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Strategies for Improving Animal Models for Regenerative Medicine

Jose Cibelli,1 Marina E. Emborg,2 Darwin J. Prockop,3 Michael Roberts,4 Gerald Schatten,5 Mahendra Rao,6 John Harding,7 and Oleg Mirochnitchenko7,* 1Michigan State University, Cellular Reprogramming Laboratory, Department of Animal Science, B270 Anthony Hall, East Lansing, MI 48824, USA 2University of Wisconsin-Madison, Department of Medical Physics and Wisconsin National Primate Research Center, 1223 Capitol Court, Madison, WI 53715, USA 3Texas A&M Health Science Center, College of Medicine Institute for Regenerative Medicine at Scott and White, Department of Medicine, 5701 Airport Road, Module C, Temple, TX 76502, USA 4University of Missouri, 240b C.S. Bond Life Sciences Center, 1201 East Rollins Street, Columbia, MO 65211-7310, USA 5University of Pittsburgh, Department of Cell Biology and Physiology, S362 Biomedical Science Towers, 3500 Terrace Street, Pittsburgh, PA 15261, USA 6Center for Regenerative Medicine, National Institutes of Health, 50 South Drive, Suite 1140, Bethesda, MD 20892, USA 7Division of Comparative Medicine/ORIP/DPCPSI/OD, National Institutes of Health, 6701 Democracy Boulevard, Suite 943/950, Bethesda, MD 20892, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.stem.2013.01.004

The field of regenerative medicine is moving toward translation to clinical practice. However, there are still knowledge gaps and safety concerns regarding stem cell-based therapies. Improving large animal models and methods for transplantation, engraftment, and imaging should help address these issues, facilitating eventual use of stem cells in the clinic.

Introduction to cancer and renal failure. They are examples of a large animal model In this Forum, we discuss the current relatively obese and hypertensive due to currently being used to study human status, challenges, and major directions the constant access to food. In compar- genetic diseases (review in Kuzmuk and for future development of animal models ison to humans, mice have small body Schook, 2011). Even without genetic to facilitate the use of stem cells in size, short lifespan, and substantially modification, minipigs and full-size regenerative medicine. The variety of different physiology. Significant progress breeds have been widely used for stem cell sources and a wide spectrum has been made in the creation and use studying infectious diseases, cardiovas- of potential applications make the devel- of humanized mice. There are, how- cular disease and atherosclerosis, wound opment of universal recommendations ever, disadvantages to using the current healing, digestive processes, diabetes, and guiding principles very challenging, strains, including complications in repro- ophthalmology, and some cancers, as yet certain common themes and ducing standard chimeras, limitations well as providing organs for xenotrans- possible solutions are emerging that in choices of human cell types that can plantation. The value of pigs as biomed- can increase the predictive validity of be used for xenotransplantation, residual ical models has been enhanced over the animal models for regenerative medicine. host immunoreactivity, and problems last decade by targeting specific genomic This report is based on discussions that with translating conditioning regiments sites for modification. Swine disease took place at a recent NIH workshop between species. The evolutionary dis- models created by targeted genetic engi- on this topic (http://dpcpsi.nih.gov/orip/ tance between donor and recipient neering include those for cystic fibrosis, documents/summary_of_the_improving_ animals will also affect the survival of Alzheimer’s and Huntington’s disease, animal_models.pdf). transplanted stem cells due to species retinitis pigmentosa, hyperlipoproteine- differences in trophic properties of mia, and muscular dystrophy. Recently, Animal Models for Stem Cell-Based tissues. creation of humanized pigs has been re- Regenerative Medicine: Mice Larger animal species, which were crit- ported (Suzuki et al., 2012), as well as versus Large Animal Models ical for developing hematopoietic stem improved preclinical disease models suit- The discovery of mouse embryonic stem cell therapies, often have an enhanced able for testing stem cell therapies in pigs cells (ESCs) in 1981 revolutionized the ability to predict clinical efficacy relative (Giraud et al., 2011; McCall et al., 2012). study of developmental biology, and to mice. The utilization of large animal Most of this work has been enhanced mice are now used extensively to study models is expected to increase and, through access to the swine genome stem cell biology. However, there are limi- therefore, further development of large sequence and the use of inbred tations to their application as models for animal stem cell technologies will also minipigs (http://www.nsrrc.missouri.edu/ regenerative medicine. Mouse models be required. It will be critical to select strainavail.asp). Work in swine will do not reproduce in full certain human the large animal that is most appropriate complement nonhuman primate research disease conditions. For example, labora- for each potential therapy in humans. for neurological treatments when ana- tory mice are insulin resistant and prone The pig has emerged as one of the best lyzing recovery of fine motor skills or

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impact on cognitive function. This related to safety, efficiency, and differen- certain cellular abnormalities of the corre- would be facilitated by equivalent tiation potential. sponding cells from patients with various advances in primate transgenesis, like Several problems remain before the Mendelian disorders. The ability to model the recent monkey models of Hunting- potential of nonrodent animal iPSCs can low-penetrance phenotypes, late-onset ton’s disease. be realized. Cell lines must be character- disorders, and genetically complex disor- Major challenges remain, however, for ized in more detail, chimerism tested, ders, however, remains to be proved. using large animals in stem cell research. and reprogramming increased in effi- Animal model systems may help solve For example, there is limited availability ciency and speed in order to enhance some of these problems because trans- of species-specific reagents, such as genome integrity. Cell surface markers plantation experiments can be performed antibodies and growth factors, and can be inconsistent among various lines using the animal as a host. The use of fully annotated expression microarrays. and cell populations may be heteroge- allogeneic iPSCs in animal model systems Authenticated ESCs have been difficult neous, probably reflecting different or xenotransplantation of human stem to generate from large domestic species, stages of reprogramming. Efficient deri- cells in immunocompromised or human- such as dog, swine, cattle, sheep, and vation of animal iPSC lines requires ized animals should facilitate analysis of goats. This has been obviated in part by further development of technologies for disease phenotypes that require cellular creation of induced pluripotent stem cells generating the cells, preferentially avoid- interactions in the tissues. Animal iPSCs (iPSCs) from these species by standard ing gene integration and potential risks can have certain advantages for testing reprogramming technologies. There is of tumorigenesis. hypotheses regarding the influence of a lack of centralized resources where An important aspect of animal studies is environmental or epigenetic components cells can be characterized and stored, the ability to test immune responses to of disease first identified in cell culture. reagents made available, and databases iPSCs and their derivatives. A number of The effects of exposures and genomic maintained for the wider biomedical animal studies have reported that iPSCs modifications can be tested at the level community. If these barriers can be over- can form teratomas and other types of of the tissue, organ, and the whole animal, come, well-characterized large animal tumors in immunodeficient, allogeneic, preserving interactions among distinct stem cells can provide an appropriate syngeneic, and xenogeneic recipients cell types in vivo. Use of animal systems choice of animal models for particular due to the inability of the host to re- also facilitates experimental design by human disease conditions and medical ject teratoma-inducing cells. The innate providing controls with matching genetic applications. These studies will comple- immune system appears capable of background, age, gender, and exposure ment the use of mice, leading to more dealing with small numbers of undifferen- history. comprehensive studies that can then be tiated iPSCs that might be present in applied to humans. grafts, despite efforts to eliminate them. Improving Stem Cell The ability of adaptive and innate immune Transplantation: Engraftment and Animal Induced Pluripotent Stem reactions to weaken engraftment of syn- Imaging Cells as Emerging Models for geneic stem cell transplants is another Two different approaches can be taken Human Therapeutic Applications important aspect of the host reaction for stem cell-based therapies. The first is The field of stem cell research experi- that can affect the efficiency of cell transplantation, in which stem cells, or enced a dramatic new direction with the transplantation. There is an urgent need derived progenitor or differentiated cells, isolation of iPSCs from humans and to test immune reactions against thera- are delivered directly to the body. The mice. Several studies on various animal peutic iPSC derivatives and a small second approach relies on activation of systems suggest that the basic pluripo- number of potentially tumorigenic cells. endogenous stem and progenitor cells tency network appears to be conserved Larger animal species have important or somatic cells reprogrammed in situ. among different species, allowing deriva- advantages as model systems in this Studies in animal model systems and in tion of iPSCs from a variety of large animal regard since their immunophysiology is humans indicate significant cell death species, including pigs, monkeys, dogs, closer to humans than to rodents. Like after transplantation and limited engraft- and several others (Plews et al., 2012). humans, larger animal models have ment and differentiation of transplanted As for the human, iPSCs from ungulates developed as outbred populations over stem cells. Retention and survival of and monkeys are of epiblast type, and time, thus shaping genomic adaptations. transplanted cells must be improved. fibroblast growth factor 2 and activin- Rodent models will remain important One of the likely reasons for low levels nodal signaling systems are critical for tools for immunological discovery and of cell survival and engraftment is the their growth in culture and for mainte- proof of concept, whereas larger animal absence of the proper cellular environ- nance of pluripotency. Animal iPSCs models will be critical for successful ment and substrate for engraftment. should be of value as a renewable source translation for clinical applications of Attempts have been made to provide of cells for testing safety after progenitor iPSCs. the transplanted cells with biomaterial cells are introduced as a tissue graft and iPSCs from patients have the potential carriers and signaling molecules, which for testing surgical and related technolo- to help identify the molecular and cellular will prevent cell death and will help to re- gies required before human trials can basis of human disease by ‘‘disease-in- establish proper cell interactions. Among proceed. Many of the challenges that a-dish’’ modeling. Though this field is still techniques that are currently under devel- face human iPSC research apply to in its infancy, reports thus far indicate the opment to address these problems in animal iPSCs as well, including issues general ability of iPSCs to recapitulate animal models are genetic modification

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of the cells and the use of tissue- such as activation, differentiation, or mation, permitting single-cell detection engineering techniques involving three- injury. These parameters in most cases in an animal model. However, current dimensional biodegradable scaffolds, cannot be obtained from patients via techniques should allow optimization of additional types of support cells, and direct sample extraction, in distinction procedures and on future improvement other bioactive molecules. Various nano- to animal studies. The development should satisfy most needs, thus facili- materials have been explored to control of noninvasive imaging approaches to tating cell tracking in humans. stem cell behavior such as adhesion monitor transplanted stem cell behavior and differentiation by modulating biomi- is critical for future translation to clinics. How Animal Models Can Address metic characteristics and mechanical Significant advances have been the Potential Challenges of Stem properties. achieved in the use of imaging modalities Cell Therapy An important goal of stem cell-based such as single-photon emission com- The challenges associated with unique therapies for treatment of damaged or puted tomography (SPECT), positron properties of stem cells that must be diseased tissues is to establish proper emission tomography (PET), and MRI to understood before moving to clinical connections between cells or with extra- monitor noninvasively transplanted cells applications have been identified and cellular signaling molecules. This has in myocardium, skin hair follicles, the continue to grow. Among these are been challenging for cardiovascular repair CNS, bone marrow, mammary glands, genetic instability, high mutation rate and even more complicated for stem cell- and retina, as well as other organs in and tumorigenic potential, epigenetic mediated transplantation in the retina rodents. The development of imaging memory of differentiated iPSCs, and the or brain, where appropriate synaptic techniques in large animals is becoming immune response after stem cell trans- connections must be created within increasing important, due to the applica- plantation. Some of these concerns the three-dimensional tissue structure. tion of protocols and equipment more became evident during experiments Knowledge of the homing and niche relevant to the clinical setting. Among when animal stem cells were tested and signals that mediate the activation, migra- complications are the need for equip- should be examined rigorously for human tion, and integration of stem cells to ment accommodating larger species, stem cells. Importantly, animal models damaged tissues is limited. Cytokines, limited tissue penetration, and the will be critical to evaluate new potential growth factors, adhesion molecules, outbred nature of large animals, which hazards and to address these problems cell-cell contacts, and small metabolic may require more animals and longer (Frey-Vasconcells et al., 2012). The stan- products are active players in these analysis time. Large animals will also dard use of preclinical animal models for processes. These mechanisms may vary demand additional knowledge of their studies that include adsorption, distribu- among different animal species and biology and careful monitoring of physio- tion, metabolism, excretion, and toxi- should be investigated and evaluated in logical parameters during imaging pro- cology is mandated by the US Food and a search for the most appropriate model cedures. MRI has been used to detect Drug Administration. The validated pro- for a particular application. Progress has nanoparticle-labeled cells in large animals cesses and regulatory requirements are been made, for example, in the use of in several studies, but the approach was well established for these studies. How- synthetic and natural guide materials, limited by label leakage and uptake ever, even in these investigations, there nerve guide coatings, topographical by macrophages. Cells have also been are issues as to whether the standard cues, special scaffolds, and support cells labeled with a variety of radionuclides. models are appropriate for cell-based to guide nerve cell engraftment (Bell Limitations of several isotopes were rela- therapy. For example, biodistribution and Haycock, 2012). Pharmacologically tively short half-life and effects on cell with pathology analysis using sequential active microcarriers, such as biodegrad- viability. Good results were obtained by sections of the entire body may not be able and noncytotoxic poly lactic-co- using a PET-herpes simplex virus thy- an appropriate model for a localized cell glycolic acid microspheres covered with midine kinase-reporter system. Stable transplant that is not expected to survive extracellular matrix molecules, have expression for an extended period of in an allogeneic setting. A number of been successfully tested in animal time was reported recently with PET or special considerations should be taken models of Parkinson’s disease and SPECT after administration of radiotracer into account when designing tumorige- infarcted myocardium in rats and mini- in combination with expression of a trans- nicity studies, such as the animal species pigs, respectively. genic sodium iodide symporter (NIS) in for testing, the type and condition of ther- Cellular imaging is a high priority for a pig model of myocardial infarction apeutic stem cells or their byproducts, basic research and clinical translation in (Templin et al., 2012). Another successful in vivo survival time, and potential distri- regenerative medicine. Imaging is impor- study has been reported in pigs using bution and migration of the transplanted tant for several applications, including iPSC-derived endothelial cells labeled cells. Several concerns were reported guiding and verifying the accuracy of with [18F]- fluorodeoxyglucose for PET based on mouse studies that employed cell injection, tracking cell migration, sur- imaging and iron particles for colocaliza- a variety of cell types, including mesen- vival, and behavior, evaluating off-target tion with MRI (Gu et al., 2012). New, chymal stem cells (MSCs). Extensive effects, and monitoring long-term cell more sophisticated PET/SPECT reporter preclinical data using MSCs from larger engraftment. Reagents should be opti- genes will require addressing potential animal species (pigs, dogs, and sheep) mized that exhibit quantitative biosensing oncogenicity and immunogenicity of for transplantation as well as clinical properties and functional readouts that transplanted cells. No single imaging studies have not detected a major risk can report changes in cell conditions, technique provides all of the desired infor- for tumor growth. The following reasons

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were proposed for the discrepancies models from larger animal species, will Frey-Vasconcells, J., Whittlesey, K.J., Baum, E., 1 between preclinical and clinical studies take place in concert with other innovative and Feigal, E.G. (2012). Stem Cells Transl Med , 353–358. at least in regard to MSCs and their poten- approaches, such as microsystem tissue tial applications for treatment of heart engineering and in vitro modeling of Giraud, S., Favreau, F., Chatauret, N., Thuillier, R., disease: substantial differences in mouse human disease pathways by using iPSCs Maiga, S., and Hauet, T. (2011). J. Biomed. 2011 and larger animals and human cell from affected individuals. These various Biotechnol. , 532127. behavior in culture, differentiation capac- approaches are complementary and are Gu, M., Nguyen, P.K., Lee, A.S., Xu, D., Hu, S., ities of particular cell preparations, sensi- highly relevant to the future of stem cell- Plews, J.R., Han, L., Huber, B.C., Lee, W.H., tivity to triggers of transformation, and based regenerative medicine. However, it Gong, Y., et al. (2012). Circ. Res. 111, 882–893. properties of the immune surveillance is also clear that extant models need to systems (Hatzistergos et al., 2011). Very be modified, new models developed, and Hatzistergos, K.E., Blum, A., Ince, T., Grichnik, 108 limited information is available regarding rigorous standards of preclinical evalua- J.M., and Hare, J.M. (2011). Circ. Res. , 1300–1303. other types of transplanted cells and tion followed to maximize their research animal model species to make a valid value. There is no single, perfect animal Kuzmuk, K.N., and Schook, L.B. (2011). Pigs as selection of the most predictable model. model that can completely predict the a model for biomedical sciences. In The Genetics Rigorous studies should be conducted outcome of clinical trials. The challenge is of the Pig, Second Edition, M.F. Rothschild and A. Ruvinsky, eds. (Wallingford, UK: CAB Interna- for each application to evaluate the onco- to collect relevant and sufficient informa- tional), pp. 426–444. genic risk of the use of stem cell products tion from as many models as are required in the most appropriate model system, to make an informed decision regarding McCall, F.C., Telukuntla, K.S., Karantalis, V., Sun- especially large animals. the potential benefits and risks to patients. cion, V.Y., Heldman, A.W., Mushtaq, M., Williams, A.R., and Hare, J.M. (2012). Nat. Protoc. 7, 1479– 1496. Conclusions ACKNOWLEDGMENTS Insufficient knowledge of human stem Plews, J.R., Gu, M., Longaker, M.T., and Wu, J.C. cell biology and the absence of animal The views and opinions of authors expressed in (2012). J. Cell. Mol. Med. 16, 1196–1202. this article do not necessarily represent those models that precisely recapitulate partic- of the National Institutes of Health. All authors Suzuki, S., Iwamoto, M., Saito, Y., Fuchimoto, D., ular human disease phenotypes, with contributed to and approved the manuscript. Sembon, S., Suzuki, M., Mikawa, S., Hashimoto, D.J.P. is chair of the scientific advisory committee comparable organ size and physiology, M., Aoki, Y., Najima, Y., et al. (2012). Cell Stem are currently significant limitations for the for Temple Thereapeutics LLC. Cell 10, 753–758. progress of regenerative medicine. Im- REFERENCES provement of existing rodent models, as Templin, C., Zweigerdt, R., Schwanke, K., Olmer, R., Ghadri, J.R., Emmert, M.Y., Mu¨ ller, E., Ku¨ est, well as development and characterization Bell, J.H., and Haycock, J.W. (2012). Tissue Eng. S.M., Cohrs, S., Schibli, R., et al. (2012). Circulation of stable stem cell lines and disease Part B Rev. 18, 116–128. 126, 430–439.

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