Nerve growth factor expression by PLG-mediated lipofection.

Biomaterials capable of efficient gene delivery provide a fundamental tool for basic and applied research models, such as promoting neural regeneration. We developed a system for the encapsulation and sustained release of plasmid DNA complexed with a cationic lipid and investigated their efficacy using in vitro models of neurite outgrowth. Sustained lipoplex release was obtained for up to 50 days, with rates controlled by the fabrication conditions. Released lipoplexes retained their activity, transfecting 48.2+/-8.3% of NIH3T3 cells with luciferase activity of 3.97x10(7)RLU/mg. Expression of nerve growth factor (NGF) was employed in two models of neurite outgrowth: PC12 and primary dorsal root ganglia (DRG) co-culture. Polymer-mediated lipofection of PC12 produced bioactive NGF, eliciting robust neurite outgrowth. An EGFP/NGF dual-expression vector identified transfected cells (GFP-positive) while neurite outgrowth verified NGF secretion. A co-culture model examined the ability of NGF secretion by an accessory cell population to stimulate DRG neurite outgrowth. Polymer-mediated transfection of HEK293T with an NGF-encoding plasmid induced outgrowth by DRG neurons. This system could be fabricated as implants or nerve guidance conduits to support cellular and tissue regeneration. Combining this physical support with the ability to locally express neurotrophic factors will potentiate regeneration in nerve injury and disease models.

Challenging Regeneration to Transform Medicine.

UNLABELLED: The aging population in the U.S. and other developed countries has led to a large increase in the number of patients suffering from degenerative diseases. Transplantation surgery has been a successful therapeutic option for certain patients; however, the availability of suitable donor organs and tissues significantly limits the number of patients who can benefit from this approach. Regenerative medicine has witnessed numerous recent and spectacular advances, making the repair or replacement of dysfunctional organs and tissues an achievable goal. Public-private partnerships and government policies and incentives would further catalyze the development of universally available donor tissues, resulting in broad medical and economic benefits. This article describes a Regenerative Medicine Grand Challenge that the Alliance for Regenerative Medicine recently shared with the White House's Office of Science and Technology Policy in response to a White House call to action in scientific disciplines suggesting that the development of "universal donor tissues" should be designated as a Regenerative Medicine Grand Challenge. Such a designation would raise national awareness of the potential of regenerative medicine to address the unmet needs of many diseases and would stimulate the scientific partnerships and investments in technology needed to expedite this goal. Here we outline key policy changes and technological challenges that must be addressed to achieve the promise of a major breakthrough in the treatment of degenerative disease. A nationalized effort and commitment to develop universal donor tissues could realize this goal within 10 years and along the way result in significant innovation in manufacturing technologies. SIGNIFICANCE: Regenerative therapies, in which dysfunctional or degenerating cells, tissues, or organs are repaired or replaced, have the potential to cure chronic degenerative diseases. Such treatments are limited by a shortage of donor organs and tissues and the need for immune suppression to prevent rejection. This article proposes a 21st Century Grand Challenge that would address this significant medical need by coordinating a national effort to convene the multidisciplinary expertise needed to manufacture functional and engraftable cells, tissues, or organs that could be made available to any patient without significant risk of rejection-so-called universal donor tissues.

US FDA outreach to the regenerative medicine community: challenges and opportunities.

Advances in the field of regenerative medicine have yielded novel approaches to developing treatments for currently unmet medical needs. The regenerative medicine field is diverse, spanning many research and clinical disciplines; no single society or organization fully represents regenerative medicine. The US FDA maintains an active dialog with a variety of stakeholders to keep abreast of the latest available science, to anticipate regulatory challenges posed by the latest scientific developments and to educate stakeholders about regulatory expectations for product development. The diversity of stakeholders in this field makes this dialog challenging. This article provides an overview of some of the FDA's current outreach activities in this area. The FDA welcomes opportunities to enhance its interactions with the regenerative medicine community.

Translation of stem cell research: points to consider in designing preclinical animal studies.

Stem cell-based therapies hold tremendous promise for the treatment of serious diseases and injuries. Although hematopoietic stem cell transplantation is routinely used as part of the treatment regime for some malignancies and genetic diseases, most stem cell-based therapeutic products are investigational and still require preclinical and clinical studies to support their many novel therapeutic uses. Because of the multiple sources of stem cells, the plethora of potential applications, and the novel mechanism of action of stem cell-based therapies, there is no single set of universal guidance documents that can be used to inform the preclinical development path for these therapeutics. Specific technical issues relating to the transplantation of human cells in animals, new delivery procedures, and laborious methods to characterize transplanted cells can present further challenges in the design and execution of preclinical animal studies for stem cell-based therapeutic products. In this article, we outline important parameters to guide the design of preclinical studies for stem cell-based therapeutics. In addition, we review the types of preclinical studies that should be considered depending on the nature and specific use of the intended stem cell therapeutic product. Finally, we describe important considerations in the design and execution of specific studies to monitor the efficacy, toxicity, biodistribution, and tumorigenicity of stem cell-based therapeutics.

Perspective: communications with the Food and Drug Administration on the development pathway for a cell-based therapy: why, what, when, and how?

Effective interaction between key stakeholders and the U.S. Food and Drug Administration (FDA) is central to successfully navigating the regulatory process and advancing new therapies into clinical trials. This is especially true when developing cell-based therapies, which pose unique challenges to demonstrating safety and effectiveness. There are numerous opportunities for developers of a new cell therapy to interact with the regulatory agency, through both formal and informal processes. It is important to understand how to maximize the productivity of dialogue with the FDA and develop an effective regulatory strategy. This article provides an overview of the types of interactions with the FDA that are available throughout the regulatory process. This article also notes some common pitfalls to avoid and directs readers to additional references and resources to help inform cell therapy researchers and product developers and enable successful regulatory interactions.

Multiple channel bridges for spinal cord injury: cellular characterization of host response.

Bridges for treatment of the injured spinal cord must stabilize the injury site to prevent secondary damage and create a permissive environment that promotes regeneration. The host response to the bridge is central to creating a permissive environment, as the cell types that respond to the injury have the potential to secrete both stimulatory and inhibitory factors. We investigated multiple channel bridges for spinal cord regeneration and correlated the bridge structure to cell infiltration and axonal elongation. Poly(lactide-co-glycolide) bridges were fabricated by a gas foaming/particulate leaching process. Channels within the bridge had diameters of 150 or 250 microm, and the main body of the bridge was highly porous with a controllable pore size. Upon implantation in a rat spinal cord hemisection site, cells infiltrated into the bridge pores and channels, with the pore size influencing the rate of infiltration. The pores had significant cell infiltration, including fibroblasts, macrophages, S-100beta-positive cells, and endothelial cells. The channels of the bridge were completely infiltrated with cells, which had aligned axially, and consisted primarily of fibroblasts, S-100beta-positive cells, and endothelial cells. Reactive astrocytes were observed primarily outside of the bridge, and staining for chondroitin sulfate proteoglycans was decreased in the region surrounding the bridge relative to studies without bridges. Neurofilament staining revealed a preferential growth of the neural fibers within the bridge channels relative to the pores. Multiple channel bridges capable of supporting cellular infiltration, creating a permissive environment, and directing the growth of neural fibers have potential for promoting and directing spinal cord regeneration.

Spatially patterned gene delivery for localized neuron survival and neurite extension.

Natural tissues can have complex architectures, which arise in part from spatial patterns in gene expression. Regenerative strategies for damaged tissue must recreate these architectures to restore function. In this article, we demonstrate spatially controlled gene delivery from a substrate for directing cellular processes. Non-viral vectors were immobilized to substrates in linear patterns using microfluidic techniques, and cells cultured on the surface had localized gene expression within the cell population. Transfection was achieved in pattern widths as low as 100 mum, with efficiencies dependent on the microchannel treatment and vector concentration. The ability of patterned expression to localize cellular processes was investigated using a neuronal co-culture model. Patterned expression of the diffusible neurotrophic factor nerve growth factor initiated neuron survival and neurite out-growth primarily within the pattern, which decreased significantly in regions directly adjacent to the pattern. Primary neurite density was significantly greater on patterned substrates than on surfaces without patterns. This approach demonstrates the basic technology to create patterns of gene expression that can direct tissue formation and could be employed in regenerative strategies to recreate the complex cellular architectures observed in tissues.

Delivery systems for small molecule drugs, proteins, and DNA: the neuroscience/biomaterial interface.

Manipulation of cellular processes in vivo by the delivery of drugs, proteins or DNA is of paramount importance to neuroscience research. Methods for the presentation of these molecules vary widely, including direct injection (either systemic or stereotactic), osmotic pump-mediated chronic delivery, or even implantation of cells engineered to indefinitely secrete a factor of interest. Biomaterial-based delivery systems represent an alternative to more traditional approaches, with the possibility of increased efficacy. Drug-releasing biomaterials, either as injectable microspheres or as three-dimensional implants, can deliver a molecule of interest (including small molecule drugs, biologically active proteins, or DNA) over a more prolonged period of time than by standard bolus injection, avoiding the need for repeated administration. Furthermore, sustained-release systems can maintain therapeutic concentrations at a target site, thus reducing the chance for toxicity. This review summarizes applications of polymer-based delivery of small molecule drugs, proteins, and DNA specifically relevant to neuroscience research. We detail the fabrication procedures for the polymeric systems and their utility in various experimental models. The biomaterial field offers unique experimental tools with downstream clinical application for the study and treatment of neurologic disease.