-based method, it is very likely that under the optimal program, similar effects on cell culture composition will be present. This suggestion is supported by a recent report from Lakshmipathy et al. showing that nucleofected ESCs have levels of expression of ESC markers identical to the nontransfected controls [48].
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 [7, 66]); (b) the other cell types of the graft (neurons and glia), which are genetically modified, produce a trophic factor to support surviving dopaminergic neurons and in the same way, the DA neurons are not altered or somehow modified (assumed that most DA cells do not survive this kind of transfection). In the previous reports from our group it was already shown that cotransplantation of Schwann cells—genetically modified to produce FGF-2—enhances survival, reinnervation, and functional recovery of intrastriatal dopamine grafts [35]. 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.
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) [70] or due to FGF-2 release from dead cells. The number of surviving cells was significantly diminished in comparison with nontransfected and sham-transfected controls. Moreover, in the first 2 days after transfection, also, the proliferation rate decreased significantly. These effects led to the reduced TH immunoreactivity of 20% after differentiation of transfected VMP cultures. Keeping in mind that excessive overproduction of the growth factor protein could interfere with functionality of the cells [36], we reduced the concentration of plasmid DNA used for transfection, resulting in a decreased expression of the FGF-2 in the transfected cells, even though it had no effect on cell proliferation. Application of exogenous FGF-2 during proliferation phase elicited a slight but significant rescue effect of TH immunoreactivity, although only in the group transfected with the highest DNA concentration. These findings suggest that excessive overexpression of FGF-2 could have some negative effect on survival of TH-positive neurons, which is supported by results of Georgievska et al. [36] but cannot explain the reduction of the cell viability and proliferation. And despite the reduced cell viability and proliferation of the FGF-2 transfected cells in proliferation phase, after differentiation the overall cell number equals that of the nontransfected group. This result indirectly shows a better cell survival in the FGF-2-transfected group. Previous studies have shown that FGF-2 can induce apoptosis by activation of the tumor necrosis factor- pathway [71]; in addition, the studies done in mutant mice showed that FGF-2 is involved in apoptotic processes mediated by FGFR3 signaling [72], and on the other hand, FGF-2 promotes neuronal survival via FGFR1/2 (for details, see the review of Grothe et al. [73].). 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.
The ventral midbrain-derived VMP cells (stage E12) were not (pre-)differentiated in vitro prior to transplantation, as done previously by Timmer et al. [24] 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.
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).
Conclusions
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.
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