Temporal changes guided by mesenchymal stem cells on a 3D microgel platform enhance angiogenesis in vivo at a low-cell dose.

Therapeutic factors secreted by mesenchymal stem cells (MSCs) promote angiogenesis in vivo. However, delivery of MSCs in the absence of a cytoprotective environment offers limited efficacy due to low cell retention, poor graft survival, and the nonmaintenance of a physiologically relevant dose of growth factors at the injury site. The delivery of stem cells on an extracellular matrix (ECM)-based platform alters cell behavior, including migration, proliferation, and paracrine activity, which are essential for angiogenesis. We demonstrate the biophysical and biochemical effects of preconditioning human MSCs (hMSCs) for 96 h on a three-dimensional (3D) ECM-based microgel platform. By altering the macromolecular concentration surrounding cells in the microgels, the proangiogenic phenotype of hMSCs can be tuned in a controlled manner through cell-driven changes in extracellular stiffness and "outside-in" integrin signaling. The softest microgels were tested at a low cell dose (5 × 104 cells) in a preclinical hindlimb ischemia model showing accelerated formation of new blood vessels with a reduced inflammatory response impeding progression of tissue damage. Molecular analysis revealed that several key mediators of angiogenesis were up-regulated in the low-cell-dose microgel group, providing a mechanistic insight of pathways modulated in vivo. Our research adds to current knowledge in cell-encapsulation strategies by highlighting the importance of preconditioning or priming the capacity of biomaterials through cell-material interactions. Obtaining therapeutic efficacy at a low cell dose in the microgel platform is a promising clinical route that would aid faster tissue repair and reperfusion in "no-option" patients suffering from peripheral arterial diseases, such as critical limb ischemia (CLI).

Mental health and wellbeing interventions in the dental sector: a systematic review.

Introduction UK dentists experience high levels of stress, anxiety and burnout. Poor mental health can lead practitioners to exit the profession, contributing to workforce and service loss. Therefore, there is a need to focus on interventions to protect the mental health and wellbeing of dental teams. Three levels of intervention can be deployed in the workplace to support mental health and wellbeing: primary prevention, secondary prevention, and tertiary prevention.Aim The aim of this systematic review was to identify evidence on interventions used to prevent, improve or tackle mental health issues among dental team members and dental profession students in countries of very high development.Methods This systematic review was conducted according to a predefined protocol and reported according to PRISMA guidelines. The MEDLINE, Embase CINAHL, DOSS, Scopus, and PsycINFO databases were searched. Prospective empirical studies were considered for inclusion. The Effective Public Health Practice Project Quality Assessment Tool (EPHPP) was used to assess the methodological quality of the included studies. The identified interventions were categorised according to level of prevention.Results The search yielded 12,919 results. Eight studies met the inclusion criteria. All of the studies concerned dentists or dental students. There were no studies for other groups of dental professionals. No primary prevention-level studies were identified. Secondary prevention-level studies (n = 4) included various psychoeducational interventions aiming to raise awareness and improve coping skills and led to significant improvements in stress levels and burnout of dentists and dental students. Tertiary prevention-level studies (n = 4) mainly employed counselling which was shown to be beneficial for dentists and students experiencing psychological ill-health.Conclusions Mental wellbeing awareness should be put at the centre of dental education and the workplace. Leadership and innovation are required to design primary-level interventions which can be implemented in the UK dental sector, with its distinct organisational and service characteristics.

Scaffold-based delivery of mesenchymal stromal cells to diabetic wounds.

Foot ulceration is a major complication of diabetes mellitus, which results in significant human suffering and a major burden on healthcare systems. The cause of impaired wound healing in diabetic patients is multifactorial with contributions from hyperglycaemia, impaired vascularization and neuropathy. Patients with non-healing diabetic ulcers may require amputation, creating an urgent need for new reparative treatments. Delivery of stem cells may be a promising approach to enhance wound healing because of their paracrine properties, including the secretion of angiogenic, immunomodulatory and anti-inflammatory factors. While a number of different cell types have been studied, the therapeutic use of mesenchymal stromal cells (MSCs) has been widely reported to improve delayed wound healing. However, topical administration of MSCs via direct injection has several disadvantages, including low cell viability and poor cell localization at the wound bed. To this end, various biomaterial conformations have emerged as MSC delivery vehicles to enhance cell viability and persistence at the site of implantation. This paper discusses biomaterial-based MSCs therapies in diabetic wound healing and highlights the low conversion rate to clinical trials and commercially available therapeutic products.

Signaling integration in the rugae growth zone directs sequential SHH signaling center formation during the rostral outgrowth of the palate.

Evolution of facial morphology arises from variation in the activity of developmental regulatory networks that guide the formation of specific craniofacial elements. Importantly, the acquisition of novel morphology must be integrated with a phylogenetically inherited developmental program. We have identified a unique region of the secondary palate associated with the periodic formation of rugae during the rostral outgrowth of the face. Rugae function as SHH signaling centers to pattern the elongating palatal shelves. We have found that a network of signaling genes and transcription factors is spatially organized relative to palatal rugae. Additionally, the first formed ruga is strategically positioned at the presumptive junction of the future hard and soft palate that defines anterior-posterior differences in regional growth, mesenchymal gene expression, and cell fate. We propose a molecular circuit integrating FGF and BMP signaling to control proliferation and differentiation during the sequential formation of rugae and inter-rugae domains in the palatal epithelium. The loss of p63 and Sostdc1 expression and failed rugae differentiation highlight that coordinated epithelial-mesenchymal signaling is lost in the Fgf10 mutant palate. Our results establish a genetic program that reiteratively organizes signaling domains to coordinate the growth of the secondary palate with the elongating midfacial complex.

Enhanced lipoplex-mediated gene expression in mesenchymal stem cells using reiterated nuclear localization sequence peptides.

BACKGROUND: Mesenchymal stem cells (MSC) are widely regarded as a promising tool for cellular therapy applications, and genetic modification by safe, liposome-based vectors may enhance their therapeutic potential. METHODS: The present study describes the use of a cationic lipid vector (Lipofectamine 2000) to deliver genes to MSC isolated from a number of species in vitro and determined the characteristics of this vector system in terms of dose, toxicity and the time course of expression. In addition, the optimal use of a nuclear localization sequence (NLS) to enhance gene expression was explored. RESULTS: Lipofection of human MSC did not adversely affect their ability to differentiate into osteogenic- and adipogenic lineages. Although human and rat MSC were found to take up lipoplexes with relative efficiency, lower levels of gene expression were detected in rabbit MSC, demonstrating a crucial effect of species. Peptides containing reiterated motifs of NLS were found to significantly improve on the level of transgene expression. Optimal gene delivery was observed when a three-fold reiterated NLS sequence was included in the liposome formulation. CONCLUSIONS: Thus, nonviral gene delivery to MSC is feasible with efficiency being species dependent and can be enhanced by use of a three-fold reiterated NLS.

Impact of time pressure on dentists' diagnostic performance.

OBJECTIVES: Although stress is prevalent amongst dentists, there is a paucity of data on the impact of stressors on dentists' clinical performance. To address this gap in the literature, the aim of the present study was to explore the role of time pressure, representing one common stressor, on dentists' radiographic diagnostic performance. METHODS: Forty dentists were randomised to examine and provide a radiographic report on two sets of radiographs (six bitewings in each set) under two conditions on a cross-over basis: time-pressure vs. no-time-pressure. The radiographic report of an experienced consultant was considered the gold standard against which participants diagnostic decisions were compared to calculate sensitivity and specificity. Participants rated their stress after each experimental condition using a 100 mm visual analogue scale (VAS). RESULTS: The VAS scores for stress were significantly higher in the time pressure condition vs. no time pressure condition (mean: 55.78 versus 10.73, p < 0.0001), indicating that the time pressure acted as a source of stress. Dentists' diagnostic performance was affected; the sensitivity was significantly lower under time pressure (median: 0.50 versus 0.80, p < 0.0001), but by contrast, the median diagnostic specificity was 1.00 under both conditions. CONCLUSIONS: Time pressure negatively impacts one aspect of dentists' diagnostic performance, namely sensitivity (increased diagnostic errors and omissions of pathology), which can potentially affect patient safety and the quality of care delivered. However, time pressure was found to have less influence on diagnostic specificity. CLINICAL SIGNIFICANCE: The present study demonstrated a significant deterioration of dentists' diagnostic performance (sensitivity) under time-pressure when examining bitewing radiographs. Diagnostic errors may put patient safety at risk, with patients potentially being harmed if pathology is missed. Such errors can have medicolegal implications on the dentists' practice.

A dosage-dependent role for Spry2 in growth and patterning during palate development.

The formation of the palate involves the coordinated outgrowth, elevation and midline fusion of bilateral shelves leading to the separation of the oral and nasal cavities. Reciprocal signaling between adjacent fields of epithelial and mesenchymal cells directs palatal shelf growth and morphogenesis. Loss of function mutations in genes encoding FGF ligands and receptors have demonstrated a critical role for FGF signaling in mediating these epithelial-mesenchymal interactions. The Sprouty family of genes encode modulators of FGF signaling. We have established that mice carrying a deletion that removes the FGF signaling antagonist Spry2 have cleft palate. We show that excessive cell proliferation in the Spry2-deficient palate is accompanied by the abnormal progression of shape changes and movements required for medially directed shelf outgrowth and midline contact. Expression of the FGF responsive transcription factors Etv5, Msx1, and Barx1, as well as the morphogen Shh, is restricted to specific regions of the developing palate. We detected elevated and ectopic expression of these transcription factors and disorganized Shh expression in the Spry2-deficient palate. Mice carrying a targeted disruption of Spry2 fail to complement the craniofacial phenotype characterized in Spry2 deletion mice. Furthermore, a Spry2-BAC transgene rescues the palate defect. However, the BAC transgenic mouse lines express reduced levels of Spry2. The resulting hypomorphic phenotype demonstrates that palate development is Spry2 dosage sensitive. Our results demonstrate the importance of proper FGF signaling thresholds in regulation of epithelial-mesenchymal interactions and cellular responses necessary for coordinated morphogenesis of the face and palate.

The characteristics of gait in Charcot-Marie-Tooth disease types I and II.

Certain typical gait characteristics such as foot-drop and foot supination are well described in Charcot-Marie-Tooth disease. These are directly related to the primary disease and due to the weakness of ankle dorsiflexors and everters characteristic of this hereditary neuropathy. We analysed 16 subjects aged 8-52 years old (11 with type I, 5 with type II Charcot-Marie-Tooth disease) using three-dimensional gait analysis and identified kinematic features previously unreported. These patients showed a combination of tight tendo achillei, foot-drop, failure of plantar flexion and increased foot supination, but also presented with excessive internal rotation of the knee and/or tibia, knee hyperextension in stance, excessive external rotation at the hips and decreased hip adduction in stance (typical of a broad based gait). These proximal features could have been an adaptation to or consequence of the disrupted ankle and foot biomechanics, however a direct relation to the neuropathy is also possible since sub-normal muscle power was observed at the proximal levels in most subjects on both manual testing and kinetic analysis. Gait analysis is a useful tool in defining the characteristic gait of patients with Charcot-Marie-Tooth disease.

Rapid Induction of Cerebral Organoids From Human Induced Pluripotent Stem Cells Using a Chemically Defined Hydrogel and Defined Cell Culture Medium.

UNLABELLED: Tissue organoids are a promising technology that may accelerate development of the societal and NIH mandate for precision medicine. Here we describe a robust and simple method for generating cerebral organoids (cOrgs) from human pluripotent stem cells by using a chemically defined hydrogel material and chemically defined culture medium. By using no additional neural induction components, cOrgs appeared on the hydrogel surface within 10-14 days, and under static culture conditions, they attained sizes up to 3 mm in greatest dimension by day 28. Histologically, the organoids showed neural rosette and neural tube-like structures and evidence of early corticogenesis. Immunostaining and quantitative reverse-transcription polymerase chain reaction demonstrated protein and gene expression representative of forebrain, midbrain, and hindbrain development. Physiologic studies showed responses to glutamate and depolarization in many cells, consistent with neural behavior. The method of cerebral organoid generation described here facilitates access to this technology, enables scalable applications, and provides a potential pathway to translational applications where defined components are desirable. SIGNIFICANCE: Tissue organoids are a promising technology with many potential applications, such as pharmaceutical screens and development of in vitro disease models, particularly for human polygenic conditions where animal models are insufficient. This work describes a robust and simple method for generating cerebral organoids from human induced pluripotent stem cells by using a chemically defined hydrogel material and chemically defined culture medium. This method, by virtue of its simplicity and use of defined materials, greatly facilitates access to cerebral organoid technology, enables scalable applications, and provides a potential pathway to translational applications where defined components are desirable.

Inefficiency in macromolecular transport of SCS-based microcapsules affects viability of primary human mesenchymal stem cells but not of immortalized cells.

Microcapsules made of sodium cellulose sulphate (SCS) and poly-diallyl-dimethyl-ammonium chloride (pDADMAC) have been employed to encapsulate a wide range of established cell lines for several applications. However, little is known about the encapsulation of primary cells including human mesenchymal stem cells (hMSCs). Human MSCs are of interest in regenerative medicine applications due to pro-angiogenic, anti-inflammatory and immunomodulatory properties, which result from paracrine effects of this cell type. In the present work we have encapsulated primary hMSCs and hMSC-TERT immortalized cells and compared their behavior and in vitro angiogenic potential. We found that, although both cell types were able to secret angiogenic factors such as VEGF, there was a marked reduction of primary hMSC viability compared to hMSC-TERT cells when cultured in these microcapsules. Moreover, this applied to other primary cell cultures such as primary human fibroblasts but not to other cell lines such as human embryonic kidney 293 (HEK293) cells. We found that the microcapsule membrane had a molecular weight cut-off below a critical size, which caused impairment in the diffusion of essential nutrients and had a more detrimental effect on the viability of primary cell cultures compared to cell lines and immortalized cells.

Nano-engineered mesenchymal stem cells as targeted therapeutic carriers.

Poor availability in deep-seated solid tumors is a significant challenge that limits the effectiveness of currently used anticancer drugs. Approaches that can specifically enhance drug delivery to the tumor tissue can potentially improve therapeutic efficacy. In our current studies, we used nano-engineered mesenchymal stem cells (nano-engineered MSCs) as tumor-targeted therapeutic carriers. In addition to their exquisite tumor homing capabilities, MSCs overexpress efflux transporters such as P-glycoprotein and are highly drug resistant. The inherent tumor-tropic and drug-resistant properties make MSCs ideal carriers for toxic payload. Nano-engineered MSCs were prepared by treating human MSCs with drug-loaded polymeric nanoparticles. Incorporating nanoparticles in MSCs did not affect their viability, differentiation or migration potential. Nano-engineered MSCs induced dose-dependent cytotoxicity in A549 lung adenocarcinoma cells and MA148 ovarian cancer cells in vitro. An orthotopic A549 lung tumor model was used to monitor the in vivo distribution of nanoengineered MSCs. Intravenous injection of nanoparticles resulted in non-specific biodistribution, with significant accumulation in the liver and spleen while nano-engineered MSCs demonstrated selective accumulation and retention in lung tumors. These studies demonstrate the feasibility of developing nano-engineered MSCs loaded with high concentration of anticancer agents without affecting their tumor-targeting or drug resistance properties.

A shape-controlled tuneable microgel platform to modulate angiogenic paracrine responses in stem cells.

Development of cell delivery platforms have been driven based on an empirical cytoprotective design. While cell-matrix and cell-cell interactions that influence biochemical effects beyond survival has been limited and overshadowed in an effort to incrementally improve biomimicking properties of the tissue-engineered constructs. Here we demonstrate fabrication of a shape controlled 3D type-I collagen-based microgel platform that can be tuned to modulate angiogenic paracrine- 'angiocrine' responses of human mesenchymal stem cells (hMSCs). Furthermore, these microgels were characterized as a 3D cell culture tool to assess optimal biological response as a function of cell-matrix and cell-cell interactions. Finally, optimised hMSC embedded microgels were shown to induce vascular repair and functional improvement in vivo in a mouse model of hind-limb ischemia. The approach described here in designing a tuneable cell delivery platform using naturally occurring extracellular matrix molecules highlights the need for highly customised matrices with an array of self-assembling proteins that dictate specific cell function resembling the native tissue of interest for repair.

Comparison of cellular architecture, axonal growth, and blood vessel formation through cell-loaded polymer scaffolds in the transected rat spinal cord.

The use of multichannel polymer scaffolds in a complete spinal cord transection injury serves as a deconstructed model that allows for control of individual variables and direct observation of their effects on regeneration. In this study, scaffolds fabricated from positively charged oligo[poly(ethylene glycol)fumarate] (OPF(+)) hydrogel were implanted into rat spinal cords following T9 complete transection. OPF(+) scaffold channels were loaded with either syngeneic Schwann cells or mesenchymal stem cells derived from enhanced green fluorescent protein transgenic rats (eGFP-MSCs). Control scaffolds contained extracellular matrix only. The capacity of each scaffold type to influence the architecture of regenerated tissue after 4 weeks was examined by detailed immunohistochemistry and stereology. Astrocytosis was observed in a circumferential peripheral channel compartment. A structurally separate channel core contained scattered astrocytes, eGFP-MSCs, blood vessels, and regenerating axons. Cells double-staining with glial fibrillary acid protein (GFAP) and S-100 antibodies populated each scaffold type, demonstrating migration of an immature cell phenotype into the scaffold from the animal. eGFP-MSCs were distributed in close association with blood vessels. Axon regeneration was augmented by Schwann cell implantation, while eGFP-MSCs did not support axon growth. Methods of unbiased stereology provided physiologic estimates of blood vessel volume, length and surface area, mean vessel diameter, and cross-sectional area in each scaffold type. Schwann cell scaffolds had high numbers of small, densely packed vessels within the channels. eGFP-MSC scaffolds contained fewer, larger vessels. There was a positive linear correlation between axon counts and vessel length density, surface density, and volume fraction. Increased axon number also correlated with decreasing vessel diameter, implicating the importance of blood flow rate. Radial diffusion distances in vessels significantly correlated to axon number as a hyperbolic function, showing a need to engineer high numbers of small vessels in parallel to improving axonal densities. In conclusion, Schwann cells and eGFP-MSCs influenced the regenerating microenvironment with lasting effect on axonal and blood vessel growth. OPF(+) scaffolds in a complete transection model allowed for a detailed comparative, histologic analysis of the cellular architecture in response to each cell type and provided insight into physiologic characteristics that may support axon regeneration.

Liposomal surface coatings of metal stents for efficient non-viral gene delivery to the injured vasculature.

Despite the widespread use of drug eluting stents (DES), in-stent restenosis (ISR), delayed arterial healing and thrombosis remain important clinical complications. Gene-eluting stents (GES) represent a potential strategy for the prevention of ISR by delivering a therapeutic gene via a vector from the stent surface to the vessel wall. To this end, a model in vitro system was established to examine whether cationic liposomes could be used for gene delivery to human artery cells. Three different formulations were compared (DOTMA/DOPE, DDAB/DOPE or DDAB/POPC/Chol) to examine the effects of different cationic and neutral lipids on the transfection efficiency of lipoplex-coatings of metal surfaces. Upon completion of the characterization and optimization of the materials for gene delivery in vitro, these coatings were examined on a range of stents and deployed in a rabbit iliac artery injury model in vivo. Maximal transfection efficiencies for all coatings were observed on day 28, followed by declining, but persisting gene expression 42 days after stent placement, thereby, presenting liposomal coatings for gene eluting stents as treatment options for clinical complications associated with stenting procedures.

High levels of ephrinB2 over-expression increases the osteogenic differentiation of human mesenchymal stem cells and promotes enhanced cell mediated mineralisation in a polyethyleneimine-ephrinB2 gene-activated matrix.

Gene therapy can be combined with tissue engineering constructs to produce gene-activated matrices (GAMs) with enhanced capacity for repair. Polyethyleneimine (PEI), a non-viral vector, has previously been optimised for high efficiency gene transfer in rat mesenchymal stem cells (rMSCs). The use of PEI to transfect human MSCs (hMSCs) with ephrinB2 is assessed here. Recently a role for the ephrinB2 ligand and EphB4 receptor duo has been proposed in bone remodelling. Herein, over-expression of the ephrinB2 ligand resulted in increased osteogenic differentiation in hMSCs. As ephrinB2 is a cell surface anchored ligand which only interacts with cells expressing the cognate EphB4 receptor through direct contact, we have shown that direct cell-cell contact between two neighbouring cells is responsible for enhanced osteogenesis. In an effort to begin to elucidate the molecular mechanisms at play downstream of ephrinB2 over-expression, RT-PCR was performed on the GAMs which revealed no significant changes in runx2 or BMP2 expression but an upregulation of osterix (Osx) and Dlx5 expression prompting the belief that the mode of osteogenesis is independent of the BMP2 pathway. This select interaction, coupled with the transient gene expression profile of PEI, makes the PEI-ephrinB2 GAM an ideal candidate matrix for a bone targeted GAM.

Depressive symptoms and diurnal salivary cortisol patterns among female caregivers of stroke survivors.

Informal caregivers of stroke survivors experience elevated chronic stress and are at risk of developing depressive symptoms. The cumulative effects of chronic stress can increase allostatic load and dysregulate biological processes, thus increasing risk of stress-related disease. Stress-induced alterations in the pattern of cortisol secretion vary with respect to stressor onset, intensity, and chronicity. Little is known about the psychoendocrine response to stress in female caregivers of stroke survivors. The purpose of this study was to examine perceived stress, caregiver burden, and the association between caregiver depressive symptoms and diurnal cortisol in 45 females caring for a significant other who experienced a stroke within the past year. Women completed the Center for Epidemiologic Studies Depression Scale (CES-D) and collected saliva for cortisol upon awakening, 30 min postawakening, noon, and bedtime for 2 consecutive days. Results revealed that women had high levels of perceived stress and caregiver burden. In women with CES-D scores ≥ 16, salivary cortisol levels were significantly lower across the day relative to women with CES-D scores < 16. This difference persisted after adjusting for age, number of caregiving hours per week, perceived social support, and quality of sleep. Younger age was associated with more depressive symptoms as well as lower levels of cortisol at awakening and 30 min postawakening. Results demonstrate that the burden of caregiving increases risk of depressive symptoms and hypocortisolism across the day. Hypocortisolism may contribute to increased risk of depressive symptoms as a result of the loss of glucocorticoid attenuation of stress-induced inflammation.

Liposomal gene delivery mediated by tissue-engineered scaffolds.

In the absence of any ideal gene delivery carrier despite the recent explosion of novel carrier systems, the current trend is to explore the complementary synergy promised by a combination of delivery systems such as liposomes, which are the most widely researched versatile non-viral carriers, and tissue-engineered scaffolds with macrostructures of defined architecture comprised of natural or synthetic macromolecules. Here, we discuss the recent advances in liposomal gene delivery and the possible benefits of a combined liposome-scaffold approach, such as long-term expression, enhanced stability, reduction in toxicity and ability to produce spatio-temporal expression patterns. This approach is generating significant impact in the field as a result of its potential for extended localised gene delivery for applications in a variety of clinical conditions.

Current tissue engineering and novel therapeutic approaches to axonal regeneration following spinal cord injury using polymer scaffolds.

This review highlights current tissue engineering and novel therapeutic approaches to axonal regeneration following spinal cord injury. The concept of developing 3-dimensional polymer scaffolds for placement into a spinal cord transection model has recently been more extensively explored as a solution for restoring neurologic function after injury. Given the patient morbidity associated with respiratory compromise, the discrete tracts in the spinal cord conveying innervation for breathing represent an important and achievable therapeutic target. The aim is to derive new neuronal tissue from the surrounding, healthy cord that will be guided by the polymer implant through the injured area to make functional reconnections. A variety of naturally derived and synthetic biomaterial polymers have been developed for placement in the injured spinal cord. Axonal growth is supported by inherent properties of the selected polymer, the architecture of the scaffold, permissive microstructures such as pores, grooves or polymer fibres, and surface modifications to provide improved adherence and growth directionality. Structural support of axonal regeneration is combined with integrated polymeric and cellular delivery systems for therapeutic drugs and for neurotrophic molecules to regionalize growth of specific nerve populations.

Comparison of viral and nonviral vectors for gene transfer to human endothelial progenitor cells.

BACKGROUND/AIMS: The ability of endothelial progenitor cells (EPCs) to home to sites of neoangiogenesis makes them attractive candidates for use in the field of gene therapy. The efficacy of this approach depends on the efficiency of the vector used for transgene delivery. METHODS/RESULTS: In this study, we have compared the efficiency of adenovirus, five serotypes of AAV2, VSVG-pseudotyped lentivirus, and nonviral plasmid/liposome DNA vectors to deliver the green fluorescence protein reporter gene to human early EPCs to determine efficacy and vector-related cell toxicity. Adenovirus proved most effective with efficiencies of up to 80% with low levels of cell death. Lower levels of expression were seen with other vectors. Electroporation proved unsuitable at the parameters tested. We have also identified at least two distinct subpopulations that exist in the heterogeneous parent EPC culture, one of which is amenable to transduction with adenovirus and one that is not. In addition, adenoviral transduction did not disrupt the ability of the cells to incorporate into endothelial structures in vitro. CONCLUSION: We have found adenovirus to be the most efficient of the vector systems tested for gene delivery to EPCs, an effect that is mediated almost entirely by one of two identified subpopulations.

Comparative evaluation of viral, nonviral and physical methods of gene delivery to normal and transformed lung epithelial cells.

Few studies have directly compared the efficiencies of gene delivery methods that target normal lung cells versus lung tumor cells. We report the first study directly comparing the efficiency and toxicity of viral [adeno-associated virus (AAV2, 5, 6) and lentivirus], nonviral (Effectene, SuperFect and Lipofectamine 2000) and physical [particle-mediated gene transfer (PMGT)] methods of gene delivery in normal mouse lung cells and in mouse adenocarcinoma cells. Lentivirus pseudotyped with the vesicular stomatitis virus glycoprotein was the most efficient gene transfer method for normal mouse airway epithelial cells [25.95 (+/-3.57) %] whereas AAV6 was most efficient for MLE-12 adenocarcinoma cells [68.2 (+/-3.2) %]. PMGT was more efficient in normal mouse airway epithelial cells than AAV5, Lipofectamine 2000 and SuperFect. AAV5 displayed the lowest transfection efficiency at less than 10% in both cell types. PMGT was the only method that resulted in significant toxicity. In summary, for all of the gene delivery methods examined here, lung tumor cells were transfected more easily than normal lung cells. Lipofectamine 2000 is potentially highly selective for lung tumor cells whereas AAV6 and lentivirus vesicular stomatitis virus glycoprotein may be useful for gene delivery strategies that require targeting of both normal and tumor cells.