Stelios T. Andreadis

Expression of multiple genes from the same target cell is required in several technological and therapeutic applications such as quantitative measurements of promoter activity or in vivo tracking of stem cells. In spite of such need, reaching independent and high-level dual-gene expression cannot be reliably accomplished by current gene transfer vehicles. To address this issue, we designed a lentiviral vector carrying two transcriptional units separated by polyadenylation, terminator and insulator sequences. With this design, the expression level of both genes was as high as that yielded from lentiviral vectors containing only a single transcriptional unit. Similar results were observed with several promoters and cell types including epidermal keratinocytes, bone marrow mesenchymal stem cells and hair follicle stem cells. Notably, we demonstrated quantitative dynamic monitoring of gene expression in primary cells with no need for selection protocols suggesting that this optimized lentivirus may be useful in high-throughput gene expression profiling studies.

DNA microarrays have the potential to revolutionize medical diagnostics and development of individualized medical treatments. However, accurate quantification of scantily expressed genes and precise measurement of small differences between different treatments is not currently feasible. A major challenge remains the understanding of physicochemical processes and rate-limiting steps of hybridization of complex mixtures of DNA targets on immobilized DNA probes. To this end, we developed a mathematical model to describe the effects of molecular orientation and transport on the kinetics and efficiency of hybridization. First, we calculated the hybridization rate constant based on the distance between the complementary nucleotides of the target and probe DNA. The surface reaction rate was then integrated with translational and rotational transport of target DNA to the surface to calculate the kinetics of hybridization. Our model predicts that hybridization of short DNA targets is diffusion limited but long targets are kinetically limited. In addition, for DNA targets with wide size distribution, it may be difficult to distinguish between specific binding of long targets from nonspecific binding of short ones. Our model provides novel insight into the process of DNA hybridization and suggests operating conditions to improve the sensitivity and accuracy of microarray experiments.

We hypothesized that hair-follicle stem cells can differentiate toward smooth contractile muscle cells, providing an autologous cell source for cardiovascular tissue regeneration. Smooth muscle progenitor cells (SMPCs) were obtained from ovine hair follicles using a tissue-specific promoter and fluorescence-activated cell sorting. Hair-follicle smooth muscle progenitor cells (HF-SMPCs) expressed several markers of vascular smooth muscle including α-actin, calponin, myosin heavy chain (MHC), caldesmon, smoothelin, and SM22. HF-SMPCs were highly proliferative and showed high clonogenic potential without any signs of chromosomal abnormalities as evidenced by karyotype analysis. HF-SMPCs compacted fibrin hydrogels to a similar extent as vascular smooth muscle cells from ovine umbilical veins (V-SMCs), indicating the development of the force-generating machinery. In addition, cylindrical tissue equivalents prepared with HF-SMPCs displayed significant contractility in response to vasoactive agonists including KCl and the thromboxane A2 mimetic U46619, suggesting that these cells had developed receptor and non-receptor-mediated pathways of contractility. Finally, transforming growth factor-β1 promoted differentiation of HF-SMPCs toward a mature SMC phenotype as suggested by increased expression of MHC and enhanced matrix compaction. Our results suggest that hair follicles may be an easily accessible, autologous, and rich source of functional SMPC for cardiovascular tissue engineering and regenerative medicine.

Objectives We recently demonstrated that fibrin-based tissue engineered blood vessels (TEV) exhibited vascular reactivity, matrix remodeling and sufficient strength for implantation into the veins of an ovine animal model, where they remained patent for 15 weeks. Here we present an approach to improve the mechanical properties of fibrin-based TEV and examine the relationship between mechanical strength and smooth muscle cell (SMC) function. Materials and Methods To this end, we prepared TEV that were composed of two layers: a cellular layer containing SMC embedded in fibrin hydrogel to provide contractility and matrix remodeling; and a second cell-free fibrin layer composed of high concentration fibrinogen to provide mechanical strength. Results The ultimate tensile force of double-layered TEV increased with FBG concentration in the cell-free layer in a dose-dependent manner. Double-layered TEV exhibited burst pressure that was ten-fold higher than single-layered tissues but vascular reactivity remained high even though the cells were constricting an additional tissue layer. Conclusion These results showed that mechanical strength results largely from the biomaterial but contractility requires active cellular machinery. Consequently, they may suggest novel approaches for engineering biomaterials that satisfy the requirement for high mechanical strength while preserving SMC function.

During wound healing, cells recreate functional structures to regenerate the injured tissue. Understanding the healing process is essential for the development of new concepts and the design of novel biomimetic approaches for delivery of cells, genes and growth factors to accelerate tissue regeneration. To this end, realistic experimental models and high-throughput diagnostics are necessary to understand the molecular mechanisms of healing and reveal the genetic networks that determine tissue repair versus regeneration. Following a brief overview of the biology of wound healing, this review covers the in vitro and in vivo models that are employed at present to study the healing process. Discussion then covers the application of high-throughput genomic and proteomic technologies in epithelial development, living skin substitutes and wound healing. Finally, this review provides a perspective on novel technologies that should be developed to facilitate the understanding of wound healing complications and the...

Tissue engineering combines the principles of cell biology, engineering and materials science to develop three-dimensional tissues to replace or restore tissue function. Tissue engineered skin is one of most advanced tissue constructs, yet it lacks several important functions including those provided by hair follicles, sebaceous glands, sweat glands and dendritic cells. Although the complexity of skin may be difficult to recapitulate entirely, new or improved functions can be provided by genetic modification of the cells that make up the tissues. Gene therapy can also be used in wound healing to promote tissue regeneration or prevent healing abnormalities such as formation of scars and keloids. Finally, gene-enhanced skin substitutes have great potential as cell-based devices to deliver therapeutics locally or systemically. Although significant progress has been made in the development of gene transfer technologies, several challenges have to be met before clinical application of genetically modified skin tissue. Engineering challenges include methods for improved efficiency and targeted gene delivery; efficient gene transfer to the stem cells that constantly regenerate the dynamic epidermal tissue; and development of novel biomaterials for controlled gene delivery. In addition, advances in regulatable vectors to achieve spatially and temporally controlled gene expression by physiological or exogenous signals may facilitate pharmacological administration of therapeutics through genetically engineered skin. Gene modified skin substitutes are also employed as biological models to understand tissue development or disease progression in a realistic three-dimensional context. In summary, gene therapy has the potential to generate the next generation of skin substitutes with enhanced capacity for treatment of burns, chronic wounds and even systemic diseases.