Stem Cell Research

There are numerous potential benefits of stem cells for the treatment of human disease, and these range from oncologic therapeutics to manipulation of the immune system response, the ability to individually tailor treatments to the specific person’s genetic haplotype, and the possibility of replacing diseased organs and tissue with genetically engineered materials. Such therapeutic promises demonstrate the importance of this relatively new area of investigation. Stem cells are also being used to study the underlying pathophysiological mechanisms of various diseases, the differences in disease manifestation between individuals, and the replacement of some animal testing by in vitro assays. This symposium highlighted areas where stem cell research could advance the understanding of human health issues and addressed some of the issues that pathologists face when trying to evaluate the use of stem cells in animals and devices.

An overview of the state of regenerative medicine was offered by Alan Trounson, past President of Californian Institute of Regenerative Medicine. The talk entitled “Where is the Field of Regenerative Medicine Going?” stated that stem cell research has changed the landscape for regenerative medicine by showing that stem cell assays and directed differentiation protocols can derive cells that are capable of repair of damaged and diseased tissues. The assays facilitate identification of cancer stem cells that are now enabling new drugs, monoclonal antibodies, and engineered cell therapies that can search and destroy dangerous metastasizing cancer stem cells. Developments in stem cell engineering also enables cure of genetic diseases by introducing true copies of genes to replace mutated gene sequences causing disease. The stem cell engineering also enables the manipulation of the immune system and the donor cells, removing alloreactive antigens and the elimination of reactive T cells. Furthermore, mutation or knockdown of key viral receptor genes will prevent blood cell infection with HIV. Such technologies are now undergoing trials as a cure for HIV/AIDS. Embryonic stem cell derivatives are also entering trials for reversing blindness and type 1 diabetes and it is expected that induced pluripotent stem cells (iPSCs) will enter clinical trials for eye disease. Fetal and embryonic stem cells are being trialed for spinal cord repair and ALS. ^1,2

There are some studies on the use of pluripotent stem derivatives for identifying toxic drugs and potential heterogeneous responses in patient populations to candidate drugs for therapeutic purposes. ³

In the future, we may see direct reprogramming of cells from one type to another (eg, heart fibroblasts to cardiomyocytes or glia to neurons) without the need for stem cell expansion and transplantation. Organogenesis will develop with cadaveric decellularized tissue or polymer scaffolds seeded with cellular products, but there are many issues to solve before widespread applications can be expected. For this it may be necessary to develop haplotyped iPSC banks for major histocompatibility antigens. ⁴

Kyle Kolaja (Cellular Dynamics International [CDI]) followed with a talk entitled “Applications of stem cell technologies in drug discovery” in which he pointed out that iPSC technology offers unprecedented opportunities to move beyond a “one size fits all” approach to pharmacology and toxicology, to a model where individual genetic and molecular profiles are used to guide diagnosis, drug development, and therapeutic decisions. Initially described in 2007, human iPSCs are derived from a patient’s somatic cells (eg, blood and skin) and have the potential to differentiate into any cell type in the human body. ^5,6 In the last 5 years, a rapidly growing body of literature has emerged demonstrating the use of iPSC-derived differentiated cells to demonstrate human-relevant toxicity as well as recapitulate human disease phenotypes in vitro. These novel human cell models are rapidly becoming the standard of choice for disease research and drug discovery as they offer better opportunities for therapeutic decision making over existing approaches, including primary cell culture and cell lines. Under a vast quality management system to uniquely ensure quantity and purity, CDI has developed a large catalog of diseased and normal iPSC lines (iCell products) including a grant from CIRM to create a bank of 3000 different diseased individuals. Several examples of the utility of iCell products were presented including their validation to predict drug-induced cardiac arrhythmia/QT prolongation with accuracy greater than 85%. ^7,8 Stem cell-derived cardiomyocytes are part of the new model being assessed by the Food and Drug Administration (FDA) as a means to reduce the extensive clinical QT assessments conducted currently. In addition, tissues derived from iPSCs have been shown to reflect the disease state of the donor, with nearly 50 examples in the literature. Furthermore, iPSC-derived tissues can be used to induce disease models such as endothelin-induced cardiac hypertrophy, amiodarone-induced steatohepatitis, and hepatitis virus infectivity. An additional published example was shown using iCell Neurons to understand the mechanism of toxicity for the Alzheimer disease-associated protein AB-42, which is to reactivate cell cycle via cdk2. This mechanism was leveraged to screen a library of compounds for new chemistry platforms. ⁹ In addition, the diseased iPSCs and tissues derived from them can be used in phenotypic screening, thus allowing for an alternative drug discovery approach. Finally, although efforts to date have focused on providing iCell products for research applications, CDI is moving into iPSC therapy as shown by efforts to make an Human Leukocyte Antigen (HLA)-homozygous Good Manufacturing Practice (GMP) iPS cell bank and the recent announcement with the National Eye Institute to make retinal-pigmented epithelium as part of an age-related macular degeneration clinical trial. The application for iPSCs and derived tissues has been established in preclinical research and the potential to change the way diseased humans are treated is just beginning.

Thomas Petersen (United Therapeutics, Research Triangle Park, North Carolina), a senior regenerative medicine scientist with a background in medicine and electrical and biomedical engineering, continued the theme with a talk entitled “Considerations for nonclinical use of stem cell therapies in animal models and toxicology studies.” This talk focused on the latest technology involved in decellularizing lung tissue scaffolds from primarily rat and pig and development of bioreactor systems to reseed these scaffolds with relevant lung cells. ¹⁰ Lung disease is the third most common cause of death annually in the United States and the demand for donor lungs is higher than the supply. Lung regenerative approaches involve the decellularization of lung tissue leaving behind the extracellular matrix. A range of detergents can be perfused through the lung to remove DNA and proteins while preserving the architecture. An important factor is that scaffold proteins such as collagen and elastin should be at least partially retained to support strength and elasticity of the lung. Scaffolds from sheep, human cadavers, pigs, and rats have been evaluated; however, the optimal source of starting lung tissue needs to be clarified. The scaffolds are reseeded in bioreactors that should mimic normal physiological development that includes oxygenation, airway ventilation, and vascular perfusion in order to induce proper cell survival, differentiation, and protein expression. Current challenges in repopulation include identifying the optimal cell source, the number and type of cells to use, and the stage of differentiation at which the stem cells should be seeded onto a scaffold. Transplantation outcomes have demonstrated short-term functionality of the lung with suitable gas exchange and dynamic compliance. Engineered lungs now last up to 7 to 14 days and failure is mostly related to decreased compliance and perfusability of the tissue with time. ¹¹ Finally, the regulatory path of approval for engineered lungs was discussed and the need to control the cell and scaffold source, the manufacturing process, and testing of the combined product prior to clinical studies was stressed. The FDA guidance indicates that scaffold materials should be evaluated for biocompatibility and toxicity. The cellular constituents should be evaluated for identity, purity, potency, sterility, and safety. Identification of appropriate nonclinical models and end points, that is, species, type of test article, immune monitoring, ventilatory mechanics, scaffold degradation, cell biodistribution, and other traditional endpoints, is ongoing in this exciting and challenging field.

Klaus Weber, (AnaPath GmbH, Oberbuchsiten, Switzerland), presented “Pathology Techniques to Aid the Development and Understanding of Advanced Therapy Medicinal Products (ATMPs)” in which he outlined the 3 major therapeutic groups of ATMPs: general therapeutics, somatic cell therapeutics, and engineered tissues. All ATMPs contain partially or consist fully of living cells or tissues. The ATMPs may also be combined with medical devices. ¹² The ATMPs are complex, and the preclinical testing differs in many aspects from “classical” testing strategies. Klaus Weber talked about how test models may differ from conventional animal models and that test models may include the use of nude mice, immunocompetent mice, hamsters, or other animal models. ^13,14 Test strategies may also be different and may require testing for virus replication, both the site and duration of transgene expression, biological activity, efficacy versus toxicity, and tumorigenicity. Testing strategies may also be quite different when using somatic cell therapeutics or stem cells, for example, xenogeneic islets will have different safety and efficacy concerns than the human stem cell-derived progenitor, β, or islet cells. Often, there is no one model serving as the gold standard. On the other hand, conventional animal models may work very well for toxicity issues associated with ATMPs. A recent example described the transplantation of microencapsulated neonatal pig islets in nondiabetic monkeys in an alginate matrix to confirm their biocompatibility. ¹⁵

Whatever the product under test is, the preclinical testing strategies include considerations on the species specificity at the molecular, cellular, and tissue levels, safety predictivity, efficacy/proof of concept (diseases models), biological relevance, biological relevance as it relates to delivery of the ATMP, animal models reliable in surgical procedures, and methods for tracking cells in vivo, among other considerations specific to the product and its intended use.

The use of many different species including disease models as well as the complex application of molecular biology approaches and the usage of an extended armamentarium to trace ATMPs in vivo may influence classical pathology evaluation. Concepts of molecular and cellular biology could dominate the evaluation strategies. Examples include understanding cellular differentiation processes or general aspects of cellular surface structures, which may preclude having samples for standard glass slide pathological evaluation.

Technologies from other sciences may become useful for the product evaluation and have to be adapted to the needs of pathology evaluation in the widest sense. Techniques from material sciences, digital technologies, and detection methods for substances and complex structures in media are of high interest for the evaluation of these ATMPs. For example, hyperspectral analysis, digital, and laser microscopy may be useful techniques in the pathological evaluation.

In addition, the pathologist should be involved in the manufacturing process where the pathologist can aid in classification of elements from engineered tissue or other ATMPs during the manufacturing process and aid in the evaluation of product stability, homogeneity, and other attributes. ¹⁶