Fluid shear stress promotes embryonic stem cell pluripotency via interplay between ß-catenin and vinculin in bioreactor culture.

The expansion of pluripotent stem cells (PSCs) as aggregates in stirred suspension bioreactors is garnering attention as an alternative to adherent culture. However, the hydrodynamic environment in the bioreactor can modulate PSC behavior, pluripotency and differentiation potential in ways that need to be well understood. In this study, we investigated how murine embryonic stem cells (mESCs) sense fluid shear stress and modulate a noncanonical Wnt signaling response to promote pluripotency. mESCs showed higher expression of pluripotency marker genes, Oct4, Sox2, and Nanog in the absence of leukemia inhibitory factor (LIF) in stirred suspension bioreactors compared to adherent culture, a phenomenon we have termed mechanopluripotency. In bioreactor culture, fluid shear promoted the nuclear translocation of the less well-known pluripotency regulator ß-catenin and concomitant increase of c-Myc expression, an upstream regulator of Oct4, Sox2, and Nanog. We also observed similar ß-catenin nuclear translocation in LIF-free mESCs cultured on E-cadherin substrate under defined fluid shear stress conditions in flow chamber plates. mESCs showed lower shear-induced expression of pluripotency marker genes when ß-catenin was inhibited, suggesting that ß-catenin signaling is crucial to mESC mechanopluripotency. Key to this process is vinculin, which is known to rearrange and associate more strongly with adherens junctions in response to fluid shear. When the vinculin gene is disrupted, we observe that nuclear ß-catenin translocation and mechanopluripotency are abrogated. Our results indicate that mechanotransduction through the adherens junction complex is important for mESC pluripotency maintenance.

Efficiency of novel shielding curtains combined with pulsed irradiation for reducing radiation exposure in an operating room: Human renal collecting system phantom study.

OBJECTIVE: To evaluate the impact of novel shielding curtains combined with pulsed irradiation mode to protect medical radiation workers from radiation exposure during ureteroscopy. METHODS: 0.25 mm Pb equivalent novel shielding curtains were mounted to the caudal and bilateral sides of the operating table in the ureteroscopy setting. C-arm was positioned as per normal in the operating room with the X-ray tube under the patient table. A water-filled anthropomorphic renal collecting system phantom was positioned in the standard position on the operating table that was set at a height of 100 cm. The ionization chambers were also positioned at a height of 100 cm and set in eight positions. We took measurements at distances of 50, 100, 150, and 200 cm from the phantom with the focus directed toward the X-ray tube. We measured the spatial distribution of the scattered radiation dose in four combinations: (1) continuous irradiation mode without novel shielding curtains; (2) pulsed irradiation mode (11 films per second) without novel shielding curtains; (3) continuous irradiation mode with novel shielding curtains; and (4) pulsed irradiation mode with novel shielding curtains. Continuous or pulsed irradiation was activated for 30 s each time. RESULTS: Pulsed irradiation mode with novel shielding curtains was a significantly more efficient method than other combinations to reduce scattered radiation exposure in this study (P < 0.001). There was approximately a 95% reduction in scattered radiation exposure with the pulsed irradiation mode with novel shielding curtains set up as compared with continuous irradiation mode without novel shielding curtains. CONCLUSION: Combining a novel shielding curtain and using a low pulse radiation setting can greatly reduce radiation exposure during ureteroscopic procedures.

Improved transfection efficiency of an aliphatic lipid substituted 2 kDa polyethylenimine is attributed to enhanced nuclear association and uptake in rat bone marrow stromal cell.

BACKGROUND: Lipid substitutions of cationic polymers are actively explored to enhance the efficiency of nonviral gene carriers. We recently took this approach to develop a novel gene carrier by grafting linoleic acid (LA) to relatively biocompatible 2 kDa polyethylenimine (PEI2). The resulting polymer (PEI2LA) displayed improved transfection efficiency over the unmodified PEI2. The intracellular kinetics and distribution of the respective polyplexes were investigated in the present study to gain a better understanding of the role of lipid modification in intracellular trafficking of gene carriers. METHODS: A Cy5-labeled plasmid DNA (pDNA) expressing the green fluorescent protein (GFP) was complexed with PEI2, PEI2LA, and 25 kDa polyethylenimine (PEI25) to transfect rat bone marrow stromal cells (BMSC). Subcellular fractionation was performed to measure the amount of nuclear associated pDNA. pDNA uptake, GFP-expression and nuclear-associated pDNA were measured by both flow cytometry and confocal laser scanning microscopy. RESULTS: PEI2LA mediated higher transgene expression and percentages of transfected cells than PEI25 and PEI2, respectively. There was a strong correlation between nuclear associated pDNA and transgene expression. PEI2LA polyplexes were significantly larger in size than PEI25. The amounts of pDNA associated with the nuclei were greater in PEI2LA than PEI25 polyplexes. The perinuclear pDNA distribution between GFP-expressing and nonGFP-expressing indicated that GFP-positive cells had a higher amount of pDNA associated with their nuclei. CONCLUSIONS: Improved transfection efficiency of PEI2LA was attributed to enhanced association with the nucleus, which may be a result of hydrophobic interaction between the lipid moieties on the modified lipopolymer and the nuclear membrane.

Using computational fluid dynamics (CFD) modeling to understand murine embryonic stem cell aggregate size and pluripotency distributions in stirred suspension bioreactors.

Computational fluid dynamics (CFD) modeling can be applied to understand hydrodynamics in stirred suspension bioreactors, which can in turn affect cell viability, proliferation, pluripotency and differentiation. In this study, we developed a CFD model to determine the effects of average shear rates and turbulent eddies on the formation and growth of murine embryonic stem cell aggregates. We found a correlation between average eddy size and aggregate size, which depended on bioreactor agitation rates. By relating these computational and biological variables, CFD modeling can predict optimal agitation rates to grow embryonic stem cell aggregates in stirred suspension bioreactors. To examine the effect of hydrodynamics on pluripotency, mESCs cultured in bioreactors under various agitation rates were tested for SSEA-1, Sox-2, and Nanog expression. Cells maintained a minimum of 95% positive expression with no change in the intensity distribution pattern between the different bioreactor conditions. This indicates that the average level of pluripotency marker expression is independent of changes in the hydrodynamic profile and resulting aggregate size distribution. The findings here can be further extended to other cell types that grow as aggregates in stirred suspension bioreactors and offer important insights necessary to realize cell therapies.

Effects of size and topology of DNA molecules on intracellular delivery with non-viral gene carriers.

BACKGROUND: Efforts to improve the efficiency of non-viral gene delivery require a better understanding of delivery kinetics of DNA molecules into clinically relevant cells. Towards this goal, three DNA molecules were employed to investigate the effects of DNA properties on cellular delivery: a circular plasmid DNA (c-DNA), a linearized plasmid DNA (l-DNA) formulated by single-site digestion of c-DNA, and smaller linear gene cassette generated by PCR (pcr-DNA). Four non-viral gene carriers were investigated for DNA delivery: polyethyleneimine (PEI), poly-L-Lysine (PLL), palmitic acid-grafted PLL (PLL-PA), and Lipofectamine-2000. Particle formation, binding and dissociation characteristics, and DNA uptake by rat bone marrow stromal cells were investigated. RESULTS: For individual carriers, there was no discernible difference in the morphology of particles formed as a result of carrier complexation with different DNA molecules. With PEI and PLL carriers, no difference was observed in the binding interaction, dissociation characteristics, and DNA uptake among the three DNA molecules. The presence of serum in cell culture media did not significantly affect the DNA delivery by the polymeric carriers, unlike other lipophilic carriers. Using PEI as the carrier, c-DNA was more effective for transgene expression as compared to its linear equivalent (l-DNA) by using the reporter gene for Enhanced Green Fluorescent Protein. pcr-DNA was the least effective despite being delivered into the cells to the same extent. CONCLUSION: We conclude that the nature of gene carriers was the primary determinant of cellular delivery of DNA molecules, and circular form of the DNA was more effectively processed for transgene expression.

Further investigation of lipid-substituted poly(L-Lysine) polymers for transfection of human skin fibroblasts.

Enabling gene expression in skin fibroblasts using safe, nonviral gene delivery has the potential to stimulate wound healing and aid in skin tissue engineering efforts. In this study, several lipid-substituted poly(L-Lysines) (PLL) were investigated for their ability to deliver a plasmid DNA (pEGFP) to human skin fibroblasts. While native and lipid-substituted PLLs showed complete complexation with pEGFP, polymers with higher lipid substitution were more resilient to dissociation after heparin treatment. All polymers showed good protection of pEGFP against DNase I and DNase II digestion in vitro. DNA delivery studies using fluorescently labeled pEGFP showed that native PLL lacked the ability to deliver pEGFP into cells, whereas most of the lipid-substituted PLLs gave efficient pEGFP delivery into the cells. Extent of lipid substitution was an important factor in DNA delivery efficiency. The intracellular pEGFP was intact after delivery with lipid-substituted polymers up to 7 days. An RT-PCR methodology indicated successful transcription of the reporter GFP gene, which was not the case when the cells were transfected with a blank plasmid containing no functional GFP gene. Further studies with flow cytometry showed that successful protein expression was obtained with PLLs substituted with myristic and stearic acid, the latter displaying a relatively lower toxicity. We conclude that substituting lipids on PLL results in effective gene carriers and the extent of substitution, rather than the individual lipid, appeared to be critical for effective plasmid delivery.

A simple and rapid nonviral approach to efficiently transfect primary tissue-derived cells using polyethylenimine.

This protocol outlines steps for optimizing the transfection of adherent primary mammalian cells using the readily available off-the-shelf cationic polymer, 25-kDa branched polyethylenimine (bPEI25). Transfection efficiency of cationic polymers varies among cell lines and is highly dependent on the conditions and environment in which complexes are formed. Factors requiring optimization include the salt concentration, volume, incubation time, mixing order and ratio of polymer to DNA. In this transfection protocol, complexes are prepared in 30 min, with analysis 24 h later; thus, experiments can be completed in 2 d. In this protocol, as an example, we describe the parameters we have optimized for the transfection of bone marrow stromal cells and normal human foreskin fibroblasts. By using this protocol, we have obtained transfection efficiencies comparable to lipofection. An appropriately optimized protocol enhances the utility of cationic polymers in transfecting mammalian cells, thereby providing an effective alternative to expensive commercial reagents.

Nucleic-acid based gene therapeutics: delivery challenges and modular design of nonviral gene carriers and expression cassettes to overcome intracellular barriers for sustained targeted expression.

The delivery of nucleic acid molecules into cells to alter physiological functions at the genetic level is a powerful approach to treat a wide range of inherited and acquired disorders. Biocompatible materials such as cationic polymers, lipids, and peptides are being explored as safer alternatives to viral gene carriers. However, the comparatively low efficiency of nonviral carriers currently hampers their translation into clinical settings. Controlling the size and stability of carrier/nucleic acid complexes is one of the primary hurdles as the physicochemical properties of the complexes can define the uptake pathways, which dictate intracellular routing, endosomal processing, and nucleocytoplasmic transport. In addition to nuclear import, subnuclear trafficking, posttranscriptional events, and immune responses can further limit transfection efficiency. Chemical moieties, reactive linkers or signal peptide have been conjugated to carriers to prevent aggregation, induce membrane destabilization and localize to subcellular compartments. Genetic elements can be inserted into the expression cassette to facilitate nuclear targeting, delimit expression to targeted tissue, and modulate transgene expression. The modular option afforded by both gene carriers and expression cassettes provides a two-tier multicomponent delivery system that can be optimized for targeted gene delivery in a variety of settings.