Rapid target validation in a Cas9-inducible hiPSC derived kidney model

AbstractRecent advances in induced pluripotent stem cells (iPSCs), genome editing technologies and 3D organoid model systems highlight opportunities to develop new in vitro human disease models to serve drug discovery programs. An ideal disease model would accurately recapitulate the relevant disease phenotype and provide a scalable platform for drug and genetic screening studies. Kidney organoids offer a high cellular complexity that may provide greater insights than conventional single-cell type cell culture models. However, genetic manipulation of the kidney organoids requires prior generation of genetically modified clonal lines, which is a time and labor consuming procedure. Here, we present a methodology for direct differentiation of the CRISPR-targeted cell pools, using a doxycycline-inducible Cas9 expressing hiPSC line for high efficiency editing to eliminate the laborious clonal line generation steps. We demonstrate the versatile use of genetically engineered kidney organoids by targeting the autosomal dominant polycystic kidney disease (ADPKD) genes: PKD1 and PKD2. Direct differentiation of the respective knockout pool populations into kidney organoids resulted in the formation of cyst-like structures in the tubular compartment. Our findings demonstrated that we can achieve > 80% editing efficiency in the iPSC pool population which resulted in a reliable 3D organoid model of ADPKD. The described methodology may provide a platform for rapid target validation in the context of disease modeling.

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Role of SHH in Patterning Human Pluripotent Cells towards Ventral Forebrain Fates

Cells ◽

10.3390/cells10040914 ◽

2021 ◽

Vol 10 (4) ◽

pp. 914

Author(s):

Melanie V. Brady ◽

Flora M. Vaccarino

Keyword(s):

Stem Cell ◽

Human Brain ◽

Human Development ◽

Disease Modeling ◽

Model Systems ◽

Advantages And Disadvantages ◽

Technical Ability ◽

Cellular Phenotypes

The complexities of human neurodevelopment have historically been challenging to decipher but continue to be of great interest in the contexts of healthy neurobiology and disease. The classic animal models and monolayer in vitro systems have limited the types of questions scientists can strive to answer in addition to the technical ability to answer them. However, the tridimensional human stem cell-derived organoid system provides the unique opportunity to model human development and mimic the diverse cellular composition of human organs. This strategy is adaptable and malleable, and these neural organoids possess the morphogenic sensitivity to be patterned in various ways to generate the different regions of the human brain. Furthermore, recapitulating human development provides a platform for disease modeling. One master regulator of human neurodevelopment in many regions of the human brain is sonic hedgehog (SHH), whose expression gradient and pathway activation are responsible for conferring ventral identity and shaping cellular phenotypes throughout the neural axis. This review first discusses the benefits, challenges, and limitations of using organoids for studying human neurodevelopment and disease, comparing advantages and disadvantages with other in vivo and in vitro model systems. Next, we explore the range of control that SHH exhibits on human neurodevelopment, and the application of SHH to various stem cell methodologies, including organoids, to expand our understanding of human development and disease. We outline how this strategy will eventually bring us much closer to uncovering the intricacies of human neurodevelopment and biology.

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Rapamycin Inhibited Pyroptosis and Reduced the Release of IL-1β and IL-18 in the Septic Response

BioMed Research International ◽

10.1155/2020/5960375 ◽

2020 ◽

Vol 2020 ◽

pp. 1-9

Author(s):

Luo Zhuo ◽

Xiaobing Chen ◽

Yan Sun ◽

Yanli Wang ◽

Yuanfeng Shi ◽

...

Keyword(s):

Therapeutic Strategy ◽

Genetic Manipulation ◽

Umbilical Vein ◽

Disease Model ◽

Positive Control ◽

Sepsis Model ◽

Autophagy Induction ◽

Immune Imbalance ◽

Umbilical Vein Endothelial Cells

Pyroptosis, an inflammatory form of programmed cell death, is the initiating event of sepsis and results in immune imbalance by releasing IL-1β and IL-18 in the early stages. Studies show that enhancing autophagy via genetic manipulation can inhibit pyroptosis and prolong the survival of a sepsis animal model, indicating a possible therapeutic strategy against sepsis. However, almost no study so far has achieved pyroptosis inhibition via pharmacological autophagy induction in a sepsis disease model. To this end, we established an in vitro sepsis model by stimulating primary human umbilical vein endothelial cells (HUVECs) with lipopolysaccharide (LPS), and analyzed the effect of the autophagy agonist rapamycin (RAPA) on pyroptosis. Phorbol 12-myristate 13-acetate- (PMA-) activated human THP-1 cells were used as the positive control. LPS significantly increased the levels of the pyroptotic protein Gasdermin D (GSDMD), cysteinyl aspartate-specific proteinase 1 (caspase-1), secreted LDH, IL-1β, and IL-18. RAPA treatment downregulated the above factors and enhanced autophagy in the LPS-stimulated HUVECs and THP-1 cells. This study shows that RAPA abrogates LPS-mediated increase in IL-1β and IL-18 by inhibiting pyroptosis and enhancing autophagy.

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Application of biomaterials to in vitro pluripotent stem cell disease modeling of the skeletal system

Journal of Materials Chemistry B ◽

10.1039/c5tb02645h ◽

2016 ◽

Vol 4 (20) ◽

pp. 3482-3489 ◽

Cited By ~ 6

Author(s):

Giuliana E. Salazar-Noratto ◽

Frank P. Barry ◽

Robert E. Guldberg

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Disease Modeling ◽

Genetic Manipulation ◽

Embryonic Stem ◽

Cell Disease ◽

Skeletal System ◽

System P ◽

Induced Pluripotent

Disease-specific pluripotent stem cells can be derived through genetic manipulation of embryonic stem cells or by reprogramming somatic cells (induced pluripotent stem cells).

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Pancreatic Progenitors and Organoids as a Prerequisite to Model Pancreatic Diseases and Cancer

Stem Cells International ◽

10.1155/2019/9301382 ◽

2019 ◽

Vol 2019 ◽

pp. 1-11 ◽

Cited By ~ 3

Author(s):

Meike Hohwieler ◽

Martin Müller ◽

Pierre-Olivier Frappart ◽

Sandra Heller

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Disease Modeling ◽

Embryonic Stem ◽

Cell Types ◽

Genetically Engineered ◽

Hereditary Diseases ◽

Pancreatic Diseases ◽

Pancreatic Progenitors

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) are characterized by their unique capacity to stepwise differentiate towards any particular cell type in an adult organism. Pluripotent stem cells provide a beneficial platform to model hereditary diseases and even cancer development. While the incidence of pancreatic diseases such as diabetes and pancreatitis is increasing, the understanding of the underlying pathogenesis of particular diseases remains limited. Only a few recent publications have contributed to the characterization of human pancreatic development in the fetal stage. Hence, most knowledge of pancreatic specification is based on murine embryology. Optimizing and understanding current in vitro protocols for pancreatic differentiation of ESCs and iPSCs constitutes a prerequisite to generate functional pancreatic cells for better disease modeling and drug discovery. Moreover, human pancreatic organoids derived from pluripotent stem cells, organ-restricted stem cells, and tumor samples provide a powerful technology to model carcinogenesis and hereditary diseases independent of genetically engineered mouse models. Herein, we summarize recent advances in directed differentiation of pancreatic organoids comprising endocrine cell types. Beyond that, we illustrate up-and-coming applications for organoid-based platforms.

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Renal Transfer of Genetically Engineered Cells

Journal of the American Society of Nephrology ◽

10.1681/asn.v11suppl_2s154 ◽

2000 ◽

Vol 11 (suppl 2) ◽

pp. S154-S158

Author(s):

MASANORI KITAMURA

Keyword(s):

Gene Transfer ◽

Cultured Cells ◽

Mesangial Cells ◽

Genetic Manipulation ◽

Ex Vivo ◽

Genetically Engineered ◽

Dominant Negative ◽

Loss Of Function ◽

Glomerular Diseases

Abstract. For many years, ex vivo gene transfer has been used for genetic manipulation of various organs. In the kidney, ex vivo gene transfer was reported using mesangial cells and macrophages. In rats, cultured cells injected into the renal artery are accumulated selectively in the glomerulus. With this approach, it is possible to transfer genetically engineered cells to normal and diseased glomeruli. The transfer of genetically engineered cells to glomeruli can be used for several purposes. With the use of resident glomerular cells engineered in vitro, it is possible to examine how the cells that overexpress certain genes behave differently in normal and diseased glomeruli. Both gain-of-function and loss-of-function strategies are useful for this purpose. For the latter, stable expression of antisense cDNA, ribosomes, or dominant-negative mutants is available. By transfer of engineered cells producing secretory, recombinant proteins, it is possible to modify glomerular microenvironment in vivo. Transfer of genes encoding therapeutically relevant molecules could be useful for therapeutic intervention. Transfer of engineered leukocytes to the glomerulus also allows investigation of cross talk between leukocytes and resident cells. Transfer of stimulated leukocytes is useful for investigation of the pathologic actions of infiltrating cells on glomerular structure and function. Leukocytes in which certain gene functions are selectively reinforced or deleted would be useful for elucidation of the exact functions of leukocyte-associated genes in glomerular diseases. This article summarizes current experience with the adoptive transfer of engineered cells to the glomerulus for investigation of and therapy for glomerular diseases.

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Maturation of Nephrons by Implanting hPSC-Derived Kidney Progenitors Under Kidney Capsules of Unilaterally Nephrectomized Mice

10.21203/rs.3.rs-847264/v1 ◽

2021 ◽

Author(s):

Xin Yu ◽

Shan Jiang ◽

Kailin Li ◽

Xianzhen Yang ◽

Zhihe Xu ◽

...

Keyword(s):

Disease Modeling ◽

Vascular System ◽

Human Kidney ◽

Target Cells ◽

Mice Model ◽

Immunodeficient Mice ◽

Filtration Barrier ◽

Kidney Organoids

Abstract Background Human pluripotent stem cell (hPSCs)-derived kidney organoids may contribute to disease modeling and generation of kidney replacement tissues. However, realization of such applications requires the induction of hPSCs into functional mature organoids. One of the key questions for this process is whether a specific vascular system exists for nephrogenesis. Our previous study showed that implantation of hPSC-derived organoids below the kidney capsules of unilaterally nephrectomized immunodeficient mice for a short-term (2 weeks) resulted in the enlargement of organoids and production of vascular cells, although signs of maturation were lacking. Methods In this study, organoids are induced in vitro during 15 days and then sub-capsularly grafted into kidneys, we used the same unilaterally nephrectomized immunodeficient mice model to examine whether a medium -term (4 weeks) implantation could improve organoid maturation and vascularization, as evaluated by immunofluorescence and transmission electron microscopy(TEM). Results We demonstrate that after 2–4 weeks implantation, implanted renal organoids can form host-derived vascularization and mature in the absence of any exogenous vascular endothelial growth factor. Glomerular filtration barrier maturation was evidenced by glomerular basement membrane deposition, perforated glomerular endothelial cell development, as well as apical to basal podocyte polarization. A polarized monolayer epithelium and extensive brush border were also observed for tubular epithelial cells. Conclusions Our results indicate that the in vivo microenvironment is important for the maturation of human kidney organoids. Stromal expansion and a reduction of nephron structures were observed following longer-term (12 weeks) implantation,suggesting effects on off-target cells during the induction process. Accordingly, induction efficiency and transplantation models should be improved in the future.

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An Integrated Approach to Studying Rare Neuromuscular Diseases Using Animal and Human Cell-Based Models

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.801819 ◽

2022 ◽

Vol 9 ◽

Author(s):

Timothy J. Hines ◽

Cathleen Lutz ◽

Stephen A. Murray ◽

Robert W. Burgess

Keyword(s):

Human Disease ◽

Neuromuscular Diseases ◽

Trna Synthetase ◽

Genetically Engineered ◽

Model Systems ◽

Model Organisms ◽

Cellular Mechanisms ◽

Disease Mechanisms

As sequencing technology improves, the identification of new disease-associated genes and new alleles of known genes is rapidly increasing our understanding of the genetic underpinnings of rare diseases, including neuromuscular diseases. However, precisely because these disorders are rare and often heterogeneous, they are difficult to study in patient populations. In parallel, our ability to engineer the genomes of model organisms, such as mice or rats, has gotten increasingly efficient through techniques such as CRISPR/Cas9 genome editing, allowing the creation of precision human disease models. Such in vivo model systems provide an efficient means for exploring disease mechanisms and identifying therapeutic strategies. Furthermore, animal models provide a platform for preclinical studies to test the efficacy of those strategies. Determining whether the same mechanisms are involved in the human disease and confirming relevant parameters for treatment ideally involves a human experimental system. One system currently being used is induced pluripotent stem cells (iPSCs), which can then be differentiated into the relevant cell type(s) for in vitro confirmation of disease mechanisms and variables such as target engagement. Here we provide a demonstration of these approaches using the example of tRNA-synthetase-associated inherited peripheral neuropathies, rare forms of Charcot-Marie-Tooth disease (CMT). Mouse models have led to a better understanding of both the genetic and cellular mechanisms underlying the disease. To determine if the mechanisms are similar in human cells, we will use genetically engineered iPSC-based models. This will allow comparisons of different CMT-associated GARS alleles in the same genetic background, reducing the variability found between patient samples and simplifying the availability of cell-based models for a rare disease. The necessity of integrating mouse and human models, strategies for accomplishing this integration, and the challenges of doing it at scale are discussed using recently published work detailing the cellular mechanisms underlying GARS-associated CMT as a framework.

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α-synuclein pathogenesis in hiPSC models of Parkinson’s Disease

Neuronal Signaling ◽

10.1042/ns20210021 ◽

2021 ◽

Author(s):

Leo R Quinlan ◽

Jara Maria Baena-Montes ◽

Sahar Avazzadeh

Keyword(s):

Parkinson’S Disease ◽

Parkinson's Disease ◽

Disease Modeling ◽

Viral Vector ◽

Neurodegenerative Disorder ◽

Current Knowledge ◽

Lewy Bodies ◽

Model Systems ◽

The Brain

α-synuclein is an increasingly prominent player in the pathology of a variety of neurodegenerative conditions. Parkinson’s disease (PD) is a neurodegenerative disorder that affects mainly the dopaminergic neurons in the substantia nigra of the brain. Typical of PD pathology is the finding of protein aggregations termed ‘Lewy bodies’ in the brain regions affected. α-synuclein is implicated in many disease states including dementia with Lewy bodies and Alzheimer’s disease. However, PD is the most common synucleinopathy and continues to be a significant focus of PD research in terms of the α-synuclein Lewy body pathology. Mutations in several genes are associated with PD development including SNCA, which encodes α-synuclein. A variety of model systems have been employed to study α-synuclein physiology and pathophysiology in an attempt to relate more closely to PD pathology. These models include cellular and animal system exploring transgenic technologies, viral vector expression and knockdown approaches, and models to study the potential prion protein-like effects of α-synuclein. The current review focuses on human induced pluripotent stem cell (iPSC) models with a specific focus on mutations or multiplications of the SNCA gene. iPSCs are a rapidly evolving technology with huge promise in the study of normal physiology and disease modeling in vitro. The ability to maintain a patient's genetic background and replicate similar cell phenotypes make iPSCs a powerful tool in the study of neurological diseases. This review focus on the current knowledge about α-synuclein physiological function as well as its role in PD pathogenesis based on human iPSC models.

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hiPSC-Derived Cardiac Tissue for Disease Modeling and Drug Discovery

International Journal of Molecular Sciences ◽

10.3390/ijms21238893 ◽

2020 ◽

Vol 21 (23) ◽

pp. 8893

Author(s):

Junjun Li ◽

Ying Hua ◽

Shigeru Miyagawa ◽

Jingbo Zhang ◽

Lingjun Li ◽

...

Keyword(s):

Drug Discovery ◽

Cardiac Tissue ◽

Disease Modeling ◽

Disease Model ◽

Induced Pluripotent Stem Cell ◽

Model Systems ◽

Toxicity Screening ◽

The Difference ◽

Induced Pluripotent ◽

Novel Drug

Relevant, predictive normal, or disease model systems are of vital importance for drug development. The difference between nonhuman models and humans could contribute to clinical trial failures despite ideal nonhuman results. As a potential substitute for animal models, human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CMs) provide a powerful tool for drug toxicity screening, modeling cardiovascular diseases, and drug discovery. Here, we review recent hiPSC-CM disease models and discuss the features of hiPSC-CMs, including subtype and maturation and the tissue engineering technologies for drug assessment. Updates from the international multisite collaborators/administrations for development of novel drug discovery paradigms are also summarized.

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Efficient and Rapid generation of neural stem cells by direct conversion Fibroblasts with A Single microRNA

10.1101/2021.08.09.452601 ◽

2021 ◽

Author(s):

yuesi wang ◽

Yuanyuan Li ◽

Jing Sun ◽

Tingting Xu ◽

Xiaobin Weng ◽

...

Keyword(s):

Stem Cells ◽

Neural Stem Cells ◽

High Efficiency ◽

Disease Modeling ◽

System Development ◽

Direct Conversion ◽

Tumor Formation ◽

Low Efficiency

Neural stem cells (NSCs) have great potential in the application of neurodegenerative disease therapy, drug screening and disease modeling. NSC can be generated by reprogramming from terminally differentiated cells with transcription factors or small molecules. However, current methods for producing NSCs involve the danger of integrating foreign genes into the genome and the problem of low efficiency. Here, we report an efficient method to generate NSCs from human skin-derived fibroblasts with microRNA (mir-302a) in 2-3 days. The induced NSCs (iNSCs) have more than 90% of purity. Their morphology is similar to regular NSCs, expressing key markers including Nestin, Pax6 and Sox2, and can be expanded for more than 20 passages in vitro. They can also differentiate into functional neuron progeny, astrocytes and oligodendrocytes as well. Those cells can elicit action potential, can be xeno-transplanted into the brain of immune-deficient mice, and can survive and differentiate in vivo without tumor formation. This study shows that a single part of pluripotency-inducing mir-302 cluster can drive fibroblasts reprogramming, providing a general platform for high-efficiency generation of individual-specific human NSCs for studies of neuron system development and regenerative cell therapy.

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