Nontoxic genetic engineering of mesenchymal stem cells using serum-compatible pullulan-spermine/DNA anioplexes

Abstract

Genetic modification of stem cells could be applied to initiate/enhance their secretion of therapeutic molecules, alter their biological properties, or label them for in vivo tracking. We recently developed a negatively charged gene carrier ("anioplex") based on pullulan-spermine, a conjugate prepared from a natural polysaccharide and polyamine. In rat mesenchymal stem cells (MSCs), anioplex-derived reporter gene activity was comparable to or exceeded that obtained using a commercial cationic lipid reagent. Transfection in the growth medium with 15% serum and antibiotics was approximately sevenfold more effective than in serum-free conditions. Cytotoxicity was essentially indiscernible after 24 h of anioplex transfection with 20 μg/mL DNA, in contrast to cationic lipid transfection that resulted in 40%-60% death of target MSCs. Anioplex-derived reporter gene activity persisted throughout the entire 3-week study, with post-transfection MSCs appearing to maintain osteogenic, adipogenic, and chondrogenic multipotency. In particular, chondrogenic pellet formation of differentiating human MSCs was significantly inhibited after lipofection but not after aniofection, which further indicates the biological inertness of pullulan-spermine/DNA anioplexes. Collectively, these data introduce a straightforward technology for genetic engineering of adult stem/progenitor cells under physiological niche-like conditions. Moreover, reporter gene activity was observed in rat spinal cords after minimally invasive intrathecal implantation, suggesting effective engraftment of donor MSCs. It is therefore plausible that anioplex-transfected MSCs or other stem/progenitor cells with autologous potential could be applied to disorders such as neurotrauma or neuropathic pain that involve the spinal cord and brain.

FIG. 1.

Transfection of rat bone marrow stromal cells (BMSCs) by pullulan-spermine/DNA anioplexes. (A) Luciferase reporter gene activity 24 h after transfection with pullulan-spermine/DNA anioplexes and Lipofectamine 2000 in OPTI-minimal essential medium (MEM) serum-reduced medium. Black columns, anioplexes; gray columns, Lipofectamine 2000. Activity levels were generally higher with anioplexes than with Lipofectamine 2000 (p < 0.05, two-way analysis of variance [ANOVA]). (B) Luciferase activity 24 h after transfection with pullulan-spermine/DNA anioplexes and Lipofectamine 2000 in OPTI-MEM medium without serum or α-MEM with 15% fetal bovine serum and antibiotics (growth medium). Gray columns, OPTI-MEM; black columns, growth medium. Anioplex transfection was significantly enhanced in the presence of serum (*p < 0.05, Neuman-Keuls post-hoc), but the difference between anioplexes and Lipofectamine 2000 did not reach significance (p = 0.058, Neuman-Keuls post-hoc). (C) Luciferase activity 24 h after transfection with pullulan-spermine/DNA anioplexes at different concentrations in the growth medium. Transfection was concentration dependent (p < 0.05, one-way ANOVA; *p < 0.05, Newman-Keuls post-hoc). (D) Luciferase activity 24 h after initiation of transfection with pullulan-spermine/DNA anioplexes (2 μg/mL DNA) in the growth medium; there was no effect of anioplex incubation time. (E) Luciferase activity 24 h after transfection with negatively and positively charged pullulan-spermine/DNA (N:P 1 and 3, respectively) in the growth medium. Luciferase activity was not detected for cells treated with naked plasmid DNA. Particles formed at N:P 3 showed a significant reduction of reporter gene activity as compared to those formed at N:P 1 (*p < 0.05, t-test) (means and standard errors depicted in all graphs; n = 3).

FIG. 2.

Uptake and intracellular trafficking of pullulan-spermine/DNA anioplexes in BMSCs. (A) Luciferase reporter gene activity was significantly decreased 24 h after anioplex transfection in OPTI-MEM medium with 1 mg/mL asialofetuin pretreatment, suggesting that uptake is glycoprotein receptor dependent (*p < 0.05, t-test). (B) Luciferase reporter gene activity was not affected by the presence of 5 mM methyl-beta-cyclodextrin during transfection, suggesting that lipid rafts are not involved in endocytosis. (C) Luciferase reporter gene activity was significantly decreased by the presence of 28 mM chlorpromazine during transfection (*p < 0.05, t-test), suggesting the use of clathrin-dependent vesicular endocytosis. (D) Luciferase reporter gene activity tended to be inhibited the presence of 65 mM colchicine during transfection, although this difference did not reach statistical significance (p = 0.069, t-test); this suggests the use of active transport for intracellular trafficking (means and standard errors depicted in all graphs; n = 3).

FIG. 3.

Osteogenic and adipogenic differentiation of BMSCs after transfection. Upon exposure to osteogenic and adipogenic media for 4 weeks, BMSCs transfected with anioplexes and Lipofectamine 2000 showed calcium deposition (Von Kossa staining) and formation of lipid vacuoles (oil red o staining), respectively, suggesting osteogenesis and adipogenesis. Culture in the growth medium for 4 weeks after transfection did not generate such effects, suggesting a lack of unintended differentiation. Color images available online at www.liebertonline.com/ten.

FIG. 4.

Chondrogenic differentiation of human BMSCs after transfection. (A) After seeding in micromass conditions for 2 h followed immediately by transfection for 6 h and exposure to the chondrogenic medium for 3 weeks, human BMSCs left untransfected (left column) or transfected with Lipofectamine 2000/DNA (middle column) or pullulan-spermine/DNA anioplexes (right column) showed formation of chondrogenic pellets with proteoglycan secretion as determined by Alcian Blue staining. Top row: imaging of intact pellets after staining in well plates. Scale bar: 500 μm. Bottom row: imaging of stained pellet sections. Scale bar: 250 μm. (B) The sizes of the intact pellets shown in the top row of (A) were measured. There was no significant difference between pullulan-spermine-transfected pellets and nontransfected pellets, even with the high plasmid DNA concentration of 10 μg/mL (p > 0.05, Tukey-Kramer post-hoc). In contrast, pellets transfected with Lipofectamine 2000/DNA showed a significant impairment of contraction compared to pellets left untransfected or transfected with pullulan-spermine (p < 0.0001, one-way ANOVA; *p = 0.003 vs. nontransfected, *p = 0.0005 vs. pullulan-spermine 2 μg/mL, *p = 0.0013 vs. pullulan-spermine 10 μg/mL, Tukey-Kramer post-hoc). (C) Top: proteoglycan secretion of micromass pellets cultured in the chondrogenic differentiation medium was quantified based on the absorbance of leached Alcian Blue stain and was not significantly different for pellets left untransfected or transfected with Lipofectamine 2000/DNA or pullulan-spermine/DNA anioplexes (p > 0.05, one-way ANOVA). Bottom: Micromass cultures incubated in the normal growth medium did not form pellets. However, they still showed a limited extent of proteoglycan secretion on Alcian Blue staining. The absorbance of the leached Alcian Blue stain was not significantly different for cultures left untransfected or transfected with Lipofectamine 2000/DNA or pullulan-spermine/DNA anioplexes (p > 0.05, one-way ANOVA) (means and standard errors depicted in all graphs; n = 3). Color images available online at www.liebertonline.com/ten.

FIG. 5.

Transfection of rat adipose-derived stromal cells (ASCs) by pullulan-spermine/DNA anioplexes. (A) Luciferase reporter gene activity 24 h after transfection with pullulan-spermine/DNA anioplexes in Medium 199 containing 10% fetal calf serum (growth medium). Transfection was concentration dependent (p < 0.05, one-way ANOVA; *p < 0.05, Newman-Keuls post-hoc). (B) Luciferase activity 24 h after initiation of transfection with pullulan-spermine/DNA anioplexes (1 μg DNA/well) in the growth medium; there was no effect of anioplex incubation time (means and standard errors depicted in all graphs; n = 3).

FIG. 6.

Substrate-mediated transfection. (A) Schematic representation of substrate-mediated transfection (subfection) strategy. Different types of charged gelatin were mixed with Pronectin F cell adhesion molecule and added to tissue culture plates. Pullulan-spermine/DNA was then applied to the coated plates. Finally, treated wells were plated with DRG neurons. (B) Comparison of substrates for ASC subfection. Suc: succinylated (negatively charged) gelatin. E50: ethylene diamine-conjugated (positively charged) gelatin. PI9: isoelectric point 9 (weakly positively charged) gelatin. Pullulan-spermine/DNA charge is given in parentheses. Anioplexes showed significantly greater subfectability when seeded on positively charged substrates (*p < 0.05 for Suc vs. E50 and Suc vs. PI9, Newman-Keuls post-hoc). (C) Time course of luciferase reporter gene expression for conventional anioplex transfection (diamonds) and PI9 anioplex subfection (squares). There were no significant differences between conventional anioplex transfection and PI9 anioplex subfection. Although there was an overall significant effect of post-transfection time point on reporter gene expression level (p < 0.05, two-way ANOVA), with a significant decrease at day 20 as compared to day 7 (p < 0.05, Bonferroni post-hoc), when conventional and subfection techniques were considered independently the attenuation of luciferase expression by day 20 was not significant. (D) ASC viability for subfection as determined by WST-8 assay. Pullulan-spermine/DNA charge is given in parentheses. PI9 anioplex subfection showed reduced viability as compared to conventional anioplex transfection, whereas Suc and E50 anioplex subfection appeared to enhance ASC proliferation (all *p < 0.05, Newman-Keuls post-hoc vs. conventional anioplex transfection condition). ASC viability with Suc subfection using positively charged pullulan-spermine/DNA was not different from conventional anioplex subfection, whereas conventional subfection using positively charged pullulan-spermine/DNA resulted in dramatically reduced ASC viability (*p < 0.05, Newman-Keuls post-hoc) (means and standard errors depicted in all graphs; n = 3). Color images available online at www.liebertonline.com/ten.

FIG. 7.

Spinal cord implantation of anioplex-transfected BMSCs. (A) Schematic depiction of injection strategy. Intrathecal injection site under L5 vertebra is indicated with an arrow. Distribution of the injected solution was observed by 30 μL injection of 1% methylene blue dye. Rat was sacrificed after 24 h and spinal cord was imaged in situ. Note subdural distribution of dye throughout the spinal cord. L4 and L5 dorsal root ganglia (DRGs) are indicated with arrows. L3–L6 and L1–L3 zones harvested for luciferase assay are outlined in green and yellow, respectively. (B) At 4 days after minimally invasive lumbar intrathecal injection with 1.2 × 10⁶ BMSCs that had been ex vivo transfected with pullulan-spermine/DNA anioplexes carrying ~40 μg of DNA, luciferase reporter gene activity was detected in spinal cord tissue both in the vicinity of and rostral to the injection site (L3–L6 and L1–L3 zones, respectively), but not in bilateral L4 and L5 DRGs. (C) Luciferase reporter gene activity at 4 days after intrathecal injection with 100 μg luciferase-expressing plasmid DNA. In this case, luciferase activity was detected in the L3–L6 and L1–L3 spinal cord, as well as in the bilateral L4 and L5 DRG. Luciferase activity near the spinal cord injection site (L3–L6) was significantly greater than that observed in the DRGs (3.9-fold, *p < 0.05, Newman-Keuls post-hoc) (means and standard errors depicted in all graphs; n = 4). Color images available online at www.liebertonline.com/ten.