HHS STTR PA-13-224

Regenerative Medicine aims to replace or regenerate human cells, tissues or organs to restore or establish normal function lost due to injury, toxicological/metabolic damage, congenital pathologies or ageing. Regenerative medicine has the potential to overcome the shortage of organs available for human transplants by replacing or stimulating the body's own repair mechanisms to heal or regrow damaged tissues or organs. The recent discovery of the reprogramming of adult cells to a pluripotent state provides opportunities to address a major problem of regenerative medicine, immune rejection of transplanted tissue. The ability to generate differentiated cells and tissues using cells from specific patients will facilitate individualized medicine and eventually will lead to specialized therapies. The field is moving toward translation to clinical practice and is becoming increasingly dependent on animal models. Generating the correct type and quantity of the specific cell types required for replacement therapy is a significant challenge, as are the problems associated with introducing these cells into the proper environment in vivo and overcoming immune reactions. Finding solutions to these problems will require extensive testing in experimental animal models.

Major advances have been made in the past several years in deriving pluripotent cells, such as embryonic stem cells (ESCs) and induced pluripotent cells (iPSCs) from both humans and animals.  In parallel, other investigations have isolated and characterized multipotent “somatic” or “adult” stem cells from various tissues, including Mesenchymal Stem Cells (MSCs) and Germinal Stem Cells (GSCs).

The discovery of mouse ESCs in 1981 revolutionized the field of developmental biology and provided new capability for genome manipulation and investigations of gene function. Isolation of human ESCs created new possibilities for the field of regenerative medicine. ES-like cells have been derived from a number of animal species, including rats, fish, cows, pigs and non-human primates. Many characteristics of animal ES-like cells, including surface markers, growth factor requirements, ability to differentiate and others can be quite different from human ESCs. 

The field of stem cell research experienced a dramatic new direction with the isolation of iPSCs, derived by reprogramming human or mouse somatic cells to a pluripotent state. Several studies on various animal systems suggest that the basic pluripotency network appears to be conserved among different species, allowing derivation of iPSCs from a variety of animals.

MSCs, a type of somatic stem cell, were originally identified as a subpopulation of bone marrow cells with osteogenic potential. The properties of MSCs have been examined extensively over the past decade. Studies using animal models have shown promising results following MSC therapy for induced injury in the musculoskeletal, cardiovascular, digestive and nervous systems. In addition, many clinical trials have demonstrated the efficacy of MSC infusion for treating various human diseases. Given the wide range of tissue sources, the recognition of subpopulations with specific properties, and the frequent production of genomic alterations upon expansion in cell culture, extensive characterization of MSCs and development of improved techniques are required. Most importantly, there is relatively limited understanding of the normal biological functions of MSCs and the mechanisms by which they participate in tissue repair.

Along with rodents, several other animal species are being developed as models for various studies in the field of regenerative medicine. Understanding the properties and capabilities of stem cells derived from animals such as rabbits, pigs, sheep, goats and monkeys will increase the potential for the use of the most appropriate systems for modeling particular human disease conditions or for other medical applications. Non-rodent mammalian species (often referred to as “large animal models”) provide important advantages for transplantation studies, including large size, similarity to human physiology and pathology and longer life span, thus facilitating translation to studies in humans. The use of animal stem cells as a model for human cells in procedures related to regenerative medicine requires in-depth understanding of common regulatory pathways as well as species-specific properties and their impact on potential therapeutic applications.

Animal experiments have historically made a significant contribution to understanding human disease.  However, animal studies need to be improved in order to better predict the efficacy of treatment strategies in clinical trials. Several possible causes of the disparity between the results of animal studies and clinical trials have been identified, including failure to acknowledge the limitations of animal models, inadequate animal data and conclusions from them, less than optimal disease models and overestimation of treatment efficacy due to the preferred publishing of positive results. These problems should be addressed in the design and execution of preclinical, animal-based studies involving stem-cell based therapies.

The potential results of investigations must be applicable to the research interests of two or more of the categorical NIH Institutes and Centers. In addition, projects that predominantly address the research interests of one NIH Institute or Center, but that are peripherally related to the research interests of other Institutes and Centers, will not be considered appropriate for this FOA. An example of an inappropriate request is one exclusively involving an animal model of cancer or some other specific disease.

Research activities that are being sought to be supported under this FOA are intended to improve existing or create new animal models for regenerative medicine applications. Highly innovative projects are encouraged and can include, but are not limited to:

• Development and genetic, phenotypic and epigenetic characterization of animal stem cells as model systems for human stem cells and their application for regenerative medicine. Stem cells from rat as well as large animal species are of particular interest.
• Development and characterization of stable, well characterized pluripotent stem cell lines from large animal species, such as rabbits, dogs, pigs, sheep and monkeys. Development of stem cell lines from this type of animal model should be justified as having direct relevance to potential uses in the field of regenerative medicine. Further improvement of the technologies for safe genetic modification of pluripotent cells and their derivatives. Creation of biomarkers, standard protocols, reporter cell lines, species-specific reagents, proteomics, transcriptomics and genetic tools to assist the use of these cells for drug screening and disease modeling is also of interest.
• Improvement of methods for testing the efficacy and potency of MSCs in animals and for controlling the MSC secretome post-transplantation. Development of definitive markers for the multipotent state of the cells. Standardization of culture conditions for scale up of production. Improvement of the methods for creation of large batches of iPSCs-derived MSCs, that would allow in vivo testing in preclinical studies.
• Development of new techniques for non-invasive guiding and verifying cell injection, tracking cell migration, and monitoring long-term integration and survival of grafted stem cells and their derivatives (reporter genes, florescence probes, animal imaging). There is particular interest in the development of technologies for large animal species, addressing problems of tissue penetration and improving imaging sensitivity and resolution.
• Development of approaches to increase retention and survival of grafted cells, using such approaches as biomaterial carriers/nanomaterials and signaling molecules, which will prevent cell death and help to re-establish appropriate cell interactions and regulation.
• Development or improvement of existing humanized animal models. Preference will be given to studies involving species other than rodents. Develop humanized animals or bioreactors for in vivo generation of complex human tissues and organs using stem cells. Models may function as cell reservoir for cell differentiation, or cell propagation/expansion, ready for biomedical use.
• Develop high throughput technologies for genetic, phenotypic and therapeutic screens to study stem cell biology and to use for drug discovery, disease modeling and pre-clinical testing in appropriate animal species, such as zebrafish. Improvement and distribution of the reagents, protocols, stem cell lines, vectors, genetically altered animal strains and disease models, suitable for such screening.
• Development of technologies to increase mammalian stem cell reprograming and somatic cell transdifferentiation efficiencies, using reproducible methods addressing safety concerns. These technologies might include, for example, the use of removable vectors and vector systems with temporal expression of transcription factors, RNA or small molecules. Large scale production of defined populations of stem-cell derived differentiated cells. Development of techniques for improving standardization and quality of stem cells and their derivatives. Decreasing the risk of contaminants and transmissible diseases. Increasing the reliability of cryopreservation and long term storage. Novel approaches for activation of endogenous stem and progenitor cells and re-programing of somatic cells in situ.
• Development of approaches for rigorous evaluation and resolving challenges associated with stem cell therapy, such as: risk of cellular genetic and epigenetic instability, high mutation rate during in vitro manipulations, epigenetic memory of differentiated iPSCs and immune responses induced after stem cell transplantation. Improvement and testing tolerance induction regimes and elucidation the underlying mechanisms of the induction, maintenance and/or loss of tolerance to transplantation in appropriate animal species, such as nonhuman primates. Of particular interest is the development of methods for sensitive detection and elimination of tumorigenic cells, and functional assays and high-throughput techniques that will predict the potential immunogenicity of transplants and the tumorigenicity or metastatic potential of different populations of stem cell derivatives.
• Develop new methods and animal models to evaluate the safety of animal and human stem cell-derived progenitor and differentiated cell transplantation in animal species, including studies of adsorption, distribution, differentiation, survival, metabolism, and toxicity of stem cell-based biomedical procedures. These models should take into account properties of the specific cell populations and should mimic the intended use of the Development of animal models for human disease conditions, which can be used for validation of stem cell-mediated therapy for regenerative medicine.  Applications should not be focused on specific disease conditions. However, a specific disease can be used as a proof of principle, if also applicable to other disease conditions. Preference will be given to disease models that best emulate physiological, cellular and molecular manifestations seen in humans. Demonstration of the functionality of specific stem cells and their derivatives and effectiveness of achieving predictive results in improved animal models.