Differentiation and Integration of Trisomy 21 iPSCs into Cerebral Tissues: Modeling Down Syndrome using Patient-specific iPSC-derived CNS Organoids and Humanized Chimeric Mice. Down syndrome (DS) is caused by trisomy 21, the triplication of human chromosome 21 (HSA21), and is the most common genetic cause of intellectual disability. We have successfully established and characterized multiple lines of iPSCs derived from DS patients. Particularly, we have established more than 50 DS Trisomy 21 iPSC lines, and obtained multiple pairs of corresponding isogenic disomy 21 control lines from these DS iPSCs. In addition, we have implemented CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated) technology in making genetic corrections in iPSCs. Modeling of human genetic diseases has previously been largely dependent upon availability of either pathological analysis of postmortem human tissue samples or recapitulation of human disease in transgenic animal models; better research tools for disease modeling are needed. Patient-specific iPSCs are excellent tools and versatile resources for this kind of translational research. As iPSCs are generated on an individual basis, iPSCs may be the optimal cellular material to use for disease modeling, drug discovery, and development of patient-specific therapies. We have already generated a significant amount of preliminary data. We have used a highly efficient CRSPR system to precisely control and normalize genes of interest on HSA21. We have also developed a system of 3- dimentional (3D) CNS organoid (CO) culture from DS iPSCs, which better recapitulates brain development and disease pathogenesis than the conventional 2-dimentional (2D) flat culture, and allows for in-depth characterization by electrophysiological assays. The CNS organoid technology represents an excellent approach for disease modeling; the cerebral organoids generated from patient iPSCs can be used as a model to recapitulate complex neural developmental diseases such as DS. In addition, we have generated a humanized chimeric mouse model, in which DS iPSC derived astrocytes are grafted to the neonatal mouse brains. The detailed genetic etiology for the various symptoms in DS remains elusive. Taking the advantage of these unique tools and resources, we will create novel in vitro and in vivo models of DS with human iPSCs derived from patients to recapitulate the defects in neural differentiation in DS. In support of the feasibility of this proposal, we have obtained the necessary materials and expertise to be used in this study, and have published a rather massive paper on DS iPSCs [Chen C, Jiang P, Xue H, Peterson SE, Tran HT, McCann AE, Parast MM, Li S, Pleasure DE, Laurent LC, Loring JF, Liu Y, Deng W. (2014) Role of astroglia in Down?s syndrome revealed by patient-derived human-induced pluripotent stem cells. Nature Communications. 5:4430 doi: 10.1038/ncomms5430 (2014)]. Our preliminary data show that both trisomy and the isogenic disomy DS astrocytes are able to repopulate the mouse brain, allowing for further interrogation of in vivo behavior of these cells and examination of their effects on neuroinflammation and cognition of the animals. Building upon prior work on multiple genes in pathways of astrocyte-mediated inflammation, we propose to produce both in vitro CNS organoids and in vivo chimeric mouse models to investigate the critical role of these astrocytic inflammatory genes (S100B, IFNAR1, IFNAR2) in development and function of DS patient-derived iPSCs. Taken together, we will use a novel platform of both an in vitro 3D organoid culture system and an in vivo humanized chimeric mouse model using DS patient-derived iPSCs. These models will provide fundamental insights into neural function in the physiological environment of 3D organoids and in early development of human cells in a living animal. The completion of the project will immensely bolster DS pathogenesis studies using patient iPSCs, as well as biochemical and molecular approaches complemented with investigation into neural network functionality. These insights will undoubtedly impact on the treatment of patients with DS.

Public Health Relevance

Down syndrome (DS), the most common birth defect, is caused by triplication of chromosome 21 (HSA21). Multiple organ defects especially intellectual disability severely affect DS patients? quality of life. Improvements by common treatments including early intervention and educational therapy are limited. Based on research in DS animal models, pharmacological interventions have also been attempted in small cohorts of patients, but with inconsistent results. Oftentimes significant improvements in learning and memory observed in DS mice could not be translated in clinical trials. Induced pluripotent stem cell (iPSC) technology provides a renewable source of patient autologous cells that not only retain identical genetic information but also give rise to many cell types of the brain including neurons and glia. Meanwhile, the rapid advancement of genetic engineering approaches such as gene targeting by genome editing tools including the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system has greatly accelerated the development of human genome manipulation at the molecular level. The significantly improved targeting efficiency allows for precise and simultaneous alteration of multiple genes in human iPSCs. These advancements have provided unprecedented opportunity to whittle down candidate causative genes/fragments for diseases that are otherwise difficult to dissect out. Current biomedical research is frequently carried out in a 2-dimensional tissue culture environment, despite the fact that human tissues are 3-dimensional structures that require the interactions of multiple cell types with one another and with the environment to maintain their shape and function. Most primary cells in 2D cultures quickly lose in vivo properties such as tissue-specific gene expression, cell polarity, and cell-cell and cell-matrix contacts. Increasing knowledge about cell biology, materials science, and microfabrication technologies enables scientists to engineer functional units of human tissues (i.e., 3D human tissue models). Further development of these highly controllable in vitro model systems would allow them to more closely mimic functions of human tissues or organs so that they could be used to study normal developmental biology or disease pathogenesis, or for drug screening. Patient-specific models could be generated to study any specific diseases, particularly diseases where appropriate animal models are not available. They would go beyond the ?flat biology? to increase the complexity and diversity of in vitro models and assays, and bridge the gap between simple cell cultures and the full complexity of animal models. They also have the potential to minimize and/or replace animal testing, and may be a better alternative than animal models to study humans. In addition to an in vitro 3D organoid culture system, we also propose to build on our preliminary studies to generate an in vivo humanized chimeric mouse model using DS patient-derived iPSCs. These novel models will provide fundamental insights into neural function in the physiological environment of 3D organoids and in early development of human cells in a living animal, and such insights will undoubtedly impact on the treatment of patients with DS. Successful completion of this project will help understand the role of astrocyte-mediated neurotoxicity in the pathogenesis of DS defects and identify novel therapeutic targets for DS, the most common cause of intellectual disability. It will also provide a proof of principle on feasibility studies using genetic approach on gene dosage in DS patient iPSCs and their neural progeny. Data from this study will provide clues to understanding molecular mechanisms of disease pathology. The abnormalities we identify occur in the early stages of disease. Thus, intervention of these processes might help slow or even prevent disease progression. Investigating important pathologies for a brain disorder by reprogramming patient cells also provides a validation of the pathologies in postmortem brains, which will critically increase the confidence that these are relevant for the disease. Using iPSC models as genetic screens to identify genetic basis and its mechanism of action against the fundamental pathology of a disease illustrates the power of the genome editing approach, and it is critical that we continue to leverage this power for reverse engineering various devastating human diseases. This application will immensely bolster studies of using patient iPSCs combined with genetic, biochemical and morphological approaches that are complemented with investigations of functionality of multiple organ systems in complex diseases. The iPSC- based models will complement transgenic animal models and patient samples as part of an extensive scientific infrastructure and tools for DS research. Similar strategies based on this conceptual framework will also benefit research of other diseases.

National Institute of Health (NIH)
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
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Bardhan, Sujata
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University of California Davis
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