Skip to main content
NCBI home page
Human Gene Therapy Methods logoLink to Human Gene Therapy Methods
. 2013 Jan 29;24(1):38–48. doi: 10.1089/hgtb.2012.185

Direct Head-To-Head Comparison of Cationic Liposome-Mediated Gene Delivery to Mesenchymal Stem/Stromal Cells of Different Human Sources: A Comprehensive Study

Joana S Boura 1, Francisco dos Santos 1, Jeffrey M Gimble 2, Carla MP Cardoso 3, Catarina Madeira 1, Joaquim MS Cabral 1, Cláudia Lobato da Silva 1,✉
PMCID: PMC4015075  PMID: 23360350

Abstract

Nonviral gene delivery to human mesenchymal stem/stromal cells (MSC) can be considered a very promising strategy to improve their intrinsic features, amplifying the therapeutic potential of these cells for clinical applications. In this work, we performed a comprehensive comparison of liposome-mediated gene transfer efficiencies to MSC derived from different human sources—bone marrow (BM MSC), adipose tissue-derived cells (ASC), and umbilical cord matrix (UCM MSC). The results obtained using a green fluorescent protein (GFP)-encoding plasmid indicated that MSC isolated from BM and UCM are more amenable to genetic modification when compared to ASC as they exhibited superior levels of viable, GFP+ cells 48 hr post-transfection, 58±7.1% and 54±3.8%, respectively, versus 33±4.7%. For all cell sources, high cell recoveries (≈50%) and viabilities (>85%) were achieved, and the transgene expression was maintained for 10 days. Levels of plasmid DNA uptake, as well as kinetics of transgene expression and cellular division, were also determined. Importantly, modified cells were found to retain their characteristic immunophenotypic profile and multilineage differentiation capacity. By using the lipofection protocol optimized herein, we were able to maximize transfection efficiencies to human MSC (maximum of 74% total GFP+ cells) and show that lipofection is a promising transfection strategy for MSC genetic modification, especially when a transient expression of a therapeutic gene is required. Importantly, we also clearly demonstrated that intrinsic features of MSC from different sources should be taken into consideration when developing and optimizing strategies for MSC engineering with a therapeutic gene.

Boura and colleagues compare liposome-mediated gene transfer efficiencies to human mesenchymal stem cells (MSC) derived from bone marrow (BM MSC), adipose tissue (ASC), or umbilical cord matrix (UCM MSC). MSC isolated from BM and UCM were more amenable to genetic modification when compared to ASC. Modified cells from all sources maintained transgene expression for 10 days and retained their characteristic immunophenotypic profile and multilineage differentiation capacity.

Introduction

In the past few years, mesenchymal stem/stromal cells (MSC) have received substantial attention by both scientific and medical communities as cell sources for regenerative medicine and cellular therapy (Ankrum and Karp, 2010). These cells are multipotent stem cells that can be readily isolated from a wide range of adult and neonatal tissues, including bone marrow (BM), adipose tissue (AT), umbilical cord matrix (UCM), and amniotic fluid (Lu et al., 2006). Though MSC isolated from BM are the most extensively studied, BM harvesting is an invasive procedure that can cause complications for the donor. Therefore, other sources with greater sample accessibility, such as UCM (Can and Karahuseyinoglu, 2007) and AT (Gimble et al., 2007), have been successfully explored as viable alternatives to BM collection for the isolation of stem cells (both umbilical cords and lipoaspirates are typically discarded by medical facilities). Similarly to BM, isolation rates of 100% have been reported in the literature for human MSC obtained from both AT (Wagner et al., 2005; Kern et al., 2006) and UCM (De Bruyn et al., 2011; Zeddou et al., 2010).

MSC present several remarkable advantageous features in the therapeutic context, namely their extensive proliferative capacity in vitro, multilineage differentiation potential into different mesodermal cell types (e.g., osteocytes, chondrocytes, adipocytes), and immunomodulatory properties (Nauta and Fibbe, 2007; Ben-Ami et al., 2011). Interestingly, after systemic administration, they are also able to migrate toward sites of tissue/organ damage, setting a local regenerative microenvironment and down-regulating inflammation through trophic activity (Stappenbeck and Miyoshi, 2009; Caplan and Correa, 2011). In the past few years, MSC have been used in clinical settings as cellular vehicles to treat or improve several medical conditions, including acute graft-versus-host disease (GvHD) and myocardial infarction (Ankrum and Karp, 2010; Caplan and Correa, 2011). Nevertheless, the medical range and therapeutic efficacy of MSC can be potentially amplified by genetically engineering these cells to overexpress therapeutic proteins, either inside or outside of their natural production spectrum (Porada and Almeida-Porada, 2010).

Despite being a very appealing and promising field, one major barrier to the success of the genetic engineering of MSC is still the lack of a safe and efficient method of gene delivery (Reiser et al., 2005; Santos et al., 2011; Hu et al., 2012). Viral-based vectors, namely retrovirus and adenovirus, are currently the most commonly used system to deliver genes in clinical trials (Edelstein et al., 2007; Park et al., 2012), as they offer a robust and sustained high gene delivery efficiency and expression. However, viral systems suffer from a number of significant limitations including: potential to induce mutagenesis and carcinogenesis and possible immunogenic reaction due to the viral infection or the presence of viral proteins (Glover et al., 2005). Consequently, nonviral vectors have generated a great deal of interest as alternatives that may circumvent the problems associated with viral vectors. In particular, nonviral vectors raise less safety concerns, are easier to produce and manufacture in large-scale, and can carry large therapeutic genes, thus being a more attractive option from a clinical point of view (Niidome and Huang, 2002). However, despite recent advances, these systems still show low to moderate gene transfer efficiency when compared with viral transduction (Xiang et al., 2012).

Among the nonviral methods available, Lipofectamine™, a cationic lipid-based reagent, has provided successful transfection to a wide range of mammalian cell lines using a simple protocol (Schenborn et al., 2000). The positively charged lipids interact electrostatically with nucleic acids (negatively charged), forming complexes that facilitate the internalization of the exogenous genetic material by the cell through endocytosis (Wasungu and Hoekstra, 2006). Though the mechanism of gene delivery inside the cells is not completely understood, it is accepted that this method is dependent on both cell division and endocytosis rate (Gresch et al., 2004). Regarding human MSC, transfection using the lipofection method has led to moderate levels of transgene-positive cells (30–40%) (Hoare et al., 2010). Higher dosage of cationic agents generally increased transfection efficacy but were also associated with low cell survival and toxicity (McMahon et al., 2006; Clements et al., 2007; Farrell et al., 2007; Gheisari et al., 2008). Recently, our group reported promising results using this reagent for gene delivery to BM MSC (Madeira et al., 2010).

In the present study, we performed a head-to-head, direct comparison of the gene transfer efficiencies to MSC derived from different human sources—BM MSC, ASC, and UCM MSC. To the best of our knowledge, this represents the first systematic study of the gene delivery performance of a nonviral vector to MSC of different human sources, which are relevant for cell/gene therapy settings. By plating cells at a lower initial cell density compared to previous studies in literature (Gheisari et al., 2008; Hoare et al., 2010; Madeira et al., 2010), a key step for the successful long-term cultivation of human MSC (Sekiya et al., 2002), lipofection efficiencies were maximized. Cell survival, cellular division kinetics, differentiation, and clonogenic potential, as well as the immunophenotype of the engineered human MSC, were also studied.

Materials and Methods

Human mesenchymal stem/stromal cell isolation and culture

All human samples were obtained from healthy donors after informed donor (BM and AT) or maternal (UCM) consent under protocols approved by the ethical review board at the respective institutions. BM MSC were obtained from BM aspirates (average age of 36±11) collected at Instituto Português de Oncologia Francisco Gentil, Lisboa, Portugal, according to the protocol previously described by dos Santos and colleagues (dos Santos et al., 2010).

ASC were isolated from adult human subcutaneous adipose tissue (average age of 30±7), as previously described at Pennington Biomedical Research Center, Baton Rouge, Louisiana (Gimble and Guilak, 2003). Human UCM MSC were obtained from umbilical cord (UC) units kindly provided by Hospital de Santa Maria, Lisboa, Portugal. Briefly, after washing UC with phosphate buffered saline (PBS) and eliminating the blood clots, arteries and veins were removed. The remaining tissue was then minced, digested with 0.1% collagenase type II (Sigma) for 4 hr at 37°C, and filtered with a 100 μm nylon mesh. After washing the cell suspension with Iscove's modified Dulbecco's media (IMDM; Gibco) supplemented with 1% penicillin-streptomycin-fungizone (Invitrogen), the cell number was determined using the trypan blue (Gibco) exclusion method. Cells were cultured in cell culture flasks precoated with CELLstart™ CTS™ (Invitrogen) using StemPro® MSC serum-free medium (SFM) (Invitrogen) supplemented with 2 mM L-glutamine (Invitrogen) and 1% penicillin-streptomycin-fungizone (Invitrogen), and kept at 37°C and 5% CO2 in a humidified atmosphere. Medium was replaced every 3–4 days.

Before transfection, cryopreserved MSC from the three cell sources were thawed and plated, at a cell density of 3000 cells/cm2, on CELLstart™ CTS™ precoated cell culture flasks using StemPro® MSC SFM supplemented with 2 mM L-glutamine and 1% penicillin-streptomycin-fungizone. Cultures were maintained at 37°C and 5% CO2 in a humidified atmosphere, and exhausted medium was changed every 3–4 days. At 70% cell confluence, MSC were detached from the flasks by adding accutase solution (Sigma) for 7 min at 37°C. Cell number and viability were determined using the trypan blue exclusion method. All experiments were performed using cells at passages 3–6.

Plasmid preparation

The enhanced green fluorescent protein (eGFP)-expressing plasmid pVAX-GFP (3697 bp) was constructed as described elsewhere (Azzoni et al., 2007). The production and purification of the plasmid was performed accordingly to Madeira and co-workers (Madeira et al., 2010). The concentration of purified pDNA solutions was assayed by spectrophotometry at 260 nm (Nanodrop, Thermo Scientific), and DNA integrity was confirmed using DNA agarose gel stained with ethidium bromide.

Liposome-mediated transfection

For transfection, cells were plated at two different cell densities, 1000 and 3000 cells/cm2, in 24-well plates and cultured in the commercially available medium StemPro® MSC SFM. After 72 hr of culture, 1 μg of plasmid DNA (pDNA, 3.5 kb size) encoding for GFP was transferred to the cells using 1 μL of Lipofectamine 2000™ (Invitrogen) according to the supplier's instructions. Briefly, plasmid and lipid were diluted in Opti-MEM® I (Gibco) and mixed together, allowing the formation of the DNA/lipid complexes. The transfection mixture was then added to the adherent human MSC and replaced with StemPro® MSC SFM after 5 hr of incubation at 37°C. GFP expression and plasmid internalization were measured at different time points until day 10 after transfection by flow cytometry and real-time polymerase chain reaction (RT-PCR), respectively. Propidium iodide (PI) was used to assess cell viability by flow cytometry. Nontransfected cells were used as a control and a condition with cells treated only with lipofectamine was also performed. The percentage of viable cells was estimated using the trypan blue dye exclusion method. Cell recovery and yield of transfection were determined using equations previously described (Madeira et al., 2011).

Flow cytometric analyses

Cells were harvested using Accutase, washed in PBS, counted, and stained for 15 min with PI (Becton Dickinson) for live/dead cell discrimination. The percentage of dead and viable GFP-expressing cells was determined by flow cytometry (FACSCalibur, Becton Dickinson Biosciences) using the CellQuest software (Becton Dickinson Biosciences). First, an FSC/SSC gate was delineated in order to define a live gate analysis, then a gate in the FL1-FL3 plot was set to exclude all PI-positive and GFP-negative cells. Nonspecific fluorescence was determined using the nontransfected cells. For each sample, 10,000 events were acquired. Transgene expression was monitored on days 1, 2, 5, 7, and 10.

Quantification of plasmid copy number

Plasmid DNA quantification was carried out in a Roche LightCycler detection system using the FastStart DNA Master SYBR Green I kit (Roche Diagnostics) as described previously in the literature (Madeira et al., 2010). Briefly, PCR reaction mixture consisted of 2.0 μL of the 10x SYBR green mixture, 0.4 μL of each primer (0.4 μM final concentration), 1.6 μL of MgCl2 solution (3.0 mM final concentration), 2–7 μL of our sample (corresponding volume to 10,000 MSC), and PCR-grade water to a final volume of 20 μl. Quantification was performed using a thermal cycling program consisting of 10 min at 95°C, followed by 40 cycles of 10 sec at 95°C, 5 seconds at 55°C, and 7 seconds at 72°C. Primers were designed to specifically amplify a 108-bp region from GFP gene (forward primer: 5′ - TCG AGC TGG ACG GCG ACG TAAA-3′; reverse primer: 5′-TGC CGG TGG TGC AGA TGA AC-3′). A calibration curve of known amounts of plasmid was used to calibrate the RT-PCR system (R2=0.999).

Cellular division kinetic studies

Upon cell seeding on the 24-well plates (BD Falcon), MSC were labeled using PKH26 Red Fluorescent Cell Linker Kit (Sigma). Cells were analyzed by flow cytometry 24 hr after plating, immediately before transfection, and on days 1, 2, 5, 7, and 10 after transfection. Since PKH dye binds irreversibly to the lipid layer of cell membrane and is equally distributed between daughter cells upon cell division, it was possible to track cell division by the continuous reduction of fluorescence throughout time in culture (da Silva et al., 2009). GFP+ and GFP− cells were discriminated by defining two different FL1/FL3 gates, whereas the percentage of cells from each generation was determined using the Proliferation Wizard of the ModFit Software (Becton Dickinson Biosciences).

Immunophenotypic analyses

The expression of surface markers was evaluated by staining the cells with the following phycoerythrin (PE)-conjugated monoclonal antibodies: CD31, CD38, CD45, CD73, CD80, CD90, CD105, and HLA-DR (all antibodies were purchased from BioLegend, San Diego, CA). Briefly, cells were incubated for 15 min in the dark with saturating concentrations of each antibody. Stained cells were then washed with PBS and analyzed by flow cytometry using the CellQuest software (Becton Dickinson Biosciences). A total of 10,000 events was acquired for each sample. Appropriate isotype (negative) controls were performed.

In vitro multilineage differentiation assays

To promote osteogenic, adipogenic, and chondrogenic differentiation, cells were cultured for 7 days in StemPro® Osteogenesis, Adipogenesis and Chondrogenesis Differentiation medium (Invitrogen). Osteogenic and adipogenic differentiation were performed as monolayers, whereas for chondrogenic differentiation, micromass cultures were generated. Specific lineage stainings were performed according to the previously described protocols (dos Santos et al., 2011). Briefly, to demonstrate osteogenesis, cultures were stained for alkaline phosphatase (ALP, Sigma) activity and mineralization was assessed using von Kossa staining (1% silver nitrate, Sigma). The adipogenic differentiation was confirmed by the determination of the accumulation of lipid droplets in the vacuoles using the 0.5% Oil Red O staining solution (in 60% isopropanol) (Sigma). Chondrocyte differentiation was assessed with 1% Alcian blue (Sigma) staining to detect sulfated glycosaminoglycans (sGAG).

Clonogenic potential—colony-forming unit fibroblasts assay

Twenty-four hours after transfection, MSC from the three different sources, as well as control cells, were harvested using accutase and reseeded onto T-12.5 cell culture flasks at 10 cells/cm2. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco) with 10% fetal bovine serum (FBS) MSC qualified (Gibco), supplemented with streptomycin (0.025 μg/ml) and penicillin (0.025 U/ml) without subsequent medium change (dos Santos et al., 2010). After 14 days, cells were stained with 0.5% crystal violet (Sigma) in methanol for 30 min at room temperature. The total number of colony-forming unit fibroblasts (CFU-F) per flask was counted under the optical microscope.

Data analysis

All data is presented as mean±standard error of the mean (SEM). Statistical significance was determined by the Mann-Whitney test and set at p<0.05.

Results

Efficiency of liposome-mediated gene transfer to MSC from different human sources

In this study, MSC derived from BM, AT, and UCM were cultured under serum-free conditions (dos Santos et al., 2011) and transfected with Lipofectamine™ 2000 using a previously optimized lipid/plasmid DNA ratio (1 μl/1 μg) (Madeira et al., 2010).

In an effort to minimize surface area limitation to the cultured cells and to ensure that the majority of the cells were actively dividing at the time of transfection, MSC were plated at lower cell densities compared to other transfection studies using human MSC (Gheisari et al., 2008; Hoare et al., 2010; Madeira et al., 2010) and lipofected 72 hr upon seeding. In particular, two initial cell densities within the range of values commonly used for the expansion of human MSC (Le Blanc et al., 2008), 1000 cells/cm2 and 3000 cells/cm2, were tested. With this approach, we anticipated that a higher number of pDNA copies should enter into the cell nucleus, maximizing the transfection efficacy. Gene delivery was evaluated by measuring the expression of the eGFP reporter gene encoded by the plasmid used, whereas cell survival was monitored by staining with PI (both by flow cytometry). Nontransfected cells were used as negative control, and cells treated with lipofectamine only were also included in the analysis.

BM and UCM-derived MSC exhibited superior levels of GFP+ cells (Fig. 1) reaching values as high as 70% within the first 48 hr after transfection; in contrast, ASC presented a significantly lower percentage of transfected cells (51±7.9%; p<0.05). Remarkably, when excluding the PI+ population, which correspond to nonviable cells, the levels of transgene expression remained high, with a maximal percentage of 58±7.1% for UCM MSC plated at 3000 cells/cm2, and 50±2.8% and 33±4.7%, for BM MSC and ASC plated at 1000 cells/cm2, respectively. After gene delivery, a time-dependent transient gene expression was maintained for 10 days (minimal levels of >4%). Interestingly, the major decrease (p<0.05) on transgene expression only occurred after 7 days in culture for the three cell sources studied. No significant differences were detected on transfection efficiencies of cells plated at 1000 cells/cm2 (Fig. 1a) and 3000 cells/cm2 (Fig. 1b), and cell viabilities were always maintained above 85% for all conditions tested.

FIG. 1.

FIG. 1.

Open in a new tab

Transgene delivery into mesenchymal stem cells (MSC) derived from BM, AT, and UCM. Quantification and long-term monitoring of the levels of total GFP+ (gray squares) and GFP+PI− (black bars) (percentage) were performed upon lipofection for each cell source. Comparison of transfection for cells plated at 1000 cells/cm2 (a) and 3000 cells/cm2 (b). Each bar and point represent the mean±standard error of mean (SEM), n=4, *p<0.05. BM, bone marrow; AT, adipose tissue; UCM, umbilical cord matrix; GFP, green fluorescent, PI, propidium iodide.

Additionally, MSC lipofection efficacy was further evaluated by calculating both cell recoveries and yields of transfection 24 hr after gene delivery (Fig. 2). Cell recovery is given by the ratio of the collected population of liposome-modified MSC and the population of nontransfected MSC, whereas the yield of transfection corresponds to the percentage of GFP+PI− cells relative to nontransfected cells. Together, these two parameters provide an accurate estimation of the number of cells that remained adherent to the tissue culture flask and expressed the transgene after lipoplex uptake. For the 1000 cells/cm2 cell density condition, BM MSC displayed values of cell recovery (56±4.4%) significantly higher (p<0.05) compared to UCM MSC (38±5.8%), and also a significantly higher (p<0.05) yield of transfection (31±0.4%) compared to both UCM MSC and ASC (15±0.1% and 13±2.5%, respectively) (Fig. 2a). In contrast, regarding the highest initial cell density tested—3000 cells/cm2—though no major differences were detected between cell sources for cell recoveries (≈50%), ASC displayed a lower yield of transfection (13±3.3%) compared to both BM MSC (26±1.8%) and UCM MSC (24±6.5%) (Fig. 2b). For UCM MSC, in particular, a lower cell survival upon lipofection was observed as the number of cells GFP+PI− was significantly reduced in comparison with the total GFP+ population within the first 48 hr after gene internalization (p<0.05), specially for the 1000 cells/cm2 condition (Fig. 1a).

FIG. 2.

FIG. 2.

Open in a new tab

Yield of transfection and cell recovery after lipofection of MSC from the different sources. Comparison of cells plated at 1000 cells/cm2 (a) and 3000 cells/cm2 (b). Yield of transfection is represented as solid bars, whereas gray squares correspond to cell recovery. Each bar and point represents the mean±standard error of mean (SEM), n=4, *p<0.05.

The plasmid copy number internalized by MSC was also assessed by quantitative RT-PCR. The results obtained indicate that MSC derived from BM (Fig. 3a) and UCM (Fig. 3c) showed higher numbers of pDNA uptake when compared to ASC (Fig. 3b), which is consistent with the previously shown data for GFP expression determined by flow cytometry. Concomitantly, cells plated at 1000 cells/cm2 were also found to internalize superior levels of pDNA. Interestingly, an accentuated decrease in the plasmid copy number content inside MSC from all sources was detected from 24 to 48 hr upon transfection (p<0.05).

FIG. 3.

FIG. 3.

Open in a new tab

Intracellular plasmid copy number present into BM- (a), AT- (b), and UCM-MSC (c) after lipofection. Two initial cell densities, 1000 cells/cm2 (black bars) and 3000 cells/cm2 (white bars) were tested. Each bar represents the mean±standard error of mean (SEM), n=4, *p<0.05.

Effect of liposome-mediated gene transfer on MSC proliferative status

Since cell division is one of the major parameters influencing plasmid uptake and loss upon nonviral gene delivery, the proliferative status of lipofectamine-treated MSC was evaluated. After 10 days of culture, the levels of expansion for modified cells plated at 1000 cells/cm2 were almost negligible, indicating that cell proliferation under these specific conditions was seriously impaired by the presence of the transgene (data not shown). In contrast, even though a clear delay in cell expansion was observed in comparison to nontransfected cells and cells treated with lipofectamine only (data not shown), MSC plated at 3000 cells/cm2 were able to expand upon liposome-mediated genetic modification (Fig. 4). After 10 days of culture, UCM MSC reached higher cell numbers when compared to BM MSC and ASC—(1.1±0.3)×105, (7.2±2.0)×104, and (6.8±1.2)×104, respectively. Considering that successful cell expansion was only achieved when an initial cell density of 3000 cells/cm2 used, the following studies were pursued with this condition.

FIG. 4.

FIG. 4.

Open in a new tab

Impact of liposome-mediated gene delivery on the proliferative status of MSC from the different human sources. The number of viable cells was determined for both transfected cells (solid lines) and nontransfected cells (dashed lines) (a). Representative microscopy images of UCM-, BM-, and AT-derived MSC plated at 3000 cells/cm2 on days 1, 2, and 3 after transfection (b), where cells were still subconfluent. Fluorescence microscopy images were merged with bright field optical microscopy images. Each point represent the mean±standard error of mean (SEM), n=4, *p<0.05. Color images available online at www.liebertpub.com/hgtb

To better understand the cellular division kinetics of transfected cells, MSC from the different sources were labeled with the membrane dye PKH26 before seeding and analyzed throughout culture by flow cytometry. The percentage of cells at each generation was calculated using the Proliferation Wizard of the ModFit Software (da Silva et al., 2009). On the day of transfection (72 hr after initial cell seeding), more than 50% of the cells were found to be at Generation 3 and only a small fraction of these (less than 5%) remained undivided, indicating that at this stage the majority of the MSC, regardless of cell source, were actively dividing (data not shown). In order to identify differences between transfected and nontransfected cells (control) throughout culture, cells at 24 hr after lipofection were reset as Generation 1 (parent) (Fig. 5). No major differences were detected between nontransfected cells and cells treated with lipofectamine only (data not shown).

FIG. 5.

FIG. 5.

Open in a new tab

Cell division kinetics of MSC from different human sources after lipofection. Cells were plated at the initial cell density of 3000 cells/cm2. Throughout culture GFP+ (black bars) and non-transfected MSC (white bars) were analyzed by flow cytometry and for each time point the percentage of cell population in each generation was determined using the Proliferation Wizard of the ModFit Software (a). GFP+ and GFP− populations were discriminated using distinct FL1/FL3 gates as shown in the representative flow cytometry dot plots of MSC expressing GFP and labeled with PKH 26 dye (b). Each bar represent the mean±standard error of mean (SEM), n=3.

It was possible to verify that cell division was highly arrested within the first 48 hr after transfection, especially for BM MSC and ASC, and that effective cell division only started upon day 2 (between day 2 and 5) (Fig. 5a). Although MSC expressing a transgene demonstrated to undergo cell division at a slower rate than unmodified MSC, they divide for more generations whereas control cells stop proliferation after reaching Generations 4–5. At day 10 of culture, modified BM MSC and UCM MSC presented a higher percentage of GFP+ cells at the latest generations (Generations 5–7), representing more than 50% of total GFP+ cell population compared to ASC (15% of the cells at Generations 5–7) (Fig. 5a).

In vitro characterization of human MSC modified by liposome-mediated gene delivery

After lipofection, cells from the different sources were characterized in vitro based on several criteria, namely, immunophenotype, differentiative potential into mesodermal lineages, and clonogenic capacity (i.e., colony forming unit-fibroblasts [CFU-F]).

Cell immunophenotyping was performed by flow cytometry and using a panel of monoclonal antibodies against specific surface makers. Modified MSC were found to be negative (<2%) for CD31, CD38, CD45, CD80, and HLA-DR and positive (>95%) for CD73, CD90, and CD105 (Fig. 6a) (Dominici et al., 2006).

FIG. 6.

FIG. 6.

Open in a new tab

Immunophenotype, multilineage diferentiative potential, and clonogenic potential of MSC from different human sources after transfection. Flow cytometric analysis of surface antigen expression showed that lipofection had no impact on MSC immunophenotype profile (a). After gene delivery, MSC were induced to differentiate and stained for osteogenesis (Von Kossa and alkaline phosphatase), adipogenesis (Oil Red-O), and chondrogenesis (Alcian blue). No major differences were noticed among MSC sources. Representative fluorescence microscopy images of transfected UCM-MSC are merged with bright field optical microscopy images (b). Clonogenic potential of genetically modified MSC isolated from BM, AT, and UCM (black bars) compared to nontransfected cells (white bars) (c). Each bar represents the mean±standard error of mean (SEM), n=2. Color images available online at www.liebertpub.com/hgtb

To test the multipotency of the transfected MSC, differentiation protocols toward osteogenic, adipogenic, and chondrogenic lineages were performed. After 7 days of culture using adipogenic-, osteogenic-, and chondrogenic-inductive medium, cells were monitored for differentiation using lineage-specific stainings. As shown in Figure 6b, adipogenesis was confirmed by the formation of intracellular lipid vacuoles stained red with Oil Red O, whereas positive staining for Alkaline phosphatase (ALP) and von Kossa were indicative osteogenesis. Chondrogenesis was verified by the presence of glycosaminoglycans stained blue using alcian blue. Thus, it is possible to conclude that engineered MSC retained their capacity to differentiate into adipocytes, chondrocytes, and osteocytes in vitro. Of notice, GFP expression was still evident after differentiation into adipocytes and chondrocytes. Additionally, no detectable differences were noticed between transfected and control cells (lipofectamine only) (data not shown), as well as among cell sources.

Cells from the three sources were also characterized by their ability to form CFU-F. After 14 days of culture (plated at an initial cell density of 10 cell/cm2), it was noticed that the clonogenic potential was negatively affected by the presence of the transgene, particularly in what concerns UCM and BM MSC, where a reduction of ≈50% in the ability to form colonies was verified (Fig. 6c).

Discussion

In the past few years, human MSC have been exploited in numerous clinical trials to treat several diseases with promising results (Ankrum and Karp, 2010; Caplan and Correa, 2011). Engineering MSC to overexpress proteins of interest is a very promising strategy to further enhance their intrinsic therapeutic features (Porada and Almeida-Porada, 2010). In contrast to viral vectors, nonviral approaches, such as cationic liposomes, are highly promising as they have less safety issues associated (such as mutagenesis, immunological reactions) and are attractive from the manufacturing point of view (Niidome and Huang, 2002). Indeed, lipofection is one of the most widely used nonviral methods to perform delivery of nucleic acids in clinical trials (Edelstein et al., 2007). In this study, we optimized and compared the transfection efficiency of cationic liposomes to MSC derived from different human sources, using a previously optimized lipid/plasmid DNA ratio (1 μl/1 μg) (Madeira et al., 2010).

Most of the studies focusing on the transfection of animal cells in general and human MSC in particular have been using initial cell densities higher than 7500 cells/cm2 mainly in order to maximize the cell numbers available for analysis (e.g., flow cytometry) (Gheisari et al., 2008; Hoare et al., 2010; Madeira et al., 2010). These plating densities are higher than those commonly used in MSC expansion studies. For instance, Sekiya and colleagues have demonstrated the crucial importance of low initial cell density (10–1000 cells/cm2) in the maximization of MSC proliferation rate and in the maintenance of their characteristics (Sekiya et al., 2002). For clinical grade expansion, the reported MSC density can range between 1000 cells/cm2 (Sensebé et al., 2011) and 4000 cells/cm2 (Le Blanc et al., 2008). These densities result from a compromise between conditions that provide the highest overall yields, in a cost-effective process, while maximizing the content of functional primitive cells. Upon our previous report on BM MSC lipofection (Madeira et al., 2010), here we hypothesize that by plating cells at initial cell densities in the aforementioned range, namely 1000 cells/cm2 and 3000 cells/cm2, prior to transfection, the uptake of plasmid DNA to the nucleus and thus transfection efficiencies could be maximized.

Though there has been much debate concerning the mechanism of uptake of exogenous DNA into the nucleus, cell division is accepted as one of the main routes for nuclear entry specially when using nonviral approaches. During mitosis, cells lose the integrity of their nuclear membrane, allowing DNA to reach the nucleus (Escriou et al., 2001; Dean et al., 2005). In fact, Tseng and colleagues have shown that mitosis enhances the uptake of plasmid delivered by cationic liposomes in HeLa cells (Tseng et al., 1999). To guarantee that most MSC were undergoing cell division upon transfection, in the present study lipofection was performed 72 hr after cell plating. Remarkably, with this strategy, we were able to increase by two- to three-fold liposome-mediated gene delivery efficiencies to human MSC when compared to previous studies, where cells are seeded at subconfluent levels (80–90%) and transfected the day after (Hoare et al., 2010; Madeira et al., 2010). In fact, the levels of gene expression obtained are comparable to the ones reported with nucleofection (approximately 70%) (Aluigi et al., 2006; Aslan et al., 2006; Flanagan et al., 2012) and microporation (60–80%) (Wang et al., 2008; Madeira et al., 2011).

Despite the fact that no major differences between the two cell densities tested were detected in what concerns transgene expression, an arrest in cell proliferation was noticed upon lipofection for the 1000 cells/cm2 condition, possibly due to a toxic effect of the DNA. Therefore, a slight increase in the initial cell density from 1000 to 3000 cells/cm2 was found to have a beneficial impact in the overall transfection process.

The characteristics of MSC from distinct human sources may account for different mechanisms of action in regenerative settings. There is evidence that MSC obtained from different tissues show specific immunological properties, as well as proliferative and differentiation abilities (Puissant et al., 2005; Dmitrieva et al., 2012). To the best of our knowledge, our work describes the first comprehensive comparison study of nonviral gene delivery to MSC obtained from different human sources, namely from adult (BM and AT) and neonatal (UCM) origin. Our results indicate that MSC isolated from BM and UCM were more amenable to transgene expression when compared to ASC, showing not only higher levels of GFP expression but also a superior plasmid copy number uptake. Nevertheless, for all MSC sources, a rapid lost of pDNA inside the cells was detected between 24 hr and 48 hr after lipofection probably as a consequence of degradation by endonucleases. However, this decrease does not correlate with the transgene expression levels measured by flow cytometry at this time point. We can hypothesize that in the first hours after transfection a high amount of pDNA is still not available for transcription, either due to the nonrelease from the lipid carrier or to the nonuptake inside the nucleus (Lechardeur et al., 2005; Ruponen et al., 2009). Additional studies, such as the quantification of the pDNA specifically inside the nucleus, would be useful to further elucidate these results.

On the other hand, in the conditions of this study, UCM MSC demonstrated to be more sensitive to the transfection process since a lower cell survival among the cell population expressing GFP for the 1000 cells/cm2 condition was noted, especially within the first 48 hr.

We have successfully demonstrated the ability to track cellular division of human MSC by using a PKH membrane dye and analyzing its expression throughout time in culture by flow cytometry (dos Santos et al., 2010). By performing cell division kinetics studies herein, it was found that the proliferative capacity of modified MSC is hampered when compared to nonmodified cells, especially within the first 48 hr upon liposome-mediated gene delivery. The metabolic burden imposed by the presence of large amounts of plasmid inside the cells within this period may be the main reason for this cell division arrest. Similar results were obtained by Madeira and colleagues after BM MSC microporation (Madeira et al., 2011). On the other hand, as control cells rapidly reach high levels of confluence, cell division is stopped due to surface area limitations approximately two generations before GFP+ cells. Comparing the three MSC sources, UCM MSC start dividing earlier, especially in comparison with ASC, indicating a faster recovery upon the transfection process. This difference might potentially result from a higher predisposition of UCM MSC to uptake genes due to their more primitive and naïve nature (Troyer and Weiss, 2008). Additionally, the results of cell division kinetics together with the plasmid copy number data revealed a correlation between the initiation of active cell division and the decrease of plasmid inside the cells.

Importantly, we also demonstrated that transfected human MSC were able to maintain transgene expression in culture over 10 days (>4% GFP+ cells), while maintaining their characteristic immunophenotype and multilineage differentiation ability into adipocytes, osteocytes, and chondrocytes. We decided to evaluate the differentiation potential of the transfected cells only after 7 days of incubation with specific media in order to enable the visualization of differentiated cells that express GFP. On the other hand, though modified cells showed a reduction in clonogenic potential when compared to nontransfected cells, it is noteworthy that the majority of clinical applications of MSC (e.g., graft-versus-host disease) are based on their paracrine function or trophic activity (secretory) and do not require cell engraftment. Therefore, complementary studies on the secretome of the transfected MSC would also be very useful to attest if the gene delivery process and the expression of a transgene affects the secretory profile of the cells.

In conclusion, we have shown that lipofection is a promising transfection strategy for MSC genetic modification, especially when a transient expression of a therapeutic gene is required. By performing lipofection after 72 hr upon plating of human MSC at an initial cell density of 3000 cell/cm2, we were able to maximize the transfection efficiency (maximum of 74% GFP+ cells) by using cationic liposomes. Importantly, our comparative study demonstrated that higher levels of liposome-mediated gene delivery were obtained with BM-and UCM-derived MSC when compared to AT-derived cells. These findings clearly indicate that the intrinsic characteristics of MSC from different sources should be taken into account when developing and optimizing strategies for MSC engineering with a therapeutic gene for clinical application. Additionally, given the recent encouraging developments in gene therapy preclinical studies and clinical trials, it is thus essential to continue developing and investing in nonviral and safer gene delivery strategies, as well as to overcome other challenges related to maximizing efficiencies and developing process-scale transfection approaches capable of meeting the large cell numbers needed for therapeutic applications.

Acknowledgments

This work was financially supported by Fundação para a Ciência e a Tecnologia (FCT), Portugal, through MIT-Portugal Program, Bioengineering Systems Focus Area, project PTDC/EQU-EQU/114231/2009. Authors also acknowledge “ISOCORD” project, led by Crioestaminal – Saúde e Tecnologia S.A., supported by the Portuguese program QREN/Mais Centro-Programa Operacional Regional do Centro and cofunded by the European Regional Development Fund (ERDF). JB and FS acknowledge FCT for grants SFRH/BD/70948/2010 and SFRH/BD/38719/2007, respectively, and CM acknowledges Fundação para a Ciência e Tecnologia (FCT) through the Compromisso para a Ciência Program (2008).

Author Disclosure Statement

No disclosure for Joana S. Boura, Francisco dos Santos, Catarina Madeira, Joaquim M.S. Cabral, and Cláudia L. da Silva. Jeffrey M. Gimble is a cofounder of LaCell, LLC. Carla M.P. Cardoso is an employee of Crioestaminal – Saúde e Tecnologia S.A.

References

  1. Aluigi M. Fogli M. Curti A, et al. Nucleofection is an efficient nonviral transfection technique for human bone marrow–derived mesenchymal stem cells. Stem Cells. 2006;24:454–461. doi: 10.1634/stemcells.2005-0198. [DOI] [PubMed] [Google Scholar]
  2. Ankrum J. Karp J.M. Mesenchymal stem cell therapy: Two steps forward, one step back. Trends Mol. Med. 2010;16:203–209. doi: 10.1016/j.molmed.2010.02.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Aslan H. Zilberman Y. Arbeli V, et al. Nucleofection-based ex vivo nonviral gene delivery to human stem cells as a platform for tissue regeneration. Tissue Eng. 2006;12:877–889. doi: 10.1089/ten.2006.12.877. [DOI] [PubMed] [Google Scholar]
  4. Azzoni A.R. Ribeiro S.C. Monteiro G.A. Prazeres D.M.F. The impact of polyadenylation signals on plasmid nuclease-resistance and transgene expression. J. Gene Med. 2007;9:392–402. doi: 10.1002/jgm.1031. [DOI] [PubMed] [Google Scholar]
  5. Ben-Ami E. Berrih-Aknin S. Miller A. Mesenchymal stem cells as an immunomodulatory therapeutic strategy for autoimmune diseases. Autoimmunity Reviews. 2011;10:410–415. doi: 10.1016/j.autrev.2011.01.005. [DOI] [PubMed] [Google Scholar]
  6. Can A. Karahuseyinoglu S. Concise review: human umbilical cord stroma with regard to the source of fetus-derived stem cells. Stem Cells. 2007;25:2886–2895. doi: 10.1634/stemcells.2007-0417. [DOI] [PubMed] [Google Scholar]
  7. Caplan A.I. Correa D. The MSC: An injury drugstore. Cell stem cell. 2011;9:11–15. doi: 10.1016/j.stem.2011.06.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Clements B.A. Incani V. Kucharski C, et al. A comparative evaluation of poly-l-lysine-palmitic acid and Lipofectamine 2000 for plasmid delivery to bone marrow stromal cells. Biomaterials. 2007;28:4693–4704. doi: 10.1016/j.biomaterials.2007.07.023. [DOI] [PubMed] [Google Scholar]
  9. Da Silva C.L. Gonçalves R. Porada C.D, et al. Differences amid bone marrow and cord blood hematopoietic stem/progenitor cell division kinetics. J. of Cell. Physiol. 2009;220:102–111. doi: 10.1002/jcp.21736. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dean D.A. Strong D.D. Zimmer W.E. Nuclear entry of nonviral vectors. Gene Ther. 2005;12:881–890. doi: 10.1038/sj.gt.3302534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. De Bruyn C. Najar M. Raicevic G, et al. A rapid, simple, and reproducible method for the isolation of mesenchymal stromal cells from Wharton's jelly without enzymatic treatment. Stem Cells Dev. 2011;20:547–557. doi: 10.1089/scd.2010.0260. [DOI] [PubMed] [Google Scholar]
  12. Dmitrieva R.I. Minullina I.R. Bilibina A.A, et al. Bone marrow- and subcutaneous adipose tissue-derived mesenchymal stem cells: Differences and similarities. Cell Cycle. 2012;11:377–383. doi: 10.4161/cc.11.2.18858. [DOI] [PubMed] [Google Scholar]
  13. Dominici M. Le Blanc K. Mueller I, et al. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8:315–317. doi: 10.1080/14653240600855905. [DOI] [PubMed] [Google Scholar]
  14. Dos Santos F. Andrade P.Z. Abecasis M.M, et al. Toward a clinical-grade expansion of mesenchymal stem cells from human sources: a microcarrier-based culture system under xeno-free conditions. Tissue Eng Part C Methods. 2011;17:1201–1210. doi: 10.1089/ten.tec.2011.0255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dos Santos F. Andrade P.Z. Boura J.S, et al. Ex vivo expansion of human mesenchymal stem cells: A more effective cell proliferation kinetics and metabolism under hypoxia. J. of Cell. Phys. 2010;223:27–35. doi: 10.1002/jcp.21987. [DOI] [PubMed] [Google Scholar]
  16. Edelstein M.L. Abedi M.R. Wixon J. Gene therapy clinical trials worldwide to 2007—an update. J. Gene Med. 2007;9:833–842. doi: 10.1002/jgm.1100. [DOI] [PubMed] [Google Scholar]
  17. Escriou V. Carrière M. Bussone F, et al. Critical assessment of the nuclear import of plasmid during cationic lipid-mediated gene transfer. J. of Gene Med. 2001;3:179–187. doi: 10.1002/jgm.174. [DOI] [PubMed] [Google Scholar]
  18. Farrell L.-L. Pepin J. Kucharski C, et al. A comparison of the effectiveness of cationic polymers poly-l-lysine (PLL) and polyethylenimine (PEI) for non-viral delivery of plasmid DNA to bone marrow stromal cells (BMSC) Eur. J. Pharm. Biopharm. 2007;65:388–397. doi: 10.1016/j.ejpb.2006.11.026. [DOI] [PubMed] [Google Scholar]
  19. Flanagan M. Gimble J. Yu G, et al. Competitive DNA transfection formulation via electroporation for human adipose stem cells and mesenchymal stem cells. Biol. Proced. 2012;14:7. doi: 10.1186/1480-9222-14-7. Online. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gheisari Y. Soleimani M. Azadmanesh K. Zeinali S. Multipotent mesenchymal stromal cells: optimization and comparison of five cationic polymer-based gene delivery methods. Cytotherapy. 2008;10:815–823. doi: 10.1080/14653240802474307. [DOI] [PubMed] [Google Scholar]
  21. Gimble J.M. Guilak F. Adipose-derived adult stem cells: isolation, characterization, and differentiation potential. Cytotherapy. 2003;5:362–369. doi: 10.1080/14653240310003026. [DOI] [PubMed] [Google Scholar]
  22. Gimble J.M. Katz A.J. Bunnell B.A. Adipose-derived stem cells for regenerative medicine. Circ. Res. 2007;100:1249–1260. doi: 10.1161/01.RES.0000265074.83288.09. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Glover D.J. Lipps H.J. Jans D.A. Towards safe, non-viral therapeutic gene expression in humans. Nat. Rev. Genet. 2005;6:299–310. doi: 10.1038/nrg1577. [DOI] [PubMed] [Google Scholar]
  24. Gresch O. Engel F.B. Nesic D, et al. New non-viral method for gene transfer into primary cells. Methods. 2004;33:151–163. doi: 10.1016/j.ymeth.2003.11.009. [DOI] [PubMed] [Google Scholar]
  25. Hoare M. Greiser U. Schu S, et al. Enhanced lipoplex-mediated gene expression in mesenchymal stem cells using reiterated nuclear localization sequence peptides. J. Gene Med. 2010;12:207–218. doi: 10.1002/jgm.1426. [DOI] [PubMed] [Google Scholar]
  26. Hu Y.-L. Huang B. Zhang T.-Y, et al. Mesenchymal stem cells as a novel carrier for targeted delivery of gene in cancer therapy based on nonviral transfection. Mol. Pharm. 2012;9:2698–2709. doi: 10.1021/mp300254s. [DOI] [PubMed] [Google Scholar]
  27. Kern S. Eichler H. Stoeve J, et al. Comparative analysis of mesenchymal stem cells from bone marrow, umbilical cord blood, or adipose tissue. Stem Cells. 2006;24:1294–1301. doi: 10.1634/stemcells.2005-0342. [DOI] [PubMed] [Google Scholar]
  28. Le Blanc K. Frassoni F. Ball L, et al. Mesenchymal stem cells for treatment of steroid-resistant, severe, acute graft-versus-host disease: a phase II study. The Lancet. 2008;371:1579–1586. doi: 10.1016/S0140-6736(08)60690-X. [DOI] [PubMed] [Google Scholar]
  29. Lechardeur D. Verkman A.S. Lukacs G.L. Intracellular routing of plasmid DNA during non-viral gene transfer. Adv Drug Deliv Rev. 2005;57:755–767. doi: 10.1016/j.addr.2004.12.008. [DOI] [PubMed] [Google Scholar]
  30. Lu L. Liu Y. Yang S, et al. Isolation and characterization of human umbilical cord mesenchymal stem cells with hematopoiesis-supportive function and other potentials. Haematologica. 2006;91:1017–1026. [PubMed] [Google Scholar]
  31. Madeira C. Mendes R.D. Ribeiro S.C, et al. Nonviral gene delivery to mesenchymal stem cells using cationic liposomes for gene and cell therapy. J Biomed Biotechnol. 2010;2010:735349. doi: 10.1155/2010/735349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Madeira C. Ribeiro S.C. Pinheiro I.S.M, et al. Gene delivery to human bone marrow mesenchymal stem cells by microporation. Journal of Biotechnology. 2011;151:130–136. doi: 10.1016/j.jbiotec.2010.11.002. [DOI] [PubMed] [Google Scholar]
  33. Mcmahon J.M. Conroy S. Lyons M, et al. gene transfer into rat mesenchymal stem cells: a comparative study of viral and nonviral vectors. Stem Cells Devel. 2006;15:87–96. doi: 10.1089/scd.2006.15.87. [DOI] [PubMed] [Google Scholar]
  34. Nauta A.J. Fibbe W.E. Immunomodulatory properties of mesenchymal stromal cells. Blood. 2007;110:3499–3506. doi: 10.1182/blood-2007-02-069716. [DOI] [PubMed] [Google Scholar]
  35. Niidome T. Huang L. Gene therapy progress and prospects: nonviral vectors. Gene therapy. 2002;9:1647–52. doi: 10.1038/sj.gt.3301923. [DOI] [PubMed] [Google Scholar]
  36. Park H.-J. Yang F. Cho S.-W. Nonviral delivery of genetic medicine for therapeutic angiogenesis. Adv Drug Deliv Rev. 2012;64:40–52. doi: 10.1016/j.addr.2011.09.005. [DOI] [PubMed] [Google Scholar]
  37. Porada C.D. Almeida-Porada G.S. Mesenchymal stem cells as therapeutics and vehicles for gene and drug delivery. Adv Drug Deliv Rev. 2010;62:1156–1166. doi: 10.1016/j.addr.2010.08.010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Puissant B. Barreau C. Bourin P, et al. Immunomodulatory effect of human adipose tissue-derived adult stem cells: comparison with bone marrow mesenchymal stem cells. Br J Haematol. 2005;129:118–129. doi: 10.1111/j.1365-2141.2005.05409.x. [DOI] [PubMed] [Google Scholar]
  39. Reiser J. Zhang X.-Y. Hemenway C.S, et al. Potential of mesenchymal stem cells in gene therapy approaches for inherited and acquired diseases. Expert Opin Biol Ther. 2005;5:1571–1584. doi: 10.1517/14712598.5.12.1571. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Ruponen M. Arkko S. Urtti A, et al. Intracellular DNA release and elimination correlate poorly with transgene expression after non-viral transfection. J Control Release. 2009;136:226–231. doi: 10.1016/j.jconrel.2009.02.012. [DOI] [PubMed] [Google Scholar]
  41. Santos J.L. Pandita D. Rodrigues J, et al. Non-viral gene delivery to mesenchymal stem cells: methods, strategies and application in bone tissue engineering and regeneration. Current Gene Therapy. 2011;11:46–57. doi: 10.2174/156652311794520102. [DOI] [PubMed] [Google Scholar]
  42. Schenborn E.T. Oler J. Tymms M.J. Humana Press; 2000. Liposome-Mediated Transfection of Mammalian Cells Transcription Factor Protocols; pp. 155–164. [DOI] [PubMed] [Google Scholar]
  43. Sekiya I. Larson B.L. Smith J.R, et al. Expansion of human adult stem cells from bone marrow stroma: conditions that maximize the yields of early progenitors and evaluate their quality. Stem Cells. 2002;20:530–541. doi: 10.1634/stemcells.20-6-530. [DOI] [PubMed] [Google Scholar]
  44. Sensebé L. Bourin P. Tarte K. Good manufacturing practices production of mesenchymal stem/stromal cells. Hum Gene Ther. 2011;22:19–26. doi: 10.1089/hum.2010.197. [DOI] [PubMed] [Google Scholar]
  45. Stappenbeck T.S. Miyoshi H. The role of stromal stem cells in tissue regeneration and wound repair. Science. 2009;324:1666–1669. doi: 10.1126/science.1172687. [DOI] [PubMed] [Google Scholar]
  46. Troyer D.L. Weiss M.L. Concise Review: Wharton's jelly-derived cells are a primitive stromal cell population. Stem Cells. 2008;26:591–599. doi: 10.1634/stemcells.2007-0439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tseng W.-C. Haselton F.R. Giorgio T.D. Mitosis enhances transgene expression of plasmid delivered by cationic liposomes. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression. 1999;1445:53–64. doi: 10.1016/s0167-4781(99)00039-1. [DOI] [PubMed] [Google Scholar]
  48. Wagner W. Wein F. Seckinger A, et al. Comparative characteristics of mesenchymal stem cells from human bone marrow, adipose tissue, and umbilical cord blood. Exp. Hematol. 2005;33:1402–1416. doi: 10.1016/j.exphem.2005.07.003. [DOI] [PubMed] [Google Scholar]
  49. Wang Y.-H. Ho M.-L. Chang J.-K, et al. Microporation is a valuable transfection method for gene expression in human adipose tissue-derived stem cells. Mol Ther. 2008;17:302–308. doi: 10.1038/mt.2008.267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wasungu L. Hoekstra D. Cationic lipids, lipoplexes and intracellular delivery of genes. J Control Release. 2006;116:255–264. doi: 10.1016/j.jconrel.2006.06.024. [DOI] [PubMed] [Google Scholar]
  51. Xiang S. Tong H. Shi Q, et al. Uptake mechanisms of non-viral gene delivery. J Control Release. 2012;158:371–378. doi: 10.1016/j.jconrel.2011.09.093. [DOI] [PubMed] [Google Scholar]
  52. Zeddou M. Briquet A. Relic B, et al. The umbilical cord matrix is a better source of mesenchymal stem cells (MSC) than the umbilical cord blood. Cell Biol. Int. 2010;34:693–701. doi: 10.1042/CBI20090414. [DOI] [PubMed] [Google Scholar]

Articles from Human Gene Therapy Methods are provided here courtesy of Mary Ann Liebert, Inc.

RESOURCES