Translation: screening for novel therapeutics with disease-relevant cell types derived from human stem cell models

Abstract

The advent of somatic cell reprogramming technologies-which enables the generation of patient-specific, induced pluripotent stem cell and other trans-differentiated human neuronal cell models-provides new means of gaining insight into the molecular mechanisms and neural substrates of psychiatric disorders. By allowing a more precise understanding of genotype-phenotype relationship in disease-relevant human cell types, the use of reprogramming technologies in tandem with emerging genome engineering approaches provides a previously "missing link" between basic research and translational efforts. In this review, we summarize advances in applying human pluripotent stem cell and reprogramming technologies to generate specific neural subtypes with a focus on the use of these in vitro systems for the discovery of small molecule-probes and novel therapeutics. Examples are given where human cell models of psychiatric disorders have begun to reveal new mechanistic insight into pathophysiology and simultaneously have provided the foundation for developing disease-relevant, phenotypic assays suitable for both functional genomic and chemical screens. A number of areas for future research are discussed, including the need to develop robust methodology for the reproducible, large-scale production of disease-relevant neural cell types in formats compatible with high-throughput screening modalities, including high-content imaging, multidimensional, signature-based screening, and in vitro network with multielectrode arrays. Limitations, including the challenges in recapitulating neurocircuits and non-cell autonomous phenotypes are discussed. Although these technologies are still in active development, we conclude that, as our understanding of how to efficiently generate and probe the plasticity of patient-specific stem models improves, their utility is likely to advance rapidly.

Keywords: Disease-relevant cell type; high-throughput screening; induced pluripotent stem cells; neural progenitors; neuropharmacology; neuroplasticity; phenotypic assays; reprogramming.

Figure 1

Overview of an Integrated Platform for Biological and Therapeutic Discovery Using Patient-Specific iPSC Models and Chemical Neurobiology.

Figure 2. Generation of Long-Term, Self-Renewing Human iPSC-Derived Neural Progenitors for Use in Chemical Neurobiology Studies and Novel Therapeutic Screening

(A) Human iPSC-derived NPCs can be derived from the manual isolation of neural rosette structures from iPSC colonies subject to neuroepithelial patterning by a variety of methods (–44), including “dual SMAD” inhibition, growth factor removal, and from spontaneous formation. Isolated NPCs can be expanded in neural progenitor selective conditions on poly-ornithine/laminin coated surfaces in the presence of the mitogens EGF and FGF2 (bFGF). (B) Example of immunocytochemical analyses of the neural progenitor markers (Nestin, SOX2 and PSA-NCAM) and (C) neuronal markers (TuJ1⁺, MAP2⁺, SMI312⁺) after differentiation for 7 days that is initiated by removal of EGF and FGF2 (bFGF) mitogens. Images adapted from (63).

Figure 3. Developing Disease-Relevant Cell-Based Assays Through Directed Differentiation of Human iPSCs to

Example of human corticogenesis based upon recent studies of human pluripotent stem cells Directed differentiation of iPSCs to cortical neurons through rosette-derived neural progenitors cells. The use of morphogens and patterning agents, including inhibitors of SMAD (e.g. Noggin, dorsomorphin, and SB431542) that attenuate bone morphogenetic protein (BMP) and transforming growth factor beta (TGF-β) signal transduction pathways, and/or retinoic acid (vitamin A), leads to the formation within neural rosettes of self-renewing, cortical progenitor cells expressing the transcription factors PAX6+, FOXG1+, OTX1/2+, and TBR2+ (40, 55). Removal of the mitogen FGF2 initiates neurogenesis with the concomitant production of intermediate/basal progenitor cells and then outer radial glia (oRG) cells that produce a diverse range of upper and lower layer cortical, glutamatergic projection neuron subtypes that follows the temporal course of neuronal production observed in vivo in the human brain over a 21–90 day period (40, 55). By addition of other patterning agents the fate of the neural progenitors can be altered. For example, addition of the hedgehog signaling agonist puromorphine can ventralize neural rosettes to generate GABAergic (GAD67+) interneurons (40). Scaling these procedures and optimizing protocols for robustness and reproducibility will be critical milestones to achieve in order to support high-throughputscreening using patient-derived neuronal subtypes that are relevant to particular psychiatric disorders.