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Nonviral Gene Delivery to Neural Stem Cells with Minicircles by Microporation Catarina Madeira,* Carlos A. V. Rodrigues, Mónica S. C. Reis, Filipa F. C. G. Ferreira, Raquel E. S. M. Correia, Maria M. Diogo, and Joaquim M. S. Cabral Department of Bioengineering and Institute for Biotechnology and Bioengineering (IBB), Instituto Superior Técnico, Technical University of Lisboa, Av. Rovisco Pais, 1049-001, Lisboa, Portugal ABSTRACT: The main purpose of this work was to evaluate the transfection of novel DNA vectors, minicircles (mC), on embryonic stem cell-derived neural stem cells (NSC). We demonstrated that by combining microporation with mC, 75% of NSC expressing a transgene is achieved without compromising cell survival, morphology, and differentiation potential. When comparing mC with their plasmid DNA (pDNA) counterparts, both gave rise to similar transfection levels but cells harboring mC showed 10% higher cell viability, maintaining 90% of survival at least for 10 days. Long-term analysis showed that NSC harbor a higher number of mC copies and consequently exhibit higher transgene expression when compared to their pDNA counterpart. Taken together, our results offer the first insights on the use of mC as a novel and safe strategy to genetically engineer NSC envisaging their use as biopharmaceuticals in clinical settings for the treatment of neurodegenerative or neurological diseases.

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CNS8 or of toxic payloads for tumor elimination.4 In 2010 started the first clinical trial in patients with recurrent high grade glioma using immortalized human NSC that have been transduced with retroviruses to express cytosine deaminase (CD) therap eutic transgene (Clinical Trial ID: NCT01172964). In this case, an oral prodrug (5-fluorocytosine), administered on the fifth day for 7 days, will be converted to the chemotherapeutic agent 5-FU by NSC expressing CD, which will then be secreted at the tumor site to produce an antitumor effect.4 The method of selection to genetically modify these cells is based on the use of viral vectors but, due to safety concerns, nonviral methodologies are gaining a major impact mainly in clinical settings. In recent years, several research groups have attempted the transient expression of transgenes in NSC using nonviral gene delivery methods such as cationic liposomes,9 polymers,9,10 magnetic particles,11,12 and physical methods as electroporation13−16 to deliver DNA into these cells. The chemical methods used so far with NSC gave rise to transfection levels below 20%.12 Using physical methods higher values have been obtained although hampering cell viability. However, a novel microporation method, used in this work, has shown promising results not only in transfection efficiency but also in terms of cell survival.17,18 When using a physical method and once inside cells, DNA vectors encounter two distinct cytosolic barriers: a diffusional and a metabolic barrier. The diffusional barrier is related to the size-dependent retardation of vectors’ mobility inside cytosol which might lead to the

INTRODUCTION Neural stem cells (NSC) are multipotent stem cells that can be isolated from embryonic, fetal, or adult brain and have the ability to self-renew and to differentiate into neurons and glia. In vivo, their morphological and phenotypic identities vary depending on the developmental stage and region occupied by these cells during the embryonic development and during adult life.1 More recently, it was also found that NSC with distinct identities can be experimentally derived from pluripotent stem cell lines from both mouse and human origin.1 As the first example of this type of NSC system, the derivation and continuous expansion, in the presence of FGF-2 and EGF, by symmetrical divisions of pure cultures of neural stem cell lines, under adherent culture conditions, was demonstrated.2 These cells have been termed NS cells to highlight the similar experimental attributes when compared with embryonic stem cells (ESC).2 Importantly, after prolonged expansion, NS cells maintain their capacity for efficient differentiation into neurons and astrocytes in vitro and upon transplantation into the adult brain they exhibit a close relationship to radial glia, a defined endogenous population of neural precursor cells. Due to these characteristics, NS cells constitute a promising tool and model for basic research and biomedical applications including the delivery of cell replacement and gene therapies toward the central nervous system (CNS). NSC hold a great potential for the development of gene and regenerative therapies for the treatment of neurodegenerative disorders,3 brain cancer,4 or regeneration of spinal cord injuries.5 Moreover, NSC can be genetically modified in vitro to express desired transgenes for an increased expansion or differentiation capacities,6,7 as well as to be used in vivo as cellular carriers of growth factors for cellular regeneration in the © XXXX American Chemical Society

Received: January 4, 2013 Revised: March 19, 2013

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Methods. Culture and Expansion of Neural Stem Cells. NS cell line CGR8-NS, derived from the mouse ES cell line CGR8 (Welcome Trust Centre for Stem Cell Research, Cambridge, U.K.) was used as cell model and will be referred throughout this work as NSC. In all the experiments, NSC were thawed from the frozen stock by swiftly submerging the criovials in a water bath at 37 °C and by resuspending the cells in DMEM/F-12 medium (Gibco) supplemented with 1% N-2 supplement (Gibco) and additional 1.6 g/mL of glucose, 20 μg/mL insulin (Gibco), and 1% of penicillin/streptomycin. The culture medium was also supplemented with 15 ng/mL of EGF and FGF-2 (both from Peprotech) and B27 (1:1000, Invitrogen). The cells were seeded in 25 cm2 T-Flasks (BD) at an initial cell density of 10000 cells/cm2 and expanded under static conditions at 37 °C and 5% CO2humidified atmosphere. When NSC reached subconfluence they were harvested using Accutase (Sigma) and the number of viable and dead cells was assessed using the trypan blue dye exclusion test (Gibco). The cells were then used directly in transfection assays or cultured in T75 flasks and grown under static conditions at 37 °C and 5% CO2humidified atmosphere. When the T75 flasks exhibited a 70−80% cell confluence, NSC were harvested and used in transfection assays. Gene Transfection of Neural Stem Cells by Microporation. NSC were resuspended in resuspension buffer (RB; provided by the equipment manufacturer (Invitrogen)) or in a sucrose-based buffer (SBB, 250 mM sucrose and 1 mM MgCl2 in Dulbecco’s phosphate buffered saline (DPBS; Gibco))13,39 at a density of 200000 CGR8-NS cells/10 μL and incubated with a specific amount of vector DNA (mC or pDNA). DNA amount is referred when appropriate and microporation was accomplished using a Microporator MP100 (Neon/Invitrogen). After microporation, the 10 μL of cell suspension was directly seeded into a well of a 24-well culture plate containing prewarmed culture medium without antibiotics, for RT-PCR assays, or added to 100 μL of prewarmed medium without antibiotics and equally divided into two wells of a 24-well culture plate containing culture medium under the same conditions, for cell proliferation kinetic analysis. Afterward, the cells were incubated under static conditions at 37 °C and a 5% CO2-humidified atmosphere for 24 h after which they were harvested or had the medium replaced by complete medium. The cells were maintained under the previously mentioned conditions for 10 days and harvested at different points in time after transfection to be further analyzed. In all experiments, nonmicroporated (NM) cells were used as control. Cell Viability, Yield of Transfection, and Protein Expression Monitoring. The number of GFP positive cells (GFP+ cells) was monitored by flow cytometry using FACSCalibur equipment (BD Biosciences) and statistically evaluated by CellQuest Software (BD Biosciences) considering a minimum of 1000-gated cells. The level of protein expression is given by the mean fluorescence intensity (MFI), also measured by flow cytometry. The percentage of viable cells (cell viability) was estimated by trypan blue exclusion method. For each microporated sample (M), yield of transfection was determined 24 h after microporation using the equation YT (%) = (GFP+ × CAM)/ CTNM, where CAM is the number of microporated cells alive and CTNM is the total number of cells in the control (NM) sample. Fluorescence Microscopy Imaging. Transfected and differentiated cells were visualized using a fluorescence optical microscope Leica DMI 3000B (Leica Microsystems GMbH) and digital images were obtained with a digital camera Nikon DXM 1200F. Fluorescence images were acquired with green, blue, or red filters, depending on the purpose, at 200× magnification. Cell Proliferation Kinetics Study. To evaluate cell proliferation kinetics, NSC were labeled using PKH26 Red Fluorescent Cell Linker Kit (Sigma)18 immediately prior to transfection. The PKH26 dye consists of a fluorophore attached to long aliphatic carbon tails that connect irreversibly to the lipid layer of the cell membrane and are evenly distributed between the daughter cells in the following generations. After centrifugation the labeled cells were resuspended with the required volume of electroporation buffer in order to achieve a concentration of 200000 cells/10 μL, incubated with 1.0 × 106 vector DNA molecules/cell, and microporated. Cells were harvested on days 1, 3, 4, 7, and 10 after transfection and further analyzed by

formation of pDNA aggregates and an increase of the cytoplasm viscosity. The metabolic barrier is related to the presence of calcium-sensitive cytosolic nucleases that degrade foreign DNA.19,20 The nuclear envelope represents the last barrier to be surpassed. To diffuse through the nuclear envelope the vectors must carry a nuclear localization sequence (NLS) to pass through the nuclear pore complex (NPC) or be transfected into dividing cells to gain access to the expression machinery after nuclear disassembly during mitosis.21 To overcome these barriers many laboratories have been committed to the development of more effective plasmid vectors. The majority of the most recent advances in plasmid technology involve the substitution of the conventional polyadenylation (polyA) signals with more effective polyA sequences,22 the production of small pDNA devoid of resistance marker,13,23 and the development of mC24−26 or CpG free-pDNA.27 Unmethylated CpG motifs are predominantly present in bacterial DNA and therefore are also common in bacterial derived pDNA. These motifs are recognized by Toll-Like receptor 9 (TLR) positive cells (e.g., Dendritic cells), which activate host defense mechanisms triggering innate and acquired immune responses.28 Transfection of CpG free-based pDNA has proven to yield high transgene expression in vivo and in vitro, hence, allowing sustained expression.29 The biomolecule under focus in our work, mC, belongs to a new generation of small sized circular DNA molecules lacking the bacterial backbone therefore with a low content of CpG dinucleotides and has been recently tested in vivo30−32 and in vitro with somatic33 or stem cells34 with superior results in terms of transfection efficiency and long-term expression of the transgene, when compared to conventional plasmids. It is commonly accepted that the bacterial backbone in the plasmid triggers immune responses23,35 and it has also been referred that it might alter gene expression by promoting silencing of the encoded transgene.30,36 When compared to regular plasmids, mC display a 10- to 10000-fold improvement in long-term transgene expression in vivo,32,37 and further improvements in their formulation, for example, addition of scaffold/matrix attachment region (S/MAR), enhance transgene expression, and persistence.27 Nevertheless, mC have not been used for transfection of NSC. Altogether, minicircles offer a new concept in highly efficient and safe nonviral gene transfer, and in the future, they might represent the preferred system for gene delivery for therapeutic applications. Actually, in gene/cell therapy, when the goal is the transient expression of a specific therapeutic protein, it may be beneficial to extend its period of expression and more importantly to ensure high levels of cell survival.

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EXPERIMENTAL SECTION

Materials. DNA vectors MC07.CMV-GFP (minicircle, 2257 bp) and pCMV-GFP (plasmid, 3487 bp) were purchased from Plasmid Factory (Bielfield, Germany) and will be referred as minicircle (mC) and plasmid (pDNA), respectively. The mC is a bacterial backbonefree vector consisting only of a mammalian expression cassette which carries a GFP gene under the control of the cytomegalovirus (CMV) promoter and the SV40 polyadenylation sequence, and has a total 129 CpG motifs. Besides the previously described expression cassette, pDNA contains an origin of replication and a Kanamycin resistance gene for prokaryotic replication and selection, respectively, and a total of 230 CpG motifs. Hence, mC used in this work contain 56% less CpG motifs and its size is reduced to around 35% compared with its conventional counterpart pDNA.38 B

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flow cytometry. The number of divisions or generations within a specific subset was determined by analyzing the decrease of fluorescence overtime on a proliferation wizard module of the ModFit Software (BD Biosciences) in the flow cytometer equipment. Real-Time PCR for Quantification of Vector DNA Content. Quantitative Real Time PCR (RT-PCR) was performed on NSC transfected with a variable amount (0.1, 0.5, 0.8, 1, and 1.5 μg) of vector DNA and harvested after 24 h of incubation and on NSC transfected with 1.0 × 106 vector DNA molecules/cell and harvested on days 1, 4, and 7 of incubation. RT-PCR reactions were carried out in a Roche LightCycler detection system using the FastStart DNA Master SYBR Green I kit (Roche, Basel) by amplification of an 80 bp sequence within the GFP gene (forward primer: 5′-TGAACGGCGTGGAGTTCGAG-3′ and reverse primer: 5′-GCTCTTCATCTTGTTGGTCATGCG-3′; STABVida). Each 20 μL of final reaction volume contained 2 μL of the 10× SYBR Green mixture, 0.4 μM of each primer, 3 mM of MgCl2, 2−5 μL of sample (corresponding volume to 20000 NSC), and 9.4 μL of PCR-grade water. The amplification of the sequence within the GFP gene was carried out at 95 °C for 10 min, followed by 40 cycles of 10 s at 94 °C, 5 s at 53 °C, and 7 s at 72 °C. Calibration curves were constructed by adding serial dilutions of vector DNA standards (mC and pDNA) to a suspension of NSC (20000 cells per reaction). These samples were then mixed with the other PCR reagents as described above. One negative control was included in the analysis containing the same amount of NM cells and PCR-grade water. Differentiation of NSC. To evaluate NSC differentiation potential, 200000 cells were microporated with 1 × 106 molecules of DNA vector/cell, and plated into 24-well plates precoated with a 1:100 diluted solution (PBS) of CELLstart (Invitrogen). Cells were first cultured in expansion medium without antibiotics, as described above, for 24 h. Afterward and for astrocyte differentiation, cells were cultured in regular expansion medium for 3 days. Culture medium was then changed to medium without growth factors (EGF and FGF-2), supplemented with 1% FBS, B27 (1:1000) and 1% penicillin/ streptomycin. Medium was changed every three days up to day 12. Neuronal differentiation was achieved by adapting a previously described protocol.40 In the first step cells were cultured in DMEM/ F-12 with N2, 1% B27, 1% Pen/Strep, and 10 ng/mL FGF-2. After three days cells were gently dissociated with Accutase and replated at a cell density of 5−7.5 × 104 cells/cm2 on laminin coated (3 μg/mL for 3−5 h at 37 °C) plates. The medium was changed to a 1:1 mixture of DMEM/F-12 and Neurobasal medium (Invitrogen) containing 0.5% N2 and 1% B27 supplements as well as FGF-2 (10 ng/mL) and BDNF (20 ng/mL). Cells were kept under these conditions for 3 days. The culture medium was then changed to medium with a similar composition but with 6.7 ng/mL FGF-2 and 30 ng/mL BDNF. On day 10 the concentration of FGF-2 was decreased to 5 ng/mL and the culture ended at day 12. Immunocytochemistry. A total of 14 days after initiating both differentiation protocols, astrocyte or neuronal generation was analyzed by fluorescence microscopy imaging after staining with antiglial fibrillary acidic protein (anti-GFAP; Milipore) or antineuronal class III β-tubulin (anti-TUJ1; Covance) antibodies, respectively, and DAPI (4′6-diamidino-2-phenylindole; Sigma-Aldrich). For this purpose, after differentiation, culture medium was removed and cells were fixed in 4% paraformaldehyde (PFA, Sigma) for 10 min, at room temperature. Cells were washed with PBS and incubated with blocking solution (10% normal goat serum (NGS), 0.1% Triton X-100 (Sigma), PBS) for 30−60 min and then with anti-GFAP (1:100) or anti-TUJ1 (1:2000) at 4 °C overnight. The antibodies were diluted in PBS with 0.1% Triton X-100 and 5% NGS. On the following day, cells were washed with PBS and incubated with appropriate secondary antibodies conjugated with Alexa Fluor 546 (dilution 1:500) for 1 h at room temperature. Afterward, cells were washed with PBS and incubated with DAPI (1.5 μg/mL in PBS) for 2 min at room temperature. Lastly, cells were washed with PBS and analyzed by fluorescence microscopy. Statistical Analysis of Data. Results are presented as mean ± standard error and were analyzed with PASW Statistics software (IBM,

U.S.A.). Significance analyses (P values 0.99) provided the relationship between the Ct and the DNA vector mass that relates to the DNA vector copy number. With this method, the first temperature cycle will disrupt the cells and a fragment of GFP gene in the DNA vectors will be amplified, allowing its direct quantification. As in our previous work, the number of DNA vector copy include episomal DNA molecules inside the nucleus and the cytosol because whole cells were used.13,50 Additionally, it has been estimated that the half-life of naked plasmid DNA in the cytosol of cells ranges between 50 min and 5 h,19 consequently, we may assume that 24 h postmicroporation, when we performed the first analysis, all the DNA molecules were inside the nuclei. Even though the same number of initial molecules of each vector has been used, 3-fold more mC molecules was measured inside cells when compared to the number of pDNA detected

pulse of 1800 V during 20 ms. The difference on the required microporation parameters when using each of these two buffers might suggest that the homemade buffer is less efficient when compared to RB. Interestingly, considering these experimental conditions for each buffer, no significant differences were observed in the percentage of cells expressing GFP (∼60%), cell viability (∼90%), and yield of transfection (∼20%) 24 h after transferring minicircles to NSC (Figure 1), and therefore,

Figure 1. Microporation of NSC with 1 × 106 molecules of minicircles/cell using the RB buffer (white bar) applying 1 pulse of 1500 V during 20 ms or the SBB buffer (black bar) using 1 pulse of 1800 V for 20 ms. Percentage of GFP+ cells, cell viability (CV), and yield of transfection (YT) 24 h after microporation are presented. Results were obtained from four independent experiments (n = 4) and as mean ± standard error.

we might suggest the homemade SBB as a suitable alternative to RB when microporating NSC, although a higher voltage is required to achieve similar transfection efficiencies. In this work we compared the effect of two different DNAbased vectors on NSC survival and level of transgene expression. Both vectors, mC and the respective pDNA, have the same eukaryotic expression cassette but differ in the amount of CpG motifs and size. The same number of DNA molecules/ cell (1 × 106) was transferred into NSC and 24h after transfection, around 60% of these cells were expressing the transgene, independently of the DNA vector used (Figure 2A). Major differences between NSC transfected with minicircles (mC-NSC) and plasmids (pDNA-NSC) were observed from the third day up to the 10th day after microporation where 10% more mC-NSC were expressing GFP when compared to pDNA-NSC. Importantly, 4 days after microporation we were still able to observe ∼75% of mC-NSC expressing the transgene against ∼50% of pDNA-NSC and in both cases, no significant differences on the number of cells expressing GFP were measured between days 1 and 4 (Figure 2A). This fact might be explained by the saturation of transgene expression on the first days after microporation. Recent studies showed that although the nucleus can take large amounts of vector copies, only at limited plasmid copy numbers, gene expression can be correlated to the number of vector copies and there is a certain dose of vector after which additional number of vector copies will not influence the level of transgene expression.44 Accordingly, upon cell division DNA molecules will be transferred to the daughter cells and an increase of transgeneexpressing cells will be observed, although predictably the level of protein expression decreases. Noteworthy and to our best D

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Figure 2. Microporation of NSC with 1 × 106 molecules of minicircles/cell (white bar; mc-NSC) or plasmids/cell (black bar; pDNA-NSC). Percentage of transfected cells (GFP+ cells) (A) and cell viability (B) along 10 days are presented. Results were obtained from four independent experiments (n = 4) and as mean ± standard error. Statistical differences are indicated with * for p < 0.05. Bright field (C, E, G) and fluorescence images (D, F, H) obtained 24 h after microporation of NSC, with minicircles (C, D; mC-NSC) plasmid (E, F; pDNA-NSC), and nonmicroporated cells (G, H; NSC).

Figure 3. Quantification of DNA vector molecules inside transfected cells using 1 × 106 molecules of DNA vector/cell. (A) Number of minicicles (white bar) and plasmids (black bar) quantified inside transfected NSC on days 1, 4, and 7 after transfection (inset is a zoom of days 4 and 7) and respective level of transgene expression (B). Results were obtained from four independent experiments (n = 4) and as mean ± standard error. Statistical differences are indicated with * for p < 0.05 and ** for p < 0.15.

(Figure 3A). In the first day after transfection, 6.0 × 105 ± 2.8 × 104 mCs were found in each nucleus of mC-NSC and 2.0 × 105 ± 9.6 × 104 pDNA inside pDNA-NSC’ nucleus. It is also important to highpoint the fact that only a percentage of DNA molecules initially provided to the cells were able to reach the

nucleus. According to our results, considering the initial amount of DNA molecules that were added to cells, 64% of mC were able to cross the nuclear envelope against only 18% of pDNA (Table 1), both corresponding to the highest percentages of entrance of DNA molecules into cells described E

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so far according to our best knowledge. Accordingly, 3.5-fold more molecules of mCs were found inside each nucleus when compared to the amount of pDNA. Recent reports determined that, depending on the used method and vector, only 1 to 10% of the delivered plasmids can be detected in the nuclei.44,51,52 For example, in B16F10 mouse carcinoma cells and A549 human lung carcinoma cells, transfected with lipoplexes or polyplexes, only 1−5% of the input DNA was detected in the

Table 1. Percentage of Vector DNA Inside the Nucleus of NSC Transfected with the Same Initial Number of Molecules/Cell of mC and pDNA (1 × 106)a initial dose

mass (μg)

molecules/cell

% DNA inside cells*

minicircle plasmid

0.5 0.8

1 × 106 1 × 106

63.67 ± 2.76 18.32 ± 9.22

a

Results were obtained from four independent experiments (n = 4) and as mean ± standard error with * for p < 0.05.

Figure 4. Cell division kinetics of transfected neural stem cells with 1 × 106 molecules of DNA vectors/cells: minicircle (mC-NSC), plasmid DNA (pDNA-NSC) and without microporation (NSC). Cells were labeled with PKH67 and data was acquired by flow cytometry at days 1, 3, 4, 7, and 10. (A) Dot-Plots of flow cytometry analysis with GFP+ cells labeled with PKH26 dye discriminated using FL1/FL2 gates. Dot-plots of day 1 include details about cell profile evolution with their content on PKH67 dye and GFP intensity (arrows). (B) Each bar represents the percentage of cells in each doubling generation. Nonmicroporated cells (gray bar; NSC), cells transfected with mC (black bar; mc-NSC), and cells transfected with pDNA (white bar; pDNA-NSC) are presented. Results were obtained from two independent assays with duplicates and as mean ± standard error. F

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Figure 5. Astrocyte and neuronal differentiation of neural stem cells after microporation with 1.0 × 106 molecules of DNA vectors/cell: minicircle (mC-NSC), plasmid DNA (pDNA-NSC), and without microporation (NSC). Prestaining images: NSC expressing GFP (green) and respective bright field image. Poststaining images: astrocyte differentiation was assessed by GFAP staining (red) and neuronal differentiation was confirmed through Tuj1 staining (red). Nuclei were labeled with DAPI (blue).

nuclei.44 The higher percentage of DNA molecules obtained by us when compared to the values reported by others might be explained by three possible factors: (i) smaller DNA molecules, (ii) more efficient delivery method, and (iii) probably higher purification levels of the DNA molecules. Unfortunately not all the authors refer to the quality or purity of the used DNA vectors and so direct comparison is impossible. However, we may highlight the fact that the DNA vectors used in our work had an amount of endotoxins (LPS) lower than 5 E.U./mg pDNA, a percentage of bacterial chromosomal DNA lower than 0.07% and supercoiled DNA above 99.5%, that comply with specifications required for plasmid purity to be used on genetherapy clinical trials.53,54 The purity of these molecules may

drastically influence the transfection efficiency as previously reported elsewhere55 and according to our own results (data not shown). It has been suggested that upon transfection there is a sizedependent correlation with the mobility of the vector, indicating that, due to the presence of organelles that impose a high molecular crowding inside the cytoplasm, the mobility of larger plasmids decreases making them more susceptible to degradation within the cell by the action of host nucleases.19,20 In agreement with these findings, other authors verified that regardless of similar levels of plasmid uptake, nuclear delivery of such molecules was strictly compromised for large DNA constructs suggesting that small plasmid molecules evade G

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degradation by rapid transit through the cytoplasm.56 Therefore, not only the lack of bacterial backbone and low CpG content, but also the smaller size of mCs molecules might contribute to their improved performance, and in fact, recent studies demonstrated that reduced sized vectors contribute to enhanced gene expression level.13 As predicted, a higher number of DNA molecules inside cells resulted in higher levels of protein expression when using mCs when compared to their pDNA counterparts (Figure 3B). Additionally, the level of expression of the reporter protein, given by the mean fluorescent intensity, showed a 3-fold decrease in the first 4 days (Figure 3B), independent of the DNA vector, and after 7 days the decrease was more obvious when using plasmids: a 31-fold decrease in NSC-pDNA was detected against 26 in mC-NSC in relation to the first day after transfection. This decrease might be associated with the decrease in the number of DNA vectors inside cells due to cell division or by silencing of the transgene. Considering the first hypothesis, no considerable differences in cell division kinetics were observed between cells harboring mC or pDNA (Figure 4). On the other hand, it was previously reported that episomal pDNA is prone to gene silencing, for example, in liver cells, due to the presence of bacterial DNA.57 These authors suggested that, within the plasmid backbone, repressive heterochromatin is formed that then spreads and inactivates the gene of interest, inducing a decrease in transgene expression.57 Additionally, several reports provided evidence that unmethylated CpG motifs in episomal plasmid DNA may play a role in transcriptional silencing of a transgene.29,36,58−60 As mentioned above, while the level of expression decreases in the first 4 days (Figure 3B), the number of cells expressing the transgene was maintained (Figure 2A). This might suggest that initially the number of DNA vectors inside the nucleus was above the saturation threshold for transcription, as previously described by others.44 Four days after microporation, a similar fold decrease (∼30) in the number of DNA molecules/cell was quantified. Nevertheless, seven days after transfection, a severe (∼300fold) decrease in plasmid DNA copy number was observed, whereas an ∼100-fold decrease in minicircles molecules was observed with mC-NSC. After establishing that different amounts of DNA molecules are found inside the cells although the same initial number of molecules was provided, we were interested in understanding whether the cell division kinetics might be affected due to the transfection with both DNA vector types. To analyze the effect of each vector on NSC proliferation rate, 2.0 × 105 cells were labeled with PKH67 (red) and then transfected with 2.0 × 1011 molecules of each vector and cultured for 10 days. Nonmicroporated cells (NSC) and transfected cells (mC-NSC and pDNA-NSC) were then compared in terms of their cell division kinetics by flow cytometry. In Figure 4A are shown dot-plots obtained with NSC labeled with the red dye (FL2, upper quadrant) and expressing GFP (FL1, left quadrant) during 10 days. Upon cell division, the membrane dye is diluted and a decrease in red fluorescence (FL2) is observed (Figure 4A). When cells are also expressing GFP, the fluorescence decrease profile (FL1) is similar between mC-NSC and pDNA-NSC, and after 10 days in culture, fluorescence intensity (both red and green) of these cell populations resembles NSC without microporation, which suggests similar division kinetics. Using a specific software, we were able to associate the decrease in red fluorescence with cells’ number of generations, and the obtained results are

shown in Figure 4B. Interestingly, nontransfected and transfected NSC showed a similar division profile, independent of the used vector. On day 3, all the cells had undergone cell division because no parent cells were detected. On day 4, most of the cells were at generation 4, and by day 7, regardless of the vector used, transfected cells were distributed around generations 3 and 8 in rates similar to the ones found in the controls. In our previous work, using other stem cell types and a different DNA vector, we observed a slowing down in division kinetics of cells harboring plasmid DNA,18 but according to the results presented herein, it seems that in NSC the proliferation kinetics is not influenced upon transfection with these mC or pDNA and by the maintenance of those inside cells’ nucleus. The potential to differentiate into astrocytes or neurons is an essential hallmark of NSC.2 Therefore, we evaluated the influence of these two DNA vectors on the multilineage differentiation of NSC. With that aim, cells were cultured in media supplemented with specific growth factors and after 12 days it was possible to detect, by immunocytochemistry, the presence of astrocytes and neurons, by using GFAP and Tuj1 antibodies, respectively (Figure 5). Nonmicroporated cells and mC-NSC or pDNA-NSC all retained the potential to differentiate and no apparent differences were detected in terms of differentiation efficiency. Moreover, GFP expression was maintained during differentiation. Accordingly, our results provide evidence that microporation and the presence of DNA vectors, mC, or pDNA do not seem to affect the differentiation capability of NSC.

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CONCLUSIONS In this work, the influence of minicircles on transgene expression and viability of NSC was evaluated. The results presented herein demonstrated for the first time that these biomolecules are optimal for sustaining higher transgene expression and NSC survival when compared to their plasmid counterpart. Moreover, no significant differences were observed in cell divison kinetics or differentiative potential between transfected and nontransfected NSC, suggesting that these biomolecules might be considered in stem-cell based therapies when the transient expression of a transgene is enough to stimulate the regeneration of brain tissues.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors gratefully acknowledge Professor Domingos Henrique, Institute of Molecular Medicine, Lisboa, Portugal, for providing the CGR8-NS cell line. M.M.D. and C.M. acknowledge Compromisso para a Ciência 2007 and 2008, respectively. This work was supported by MIT-Portugal Ph.D. program. C.A.V.R. is a recipient of a FCT fellowship (SFRH/ BPD/82056/2011).

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REFERENCES

(1) Conti, L.; Cattaneo, E. Nat. Rev. Neurosci. 2010, 11, 176−187. (2) Conti, L.; Pollard, S. M.; Gorba, T.; Reitano, E.; Toselli, M.; Biella, G.; Sun, Y. R.; Sanzone, S.; Ying, Q. L.; Cattaneo, E.; Smith, A. Plos Biol. 2005, 3, 1594−1606. (3) Taupin, P. Curr. Alzheimer Res. 2009, 6, 461−470.

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dx.doi.org/10.1021/bm400015b | Biomacromolecules XXXX, XXX, XXX−XXX