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Short Course I SHORT COURSE I Using iPS Cells and Reprogramming to Model Neural Development and Disease Organized by Kevin Eggan, PhD Short Course I Using iPS Cells and Reprogramming to Model Neural Development and Disease Organized by Kevin Eggan, PhD Please cite articles using the model: [AUTHOR’S LAST NAME, AUTHOR’S FIRST & MIDDLE INITIALS] (2015) [CHAPTER TITLE] In: Using iPS Cells and Reprogramming to Model Neural Development and Disease. (Eggan K, ed) pp. [xx-xx]. Chicago, IL: Society for Neuroscience. All articles and their graphics are under the copyright of their respective authors. Cover graphics and design © 2015 Society for Neuroscience. Table of Contents Probing Disorders of the Nervous System Using Reprogramming Approaches Evangelos Kiskinis, PhD . 7 Stem Cells As a Tool for Studying the Developmental Regulation of Gene Expression Hynek Wichterle, PhD, Michael Closser, BS, Esteban O . Mazzoni, PhD, Shaun Mahony, PhD, Yuchun Guo, PhD, Rachel Kopunova, and David K . Gifford, PhD . 17 Generating a Functional Human Cortex In Vitro From Induced Pluripotent Stem Cells Sergiu P . Pas¸ca, MD . 27 Generating 3D Cerebral Organoids From Human Pluripotent Stem Cells to Model Cortical Development and Disease Paola Arlotta, PhD . 41 Drug-Based Modulation of Endogenous Stem Cells Promotes Functional Remyelination In Vivo Fadi J . Najm, MBA, Mayur Madhavan, PhD, Anita Zaremba, BA, Elizabeth Shick, BS, Robert T . Karl, BS, Daniel C . Factor, BA, Tyler E . Miller, BS, Zachary S . Nevin, BS, Christopher Kantor, Alex Sargent, Kevin L . Quick, Daniela M . Schlatzer, Hong Tang, Ruben Papoian, PhD, Kyle R . Brimacombe, MS, Min Shen, Matthew B . Boxer, Ajit Jadhav, Andrew P . Robinson, Joseph R . Podojil, PhD, Stephen D . Miller, Robert H . Miller, and Paul J . Tesar, PhD . 51 Developing Stem Cell Models to Study Neuropsychiatric Diseases Lindy Barrett, PhD, and Kevin Eggan, PhD . 63 Modeling Predisposition to Schizophrenia, a Genetically Heterogeneous Neuropsychiatric Disorder, Using Induced Pluripotent Stem Cells Seok-Man Ho, BSc, Erin Flaherty, BSc, and Kristen J . Brennand, PhD . 71 Probing Disorders of the Nervous System Using Reprogramming Approaches Evangelos Kiskinis, PhD Assistant Professor of Neurology and Physiology Feinberg School of Medicine Northwestern University Chicago, Illinois © 2015 Kiskinis Probing Disorders of the Nervous System Using Reprogramming Approaches 9 Introduction differentiation protocols allow for the generation NOTES Diseases of the nervous system represent an enormous of the particular neuronal subtype that is most burden for society in terms of human suffering and vulnerable to the particular disease, such as spinal financial cost. While significant advancements have motor neurons (Davis-Dusenbery et al., 2014) and been achieved over the last few decades particularly dopaminergic neurons (Kriks et al., 2011). These in terms of genetic linkage, clinical classification, neurons can be produced in abundance from variable and patient care, effective treatments are lacking. genetic backgrounds and could provide useful The inaccessibility of the relevant tissues and cell platforms for drug discovery. types in the CNS and the complex, multifactorial nature of most neurological disorders have hampered The concept of using iPSCs and lineage conversion research progress. Animal models have been crucial to study neurological disease appears straightforward: in the investigation of disease mechanisms, but both approaches allow for the generation of patient- fundamental developmental, biochemical, and specific neurons, which are relevant to the disease physiological differences exist between animals and of interest. In addition, when these neurons are humans. The importance of utilizing human cells compared with neurons generated from healthy for these purposes is evident by the large number of controls, any differences identified could be related drugs that show efficacy and safety in rodent models to the disease. In practice, however, this approach of diseases but subsequently fail in human clinical has proven to be more challenging than initially trials, failures that are attributed partly to these believed. What is the right cell type to make and species differences (Rubin, 2008). Furthermore, study? How should quality control of neurons be the overwhelming majority of neurological disease performed? What are the right controls to use when is of a sporadic nature, rendering animal modeling assessing a disease-related phenotype? How do ineffective, while it remains unclear whether the phenotypes identified in vitro relate to the clinical relatively rare monogenic forms of disease truly presentation of patients? These are just some of the represent the vast majority of sporadic cases. questions that the community has struggled with since the initial description of iPSCs and the onset The simultaneous development of methods for of the development of in vitro patient-specific disease reprogramming adult cells into induced pluripotent models. Perhaps the seemingly biggest advantage of stem cells (iPSCs) (Takahashi et al., 2007; Yu this approach—the ability to study disease in the et al., 2007; Park et al., 2008) and the directed genetic background of the patient—has created the differentiation of pluripotent stem cells into distinct biggest challenge, as genetic background contributes neuronal subtypes (Williams et al., 2012) suggested to high variability in the properties of the patient- an attractive route to a novel model system for the derived cells. This variability is a reality that study of neurological disorders. Patient-specific iPSCs neurologists have been facing for years, as often, two can be generated using epigenetic reprogramming of patients diagnosed with the same condition might various adult cell types, such as skin fibroblasts and present with very different clinical profiles. The blood mononuclear cells, and just like embryonic technology of cellular reprogramming has brought stem cells (ESCs), self-renew indefinitely and retain this reality of clinical heterogeneity seen in patients the potential to give rise to all cell types in the from the bedside to the lab bench. human body (Takahashi et al., 2007). More recently, sophisticated lineage-conversion approaches have Since the initial description of reprogramming allowed for the direct generation of neurons and technologies, neuroscientists, neurologists, and stem neural cell types from adult cells by means of cell researchers have generated and characterized overexpressing key transcription factors (Tsunemoto hundreds of patient-specific stem cell lines as well as et al., 2014). These methods have overcome some of neuronal cells derived from them. The first “wave” the limitations of directed differentiation and have of disease-modeling studies focused on generating enabled the generation of cell types that, in many patient-specific human neurons and confirming cases, were previously unattainable. previously described pathologies (Dimos et al., 2008; Ebert et al., 2009; Marchetto et al., 2010; Brennand The overwhelming advantages of using iPSCs and et al., 2011; Seibler et al., 2011; Bilican et al., 2012; lineage conversion to develop models of diseases Israel et al., 2012). More recent studies have revealed of the nervous system are that they allow one to novel insights into disease mechanisms and employed study disease mechanisms in the context of human gene editing approaches to clearly demonstrate the neurons and in the context of each patient’s unique association of identified phenotypes with known genetic constellation. In many cases, established genetic variants that contribute to disease (An © 2015 Kiskinis 10 NOTES et al., 2012; Corti et al., 2012; Fong et al., 2013; single-stranded oligodeoxynucleotide containing the Reinhardt et al., 2013; Kiskinis et al., 2014; Wainger desired edit, or a targeting plasmid with larger desired et al., 2014; Wen et al., 2014). At the same time, sequence alterations, the genomic sequence can be our ability to generate neuronal subtypes via directed precisely edited via the cells’ own endogenous repair differentiation and the exogenous expression of mechanism, homologous recombination. Given the transcription factors has made tremendous progress. incredible versatility of the CRISPR/Cas9 system and the continuous evolvement of the technical Specificity of Phenotypes: aspects of this approach, it should be expected that every iPSC study that focuses on genetic forms of The Importance of Controls disease should include an isogenic control cell line. Significant technical advancements achieved during The rescue of a phenotype by genetic correction the past few years have allowed for the generation can lead to the conclusion that the genetic lesion of patient-specific iPSCs that are free from genomic is necessary for the onset of the phenotype. The integration of the reprogramming factors (Malik same technique can be used to introduce a disease- and Rao, 2013). The essential quality of any associated mutation in a healthy iPSC line in order newly derived iPSC can be easily assessed by (1) to assess whether the mutation in itself is sufficient immunocytochemistry for pluripotency markers (e.g., for the onset of particular phenotypes. NANOG/SSEA3); (2) a quantitative pluripotency ® assay, such as TaqMan hPSC Scorecard™ Assay An alternative approach to the concern of variation (ThermoFisher Scientific, Waltham, MA) or would be to utilize multiple stem cell clones from PluriTest™ (Scripps Research Institute, available
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