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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.
The field of stem cell-based medicine is advancing rapidly. Hematopoietic stem cell transplantation is routinely included during treatment of some malignancies or genetic disorders. Many adult stem cell and even embryonic stem cell-based therapeutics are being tested in clinical trials. Such potential therapeutics include adult stem cells to treat heart disease  and stroke  and human embryonic stem cell-derived products to treat retinal disease  and spinal cord injury . Commonly used stem cell products, such as minimally manipulated hematopoietic stem cells, may not require animal studies prior to use in human clinical studies in certain circumstances. Stem cells from more recently discovered sources, however, require activity and safety assessment in relevant animal models. Such testing is especially important when the intended use or injection site of the cells is different from their normal function or environment (nonhomologous use). Careful attention must be paid to the survival, migration, phenotype, function, and potential toxicities of the implanted cells, and in Table 1 we summarize some of the unique challenges posed by preclinical testing of cell-based therapies. Preclinical studies should provide evidence that it is reasonably safe to conduct a proposed clinical investigation with a particular product . In addition, preclinical studies should establish the activity, effective dose, dosing regimen, and route of administration of a therapeutic product.
When designing preclinical animal studies that will enable clinical translation of a stem cell-derived product, it is important to ascertain regulatory requirements. The unique nature of each particular cell therapy and clinical application requires special consideration by the Food and Drug Administration (FDA) in the U.S. or by other regulatory agencies abroad. In the U.S., cell therapies are regulated by the Office of Cellular, Tissue, and Gene Therapies (OCTGT) in the FDA's Center for Biologics Evaluation and Research. Although OCTGT has not issued guidance specific to preclinical models for stem cell-derived products, numerous guidance documents contain recommendations that provide a general framework for conducting preclinical animal studies [6–8]. In addition, OCTGT developed a webinar series that includes a module dedicated to preclinical study considerations .
This article outlines important parameters to guide the design of preclinical studies and reviews the types of preclinical studies that should be considered depending on the nature and specific use of the intended stem cell therapeutic product (Table 2). Some of the design features that should be contemplated for those studies are also discussed. It should be noted that the suggestions below may not be appropriate in all instances. Investigators are encouraged to contact the FDA or the appropriate regulatory body to review their preclinical development plans prior to execution.
Preclinical studies should be designed to address the activity and safety of an investigational stem cell-based product for the intended clinical use. Insights into the potential mechanism of action of the therapeutic cells in the disease indication, the timing of intervention with respect to disease course, and the mode of delivery to the site of action must be investigated in preclinical models. Ideally the preclinical animal model studies should recapitulate human disease pathogenesis so that the therapeutic intervention in animals mimics the intended clinical application. The type, duration, and scope of preclinical studies will depend on the nature and anticipated duration of survival of the stem cell therapeutic product. To the extent possible, differences between the animal model and human disease should be determined and limitations of the model considered. For instance, xenograft rejection can make long-term assessment of human cells in immunocompetent animal models problematic. In some cases, alternative animal models or protocols may be required to overcome limitations of primary animal models. Some stem cell-derived therapeutic products may require more than one animal model to obtain a more complete picture of the delivery, safety, and activity of the cell product prior to initiating clinical studies. Published literature and data from relevant investigational studies are useful in helping define the scope of a preclinical program and the types of studies that may or may not be required to enable clinical trials.
General consideration must also be paid to methodologies for manufacturing the stem cell product for the final preclinical studies. Given the numerous sources, differentiation states, and production protocols for stem cell-based products, it is generally best to select the protocol that will be used to produce the clinical product prior to the initiation of final preclinical studies. As a result, the stem cell product should be manufactured using protocols and procedures comparable to those intended for cGMP clinical production. Provisional release criteria should be implemented for the stem cell product. Results from the final preclinical studies can be used to refine and finalize release criteria for the cGMP therapeutic product.
There can be significant debate on the appropriate animal models needed for preclinical studies. The International Conference on Harmonization's “ICH Guideline S6 (R1): Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals” provides practical principles on the selection of animal models, although it does not directly address cell therapy products . There are no requirements for the number of animal models that must be used for preclinical studies; however, the relevance of the model(s) must be justifiable. In many cases, there are no reliable animal models that are predictive of the human disease. Under such circumstances, using the model that most closely represents critical features of the intended indication is the best alternative. Depending on the specific stem cell product and targeted disease indication, multiple animal models may be required to address questions about the delivery, efficacy, toxicity, and tumorigenicity of the product. Some circumstances may require large animal models to demonstrate scalability of a therapy or safety and/or activity in an animal with physiology closer to humans. For instance, a specialized delivery device that will be used for clinical administration of the product may logistically preclude using small animals. Similarly, determining the safety of a cellular product may require administering the equivalent human dose, which may not be possible with small animals. In some cases, the use of a large animal model, if feasible, may be more representative of the anatomy, function, and pathophysiology of the human condition. Such is the case for orthopedic indications, where large animals, such as goats and sheep, more closely represent human joint anatomy and load-bearing function. In all cases, animal study design should be optimized to limit the number of animals to that which is necessary to achieve reliable information and enable practical and thorough execution of the studies.
Whenever possible, the intended human stem cell product should be used for activity and safety studies. The product should have activity in the animal model chosen for efficacy and toxicity studies to enable assessment of potential benefits and untoward effects. Models to consider for preclinical studies may include either diseased or healthy animals. Additionally, immune-compromised or immune-suppressed animals should be considered, if necessary, to prevent rejection of the human cellular product . Different immune-compromised rodent models (e.g., NOD-SCID, NOD-SCID/IL-2Rγnull, Nude, RAG1) exist, and it is important to understand how an immune-compromised animal may influence and potentially improve the ability to assess beneficial and adverse effects of the product. For instance, testing of a human stem cell-based product in an immune-compromised model may enable long-term survival of the cells, providing a greater ability to assess ectopic tissue or tumor-forming potential. Pilot studies may be needed to test different immune-compromised models to determine which best supports assessment of the investigational product.
Diseased or injured animal models are typically selected for proof-of-concept studies to demonstrate clinical activity and potential mechanism of action for the stem cell-based product. Diseased, injured, or healthy animal models can also be used for toxicity studies. If diseased or injured animals are used for toxicology studies, appropriate control animals should be included to distinguish events associated with the investigational product from those associated with disease or injury progression.
In some cases, generally accepted animal models of disease may not support adequate testing of the human product. For example, the human stem cell product may not survive as a xenograft in animal models, but high doses of immunosuppressive agents may compromise medium- to long-term health and survival of the animal. In this circumstance, it is particularly important to determine the exact information that is sought in the animal model and whether available alternative models can provide sufficient preclinical information. If alternative models are not feasible, one approach to consider is developing an analogous animal stem cell product for preclinical studies. In such instances, it is important to understand the similarities and differences between the human clinical and animal surrogate products and potential limitations of such an approach.
Preclinical studies typically provide the basis for determining a starting human dose. The safety signals seen in animals allow extrapolation of a safe human starting dose. The extrapolation method should consider the route of administration and whether the product is expected to have local or systemic effects. Although preclinical studies are not always feasible, they can provide insight into the safe human dose escalation range. Ideally, preclinical studies will also yield information regarding potentially efficacious doses and dosing frequency in animals, providing further support for starting doses in humans. For novel, specialized stem cell therapeutics that are administered invasively and hence have increased risks, using clinical starting doses that have some potential for efficacy is desirable. Limitations on the concentration and/or volume of cells that can be delivered can impart practical considerations on the potential utility of a stem cell-based therapeutic, so these should be investigated in preclinical animal studies with respect to the route and mode of administration that will be used in the proposed clinical trials.
It is standard practice in developing a new therapeutic to use diseased or injured animal models to establish potential benefits of the investigational therapy. Proof-of-concept or potential efficacy for stem cell therapies is ideally established by one of a number of clinically relevant outcomes in animal studies, such as overall survival, recovery of organ function, or improvements in behavioral activity. If this is not feasible, it is good practice to seek evidence of in vivo activity of the cells based on cell engraftment, differentiation, stimulation of endogenous repair, production of paracrine factors, induction of immunomodulation, improvement in tissue architecture, and/or prevention or repair of damage. The durability of activity of transplanted cells should also be investigated within practical limitations of the studies. When designing proof-of-concept studies, the route, dose, concentration, and dosing regimen intended for clinical trials should be mimicked as closely as possible. Information gained in proof-of-concept studies provides a scientific basis for the potential endpoints, safety monitoring parameters, and length of monitoring that may be required to protect human clinical trials subjects.
Unlike pharmaceutical drugs, stem cell-based therapeutics may survive long after administration. Preclinical proof-of-concept studies should follow cell survival, migration, phenotype, and fate following administration. Methodologies including histology and gene expression should be used to examine survival, phenotype, differentiation state, and proliferative status of the transplanted cells. Such studies can be challenging because of immunological rejection in some models. In addition, limitations in methodologies to sensitively track and phenotype cells over time can require numerous animals in these studies. Advanced technologies that facilitate such studies are needed. The California Institute for Regenerative Medicine (CIRM) and other organizations are funding projects to develop magnetic resonance imaging (MRI)- and magnetic particle-based methods, positron emission-based methods, and other approaches that may help overcome this challenge.
As stated above, there are circumstances in which animal models of disease or injury do not support short- or long-term efficacy studies of stem cell therapeutics. In other situations, animal models may not reflect the true pathophysiology of the human disease. Under these circumstances, results from alternative animal models and in vitro studies, along with consideration of the severity of the targeted clinical indication and design of carefully monitored clinical studies, could be contemplated in proposing advancement of the product. Early consultation with the FDA is highly recommended.
Depending on the route of administration, stem cell-based therapeutic products can be distributed locally or widely disseminated throughout the body. In addition, stem cells and their differentiation products have the potential to migrate depending on their location and particular state of activation or maturation. Biodistribution studies are important for many stem cell therapeutic products to understand where the transplanted cells can be found acutely and at later time points after administration. The results can establish potential sites of toxicity and the propensity of the investigational cells to accumulate in sufficient numbers at the targeted site of action.
Biodistribution studies are performed in diseased, injured, or normal and healthy animal models, as appropriate for the therapeutic intervention, using doses and route of administration that closely mimic those intended for the clinical trial (see section below on safety studies for more details). At acute and chronic time points post-administration, the animals are examined for the location and presence of cells derived from the therapeutic product. Traditionally, individual organs are harvested and examined for the presence of transplanted cells. A variety of target and nontarget tissues, including the gonads, are examined for presence of the cellular product. Methods to track human cells in rodent tissues usually involve quantitative polymerase chain reaction-based detection of human Alu sequences or detection methods for labeled cells. Currently, MRI and fluoroscopic methodologies are being improved to detect labeled cells in animals without the need for sacrifice, enabling longitudinal examination of the migration of implanted cells.
Preclinical toxicology animal studies are fundamental for determining the potential for untoward effects of a novel therapeutic or a new application of a therapeutic product. Preclinical toxicology studies should provide safety data to determine whether the investigational product has an acceptable safety profile to justify clinical trials. In addition, results from the preclinical toxicology studies may provide important information regarding specific toxicities that should be more closely monitored in clinical trials.
Dose ranging is a key design parameter in preclinical toxicology studies to establish the margin of safety between beneficial and toxic doses of the investigational product. Examination of a broad range of doses of a stem-cell therapeutic product can be challenging in some cases because of the size of the animal, delivery methods, and implant rejection in many large animal models. In such case, one approach may be to use doses investigated in the toxicology study that span the minimum dose demonstrated to have activity in proof-of-concept studies through to a highest feasible dose.
It is important to consider including both early and late time points in the design of the preclinical toxicology study to examine short- and long-term toxicity. Time points should be guided by the timing of administration of the stem cell-derived therapeutic in the disease process, the estimated time for activity of the implanted cells, and the potential health and life span of the animals. As mentioned previously, use of the stem cell product in preclinical toxicology studies should ideally mimic the intended clinical use. As such, if repeated dosing will be used in the desired human trial, repeated dosing should be investigated in the preclinical toxicology studies within practical limitations. Dose escalation cohorts may need to be incorporated in clinical trial designs to accommodate any inability to carry out repeated dosing in preclinical toxicology studies. Depending on the animal model, a sufficient number of animals for each sex, dose group, and time point should be included in the preclinical toxicology study. Although many toxicology studies use 10 animals of each sex per group per time point, mortality at later time points inherent in the animal model or at higher doses may require more animals for those conditions. Furthermore, animal studies should be randomized to reduce bias. In some cases, measurement of efficacy and safety can be combined in the same study, but such a study must consider the number of animals required for all terminal endpoints, such as histological, toxicological, and final efficacy measurements, and include appropriate controls. At each time point, it is good practice to assess animals for general health, serum biochemistry, clinical chemistries, and hematologic profiles. Both macropathological examination of gross tissue upon necropsy and micropathological histological examination of each animal should be considered to assess changes in target and other tissues. Such examination is important to determine the propensity of the stem cell-based product to form ectopic tissue or tumors and their potential consequences. In cases where ectopic tissue is found, assays can determine whether such tissue originated from the transplanted stem cell-based product, since some animal models, especially immune-compromised models, have a propensity for spontaneous endogenous tumor formation.
The potential for formation of ectopic tissue or tumors can be a major concern for stem cell-based therapies. Concerns range from the formation of benign tissue to expanding or malignant tumors upon transplantation of stem cell products. As stated above, in many cases ectopic tissue and tumors originating from the stem cell product can be monitored in preclinical toxicology studies. Although no regulatory guidance has been issued directly addressing tumorigenicity studies for stem cell-based products, two FDA guidance documents provide study design advice and general principles for when to consider tumorigenicity testing [11, 12]. Considerations regarding the need for and design of tumorigenicity studies include the following: (a) duration of culture of the source stem cells, (b) the type of stem cells used to generate the product, (c) the differentiation state of the final cell product, (d) the expected in vivo survival duration of the transplanted cell product, (e) the distribution and migration potential of the transplanted cells, (f) the potential clinical consequences should ectopic tissue or tumors form, and (g) prior clinical experience with the investigational product. For instance, human embryonic stem cells (hESCs) or induced pluripotent stem cells have higher propensities to form benign teratomas than many stem cell types from fetal or adult tissues. Furthermore, stem cells cultured for extended periods may accumulate genetic and/or epigenetic changes that increase their propensity to form ectopic tissue or tumors. Stem cell therapies designed to survive for long periods of time in vivo may also have higher risk for producing undesirable tissue than short-lived cells. Stem cell products that are injected and remain local may, depending on the site of injection, pose less safety risk, especially if any potential tumor could be easily resected without severe clinical consequences. Finally, stem cell-based products with an established history of safe clinical use may not warrant further tumorigenicity studies for new indications under investigation, assuming that manufacturing processes have not changed substantially.
When tumorigenicity studies are conducted, there are numerous experimental design parameters to consider. These parameters include the following: (a) choice of the animal model, (b) study duration, (c) route of administration, (d) number of cells tested, (e) positive control selection, and (f) definition of a positive result. The selected animal model should allow sufficient survival of the cells to enable assessment of potential tumorigenicity. Immune-compromised rodents are frequently used for this purpose. Likewise, the study duration should be sufficient to permit detection of potential tumors. Tumorigenicity studies lasting 9–12 months have been requested by regulatory agencies.
In designing tumorigenicity studies, implantation at the clinical delivery site is important since the local microenvironment may influence the concentration, aggregation state, and biological activation of the transplanted cells. In such cases, dose selection can be especially challenging since ideally the intended clinical dose would be tested. If the human clinical dose cannot be tested using the clinically relevant delivery route, the maximum feasible dose is commonly used in tumorigenicity studies. Supplemental data using other routes of delivery may be required. Investigators are encouraged to confer with appropriate regulatory authorities for such investigations. Pilot studies may be necessary to address all of the considerations above.
Positive tumorigenicity results might be ectopic tissue formation, gross tumor development, or even the observation of micrometastases. As for other studies, histological, gene expression, or genotype analyses should be performed to determine whether any observed ectopic or tumor tissue originated from the transplanted stem cell-based product. Histological or other evaluations should also assess the proliferative status of the ectopic or tumor tissue compared with positive controls. In human embryonic stem cell-based therapies, hESCs themselves can serve as the positive control as they can form benign teratomas, depending on their number and aggregation state. For tumorigenicity studies, hESCs injected alone or directly spiked into the final therapeutic product can not only serve as the positive control but provide some indication of the level of hESC impurities required to produce a teratoma. It should be noted that it is desirable to use the actual human stem cell-based product and not an analogous animal-derived product for tumorigenicity studies.
As outlined above, there are many questions and challenges associated with developing preclinical models for stem cell therapy applications and how to successfully navigate a regulatory pathway through this emerging landscape. Since each stem cell-based therapeutic approach is unique, there is not a single, fixed set of requirements for preclinical studies. Each preclinical program must therefore be tailored to the intended clinical application and take into consideration numerous factors specific to the proposed therapeutic approach. As more stem cell-derived therapies progress into clinical trials, the field will develop a better understanding of appropriate preclinical models and the regulatory expectations for those studies.
CIRM believes that openly discussing outstanding preclinical issues and promoting data sharing and transparency are critical so that stakeholders may learn from one another and collectively advance the science. As such, CIRM is engaged in dialogue with industry, academia, and patient advocacy groups to better understand the science and challenges in the field. Through interactions such as roundtables and workshops, CIRM and its network of industry and academic researchers are working to maintain forums through which stakeholders are able to provide input to the FDA and stay abreast of the FDA's current thinking. Furthermore, recent webinars have addressed preclinical model considerations for stem cell therapies, as well as imaging technologies that may enable translation of cell therapies .
Although many resources are available to inform preclinical study design for stem cell-derived products, many questions remain. Using the intended clinical study to inform preclinical design, having a strong understanding of the cellular product, and using scientifically rigorous methods are core requirements of a sound preclinical animal testing program. Those principles combined with efforts to promote collaboration and communication among the regenerative medicine field will inform and clarify the regulatory pathway and requirements as the field grows and matures.
We thank Jane Lebkowski for helpful discussions and editorial assistance. We also thank Mahendra Rao and Mitch Finer for discussions and critical review and Cynthia Schaffer for assistance in manuscript preparation.
The authors indicate no potential conflicts of interest.
J.F.-V.: conception and design, collection and assembly of data, manuscript writing; K.J.W.: conception and design, collection and assembly of data, manuscript writing, administrative support; E.B. and E.G.F.: conception and design, final approval of manuscript.