Brief Article pubs.acs.org/molecularpharmaceutics
Dendrimer-Driven Neurotrophin Expression Differs in Temporal Patterns between Rodent and Human Stem Cells Antos Shakhbazau,*,†,‡ Dzmitry Shcharbin,§ Ihar Seviaryn,∥ Natalya Goncharova,∥ Svetlana Kosmacheva,∥ Mihail Potapnev,∥ Maria Bryszewska,⊥ Ranjan Kumar,†,‡,# Jeffrey Biernaskie,‡,# and Rajiv Midha†,‡ †
Department of Clinical Neurosciences, Faculty of Medicine, University of Calgary, Calgary, Canada Hotchkiss Brain Institute, University of Calgary, Calgary, Canada § Institute of Biophysics and Cell Engineering, National Academy of Sciences of Belarus, Minsk, Belarus ∥ Republic Centre for Hematology and Transfusiology, Minsk, Belarus ⊥ Department of General Biophysics, University of Lodz, Lodz, Poland # Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada ‡
S Supporting Information *
ABSTRACT: This study reports the use of a nonviral expression system based on polyamidoamine dendrimers for time-restricted neurotrophin overproduction in mesenchymal stem cells and skin precursor-derived Schwann cells. The dendrimers were used to deliver plasmids for brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3) expression in both rodent and human stem cells, and the timelines of expression were studied. We have found that, despite the fact that transfection efficiencies and protein expression levels were comparable, dendrimer-driven expression in human mesenchymal stem cells was characterized by a more rapid decline compared to rodent cells. Transient expression systems can be beneficial for some neurotrophins, which were earlier reported to cause unwanted side effects in virus-based long-term expression models. Nonviral neurotrophin expression is a biologically safe and accessible alternative to increase the therapeutic potential of autologous adult stem cells and stem cellderived functional differentiated cells. KEYWORDS: BDNF, NT-3, transfection, PAMAM dendrimers, mesenchymal stem cells, skin precursor-derived Schwann cells
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effectiveness of cell therapies in some models of central7−9 (CNS) and peripheral nervous system (PNS)10 repair. NTFs comprise several families of proteins essential for the survival, maintenance, and regeneration of neurons.11−15 The engineered expression of NTFs facilitates trophic support and induces axonal outgrowth, resulting in neuroprotection and improved regeneration.10,16,17 Genetic modification eliminates the need for continuous exogenous NTF application, thus reducing the risk of additional trauma or infection. However, it was found that long-term virus-based NTF expression caused significant negative side effects in both CNS and PNS regeneration models,18−23 such as aberrant sprouting and trapping of regenerating axons. The undesired effects of longterm expression together with biosafety considerations appeal to the use of time-restricted expression systems, which can be provided by nonviral gene carries. The latter are represented by
INTRODUCTION Adult autologous stem cells from different sources are currently viewed as a potent tool in the emerging field of cell therapies. The expected high therapeutic potential of stem cells called for the development of numerous technologies for stem cell culture, differentiation, matrix embedding, and genetic modification.1−3 Most of the stem cell-related techniques and protocols are first optimized in rodent models, with a perspective to translate the results to the respective human stem cells. However, a number of rodent-vs-human expression studies revealed species-specific differences with critical impact onto the pharmaceutical trials.4−6 Understanding the differences between rodent and human stem cells is required to predict the success of the above-mentioned translation. One of the technologies with a considerable promise to improve cell therapies is genetic engineering. In addition to their own repertoire of secreted molecules, stem cells can be modified for elevated expression and supply of trophic, differentiation, antitumor, immunomodulatory, or other factors. In particular, ex vivo modification for the overexpression of neurotrophic factors (NTFs) was shown to increase the © 2012 American Chemical Society
Received: Revised: Accepted: Published: 1521
January 22, 2012 April 2, 2012 April 5, 2012 April 5, 2012 dx.doi.org/10.1021/mp300041k | Mol. Pharmaceutics 2012, 9, 1521−1528
Molecular Pharmaceutics
Brief Article
charged DNA, provided by multiple interactions between surface amines and nucleic acid phosphates. Earlier we have tested a variety of dendrimers for transfection efficiency and cytotoxicity, with PAMAM-NH2 G4 found to be most effective.31 Our aim in this study was to explore the feasibility and time patterns of dendrimer-driven expression of BDNF and NT-3 neurotrophins in rodent and human adult bone marrow-derived mesenchymal stem cells (MSC). We have also examined for the first time the potential of PAMAM dendrimers as gene carriers into skin precursor-derived differentiated Schwann cells (SKP-SC), being another perspective tool in CNS and PNS regeneration strategies.32
a variety of polycations, lipophilic compounds, magnetic particles, dendrimers, and so forth.24−28 Polyamidoamine (PAMAM) dendrimers used in this study are macromolecules with a highly branched three-dimensional structure, based on an ethylenediamine core and branched units built from methyl acrylate and ethylenediamine (Figure 1A).
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EXPERIMENTAL SECTION Vector Construction and Preparation. Expression cassettes for NTFs BDNF and NT-3 were delivered into the target cells using the previously described31,33 expression system on the base of pAAV-IRES-hrGFP plasmid (Stratagene), which also served as a control vector in transfection optimization experiments (referred to as pGFP). This plasmid provides an effective bicistronic expression cassette with a reliable reporter genehuman-recombinant green fluorescent protein (hrGFP), located downstream of the internal ribosome entry site (IRES). Human bdnf or ntf 3 sequences (791 and 821 bp, respectively) were cloned into pGFP multiple cloning site downstream of CMV promoter at the restriction sites ClaI and EcoRI by routine molecular cloning procedures. The endonuclease recognition sequences were added to the 5′ termini of amplification primers: 5′-tgaattcatcgatgccaccatgaccatccttttccttac-3′ and 5′-tgaattcctatcttccccttttaatgg-3′ for bdnf and 5′-tgaattcatcgatgccaccatgtccatcttgttttatgt-3′ and 5′-tgaattctcatgttcttccgatttttc-3′ for ntf 3. The vectors generated (referred to as plasmids pBDNF and pNT3, respectively) were confirmed by restriction digest mapping and sequence analysis. All plasmids were propagated in E. coli strain DH5α and isolated by Plasmid Maxi kits (Qiagen) according to the manufacturer's instructions. Purified plasmid DNA with an A260/A280 ratio of 1.8 was used for transfection. Ethidium Bromide Intercalation Assay. For the biophysical characterization of plasmid/dendrimer complexation, ethidium bromide (EB) was added to the plasmid solution (1 dye molecule per 1 bp), and its fluorescence was monitored with a JASCO-FP 6300 spectrofluorimeter (JASCO GmbH, Germany) at the excitation wavelength of 477 nm. The emission spectra were recorded between 500 and 800 nm, and the emission maxima were determined. Then “dye/plasmid” complex was titrated with a dendrimer, and the changes in fluorescence parameters (intensity and λem max) were recorded. The data graphs were modified so that the changes in fluorescence intensity of the EB-plasmid complex when dendrimers were added were presented as
Figure 1. A, PAMAM-NH2 dendrimer of the fourth generation; B, schematic of the pBDNF expression cassette; C, schematic of the pNT3 expression cassette; D, the changes of EB fluorescence complexed with pGFP, pBDNF, and pNT3 plasmids upon the addition of PAMAM G4 dendrimers at different charge ratios (for visualization, the intensities of pure EB and EB-plasmid complex were presented at point X = 0.01 instead of point X = 0).
F rev =
F complex − F pureEB F0complex − F pureEB
(1)
where F is the fluorescence of EB-plasmid in the presence of dendrimer, FpureEB is the fluorescence of pure (free) EB, and F0complex is the fluorescence of the EB-plasmid complex in the absence of dendrimer when EB is fully bound by the plasmid. Cell Culture. Mesenchymal stem cells (MSC) were isolated from rat (rMSC) or human (hMSC) bone marrow and grown in DMEM-Glutamax (Gibco) or α-MEM with 10% heatinactivated FBS (HyClone), 100 U/mL penicillin, and 100 mg/ complex
Such a structure provides the dendrimers with a high degree of surface functionality, versatility, and multivalency, which makes these nanoparticles a unique tool in biomedical applications such as gene, oligonucleotide, and drug delivery, as well as cell culture on functionalized surfaces.29,30 In particular, the application of the dendrimers as gene delivery vectors is based on their ability to form complexes with negatively 1522
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using the 85L3R-1KT alkaline phosphatase staining kit (SigmaAldrich). Transfection Experiments. Transfections (n = 3 for each cell type) were carried out as described earlier31,33 with the use of fourth generation (G4) polyamidoamine dendrimers PAMAM-NH2. Dendrimers were diluted to a concentration of 20 mM in terms of nitrogen residues, to allow uniform conditions for comparative analysis of their DNA condensing and gene-delivering properties. The cell density was 5 × 104 for each cell type, and the final plasmid concentration was 2 μg/ mL. For rMSC, hMSC, and SKP-SC transfections, complexes of plasmid DNA and the dendrimers at a charge ratio of 1:1 to 1:1.2 were prepared in 50 μL of 150 mM NaCl solution, and the mixtures were vortexed and incubated for 15 min at room temperature. Transfections were carried out in 24-well or 6-well plates in DMEM or SC culture medium, respectively, at ∼65− 70% confluence by 3 h or overnight exposure to PAMAM− plasmid complexes. Dendrimer or plasmid alone were used in control experiments. hrGFP fluorescence was monitored by microscopy, and the percentage of GFP-positive cells was determined using a manual counting or FACScan analytical flow cytometer (Becton Dickinson) after fixation with 2% paraformaldehyde. MTT Assay. Target cells were seeded in 24 well plates allowed to grow to 60−70% confluence before transfection and subjected to the exposure of dendrimer alone or dendrimer− plasmid complex, as described above. Then, 24 h later, 200 μL of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium-bromide) substrate solution (5 mg/mL) was added to the cells to measure mitochondrial activity. After 2 h, the supernatant was removed, and the formed formazan crystals were dissolved in 650 μL of DMSO and transferred to 96-well plate for the readout. The absorbance was measured at 570 nm with a reference wavelength of 690 nm. All points were performed in triplicate, and the results were calculated as a percentage related to control cells. Neurotrophin Release. The release of BDNF or NT-3 proteins into the culture medium was assessed in vitro using an enzyme-linked immunosorbent assay (ELISA). Supernatant samples were collected at three time points: 1 day, 1 week, and 2 weeks post-transfection, and analyzed using BDNF and NT-3 Emax ImmunoAssay Systems (Promega, USA) according to the manufacturer's instructions. Three replicate samples from each well were pooled for statistical analysis. Statistics. Results are presented as mean ± SD (standard deviation). Data were analyzed by one-way analysis of variance (ANOVA) with the posthoc Newman-Keuls test.
mL streptomycin. The cells were routinely maintained on plastic tissue culture flasks and plates (Falcon) at 37 °C in a humidified atmosphere containing 5% CO2/95% air. Rat bone marrow mesenchymal stem cells were isolated as described earlier.34,35 Adult human bone marrow was harvested from routine surgical procedures (femoral osteotomies), diluted 10fold in phosphate-buffered saline (PBS), and separated by centrifugation on a Ficoll-Paque layer. After centrifugation at 3000 g for 30 min, the mononuclear cell layer was recovered from the gradient interface and washed with PBS. The cells were then centrifuged at 1500 g for 30 min and resuspended in complete culture medium. The hMSC phenotype was confirmed by flow cytometry with a FACScan analytical flow cytometer (Becton Dickinson), using CD90 and CD105 (positive) and CD34 and CD45 (negative) as markers. All experiments with human cells were approved by Research Ethical Board of the Republic Centre for Hematology and Transfusiology. Rat skin precursor cells (SKPs) were obtained from dermis of postnatal day 2 Lewis rats and cultured according to published protocols.3,32,36 Briefly, skin from the dorsal torso was minced in HBSS (Gibco) on ice, incubated for 45 min in 0.1% collagenase XI at 37 °C, dissociated mechanically, washed in cold DMEM, and passed through a 40 μm cell strainer. Filtrate was centrifuged at 1200 rpm, and the pellet was triturated and resuspended in culture media (DMEM/F12 3:1, 100 U/mL penicillin and 100 mg/mL streptomycin, 0,25 μg/ mL Fungizon, 1× B27 supplement, 20 ng/mL EGF, and 40 ng/ mL bFGF (all from Gibco). SKPs were cultured and passaged as undifferentiated spheres at 37 °C in a humidified atmosphere containing 5% CO2/95% air. The progenitor state of the spheres was confirmed with nestin immunolabeling. To induce differentiation toward Schwann cells, spheres were triturated and replated on poly-D-lysine/laminin coated culture dishes (Corning) in DMEM/F12 media supplemented with 4 μM forskolin, 10 ng/mL heregulin-1β, and 1% N2 supplement (Gibco). Following 1−2 weeks incubation, cells appearing under phase contrast to have bipolar SC morphology were isolated with cloning cylinders, and their fate specification was confirmed with GFAP and p75-NTR antibody staining. The culture was further enriched for p75-NTR-positive cells by means of flow cytometry-assisted sorting using flow cytometer FACSAria II (Beckton Dickinson, USA) and expanded in the differentiation medium until ∼95% purity was achieved. These cells are subsequently referred to as SKP-derived Schwann cells, or SKP-SCs. Induction of Osteogenic and Chondrogenic Differentiation of MSC. Cultured MSC of P2-3 were seeded at a density of 8000 cells/cm2 and maintained in growth medium until they reached 70% confluence. The medium was then replaced by differentiation medium containing α-MEM, 2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin, and additionally either 10% FBS, 0,1 μM dexamethasone, 50 μM L-ascorbic acid, and 10 mM β-glycerophosphate (for osteogenic differentiation, 3 weeks) or 2% FBS, 0.1 μM dexamethasone, 60 μM L-ascorbic acid, 10 ng/mL TGF-β3, and 1× ITS (insulin, transferrin, selenous acid) premix (for chondrogenic differentiation, 5 weeks). Media were changed every 4 days. After 3−5 weeks, differentiated cells were visualized by staining with either hematoxylin/eosin (chondrogenic differentiation) or Alizarin Red S (osteogenic differentiation). Differentiation of hMSC into osteoblasts was also confirmed histochemically by alkaline phosphatase activity
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RESULTS AND DISCUSSION Three cell types, including adult stem cells and stem cellderived functional differentiated cells, were used in this study. All of the cell populations were isolated or differentiated according to the established protocols, and their morphology, differentiation potential, and antigenic profile were in good agreement with previously published results.32,37 Primary bone marrow culture from rat and human femurs contained a heterogeneous population of cells, where colony-forming MSC were found at days 3−8. In cultures of P2, which were used for the experiments, more than 90% of the cells showed a MSClike morphology. Consistent with previous reports,37,38 flow cytometry indicated that more than 93% of hMSCs expressed CD90 and CD105, but not CD34 and CD45. MSCs were shown to differentiate into mesodermal cell types (Supporting 1523
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indicates the effective formation of dendriplexes with the base vector pGFP and its derivatives carrying neurotrophin expression cassettes (pBDNF and pNT3) at the charge ratio ∼1:(1−2). The data obtained on DNA/dendrimer charge ratios in dendriplex formation was used for further transfection optimization. To deliver DNA/dendrimer nanocomplexes into SKP-SC, rMSC, and hMSCs, we used a previously described transfection approach.33 Incubation with dendrimer/plasmid complexes allowed transgene expression in a subsets of each type of transfected cells (Figure 2A−C), according to the expression of
Information, Figure 1A,B). Dermal cells from rat skin formed characteristic nestin-positive spheres3,32 after 1 week of culture in medium containing EGF and bFGF. Then 1−2 weeks following transfer into DMEM/F12 media supplemented by forskolin and heregulin 1β, dissociated SKP spheres differentiated into small colonies of bipolar cells with the morphological characteristics of SCs positive for Schwann cell markers p75 and GFAP. Previous studies have shown that supplementation of stem cell therapies with the expression of NTFs, including BDNF and NT-3, markedly increases the therapeutic value of the cell transplantation approach in different models of nerve injuries.39−41 To provide enhanced NTF secretion by our target cells, we constructed plasmid vectors pBDNF and pNT3 (see Figure 1B,C for schematics), allowing NTF expression under a potent CMV promoter with chicken β-globin intron as an enhancer. The expression of humanized recombinant green fluorescent protein (hrGFP) from the same transcript via the IRES-driven second open reading frame serves as a marker for both transfection efficiency and the transcription of neurotrophin-encoding gene (bdnf or ntf 3) from bicistronic cassette. In the present work, we aimed for genetic modification of SKP-SC, rMSC, and hMSC using nonviral vehicles. Viral systems, having a number of their own advantages, still have such drawbacks as immunogenicity, toxicity, risk of genome destabilization, and oncogene activation due to transgene integration and the potential of virus reversion to the wild type.42−44 In the search for virus-free alternatives for stem cell genetic modification, a number of vehicles with unique transfection properties have been proposed and tested.25,45,46 Of these, cationic polymers are being researched extensively as a tool for gene delivery25,26 and considered superior to lipidbased lipoplexes in terms of toxicity, reproducibility, and stability.26,47 Polyamidoamine (PAMAM) dendrimers are treelike molecules branching from the central core, with a multivalent surface represented by amine termini which make it cationic at the physiological pH. The molecular structure of dendrimers is characterized by a large proportion of the active groups exposed at the surface, conferring these nanoparticles with very high molecular surface/volume ratios. The fourth generation of PAMAM-NH2 dendrimers used in our study possesses 64 surface amino groups, which self-assemble electrostatically with plasmid DNA, forming nanometer-scale complexes. The molecular weight of PAMAM-NH2 G4 dendrimers is 14.2 Da, with corresponding molecular diameters of 4 nm.48 PAMAM dendrimers have been shown to effectively bind, compact, and deliver plasmid DNA into a high variety of targets, including stem cells.2,49,50 To assess the formation of plasmid DNA/dendrimer nanocomplexes, we used a previously described biophysical approachethidium bromide (EB) intercalation assay.31 EB is known to intercalate into double-stranded DNA or RNA, occupying an effective binding site of several base pairs, which leads to a significant increase of its fluorescence intensity and to em a blue-shift of its maximum emission wavelength λmax . Compounds with higher affinity for DNA, including dendrimers, displace the dye, quench its fluorescence, and induce a red-shift of its λem max. Changes in fluorescence emission intensity of EB complexed with pGFP, pBDNF, or pNT3 plasmids after addition of PAMAM G4 dendrimers at different charge ratios can be seen in Figure 1D. The addition of the dendrimers in charge ratios varying from 0.1 to 2 led to a decrease of fluorescence intensity to the level of pure EB. This
Figure 2. Dendrimer-transfected cells, magnification 100×: A, rMSC; B, hMSC; C and D, rSKP-SC labeled with Schwann cell marker p75 and Hoechst nuclear stain. Arrow indicates representative GFPexpressing SKP-SC cell positive for p75.
hrGFP as a reporter gene. Transfection efficiencies varied within 1−4% in SKP-SC and hMSC, being somewhat lower in rMSC (usually
