iPSC-derived hepatocytes generated from NASH donors provide a valuable platform for disease modeling and drug discovery

ABSTRACTNon-alcoholic fatty liver disease (NAFLD) affects 30–40% of adults and 10% of children in the US. About 20% of people with NAFLD develop non-alcoholic steatohepatitis (NASH), which may lead to cirrhosis and liver cancer, and is projected to be a leading cause of liver transplantation in the near future. Human induced pluripotent stem cells (iPSC) from NASH patients are useful for generating a large number of hepatocytes for NASH modeling applications and identification of potential drug targets. We developed a novel defined in vitro differentiation process to generate cryopreservable hepatocytes using an iPSC panel of NASH donors and apparently healthy normal (AHN) controls. iPSC-derived hepatocytes displayed stage specific phenotypic markers, hepatocyte morphology, with bile canaliculi. Importantly, both fresh and cryopreserved definitive endoderm and hepatoblasts successfully differentiated to pure and functional hepatocytes with increased CYP3A4 activity in response to rifampicin and lipid accumulation upon fatty acid (FA) treatment. End-stage hepatocytes integrated into three-dimensional (3D) liver organoids and demonstrated increased levels of albumin secretion compared to aggregates consisting of hepatocytes alone. End-stage hepatocytes derived from NASH donors demonstrated spontaneous lipidosis without FA supplementation, recapitulating a feature of NASH hepatocytes in vivo. Cryopreserved hepatocytes generated by this protocol across multiple donors will provide a critical cell source to facilitate the fundamental understanding of NAFLD/NASH biology and potential high throughput screening applications for preclinical evaluation of therapeutic targets.

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Liver Organoids: Updates on Disease Modeling and Biomedical Applications

Biology ◽

10.3390/biology10090835 ◽

2021 ◽

Vol 10 (9) ◽

pp. 835

Author(s):

Carmen Caiazza ◽

Silvia Parisi ◽

Massimiliano Caiazzo

Keyword(s):

Stem Cells ◽

Disease Modeling ◽

Embryonic Stem ◽

Low Complexity ◽

In Vivo Models ◽

Innovative Tool ◽

Induced Pluripotent ◽

Liver Organoids

Liver organoids are stem cell-derived 3D structures that are generated by liver differentiation signals in the presence of a supporting extracellular matrix. Liver organoids overcome low complexity grade of bidimensional culture and high costs of in vivo models thus representing a turning point for studying liver disease modeling. Liver organoids can be established from different sources as induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), hepatoblasts and tissue-derived cells. This novel in vitro system represents an innovative tool to deeper understand the physiology and pathological mechanisms affecting the liver. In this review, we discuss the current advances in the field focusing on their application in modeling diseases, regenerative medicine and drug discovery.

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Toward the Effective Bioengineering of a Pathological Tissue for Cardiovascular Disease Modeling: Old Strategies and New Frontiers for Prevention, Diagnosis, and Therapy

Frontiers in Cardiovascular Medicine ◽

10.3389/fcvm.2020.591583 ◽

2021 ◽

Vol 7 ◽

Author(s):

Laura Iop

Keyword(s):

Aortic Valve ◽

Disease Modeling ◽

Three Dimensional ◽

Induced Pluripotent Stem Cell ◽

Current Treatment ◽

Advantages And Disadvantages ◽

Three Dimensional Modeling ◽

Induced Pluripotent

Cardiovascular diseases (CVDs) still represent the primary cause of mortality worldwide. Preclinical modeling by recapitulating human pathophysiology is fundamental to advance the comprehension of these diseases and propose effective strategies for their prevention, diagnosis, and treatment. In silico, in vivo, and in vitro models have been applied to dissect many cardiovascular pathologies. Computational and bioinformatic simulations allow developing algorithmic disease models considering all known variables and severity degrees of disease. In vivo studies based on small or large animals have a long tradition and largely contribute to the current treatment and management of CVDs. In vitro investigation with two-dimensional cell culture demonstrates its suitability to analyze the behavior of single, diseased cellular types. The introduction of induced pluripotent stem cell technology and the application of bioengineering principles raised the bar toward in vitro three-dimensional modeling by enabling the development of pathological tissue equivalents. This review article intends to describe the advantages and disadvantages of past and present modeling approaches applied to provide insights on some of the most relevant congenital and acquired CVDs, such as rhythm disturbances, bicuspid aortic valve, cardiac infections and autoimmunity, cardiovascular fibrosis, atherosclerosis, and calcific aortic valve stenosis.

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iPSC-Derived Liver Organoids: A Journey from Drug Screening, to Disease Modeling, Arriving to Regenerative Medicine

International Journal of Molecular Sciences ◽

10.3390/ijms21176215 ◽

2020 ◽

Vol 21 (17) ◽

pp. 6215

Author(s):

Cristina Olgasi ◽

Alessia Cucci ◽

Antonia Follenzi

Keyword(s):

Regenerative Medicine ◽

Disease Modeling ◽

Three Dimensional ◽

Cell Types ◽

High Rate ◽

Limiting Factor ◽

Congenital Diseases ◽

Induced Pluripotent ◽

Liver Organoids ◽

Bioartificial Livers

Liver transplantation is the most common treatment for patients suffering from liver failure that is caused by congenital diseases, infectious agents, and environmental factors. Despite a high rate of patient survival following transplantation, organ availability remains the key limiting factor. As such, research has focused on the transplantation of different cell types that are capable of repopulating and restoring liver function. The best cellular mix capable of engrafting and proliferating over the long-term, as well as the optimal immunosuppression regimens, remain to be clearly well-defined. Hence, alternative strategies in the field of regenerative medicine have been explored. Since the discovery of induced pluripotent stem cells (iPSC) that have the potential of differentiating into a broad spectrum of cell types, many studies have reported the achievement of iPSCs differentiation into liver cells, such as hepatocytes, cholangiocytes, endothelial cells, and Kupffer cells. In parallel, an increasing interest in the study of self-assemble or matrix-guided three-dimensional (3D) organoids have paved the way for functional bioartificial livers. In this review, we will focus on the recent breakthroughs in the development of iPSCs-based liver organoids and the major drawbacks and challenges that need to be overcome for the development of future applications.

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THEORETICAL APPROACH ON TARGETING PLANT FUNGAL PATHOGENIC PROTEINS AGAINST NATURALLY ISOLATED COMPOUNDS FROM CHITINIPHILUS SHINANONENSIS

Asian Journal of Pharmaceutical and Clinical Research ◽

10.22159/ajpcr.2019.v12i12.35639 ◽

2019 ◽

pp. 138-142

Author(s):

KRITHIKA S ◽

CHELLARAM C

Keyword(s):

Drug Targets ◽

Fungal Pathogens ◽

Interaction Analysis ◽

Pathogenic Fungus ◽

Three Dimensional ◽

Data Bank ◽

Asiatic Acid ◽

Literature Study

Objective: The objective of this study was to find the potency and bioefficacy of Asiatic acid and triterpene against four different plant fungal pathogens using a structure-based drug designing approach. Methods: The pathogenic fungus which causes a dreadful effect on plants is reviewed from literature study, and its three-dimensional structures are retrieved from the protein data bank database. On the other hand, ligands are prepared. Finally, prepared fungal drug targets are docked with naturally isolated compounds using AutoDock tools. Results: Both compounds Asiatic acid and triterpene structures are complementary to bind at the active site of four different drug targets. Comparatively, it is more favorable for Avr2 effector protein from Fusarium oxysporum with Ki value of 126.60 μM, 1.76 μM, and dock score value of −5.32 kcal/mol and −7.85 kcal/mol for Asiatic acid and triterpene, respectively. Thus, interaction analysis was carried out only for these protein-ligand complexes. Conclusion: The computational biology study states that these two compounds can be the lead candidate for treating disease caused by plant fungal pathogen F. oxysporum. However, further study has to be done in vitro and in vivo to prove its same efficacy.

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Genomics Analysis of Metabolic Pathways of Human Stem Cell-Derived Microglia-Like Cells and the Integrated Cortical Spheroids

Stem Cells International ◽

10.1155/2019/2382534 ◽

2019 ◽

Vol 2019 ◽

pp. 1-21 ◽

Cited By ~ 7

Author(s):

Julie Bejoy ◽

Xuegang Yuan ◽

Liqing Song ◽

Thien Hua ◽

Richard Jeske ◽

...

Keyword(s):

Brain Tissue ◽

Metabolic Pathways ◽

Disease Modeling ◽

Aerobic Glycolysis ◽

Three Dimensional ◽

Initiation Factor ◽

Human Brain Tissue ◽

P53 Signaling

Brain spheroids or organoids derived from human pluripotent stem cells (hiPSCs) are still not capable of completely recapitulating in vivo human brain tissue, and one of the limitations is lack of microglia. To add built-in immune function, coculture of the dorsal forebrain spheroids with isogenic microglia-like cells (D-MG) was performed in our study. The three-dimensional D-MG spheroids were analyzed for their transcriptome and compared with isogenic microglia-like cells (MG). Cortical spheroids containing microglia-like cells displayed different metabolic programming, which may affect the associated phenotype. The expression of genes related to glycolysis and hypoxia signaling was increased in cocultured D-MG spheroids, indicating the metabolic shift to aerobic glycolysis, which is in favor of M1 polarization of microglia-like cells. In addition, the metabolic pathways and the signaling pathways involved in cell proliferation, cell death, PIK3/AKT/mTOR signaling, eukaryotic initiation factor 2 pathway, and Wnt and Notch pathways were analyzed. The results demonstrate the activation of mTOR and p53 signaling, increased expression of Notch ligands, and the repression of NF-κB and canonical Wnt pathways, as well as the lower expression of cell cycle genes in the cocultured D-MG spheroids. This analysis indicates that physiological 3-D microenvironment may reshape the immunity of in vitro cortical spheroids and better recapitulate in vivo brain tissue function for disease modeling and drug screening.

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Development of a Scalable, High-Throughput-Compatible Assay to Detect Tau Aggregates Using iPSC-Derived Cortical Neurons Maintained in a Three-Dimensional Culture Format

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057116638029 ◽

2016 ◽

Vol 21 (8) ◽

pp. 804-815 ◽

Cited By ~ 28

Author(s):

X. Medda ◽

L. Mertens ◽

S. Versweyveld ◽

A. Diels ◽

L. Barnham ◽

...

Keyword(s):

High Throughput ◽

Cortical Neurons ◽

High Throughput Screening ◽

Three Dimensional ◽

Culture Conditions ◽

Novel Treatments ◽

Entry Points ◽

Induced Pluripotent ◽

Tau Aggregation

Tau aggregation is the pathological hallmark that best correlates with the progression of Alzheimer’s disease (AD). The presence of neurofibrillary tangles (NFTs), formed of hyperphosphorylated tau, leads to neuronal dysfunction and loss, and is directly associated with the cognitive decline observed in AD patients. The limited success in targeting β-amyloid pathologies has reinforced the hypothesis of blocking tau phosphorylation, aggregation, and/or spreading as alternative therapeutic entry points to treat AD. Identification of novel therapies requires disease-relevant and scalable assays capable of reproducing key features of the pathology in an in vitro setting. Here we use induced pluripotent stem cells (iPSCs) as a virtually unlimited source of human cortical neurons to develop a robust and scalable tau aggregation model compatible with high-throughput screening (HTS). We downscaled cell culture conditions to 384-well plate format and used Matrigel to introduce an extra physical protection against cell detachment that reduces shearing stress and better recapitulates pathological conditions. We complemented the assay with AlphaLISA technology for the detection of tau aggregates in a high-throughput-compatible format. The assay is reproducible across users and works with different commercially available iPSC lines, representing a highly translational tool for the identification of novel treatments against tauopathies, including AD.

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Intestinal Organoids—Current and Future Applications

10.20944/preprints201608.0090.v1 ◽

2016 ◽

Author(s):

Andre M. C. Meneses ◽

Kerstin Schneeberger ◽

Hedwig S. Kruitwagen ◽

Louis C. Penning ◽

Frank G. van Steenbeek ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Pluripotent Stem Cells ◽

Disease Modeling ◽

Three Dimensional ◽

Intestinal Stem Cells ◽

Complex Structures ◽

Technical Advances ◽

Induced Pluripotent

Recent technical advances in the stem cell field have enabled the in vitro generation of complex structures resembling whole organs termed organoids. Most of these approaches employ culture systems that allow stem cell-derived or tissue progenitor cells to self-organize into three-dimensional (3D)-structures. Since organoids can be grown from various species, organs and from patient-derived induced pluripotent stem cells, they create significant prospects for modelling development and diseases, for toxicology and drug discovery studies, and in the field of regenerative medicine. Here, we report on intestinal stem cells, organoid culture, organoid disease modeling, transplantation, current and future uses of this exciting new insight model to veterinary medicine field.

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Human induced pluripotent stem cell-derived three-dimensional cardiomyocyte tissues ameliorate the rat ischemic myocardium by remodeling the extracellular matrix and cardiac protein phenotype

PLoS ONE ◽

10.1371/journal.pone.0245571 ◽

2021 ◽

Vol 16 (3) ◽

pp. e0245571

Author(s):

Junya Yokoyama ◽

Shigeru Miyagawa ◽

Takami Akagi ◽

Mitsuru Akashi ◽

Yoshiki Sawa

Keyword(s):

Myocardial Infarction ◽

Extracellular Matrix ◽

Pluripotent Stem Cell ◽

Heart Function ◽

Three Dimensional ◽

Induced Pluripotent Stem Cell ◽

Left Ventricular ◽

Induced Pluripotent

The extracellular matrix (ECM) plays a key role in the viability and survival of implanted human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). We hypothesized that coating of three-dimensional (3D) cardiac tissue-derived hiPSC-CMs with the ECM protein fibronectin (FN) would improve the survival of transplanted cells in the heart and improve heart function in a rat model of ischemic heart failure. To test this hypothesis, we first explored the tolerance of FN-coated hiPSC-CMs to hypoxia in an in vitro study. For in vivo assessments, we constructed 3D-hiPSC cardiac tissues (3D-hiPSC-CTs) using a layer-by-layer technique, and then the cells were implanted in the hearts of a myocardial infarction rat model (3D-hiPSC-CTs, n = 10; sham surgery control group (without implant), n = 10). Heart function and histology were analyzed 4 weeks after transplantation. In the in vitro assessment, cell viability and lactate dehydrogenase assays showed that FN-coated hiPSC-CMs had improved tolerance to hypoxia compared with the control cells. In vivo, the left ventricular ejection fraction of hearts implanted with 3D-hiPSC-CT was significantly better than that of the sham control hearts. Histological analysis showed clear expression of collagen type IV and plasma membrane markers such as desmin and dystrophin in vivo after implantation of 3D-hiPSC-CT, which were not detected in 3D-hiPSC-CMs in vitro. Overall, these results indicated that FN-coated 3D-hiPSC-CT could improve distressed heart function in a rat myocardial infarction model with a well-expressed cytoskeletal or basement membrane matrix. Therefore, FN-coated 3D-hiPSC-CT may serve as a promising replacement for heart transplantation and left ventricular assist devices and has the potential to improve survivability and therapeutic efficacy in cases of ischemic heart disease.

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Induced Pluripotency: A Powerful Tool for In Vitro Modeling

International Journal of Molecular Sciences ◽

10.3390/ijms21238910 ◽

2020 ◽

Vol 21 (23) ◽

pp. 8910 ◽

Cited By ~ 1

Author(s):

Romana Zahumenska ◽

Vladimir Nosal ◽

Marek Smolar ◽

Terezia Okajcekova ◽

Henrieta Skovierova ◽

...

Keyword(s):

Drug Testing ◽

Disease Modeling ◽

Three Dimensional ◽

Induced Pluripotent Stem Cell ◽

Cellular Mechanisms ◽

Major Health ◽

Induced Pluripotency ◽

Developmental Capacity ◽

Induced Pluripotent

One of the greatest breakthroughs of regenerative medicine in this century was the discovery of induced pluripotent stem cell (iPSC) technology in 2006 by Shinya Yamanaka. iPSCs originate from terminally differentiated somatic cells that have newly acquired the developmental capacity of self-renewal and differentiation into any cells of three germ layers. Before iPSCs can be used routinely in clinical practice, their efficacy and safety need to be rigorously tested; however, iPSCs have already become effective and fully-fledged tools for application under in vitro conditions. They are currently routinely used for disease modeling, preparation of difficult-to-access cell lines, monitoring of cellular mechanisms in micro- or macroscopic scales, drug testing and screening, genetic engineering, and many other applications. This review is a brief summary of the reprogramming process and subsequent differentiation and culture of reprogrammed cells into neural precursor cells (NPCs) in two-dimensional (2D) and three-dimensional (3D) conditions. NPCs can be used as biomedical models for neurodegenerative diseases (NDs), which are currently considered to be one of the major health problems in the human population.

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Organoid: Current Implications and Pharmaceutical Applications in Liver Diseases

Current Molecular Pharmacology ◽

10.2174/1874467213666201217115854 ◽

2020 ◽

Vol 13 ◽

Author(s):

Mengqi Zhu ◽

Yuting Huang ◽

Saiyan Bian ◽

Qianqian Song ◽

Jie Zhang ◽

...

Keyword(s):

Structural Characteristics ◽

Three Dimensional ◽

3D Culture ◽

Disease Models ◽

In Vivo Models ◽

Construction Methods ◽

Full Picture ◽

Liver Organoids

Background: Understanding organogenesis, disorders, and repairing processes particularly important for understanding disease occurrence and developing treatment approaches. At present, liver-related studies are mainly conducted using in vivo models and cell lines, making it difficult to generalize the full picture of the structural characteristics and functions of human organs. Organoid is a three-dimensional (3D) culture system in vitro, which holds the promise to establish various disease models and conduct in-depth research by generating organ-like tissues in a dish. Recent advances of human liver organoids have provided us a deeper understanding of this complex organ. Conclusion: In this review, we provide a systematic overview of the construction methods of organoids, focusing on their applications in the hepatic organogenesis and various liver disease models, as well as the limitations of current models. The development of organoid models is proving to be crucial in future liver research.

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