Stem Cells For Disease Modeling Research Articles

Genome-wide CRISPR screen identifies neddylation as a regulator of neuronal aging and AD neurodegeneration

Aging is the biggest risk factor for the development of Alzheimer's disease (AD). Here, we performed a whole-genome CRISPR screen to identify regulators of neuronal age and show that the neddylation pathway regulates both cellular age and AD neurodegeneration in a human stem cell model. Specifically, we demonstrate that blocking neddylation increased cellular hallmarks of aging and led to an increase in Tau aggregation and phosphorylation in neurons carrying the APPswe/swe mutation. Aged APPswe/swe but not isogenic control neurons also showed a progressive decrease in viability. Selective neuronal loss upon neddylation inhibition was similarly observed in other isogenic AD and in Parkinson's disease (PD) models, including PSENM146V/M146V cortical and LRRK2G2019S/G2019S midbrain dopamine neurons, respectively. This study indicates that cellular aging can reveal late-onset disease phenotypes, identifies new potential targets to modulate AD progression, and describes a strategy to program age-associated phenotypes into stem cell models of disease.

Heparan Sulfate Chain-Conjugated Laminin-E8 Fragments Advance Paraxial Mesodermal Differentiation Followed by High Myogenic Induction from hiPSCs.

Human-induced pluripotent stem cells (hiPSCs) have great therapeutic potential. The cell source differentiated from hiPSCs requires xeno-free and robust methods for lineage-specific differentiation. Here, a system is described for differentiating hiPSCs on new generation laminin fragments (NGLFs), a recombinant form of a laminin E8 fragment conjugated to the heparan sulfate chains (HS) attachment domain of perlecan. Using NGLFs, hiPSCs are highly promoted to direct differentiation into a paraxial mesoderm state with high-efficiency muscle lineage generation. HS conjugation to the C-terminus of Laminin E8 fragments brings fibroblast growth factors (FGFs) bound to the HS close to the cell surface of hiPSCs, thereby facilitating stronger FGF signaling pathways stimulation and initiating HOX gene expression, which triggers the paraxial mesoderm differentiation of hiPSCs. This highly efficient differentiation system can provide a roadmap for paraxial mesoderm development and an infinite source of myocytes and muscle stem cells for disease modeling and regenerative medicine.

Crispr-Based Editing of Human Pluripotent Stem Cells for Disease Modeling.

The CRISPR system, as an effective genome editing technology, has been extensively utilized for the construction of disease models in human pluripotent stem cells. Establishment of a gene mutant or knockout stem cell line typically relies on Cas nuclease-generated double-stranded DNA breaks and exogenous templates, which can produce uncontrollable editing byproducts and toxicity. The recently developed adenine base editors (ABE) have greatly facilitated related research by introducing A/T > G/C mutations in the coding regions or splitting sites (AG-GT) of genes, enabling mutant gene knock-in or knock-out without introducing DNA breaks. In this study, we edit the AG bases in exons anterior to achieve gene knockout via the ABE8e-SpRY, which recognizes most expanded protospacer adjacent motif to target the genome. Except for gene-knockout, ABE8e-SpRY can also efficiently establish disease-related A/T-to-G/C variation cell lines by targeting coding sequences. The method we generated is simple and time-saving, and it only takes two weeks to obtain the desired cell line. This protocol provides operating instructions step-by-step for constructing knockout and point mutation cell lines.

Differentiation of Pluripotent Stem Cells for Disease Modeling: Learning from Heart Development.

Heart disease is a pressing public health problem and the leading cause of death worldwide. The heart is the first organ to gain function during embryogenesis in mammals. Heart development involves cell determination, expansion, migration, and crosstalk, which are orchestrated by numerous signaling pathways, such as the Wnt, TGF-β, IGF, and Retinoic acid signaling pathways. Human-induced pluripotent stem cell-based platforms are emerging as promising approaches for modeling heart disease in vitro. Understanding the signaling pathways that are essential for cardiac development has shed light on the molecular mechanisms of congenital heart defects and postnatal heart diseases, significantly advancing stem cell-based platforms to model heart diseases. This review summarizes signaling pathways that are crucial for heart development and discusses how these findings improve the strategies for modeling human heart disease in vitro.

Advances in the Generation of Constructed Cardiac Tissue Derived from Induced Pluripotent Stem Cells for Disease Modeling and Therapeutic Discovery.

The human heart lacks significant regenerative capacity; thus, the solution to heart failure (HF) remains organ donation, requiring surgery and immunosuppression. The demand for constructed cardiac tissues (CCTs) to model and treat disease continues to grow. Recent advances in induced pluripotent stem cell (iPSC) manipulation, CRISPR gene editing, and 3D tissue culture have enabled a boom in iPSC-derived CCTs (iPSC-CCTs) with diverse cell types and architecture. Compared with 2D-cultured cells, iPSC-CCTs better recapitulate heart biology, demonstrating the potential to advance organ modeling, drug discovery, and regenerative medicine, though iPSC-CCTs could benefit from better methods to faithfully mimic heart physiology and electrophysiology. Here, we summarize advances in iPSC-CCTs and future developments in the vascularization, immunization, and maturation of iPSC-CCTs for study and therapy.

Integration of xeno-free single-cell cloning in CRISPR-mediated DNA editing of human iPSCs improves homogeneity and methodological efficiency of cellular disease modeling

The capability to generate induced pluripotent stem cell (iPSC) lines, in tandem with CRISPR-Cas9 DNA editing, offers great promise to understand the underlying genetic mechanisms of human disease. The low efficiency of available methods for homogeneous expansion of singularized CRISPR-transfected iPSCs necessitates the coculture of transfected cells in mixed populations and/or on feeder layers. Consequently, edited cells must be purified using labor-intensive screening and selection, culminating in inefficient editing. Here, we provide a xeno-free method for single-cell cloning of CRISPRed iPSCs achieving a clonal survival of up to 70% within 7-10days. This is accomplished through improved viability of the transfected cells, paralleled with provision of an enriched environment for the robust establishment and proliferation of singularized iPSC clones. Enhanced cell survival was accompanied by a high transfection efficiency exceeding 97%, and editing efficiencies of 50%-65% for NHEJ and 10% for HDR, indicative of the method's utility in stem cell disease modeling.

Induced pluripotent stem cells derived from patients carrying mitochondrial mutations exhibit altered bioenergetics and aberrant differentiation potential

BackgroundHuman mitochondrial DNA mutations are associated with common to rare mitochondrial disorders, which are multisystemic with complex clinical pathologies. The pathologies of these diseases are poorly understood and have no FDA-approved treatments leading to symptom management. Leigh syndrome (LS) is a pediatric mitochondrial disorder that affects the central nervous system during early development and causes death in infancy. Since there are no adequate models for understanding the rapid fatality associated with LS, human-induced pluripotent stem cell (hiPSC) technology has been recognized as a useful approach to generate patient-specific stem cells for disease modeling and understanding the origins of the phenotype.MethodshiPSCs were generated from control BJ and four disease fibroblast lines using a cocktail of non-modified reprogramming and immune evasion mRNAs and microRNAs. Expression of hiPSC-associated intracellular and cell surface markers was identified by immunofluorescence and flow cytometry. Karyotyping of hiPSCs was performed with cytogenetic analysis. Sanger and next-generation sequencing were used to detect and quantify the mutation in all hiPSCs. The mitochondrial respiration ability and glycolytic function were measured by the Seahorse Bioscience XFe96 extracellular flux analyzer.ResultsReprogrammed hiPSCs expressed pluripotent stem cell markers including transcription factors POU5F1, NANOG and SOX2 and cell surface markers SSEA4, TRA-1-60 and TRA-1-81 at the protein level. Sanger sequencing analysis confirmed the presence of mutations in all reprogrammed hiPSCs. Next-generation sequencing demonstrated the variable presence of mutant mtDNA in reprogrammed hiPSCs. Cytogenetic analyses confirmed the presence of normal karyotype in all reprogrammed hiPSCs. Patient-derived hiPSCs demonstrated decreased maximal mitochondrial respiration, while mitochondrial ATP production was not significantly different between the control and disease hiPSCs. In line with low maximal respiration, the spare respiratory capacity was lower in all the disease hiPSCs. The hiPSCs also demonstrated neural and cardiac differentiation potential.ConclusionOverall, the hiPSCs exhibited variable mitochondrial dysfunction that may alter their differentiation potential and provide key insights into clinically relevant developmental perturbations.

Mitochondrial dysfunction characterized in human induced pluripotent stem cell disease models in MELAS syndrome: A brief summary

Human induced pluripotent stem cells (hiPSCs) for MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis, stroke-like episodes) may allow deeper understanding of how tissue-specific mitochondrial dysfunction result in multi-systemic disease. Here, we summarize how the m.3243G mtDNA mutation affects mitochondrial function in different tissues using iPSC and iPSC-differentiated cell type disease models and what significant findings have been replicated in the independent studies. Through this brief review and with a focus on mitochondrial dysfunction in iPSC-differentiated cell types, namely fibroblast, neuron, and retinal pigment epithelium cells, we aim to bring awareness of hiPSC as a robust mitochondrial disease model even if many unanswered questions remain.

Kidney Organoid Derived from Human Pluripotent and Adult Stem Cells for Disease Modeling.

Kidney disease affects a significant portion of the global population, yet effective therapies are lacking despite advancements in identifying genetic causes. This limitation can be attributed to the absence of adequate in vitro models that accurately mimic human kidney disease, hindering targeted therapeutic development. However, the emergence of human induced pluripotent stem cells (PSCs) and the development of organoids using them have opened up a way to model kidney development and disease in humans, as well as validate the effects of new drugs. To fully leverage their capabilities in these fields, it is crucial for kidney organoids to closely resemble the structure and functionality of adult human kidneys. In this review, we aim to discuss the potential of using human PSCs or adult kidney stem cell-derived kidney organoids to model genetic kidney disease and renal cancer.

"Exploring the Applications of Pluripotent Stem Cells in Disease Modeling and Precision Medicine"

"Exploring the Applications of Pluripotent Stem Cells in Disease Modeling and Precision Medicine"

A New Approach to Generate Gastruloids to Develop Anterior Neural Tissues.

Embryonic development is a complex process integrating cell fate decisions and morphogenesis in a spatiotemporally controlled manner. Previous studies with model organisms laid the foundation of our knowledge on post-implantation development; however, studying mammalian embryos at this stage is a difficult and laborious process. Early attempts to recapitulate mammalian development in vitro begun with embryoid bodies (EBs), in which aggregates of mouse embryonic stem cells (mESCs) were shown to differentiate into spatially arranged germ layers. A more revised version of EBs, gastruloids, improved the germ layer differentiation efficiency and demonstrated cell fate patterning on multiple axes. However, gastruloids lack anterior neural progenitors that give rise to brain tissues in the embryo. Here, we report a novel culture protocol to coax mESCs into post-implantation epiblast-like (EPI) aggregates in high throughput on bioengineered microwell arrays. We show that upon inhibition of the Wnt signaling pathway, EPI aggregates establish an extended axial patterning, leading to co-derivation of anterior neural progenitors and posterior tissues. Our approach is amenable to large-scale studies aimed at identifying novel regulators of gastrulation and anterior neural development that is currently out of reach with existing embryoid models. This work should contribute to the advancement of the nascent field of synthetic embryology, opening up exciting perspectives for various applications of pluripotent stem cells in disease modeling and tissue engineering. Key features A new gastruloid culture system to model post-implantation mouse embryonic development in vitro High-throughput formation of epiblast-like aggregates on hydrogel microwells Builds upon conventional gastruloid cultures and provides insight into the role of Wnt signaling for the formation of anterior neural tissues Graphical overview.

Development of a scalable method to isolate subsets of stem cell-derived pancreatic islet cells.

SummaryCell replacement therapy using β cells derived from stem cells is a promising alternative to conventional diabetes treatment options. Although current differentiation methods produce glucose-responsive β cells, they can also yield populations of undesired endocrine progenitors and other proliferating cell types that might interfere with long-term islet function and safety of transplanted cells. Here, we describe the generation of an array of monoclonal antibodies against cell surface markers that selectively label stem cell-derived islet cells. A high-throughput screen identified promising candidates, including three clones that mark a high proportion of endocrine cells in differentiated cultures. A scalable magnetic sorting method was developed to enrich for human pluripotent stem cell (hPSC)-derived islet cells using these three antibodies, leading to the formation of islet-like clusters with improved glucose-stimulated insulin secretion and reduced growth upon transplantation. This strategy should facilitate large-scale production of functional islet clusters from stem cells for disease modeling and cell replacement therapy.

IPS-derived neural stem cells for disease modeling and evaluation of therapeutics for mucopolysaccharidosis type II

Mucopolysaccharidosis type II (MPS II), also known as Hunter syndrome, is a rare, lysosomal disorder caused by mutations in a gene encoding iduronate-2-sulfatase (IDS). IDS deficiency results in an accumulation of glycosaminoglycans (GAGs) and secondary accumulations of other lipids in lysosomes. Symptoms of MPS II include a variety of soft and hard tissue problems, developmental delay, and deterioration of multiple organs. Enzyme replacement therapy is an approved treatment for MPS II, but fails to improve neuronal symptoms. Cell-based neuronal models of MPS II disease are needed for compound screening and drug development for the treatment of the neuronal symptoms in MPS II. In this study, three induced pluripotent stem cell (iPSC) lines were generated from three MPS II patient-derived dermal fibroblast cell lines that were differentiated into neural stem cells and neurons. The disease phenotypes were measured using immunofluorescence staining and Nile red dye staining. In addition, the therapeutic effects of recombinant human IDS enzyme, delta-tocopherol (DT), and hydroxypropyl-beta-cyclodextrin (HPBCD) were determined in the MPS II disease cells. Finally, the neural stem cells from two of the MPS II iPSC lines exhibited typical disease features including a deficiency of IDS activity, abnormal glycosaminoglycan storage, and secondary lipid accumulation. Enzyme replacement therapy partially rescued the disease phenotypes in these cells. DT showed a significant effect in reducing the secondary accumulation of lipids in the MPS II neural stem cells. In contrast, HPBCD displayed limited or no effect in these cells. Our data indicate that these MPS II cells can be used as a cell-based disease model to study disease pathogenesis, evaluate drug efficacy, and screen compounds for drug development.

Application of Induced Pluripotent Stem Cells for Disease Modeling and 3D Model Construction: Focus on Osteoarthritis.

Since their discovery in 2006, induced pluripotent stem cells (iPSCs) have shown promising potential, specifically because of their accessibility and plasticity. Hence, the clinical applicability of iPSCs was investigated in various fields of research. However, only a few iPSC studies pertaining to osteoarthritis (OA) have been performed so far, despite the high prevalence rate of degenerative joint disease. In this review, we discuss some of the most recent applications of iPSCs in disease modeling and the construction of 3D models in various fields, specifically focusing on osteoarthritis and OA-related conditions. Notably, we comprehensively reviewed the successful results of iPSC-derived disease models in recapitulating OA phenotypes for both OA and early-onset OA to encompass their broad etiology. Moreover, the latest publications with protocols that have used iPSCs to construct 3D models in recapitulating various conditions, particularly the OA environment, were further discussed. With the overall optimistic results seen in both fields, iPSCs are expected to be more widely used for OA disease modeling and 3D model construction, which could further expand OA drug screening, risk assessment, and therapeutic capabilities.

Using CRISPR to understand and manipulate gene regulation.

Understanding how genes are expressed in the correct cell types and at the correct level is a key goal of developmental biology research. Gene regulation has traditionally been approached largely through observational methods, whereas perturbational approaches have lacked precision. CRISPR-Cas9 has begun to transform the study of gene regulation, allowing for precise manipulation of genomic sequences, epigenetic functionalization and gene expression. CRISPR-Cas9 technology has already led to the discovery of new paradigms in gene regulation and, as new CRISPR-based tools and methods continue to be developed, promises to transform our knowledge of the gene regulatory code and our ability to manipulate cell fate. Here, we discuss the current and future application of the emerging CRISPR toolbox toward predicting gene regulatory network behavior, improving stem cell disease modeling, dissecting the epigenetic code, reprogramming cell fate and treating diseases of gene dysregulation.

Generation of cryopreserved macrophages from normal and genetically engineered human pluripotent stem cells for disease modelling.

Macrophages are innate immune cells that play critical roles in tissue homeostasis, inflammation, and immune oncology. Macrophages differentiated from human induced pluripotent stem cells (iPSCs) overcome many limitations of using peripheral blood derived macrophages. The ability to scale up and cryopreserve a large amount of end stage macrophages from single clonal iPSCs from normal and disease specific donors offers a unique opportunity for genomic analysis and drug screening. The present study describes the step wise generation and characterization of macrophages from iPSCs using a defined serum free method amenable to scale up to generate a large batch of pure end stage cryopreservable macrophages expressing CD68, CD33, CD11c, CD11b, CD1a, HLA-DR, CD86, CD64, CD80, CD206, CD169, CD47, HLA-ABC, and CX3CR. The end stage macrophages pre and post cryopreservation retain purity, morphology, responsiveness to stimuli and display robust phagocytic function coming right out of cryopreservation. The same differentiation process was used to generate end stage macrophages from isogenic iPSCs engineered to mimic mutations associated with Parkinson's disease (SNCA A53T), neuronal ceroid lipofuscinosis (GRN2/GRN R493X), and Rett syndrome (MECP2-Knockout). End stage macrophages from isogenic engineered clones displayed differential macrophage-specific purity markers, phagocytic function, and response to specific stimuli. Thus, generating a panel of functional, physiologically relevant iPSC-derived macrophages can potentially facilitate the understanding of neural inflammatory responses associated with neurodegeneration.

Machine learning for medicine

Heart Valve Disease Small-molecule screens aimed at identifying therapeutic candidates traditionally search for molecules that affect one to several outputs at most, limiting discovery of true disease-modifying drugs. Theodoris et al. developed a machine-learning approach to identify small molecules that broadly correct gene networks dysregulated in a human induced pluripotent stem cell disease model of a common form of heart disease involving the aortic valve. Gene network correction by the most efficacious therapeutic candidate generalized to primary aortic valve cells derived from more than 20 patients with sporadic aortic valve disease and prevented aortic valve disease in vivo in a mouse model. Science , this issue p. [eabd0724][1] [1]: /lookup/doi/10.1126/science.abd0724

Chapter 25 - Hypothalamus and neuroendocrine diseases: The use of human-induced pluripotent stem cells for disease modeling

The hypothalamus, which is part of the brain of all vertebrate animals, is considered the link between the central nervous system (CNS) and (i) the endocrine system via the pituitary gland and (ii) with our organs via the autonomic nervous system. It synthesizes and releases neurohormones, which in turn stimulate or inhibit the secretion of other hormones within the CNS, and sends and receives signals to and from the peripheral nervous and endocrine systems. As the brain region responsible for energy homeostasis, the hypothalamus is the key regulator of thermoregulation, hunger and satiety, circadian rhythms, sleep and fatigue, memory and learning, arousal and reproductive cycling, blood pressure, and heart rate and thus orchestrates complex physiological responses in order to maintain metabolic homeostasis. These critical roles implicate the hypothalamus in neuroendocrine disorders such as obesity, diabetes, anorexia nervosa, bulimia, and others. In this chapter, we focus on the use of human-induced pluripotent stem cells (hiPSCs) and their differentiation into hypothalamic neurons in order to model neuroendocrine disorders such as extreme obesity in a dish. To do so, we discuss important steps of human hypothalamus development, neuroendocrine diseases related to the hypothalamus, multiple protocols to differentiate hiPSCs into hypothalamic neurons, and severe obesity modeling in vitro using hiPSCs-derived hypothalamic neurons.

Pluripotent Stem Cells for Cell Therapy.

In an increasingly geriatric population, in which elderly people frequently face chronic diseases and degenerative conditions, cell therapies as part of novel regenerative medicine approaches are of great interest. Even though today's cell therapies mostly rely on adult stem cells like the mesenchymal stem cells or primary somatic cells, pluripotent stem cells represent an enormously versatile cell model to explore possible new avenues in the field of regenerative medicine due to their capacity to grow indefinitely and to differentiate into the desired cell types. The discovery of reprogramming somatic cells into induced pluripotent stem cells augmented the pool of applicable cell entities so that researchers nowadays can resort to embryonic stem cells, but also to a plethora of patient- and disease-specific induced pluripotent stem cells. The ease of targeted genome engineering is an additional benefit that allows using pluripotent stem cells for disease modeling, drug discovery, and the development of cell therapies. However, the task is still demanding as the generation of subpopulations and a sufficient cell maturation for some cell entities have yet to be achieved. Likewise, even though for some applications the cells of interest can be produced in the large-scale dimensions and purity that are required for clinical purposes, proper integration, and function in the host tissue remain challenging. Nonetheless, the immense progress that has been made over the last decades warrants the prominent role of pluripotent stem cells in regenerative medicine as in vitro models to broaden our knowledge of disease onset/progression and treatment as well as in vivo as a substitution of damaged/aged tissue.

Efficient Generation of Functional Hepatocytes from Human Induced Pluripotent Stem Cells for Disease Modeling and Disease Gene Discovery.

In vitro hepatocyte cell models are being used to study the pathogenesis of liver disease and in the discovery and preclinical stages of drug development. The culture of hepatic cell lines and primary hepatocytes as in vitro cell models has been carried out for several decades. However, hepatic cell lines (hepatic carcinoma generated or immortalized) have limited accuracy when recapitulating complex physiological functions of the liver. Additionally, primary hepatocytes sourced from human cadavers or medical biopsies are difficult to obtain due to sourcing limitations, particularly for large-scale population studies or in applications requiring large number of cells. Hepatocyte cultures differentiated from human embryonic stem cells (ESCs) and induced pluripotent stem cell (iPSCs) overcome in large part the limitations of traditional hepatocyte in vitro models. In this chapter, we described an efficient protocol routinely used in our laboratory to differentiate human iPSCs into functional hepatocyte cultures for in vitro modeling of liver function and disease. The protocol uses a three-stage differentiation strategy to generate functional hepatocytes from human iPSCs. The differentiated cells show characteristic hepatocyte morphology including flat and polygonal shape, distinct round nuclei, and presence of biliary canaliculi and they express hepatic markers alpha-fetoprotein (AFP), albumin (ALB), E-cadherin (CHD1), hepatocyte nuclear factor 4 alpha (HNF4α), and actin.