iPSC Disease Modeling Research Articles

Diminished GALNS activity in induced pluripotent stem cells of mucopolysaccharidosis IVA caused by compound p. S162Y and p. C165F mutation.

Mucopolysaccharidosis (MPS) IVA is a lysosomal storage disorder caused by mutations in the gene encoding the galactosamine (N-acetyl)-6-sulfatase (GALNS) enzyme. Children with MPS IVA usually develop pectus carinatum, genu valgum, and multiple skeletal abnormalities. To establish a patient-derived induced pluripotent stem cell (iPSC) disease model to investigate the effects of two GALNS missense mutations. The medical history and clinical manifestations of a patient with MPS IVA were first inspected. The effects of the identified GALNS mutations were predicted through bioinformatic analysis. iPSCs were then generated by using Sendai virus to introduce Yamanaka reprogramming factors to urinary cells isolated from the patient. The pluripotency, karyotypic integrity, genetic mutations, and differentiation ability of the iPSCs were tested. The effects of the GALNS mutations were further experimentally characterized using patient-derived cells. The patient exhibited a typical MPS IVA phenotype. Enzyme replacement therapy could not correct her skeletal abnormalities. GALNS c.485C>A (p.S162Y) and c.494G>T (p.C165F) mutations, inherited from her father and mother respectively, were identified in the patient. These two mutations were predicted to disturb the hydrophobic core of the GALNS catalytic domain. Patient-derived iPSCs were successfully generated, and further characterization indicated that the two missense mutations significantly diminished GALNS activity without affecting its amount at both the RNA and protein levels. We established a novel clinically relevant MPS IVA disease model that will be useful not only for investigating the pathogenic mechanisms of MPS IVA variants but also for drug screening and preclinical evaluation of novel therapies.

Intracellular calcium dysregulation in heart and brain diseases: Insights from induced pluripotent stem cell studies.

The central nervous system (CNS) plays a role in regulating heart rate and myocardial contractility through sympathetic and parasympathetic nerves, and the heart can impact the functional equilibrium of the CNS through feedback signals. Although heart and brain diseases often coexist and mutually influence each other, the potential links between heart and brain diseases remain unclear due to a lack of reliable models of these relationships. Induced pluripotent stem cells (iPSCs), which can differentiate into multiple functional cell types, stem cell biology and regenerative medicine may offer tools to clarify the mechanisms of these relationships and facilitate screening of effective therapeutic agents. Because calcium ions play essential roles in regulating both the cardiovascular and nervous systems, this review addresses how recent iPSC disease models reveal how dysregulation of intracellular calcium might be a common pathological factor underlying the relationships between heart and brain diseases.

Generation of Alzheimer's Disease Model Derived from Induced Pluripotent Stem Cells with APP Gene Mutation.

Alzheimer's disease (AD), the most common cause of dementia, is characterized by disruptions in memory, cognition, and personality, significantly impacting morbidity and mortality rates among older adults. However, the exact pathophysiological mechanism of AD remains unknown, and effective treatment options for AD are still lacking. Human induced pluripotent stem cells (iPSC) are emerging as promising platforms for disease research, offering the ability to model the genetic mutations associated with various conditions. Patient-derived iPSCs are useful for modeling neurodegenerative and neurodevelopmental disorders. In this study, we generated AD iPSCs from peripheral blood mononuclear cells obtained from a 65-year-old patient with AD carrying the E682K mutation in the gene encoding the amyloid precursor protein. Cerebral organoids derived from AD iPSCs recapitulated the AD phenotype, exhibiting significantly increased levels of tau protein. Our analysis revealed that an iPSC disease model of AD is a valuable assessment tool for pathophysiological research and drug screening.

Functional genomics in stem cell models: considerations and applications.

Protocols to differentiate human pluripotent stem cells have advanced in terms of cell type specificity and tissue-level complexity over the past 2decades, which has facilitated human disease modeling in the most relevant cell types. The ability to generate induced PSCs (iPSCs) from patients further enables the study of disease mutations in an appropriate cellular context to reveal the mechanisms that underlie disease etiology and progression. As iPSC-derived disease models have improved in robustness and scale, they have also been adopted more widely for use in drug screens to discover new therapies and therapeutic targets. Advancement in genome editing technologies, in particular the discovery of CRISPR-Cas9, has further allowed for rapid development of iPSCs containing disease-causing mutations. CRISPR-Cas9 technologies have now evolved beyond creating single gene edits, aided by the fusion of inhibitory (CRISPRi) or activation (CRISPRa) domains to a catalytically dead Cas9 protein, enabling inhibition or activation of endogenous gene loci. These tools have been used in CRISPR knockout, CRISPRi, or CRISPRa screens to identify genetic modifiers that synergize or antagonize with disease mutations in a systematic and unbiased manner, resulting in identification of disease mechanisms and discovery of new therapeutic targets to accelerate drug discovery research. However, many technical challenges remain when applying large-scale functional genomics approaches to differentiated PSC populations. Here we review current technologies in the field of iPSC disease modeling and CRISPR-based functional genomics screens and practical considerations for implementation across a range of modalities, applications, and disease areas, as well as explore CRISPR screens that have been performed in iPSC models to-date and the insights and therapies these screens have produced.

Small Molecule Modulators of Metabolic Dysfunction as a Therapeutic Strategy For ALS (P10-8.014)

<h3>Objective:</h3> To develop small molecules that correct metabolic dysfunction, specifically aberrant Fatty Acid Oxidation (FAO), which is a major pathophysiological feature of ALS. <h3>Background:</h3> Our drug discovery effort is nucleated around a new disease hypothesis for ALS – that when faced with a routine energy deficit, ALS-predisposed cells fail to respond correctly, and send themselves into a deadly spiral that is exacerbated by signals misconstrued as starvation, and which in turn lead to increased starvation. The correct response to an energy deficit (in neurons and in muscle cells) is to temporarily switch to burning Fatty Acids for energy, but to temper this process by initiating its shutdown almost as soon as it is activated. The incorrect response is to open the Fatty Acid import valve and forget to close it. We discovered that Stress Granules (SGs) are responsible for closing the Fatty Acid import valve, and as their function is disrupted in ALS, so is this critical metabolic response mechanism. This dysfunction can come about either because of a loss of function OR toxic gain of function of key SG and metabolic regulators, particularly TDP-43. By designing a metabolic “fingerprint” for ALS-related dysfunction, our therapeutic efforts can span the entire gain/loss of function spectrum of related mutations. <h3>Design/Methods:</h3> We investigated the metabolic response to fuel-deficit stress in ALS model cell lines and ALS patient-derived iPSCs differentiated to motor neurons. <h3>Results:</h3> Stress Granules associate with the mitochondrial porin, VDAC2, and regulate the gating of Fatty Acid import, the limiting factor for FAO. Stress Granule pathology and TDP-43 pathology disrupt the gating and induce constitutive FAO. <h3>Conclusions:</h3> The mitochondrial Fatty Acid import machinery is a promising, unexplored therapeutic target for ALS. Studies in patient-derived iPSC disease models provide important validation for this new therapeutic hypothesis. <b>Disclosure:</b> Daniel Kaganovich has received personal compensation for serving as an employee of Wren Therapeutics. Dr. Thomson has received personal compensation for serving as an employee of Wren Therapeutics. Dr. Thomson has stock in Amylyx. Dr. Thomson has received intellectual property interests from a discovery or technology relating to health care. Dr. Thomson has received intellectual property interests from a discovery or technology relating to health care.

Investigation of PTC124-mediated translational readthrough in a retinal organoid model of AIPL1-associated Leber congenital amaurosis.

SummaryLeber congenital amaurosis type 4 (LCA4), caused by AIPL1 mutations, is characterized by severe sight impairment in infancy and rapidly progressing degeneration of photoreceptor cells. We generated retinal organoids using induced pluripotent stem cells (iPSCs) from renal epithelial cells obtained from four children with AIPL1 nonsense mutations. iPSC-derived photoreceptors exhibited the molecular hallmarks of LCA4, including undetectable AIPL1 and rod cyclic guanosine monophosphate (cGMP) phosphodiesterase (PDE6) compared with control or CRISPR-corrected organoids. Increased levels of cGMP were detected. The translational readthrough-inducing drug (TRID) PTC124 was investigated as a potential therapeutic agent. LCA4 retinal organoids exhibited low levels of rescue of full-length AIPL1. However, this was insufficient to fully restore PDE6 in photoreceptors and reduce cGMP. LCA4 retinal organoids are a valuable platform for in vitro investigation of novel therapeutic agents.

RNA-Binding Proteins: Emerging Therapeutics for Vascular Dysfunction.

Vascular diseases account for a significant number of deaths worldwide, with cardiovascular diseases remaining the leading cause of mortality. This ongoing, ever-increasing burden has made the need for an effective treatment strategy a global priority. Recent advances in regenerative medicine, largely the derivation and use of induced pluripotent stem cell (iPSC) technologies as disease models, have provided powerful tools to study the different cell types that comprise the vascular system, allowing for a greater understanding of the molecular mechanisms behind vascular health. iPSC disease models consequently offer an exciting strategy to deepen our understanding of disease as well as develop new therapeutic avenues with clinical translation. Both transcriptional and post-transcriptional mechanisms are widely accepted to have fundamental roles in orchestrating responses to vascular damage. Recently, iPSC technologies have increased our understanding of RNA-binding proteins (RBPs) in controlling gene expression and cellular functions, providing an insight into the onset and progression of vascular dysfunction. Revelations of such roles within vascular disease states have therefore allowed for a greater clarification of disease mechanisms, aiding the development of novel therapeutic interventions. Here, we discuss newly discovered roles of RBPs within the cardio-vasculature aided by iPSC technologies, as well as examine their therapeutic potential, with a particular focus on the Quaking family of isoforms.

Multielectrode Arrays for Functional Phenotyping of Neurons from Induced Pluripotent Stem Cell Models of Neurodevelopmental Disorders.

Simple SummaryMultielectrode array technology allows researchers to record the spontaneous firing activity of cultured neurons over a period of multiple weeks or months. These data can be valuable for understanding how the functional relationships between neurons evolve as they begin to form connections and wire into a functional network. This technology has been adopted by researchers using stem cells to produce human neurons in culture to study neurodevelopmental disorders. However, the dizzying complexity and scale of the data generated have posed some challenges with the analysis and interpretation of experimental results. Here, we first provide historical context as to why multielectrode array platforms were originally developed, and use this perspective to explore some of the challenges currently facing the field. We then highlight new analysis methods, provide some guidance for improving the analysis of multielectrode array data, and discuss standardizing how these findings are communicated in scientific publications.In vitro multielectrode array (MEA) systems are increasingly used as higher-throughput platforms for functional phenotyping studies of neurons in induced pluripotent stem cell (iPSC) disease models. While MEA systems generate large amounts of spatiotemporal activity data from networks of iPSC-derived neurons, the downstream analysis and interpretation of such high-dimensional data often pose a significant challenge to researchers. In this review, we examine how MEA technology is currently deployed in iPSC modeling studies of neurodevelopmental disorders. We first highlight the strengths of in vitro MEA technology by reviewing the history of its development and the original scientific questions MEAs were intended to answer. Methods of generating patient iPSC-derived neurons and astrocytes for MEA co-cultures are summarized. We then discuss challenges associated with MEA data analysis in a disease modeling context, and present novel computational methods used to better interpret network phenotyping data. We end by suggesting best practices for presenting MEA data in research publications, and propose that the creation of a public MEA data repository to enable collaborative data sharing would be of great benefit to the iPSC disease modeling community.

Edaravone activates the GDNF/RET neurotrophic signaling pathway and protects mRNA-induced motor neurons from iPS cells

BackgroundSpinal cord motor neurons (MNs) from human iPS cells (iPSCs) have wide applications in disease modeling and therapeutic development for amyotrophic lateral sclerosis (ALS) and other MN-associated neurodegenerative diseases. We need highly efficient MN differentiation strategies for generating iPSC-derived disease models that closely recapitulate the genetic and phenotypic complexity of ALS. An important application of these models is to understand molecular mechanisms of action of FDA-approved ALS drugs that only show modest clinical efficacy. Novel mechanistic insights will help us design optimal therapeutic strategies together with predictive biomarkers to achieve better efficacy.MethodsWe induce efficient MN differentiation from iPSCs in 4 days using synthetic mRNAs coding two transcription factors (Ngn2 and Olig2) with phosphosite modification. These MNs after extensive characterization were applied in electrophysiological and neurotoxicity assays as well as transcriptomic analysis, to study the neuroprotective effect and molecular mechanisms of edaravone, an FDA-approved drug for ALS, for improving its clinical efficacy.ResultsWe generate highly pure and functional mRNA-induced MNs (miMNs) from control and ALS iPSCs, as well as embryonic stem cells. Edaravone alleviates H2O2-induced neurotoxicity and electrophysiological dysfunction in miMNs, demonstrating its neuroprotective effect that was also found in the glutamate-induced miMN neurotoxicity model. Guided by the transcriptomic analysis, we show a previously unrecognized effect of edaravone to induce the GDNF receptor RET and the GDNF/RET neurotrophic signaling in vitro and in vivo, suggesting a clinically translatable strategy to activate this key neuroprotective signaling. Notably, edaravone can replace required neurotrophic factors (BDNF and GDNF) to support long-term miMN survival and maturation, further supporting the neurotrophic function of edaravone-activated signaling. Furthermore, we show that edaravone and GDNF combined treatment more effectively protects miMNs from H2O2-induced neurotoxicity than single treatment, suggesting a potential combination strategy for ALS treatment.ConclusionsThis study provides methodology to facilitate iPSC differentiation and disease modeling. Our discoveries will facilitate the development of optimal edaravone-based therapies for ALS and potentially other neurodegenerative diseases.Graphical abstract

Mitochondrial Phenotypes in Parkinson’s Diseases—A Focus on Human iPSC-Derived Dopaminergic Neurons

Established disease models have helped unravel the mechanistic underpinnings of pathological phenotypes in Parkinson’s disease (PD), the second most common neurodegenerative disorder. However, these discoveries have been limited to relatively simple cellular systems and animal models, which typically manifest with incomplete or imperfect recapitulation of disease phenotypes. The advent of induced pluripotent stem cells (iPSCs) has provided a powerful scientific tool for investigating the underlying molecular mechanisms of both familial and sporadic PD within disease-relevant cell types and patient-specific genetic backgrounds. Overwhelming evidence supports mitochondrial dysfunction as a central feature in PD pathophysiology, and iPSC-based neuronal models have expanded our understanding of mitochondrial dynamics in the development and progression of this devastating disorder. The present review provides a comprehensive assessment of mitochondrial phenotypes reported in iPSC-derived neurons generated from PD patients’ somatic cells, with an emphasis on the role of mitochondrial respiration, morphology, and trafficking, as well as mitophagy and calcium handling in health and disease. Furthermore, we summarize the distinguishing characteristics of vulnerable midbrain dopaminergic neurons in PD and report the unique advantages and challenges of iPSC disease modeling at present, and for future mechanistic and therapeutic applications.

Human Induced Pluripotent Stem Cell Models of Frontotemporal Dementia With Tau Pathology.

Neurodegenerative dementias are the most common group of neurodegenerative diseases affecting more than 40 million people worldwide. One of these diseases is frontotemporal dementia (FTD), an early onset dementia and one of the leading causes of dementia in people under the age of 60. FTD is a heterogeneous group of neurodegenerative disorders with pathological accumulation of particular proteins in neurons and glial cells including the microtubule-associated protein tau, which is deposited in its hyperphosphorylated form in about half of all patients with FTD. As for other patients with dementia, there is currently no cure for patients with FTD and thus several lines of research focus on the characterization of underlying pathogenic mechanisms with the goal to identify therapeutic targets. In this review, we provide an overview of reported disease phenotypes in induced pluripotent stem cell (iPSC)-derived neurons and glial cells from patients with tau-associated FTD with the aim to highlight recent progress in this fast-moving field of iPSC disease modeling. We put a particular focus on genetic forms of the disease that are linked to mutations in the gene encoding tau and summarize mutation-associated changes in FTD patient cells related to tau splicing and tau phosphorylation, microtubule function and cell metabolism as well as calcium homeostasis and cellular stress. In addition, we discuss challenges and limitations but also opportunities using differentiated patient-derived iPSCs for disease modeling and biomedical research on neurodegenerative diseases including FTD.

Harnessing the Power of Induced Pluripotent Stem Cells and Gene Editing Technology: Therapeutic Implications in Hematological Malignancies.

In vitro modeling of hematological malignancies not only provides insights into the influence of genetic aberrations on cellular and molecular mechanisms involved in disease progression but also aids development and evaluation of therapeutic agents. Owing to their self-renewal and differentiation capacity, induced pluripotent stem cells (iPSCs) have emerged as a potential source of short in supply disease-specific human cells of the hematopoietic lineage. Patient-derived iPSCs can recapitulate the disease severity and spectrum of prognosis dictated by the genetic variation among patients and can be used for drug screening and studying clonal evolution. However, this approach lacks the ability to model the early phases of the disease leading to cancer. The advent of genetic editing technology has promoted the generation of precise isogenic iPSC disease models to address questions regarding the underlying genetic mechanism of disease initiation and progression. In this review, we discuss the use of iPSC disease modeling in hematological diseases, where there is lack of patient sample availability and/or difficulty of engraftment to generate animal models. Furthermore, we describe the power of combining iPSC and precise gene editing to elucidate the underlying mechanism of initiation and progression of various hematological malignancies. Finally, we discuss the power of iPSC disease modeling in developing and testing novel therapies in a high throughput setting.

An induced pluripotent stem cell line (NCATS-CL9075) from a patient carrying compound heterozygote mutations, p.R390P and p.L318P, in the NGLY1 gene

NGLY1 deficiency is a rare disorder caused by mutations in the NGLY1 gene which codes for the highly conserved N-glycanase1 (NGLY1). This enzyme functions in cytosolic deglycosylation of N- linked glycoproteins. An induced pluripotent stem cell (iPSC) line was generated from the dermal fibroblasts of a 2-year-old patient carrying compound heterozygous mutations, p.R390P and p.L318P in the NGLY1 gene. This cell-based iPSC disease model provides a resource to study disease pathophysiology and to develop a cell-based disease model for drug development for NGLY1 patients.

Network-based screen in iPSC-derived cells reveals therapeutic candidate for heart valve disease.

Mapping the gene-regulatory networks dysregulated in human disease would allow the design of network-correcting therapies that treat the core disease mechanism. However, small molecules are traditionally screened for their effects on one to several outputs at most, biasing discovery and limiting the likelihood of true disease-modifying drug candidates. Here, we developed a machine-learning approach to identify small molecules that broadly correct gene networks dysregulated in a human induced pluripotent stem cell (iPSC) disease model of a common form of heart disease involving the aortic valve (AV). Gene network correction by the most efficacious therapeutic candidate, XCT790, generalized to patient-derived primary AV cells and was sufficient to prevent and treat AV disease in vivo in a mouse model. This strategy, made feasible by human iPSC technology, network analysis, and machine learning, may represent an effective path for drug discovery.

Quantitative proteomics reveal lineage-specific protein profiles in iPSC-derived Marfan syndrome smooth muscle cells

Marfan syndrome (MFS) is a connective tissue disorder caused by mutations in the FBN1 gene that produces wide disease phenotypic variability. The lack of ample genotype–phenotype correlation hinders translational study development aimed at improving disease prognosis. In response to this need, an induced pluripotent stem cell (iPSC) disease model has been used to test patient-specific cells by a proteomic approach. This model has the potential to risk stratify patients to make clinical decisions, including timing for surgical treatment. The regional propensity for aneurysm formation in MFS may be related to distinct smooth muscle cell (SMC) embryologic lineages. Thus, peripheral blood mononuclear cell (PBMC)-derived induced pluripotent stem cells (iPSC) were differentiated into lateral mesoderm (LM, aortic root) and neural crest (NC, ascending aorta/transverse arch) SMC lineages to model MFS aortic pathology. Isobaric Tags for Relative and Absolute Quantitation (iTRAQ) proteomic analysis by tandem mass spectrometry was applied to profile LM and NC iPSC SMCs from four MFS patients and two healthy controls. Analysis revealed 45 proteins with lineage-dependent expression in MFS patients, many of which were specific to diseased samples. Single protein-level data from both iPSC SMCs and primary MFS aortic root aneurysm tissue confirmed elevated integrin αV and reduced MRC2 in clinical disease specimens, validating the iPSC iTRAQ findings. Functionally, iPSC SMCs exhibited defective adhesion to a variety of extracellular matrix proteins, especially laminin-1 and fibronectin, suggesting altered cytoskeleton dynamics. This study defines the aortic embryologic origin-specific proteome in a validated iPSC SMC model to identify novel protein markers associated with MFS aneurysm phenotype. Translating iPSC findings into clinical aortic aneurysm tissue samples highlights the potential for iPSC-based methods to model MFS disease for mechanistic studies and therapeutic discovery in vitro.

Elimination of Mutant mtDNA by an Optimized mpTALEN Restores Differentiation Capacities of Heteroplasmic MELAS-iPSCs

Various mitochondrial diseases, including mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS), are associated with heteroplasmic mutations in mitochondrial DNA (mtDNA). Herein, we refined a previously generated G13513A mtDNA-targeted platinum transcription activator-like effector nuclease (G13513A-mpTALEN) to more efficiently manipulate mtDNA heteroplasmy in MELAS-induced pluripotent stem cells (iPSCs). Introduction of a nonconventional TALE array at position 6 in the mpTALEN monomer, which recognizes the sequence around the m.13513G>A position, improved the mpTALEN effect on the heteroplasmic shift. Furthermore, the reduced expression of the new Lv-mpTALEN(PKLB)/R-mpTALEN(PKR6C) pair by modifying codons in their expression vectors could suppress the reduction in the mtDNA copy number, which contributed to the rapid recovery of mtDNA in mpTALEN-applied iPSCs during subsequent culturing. Moreover, MELAS-iPSCs with a high proportion of G13513A mutant mtDNA showed unusual properties of spontaneous, embryoid body-mediated differentiation in vitro, which was relieved by decreasing the heteroplasmy level with G13513A-mpTALEN. Additionally, drug-inducible, myogenic differentiation 1 (MYOD)-transfected MELAS-iPSCs (MyoD-iPSCs) efficiently differentiated into myosin heavy chain-positive myocytes, with or without mutant mtDNA. Hence, heteroplasmic MyoD-iPSCs controlled by fine-tuned mpTALENs may contribute to a detailed analysis of the relationship between mutation load and cellular phenotypes in disease modeling.

2050-P: A Multiomics Investigation of a Patient-Derived HNF-1A MODY IPSC Disease Model Reveals Insights into Its Role in Pancreatic Islet Development and Function

Heterozygous loss of function mutations in HNF1A, encoding the transcription factor hepatocyte nuclear factor 1 alpha (HNF-1A) are the most common cause of monogenic diabetes. HNF-1A is involved in both beta cell development and mature cell function. However, model organisms have often failed to faithfully recapitulate the human phenotypes. As such, we used a patient-derived HNF1A Pro291fsinsC iPSC model and CRISPR-Cas9 genome editing to correct the mutation and generate an isogenic control. iPSC clones (n=6) were differentiated towards the endocrine lineage in triplicate, with RNA-seq (bulk and single-cell) and ATAC-seq performed at definitive endoderm (DE), pancreatic endoderm (PE) and beta-like cells (BLC) stages. RNA-seq data confirmed HNF1A haploinsuffiency in the patient cell lines, with corrected cell lines demonstrating restored HNF1A expression at both transcript and protein level. At the BLC stage, there was a significant overlap in differentially expressed genes and HNF1A targets identified in an independent HNF1A knock-down experiment in the EndoC-βh1 cell line (p=4.4e-132). Corrected clones demonstrated a higher expression of INS (p=7.2e-03), insulin secretion genes and effects on pancreas progenitor cell genes indicating that HNF1A may mediate the cellular composition of the pancreatic islet. Bulk RNA-seq data were also supported in the preliminary analysis of the single-cell RNA-seq data. Open chromatin, assessed by ATAC-seq was significantly different between patient and corrected lines, with &amp;gt;90% of the sites being more accessible at both PE and BLC stages in the corrected clones. These sites were enriched for HNF1A binding motifs, implicating HNF1A in the establishment of open chromatin. In conclusion, our patient-derived HNF1A-MODY iPSC model recapitulates a number of features of HNF1A-MODY, as well as providing insights into the cellular and molecular phenotypes caused by HNF-1A deficiency. Disclosure C. Duff: Research Support; Self; Novo Nordisk A/S. R. Jensen: None. M. Perez-Alcantara: None. M. Rasmussen: None. A. Grotz: None. M. Jansen: None. B. Holst: None. C. Clausen: None. N.A.J. Krentz: None. M. Hansson: Employee; Self; Novo Nordisk A/S. M.I. McCarthy: Advisory Panel; Self; Illumina. Consultant; Self; Eli Lilly and Company, Novo Nordisk Inc., Zoe Global Ltd. Employee; Self; Genentech, Inc. Research Support; Self; AbbVie Inc., Merck &amp; Co., Inc., Sanofi-Aventis, Servier, Takeda UK. A.L. Gloyn: Consultant; Self; Merck Sharp &amp; Dohme Corp. Employee; Spouse/Partner; Genentech, Inc. Research Support; Self; Novo Nordisk A/S. A. Wesolowska-Andersen: None. C. Honore: Employee; Self; Novo Nordisk A/S. Stock/Shareholder; Self; Novo Nordisk A/S. Funding Innovative Medicines Initiative (115439)

Induced pluripotent stem cells in disease modelling and drug discovery.

The derivation of induced pluripotent stem cells (iPSCs) over a decade ago sparked widespread enthusiasm for the development of new models of human disease, enhanced platforms for drug discovery and more widespread use of autologous cell-based therapy. Early studies using directed differentiation of iPSCs frequently uncovered cell-level phenotypes in monogenic diseases, but translation to tissue-level and organ-level diseases has required development of more complex, 3D, multicellular systems. Organoids and human-rodent chimaeras more accurately mirror the diverse cellular ecosystems of complex tissues and are being applied to iPSC disease models to recapitulate the pathobiology of a broad spectrum of human maladies, including infectious diseases, genetic disorders and cancer.

From Genetic Association To Disease Biology: 2d And 3d Human Ipsc Models of Neuropsychiatric Disorders and Crispr/Cas9 Genome Editing

Overall Abstract Recent genome-wide association studies (GWAS) under the framework of the Psychiatric Genomics Consortium (PGC), along with large-scale sequencing efforts, have identified a plethora of disease risk loci with common and/or rare risk variants. Translating these exciting genomic findings into causation and disease biology offers the promise of developing more tailored therapies in psychiatry. Large-scale transcriptomic studies in postmortem brains (e.g., CommonMind Consortium) and in lymphoblastoid cell lines (e.g., the Molecular Genetics of Schizophrenia Consortium) have re-confirmed the polygenic nature of psychiatric disease and provided novel biological insights for some risk loci. However, understanding the disease biology underlying most GWAS findings remains challenging. An important challenge is the paucity of disease-relevant biological materials for assaying molecular and cellular phenotypes associated with risk loci. Human neurons derived from induced pluripotent stem cells (iPSCs), both monolayer cultures (2D model) and the emerging brain organoids or spheroids (3D model), provide a promising alternative to human brains for recapitulating cellular phenotypes of psychiatric disorders. CRISPR/Cas9 editing could further strengthens these models by enabling the generation of isogenic lines with essentially the same genetic background on which allelic effects of a risk variant can be directly compared, thus increasing the sensitivity to detect typically small effects of a GWAS variant. Proof-of-principle studies from NextGen Genetic Association Studies (Cell Stem Cell special issue, April 2017) have demonstrated the promise of molecular profiling and genome editing of iPSC models in identifying causal variants/genes of heart, lung, and blood diseases. Our symposium aims to present similar studies that use iPSC-derived neurons or brain organoids to model molecular/cellular phenotypes, as well as developmental aspects, of psychiatric disorders. Dr. Joachim Hallmayer will first review the current status of using iPSCs for disease modeling, with autism as example to demonstrate how to tackle both monogenic and idiopathic forms of autism. Dr. Jubao Duan will then present the prioritization of putatively functional noncoding schizophrenia risk variants based on open chromatin profiles of iPSC-derived neurons, showing that a common noncoding GWAS risk SNP has detectable biological effects in CRISPR/Cas9-edited isogenic iPSC neurons. The 3rd talk by Dr. Sergiu Pasca, who is an expert in 3D iPSC modeling (Nature, 2017), will present exciting progress on building 3D models of human nervous system to study neurodevelopmental aspects of psychiatric disorders. In the 4th talk, Dr. Joseph Buxbaum will present gene expression profiling in iPSC-derived neurons of case-siblings pairs of rare genetic disorders associated with autism, and provide an approach to address the heterogeneous nature of psychiatric disorders in iPSC disease modeling by scaling up the sample size for transcriptomic profiling via robotic reprogramming and differentiation. Lastly, Dr. Pablo Gejman (discussant) will discuss challenges and limitations of using iPSC models for psychiatric disorders. Together, this symposium will provide novel insights into how iPSC models, empowered by genome-editing technology and/or automation of iPSC reprograming, can bridge gaps in understanding the disease biology and mechanisms underlying the genetic associations.

Cell Fate Engineering Tools for iPSC Disease Modeling.

The field of cell fate engineering is contingent on tools that can quantitatively assess the efficacy of cell fate engineering protocols and experiments. CellNet is such a cell fate assessment tool that utilizes network biology to both evaluate and suggest candidate transcriptional regulatory modifications to improve the similarity of an engineered population to its corresponding in vivo target population. CellNet takes in expression profiles in the form of RNA-sequencing data and generates several metrics of cell identity and protocol efficacy. In this chapter, we demonstrate how to (1) preprocess raw RNA-sequencing data to generate an expression matrix, (2) train CellNet using preprocessed expression matrices, and (3) apply CellNet to a query study and interpret its results. We demonstrate the utility of CellNet for analysis of iPSC disease modeling studies, which we evaluate through the lens of cell fate engineering.