ReviewThe potential of induced pluripotent stem cells as a translational model for neurotoxicological risk
Introduction
The field of toxicology has seen rapid innovation in the past two decades by the advent of stem cell technology. Perhaps the first major successful use of stem cells for the study of toxicity was the embryonic stem cell test (EST) developed by Spielmann and colleagues (Heuer et al., 1993, Spielmann et al., 1997). This approach differentiates mouse embryonic stem cells (ESCs) into cardiomyocytes in the presence of potential developmentally toxic agents (Heuer et al., 1993, Seiler and Spielmann, 2011). Although this method utilizes mouse stem cells, and focuses on differentiation into beating cardiomyocytes, the method has been broadly hailed for its ingenuity (Laustriat et al., 2010, Scholz et al., 1999, Wobus and Loser, 2011). However, the method has notable shortcomings in its application to neurotoxicology. For example, although the EST correctly classified the majority of known embryotoxic chemicals tested, it is known that the EST in some cases failed to correctly classify methylmercury as a developmental toxicant (Genschow et al., 2004). There are several potential reasons for these shortcomings of the EST – including species-specific toxicities and tissue-type specific toxicities. Recently, Bremer et al. sought to address both of these issues by adapting the principles of the EST to toxicity testing in human ESCs (hESCs) undergoing neuronal differentiation (Stummann et al., 2009). Their study showed greater sensitivity of early-developing neural precursors over maturing neuronal cells to methylmercury toxicity (i.e. greater changes in expression of key early neurodevelopmental markers versus more mature neuronal markers) (Stummann et al., 2009). Other groups have also provided proof-of-principle experiments demonstrating the potential of hESCs to evaluate developmental toxicity (Pal et al., 2011). However, ethical and regulatory concerns about the use of cells derived from human embryos have limited adoption of hESC based toxicity testing (Leist et al., 2008, Vojnits and Bremer, 2010).
Pioneering studies have revealed both the feasibility as well as clear advantages for use of stem cell based approaches for neurotoxicological risk assessment. Although the fundamentals of stem cell culture are outside the scope of this review, a number of book chapters and review articles are available on this topic (Neely et al., 2011, Park et al., 2008, Takahashi et al., 2007). Studies using murine stem cells have identified mRNA based expression markers for assessment of neurodevelopmental toxicity (Kuegler et al., 2010, Theunissen et al., 2011). Comparative studies using hESC derived neurons versus rodent primary neuronal cultures have revealed important differences in sensitivity, reproducibility, and dynamic ranges by toxicity measures examining neurite outgrowth and cytotoxicity; suggesting further work is needed in developing and interpreting hESC-derived neurotoxicity tests (Harrill et al., 2011). Indeed, toxicogenomic approaches revealed key differences on the influence of a developmental neurotoxicant on expression profiles between in vivo models, stem-cell based in vitro models and primary tissue/cell culture based models – yet also identified examples of coherent responses from the in vitro ESC-based models and in vivo measures (Robinson et al., 2011). Furthermore, predictive neurotoxicity testing by hESC-based neuronal differentiation approaches has proven successful in discriminating chemicals and pharmaceuticals with known developmental neurotoxicity (Buzanska et al., 2009). A related approach to hESC-based neurotoxicology has been to start developmentally down-stream of the pluripotent state and utilize multipotent human neuroprogenitors as a starting point for developmental neurotoxicity testing (Breier et al., 2008, Harrill et al., 2010, Harrill et al., 2011, Moors et al., 2009, Schreiber et al., 2010, Tofighi et al., 2011a, Tofighi et al., 2011b). Neuralization of pluripotent stem cells or neuroprogenitors can be accomplished either by adherent culture-based neuronal differentiation or a neurosphere suspension culture, which may be followed by subsequent plating, differentiation and migration. A discussion of the advantages and disadvantages of these two approaches has been recently reviewed by Breier and colleagues (Breier et al., 2010).
In this review, we seek to describe the methods of generating hiPSCs, explore the utility of this technology in the field of neurotoxicology, and discuss technical challenges for these applications. In addition, we will outline the process of generating and maintaining hiPSCs for toxicity testing, characterize multiple exposure paradigms, and attempt to predict the future of the field.
Section snippets
The promise of iPSC technology for neurotoxicology
A number of recent reviews have described potential applications of hESC and hiPSC technology to toxicology, pharmacology and the study of human diseases that have environmental contributions to their etiology (Anson et al., 2011, Heng et al., 2009, Marchetto et al., 2011, Saha and Jaenisch, 2009, Vojnits and Bremer, 2010, Winkler et al., 2009, Wobus and Loser, 2011). Here we focus on the promise and roadblocks specifically for neurotoxicological applications. An important advantage of a
Methods of hiPSC generation
In 2007 Yamanaka showed for the first time the possibility of transforming adult human fibroblasts to pluripotent stem cells using four defined transcription factors (Takahashi et al., 2007). This ground breaking discovery led the way to ample research in an attempt to both understand the molecular basis of stem cell induction and possible ways to improve it. Induced pluripotent stem cells (iPSCs) exhibit the typical characteristics of the inner mass-derived human embryonic stem cells,
Special considerations and technical challenges for iPSC-based neurotoxicological applications
A number of obstacles stand between these promises of iPSC technology and their practical application. This review seeks to find a realistic optimism for what can reasonably be accomplished in the coming years as well as detail some of the key roadblocks that must be addressed before the hope of such applications can be realized.
The potential hiPSC technology for personalized medicine and risk assessment
As discussed above, hiPSCs have a wide-range of potential clinical applications, particularly in the field of personalized medicine. These applications include generation of cells for transplantation therapy and as a model of human disease (Hankowski et al., 2011). The major advantage of iPSCs over other disease models is that it has the potential to model any disease and toxicological phenotype, especially those that do not currently have an animal model. Furthermore, hiPSCs permit the study
Conclusions
The ability of iPSC lines to be isolated from patients with any neurological disease provides an important tool for characterization of susceptibility to various toxicants. Neurotoxicological studies of iPSC-derived cells are readily adaptable to existing toxicity assays, which allows experiments to be controlled against primary neuronal cultures. Validation of iPSC-based findings will likely be dependent on appropriate validation of the iPSCs themselves. For example, expressions of surface
Conflict of interest
The authors declare that there are no conflicts of interest.
Acknowledgments
K.K.K. acknowledges support by the Public Health Service award T32 GM07347 from the National Institute of General Medical Studies for the Vanderbilt Medical Scientist Training Program. This work was supported by grants from the NIH P30 ES000267 and NIH RO1 ES016931.
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