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Advances in iPSC-Based Disease Modeling: From Two Dimensional Monolayers to...
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Advances in iPSC-Based Disease Modeling: From Two Dimensional Monolayers to Organoids

A special issue of Cells (ISSN 2073-4409). This special issue belongs to the section "Stem Cells".

Deadline for manuscript submissions: closed (30 March 2023) | Viewed by 41987

Special Issue Editors

Prof. Dr. Giovanni Cuda

E-Mail Website
Guest Editor
Department of Experimental and Clinical Medicine, Magna Græcia University, 88100 Catanzaro, Italy
Interests: human Induced pluripotent stem cells (iPSCs); organoids; disease modeling; drug testing and discovery
Special Issues, Collections and Topics in MDPI journals
Dr. Elvira Immacolata Parrotta

E-Mail Website
Guest Editor
Department of Medical and Surgical Sciences, Magna Græcia Univeristy, 88100 Catanzaro, Italy
Interests: human Induced pluripotent stem cells (iPSCs); organoids; disease modeling; drug testing and discovery

Special Issue Information

Dear Colleagues, 

Understanding the mechanisms underlying human diseases is important for the development of effective therapeutic strategies. Progress made during the last decade in stem cell biology with the discovery of induced pluripotent stem cell (iPSC) technology offers unprecedented opportunities to model human diseases. Disease-relevant cell types differentiated from iPSCs in two-dimensional (2D) monolayers have proven to be an extraordinary tool for uncovering diseases’ underlying molecular mechanisms. Interestingly, recent advances in this direction have led to the development of iPSC-derived 3D organoids that can more accurately recapitulate organ architecture, serving as a biological platform for investigating organs and tissues’ developmental processes under both normal and pathological conditions and paving the way for disease modeling, therapeutics, and regenerative medicine.

In this Special Issue of Cells, we invite you to contribute original research articles, reviews, or shorter perspective articles on all aspects related to the theme of Disease Modeling with Human Induced Pluripotent Stem Cells. Articles describing mechanistic, functional, and cellular aspects of disease models based on iPSCs and iPSC-derived organoids are highly welcome.

Prof. Dr. Giovanni Cuda
Dr. Elvira Immacolata Parrotta
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Cells is an international peer-reviewed open access semimonthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • induced pluripotent stem cells (iPSCs)
  • disease modeling
  • organoids
  • drug development
  • regenerative medicine

Published Papers (17 papers)

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Research

Jump to: Review, Other

13 pages, 3538 KiB  
Article
Proteomic Analysis of Human iPSC-Derived Neural Stem Cells and Motor Neurons Identifies Proteasome Structural Alterations
by Iñaki Álvarez, Adrián Tirado-Herranz, Belén Alvarez-Palomo, Jordi Requena Osete and Michael J. Edel
Cells 2023, 12(24), 2800; https://doi.org/10.3390/cells12242800 - 08 Dec 2023
Viewed by 1215
Abstract
Background: Proteins targeted by the ubiquitin proteasome system (UPS) are identified for degradation by the proteasome, which has been implicated in the development of neurodegenerative diseases. Major histocompatibility complex (MHC) molecules present peptides broken down by the proteasome and are involved in neuronal [...] Read more.
Background: Proteins targeted by the ubiquitin proteasome system (UPS) are identified for degradation by the proteasome, which has been implicated in the development of neurodegenerative diseases. Major histocompatibility complex (MHC) molecules present peptides broken down by the proteasome and are involved in neuronal plasticity, regulating the synapse number and axon regeneration in the central or peripheral nervous system during development and in brain diseases. The mechanisms governing these effects are mostly unknown, but evidence from different compartments of the cerebral cortex indicates the presence of immune-like MHC receptors in the central nervous system. Methods: We used human induced pluripotent stem cells (iPSCs) differentiated into neural stem cells and then into motor neurons as a developmental model to better understand the structure of the proteasome in developing motor neurons. We performed a proteomic analysis of starting human skin fibroblasts, their matching iPSCs, differentiated neural stem cells and motor neurons that highlighted significant differences in the constitutive proteasome and immunoproteasome subunits during development toward motor neurons from iPSCs. Results: The proteomic analysis showed that the catalytic proteasome subunits expressed in fibroblasts differed from those in the neural stem cells and motor neurons. Western blot analysis confirmed the proteomic data, particularly the decreased expression of the β5i (PSMB8) subunit immunoproteasome in MNs compared to HFFs and increased β5 (PSMB5) in MNs compared to HFFs. Conclusion: The constitutive proteasome subunits are upregulated in iPSCs and NSCs from HFFs. Immunoproteasome subunit β5i expression is higher in MNs than NSCs; however, overall, there is more of a constitutive proteasome structure in MNs when comparing HFFs to MNs. The proteasome composition may have implications for motor neuron development and neurodevelopmental diseases that warrant further investigation. Full article
(This article belongs to the Special Issue Advances in iPSC-Based Disease Modeling: From Two Dimensional Monolayers to Organoids)
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16 pages, 4379 KiB  
Article
Measuring Single-Cell Calcium Dynamics Using a Myofilament-Localized Optical Biosensor in hiPSC-CMs Derived from DCM Patients
by Cara Hawey, Kyla Bourque, Karima Alim, Ida Derish, Elise Rody, Kashif Khan, Natalie Gendron, Renzo Cecere, Nadia Giannetti and Terence E. Hébert
Cells 2023, 12(21), 2526; https://doi.org/10.3390/cells12212526 - 26 Oct 2023
Viewed by 1880
Abstract
Synchronized contractions of cardiomyocytes within the heart are tightly coupled to electrical stimulation known as excitation-contraction coupling. Calcium plays a key role in this process and dysregulated calcium handling can significantly impair cardiac function and lead to the development of cardiomyopathies and heart [...] Read more.
Synchronized contractions of cardiomyocytes within the heart are tightly coupled to electrical stimulation known as excitation-contraction coupling. Calcium plays a key role in this process and dysregulated calcium handling can significantly impair cardiac function and lead to the development of cardiomyopathies and heart failure. Here, we describe a method and analytical technique to study myofilament-localized calcium signaling using the intensity-based fluorescent biosensor, RGECO-TnT. Dilated cardiomyopathy is a heart muscle disease that negatively impacts the heart’s contractile function following dilatation of the left ventricle. We demonstrate how this biosensor can be used to characterize 2D hiPSC-CMs monolayers generated from a healthy control subject compared to two patients diagnosed with dilated cardiomyopathy. Lastly, we provide a step-by-step guide for single-cell data analysis and describe a custom Transient Analysis application, specifically designed to quantify features of calcium transients. All in all, we explain how this analytical approach can be applied to phenotype hiPSC-CM behaviours and stratify patient responses to identify perturbations in calcium signaling. Full article
(This article belongs to the Special Issue Advances in iPSC-Based Disease Modeling: From Two Dimensional Monolayers to Organoids)
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14 pages, 17035 KiB  
Article
Modeling of FAN1-Deficient Kidney Disease Using a Human Induced Pluripotent Stem Cell-Derived Kidney Organoid System
by Sun Woo Lim, Dohyun Na, Hanbi Lee, Xianying Fang, Sheng Cui, Yoo Jin Shin, Kang In Lee, Jae Young Lee, Chul Woo Yang and Byung Ha Chung
Cells 2023, 12(18), 2319; https://doi.org/10.3390/cells12182319 - 20 Sep 2023
Viewed by 1204
Abstract
Karyomegalic interstitial nephritis (KIN) is a genetic kidney disease caused by mutations in the FANCD2/FANCI-Associated Nuclease 1 (FAN1) gene on 15q13.3, which results in karyomegaly and fibrosis of kidney cells through the incomplete repair of DNA damage. The aim of this [...] Read more.
Karyomegalic interstitial nephritis (KIN) is a genetic kidney disease caused by mutations in the FANCD2/FANCI-Associated Nuclease 1 (FAN1) gene on 15q13.3, which results in karyomegaly and fibrosis of kidney cells through the incomplete repair of DNA damage. The aim of this study was to explore the possibility of using a human induced pluripotent stem cell (hiPSC)-derived kidney organoid system for modeling FAN1-deficient kidney disease, also known as KIN. We generated kidney organoids using WTC-11 (wild-type) hiPSCs and FAN1-mutant hiPSCs which include KIN patient-derived hiPSCs and FAN1-edited hiPSCs (WTC-11 FAN1+/−), created using the CRISPR/Cas9 system in WTC-11-hiPSCs. Kidney organoids from each group were treated with 20 nM of mitomycin C (MMC) for 24 or 48 h, and the expression levels of Ki67 and H2A histone family member X (H2A.X) were analyzed to detect DNA damage and assess the viability of cells within the kidney organoids. Both WTC-11-hiPSCs and FAN1-mutant hiPSCs were successfully differentiated into kidney organoids without structural deformities. MMC treatment for 48 h significantly increased the expression of DNA damage markers, while cell viability in both FAN1-mutant kidney organoids was decreased. However, these findings were observed in WTC-11-kidney organoids. These results suggest that FAN1-mutant kidney organoids can recapitulate the phenotype of FAN1-deficient kidney disease. Full article
(This article belongs to the Special Issue Advances in iPSC-Based Disease Modeling: From Two Dimensional Monolayers to Organoids)
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22 pages, 3755 KiB  
Article
Guiding Hepatic Differentiation of Pluripotent Stem Cells Using 3D Microfluidic Co-Cultures with Human Hepatocytes
by Pouria Fattahi, Jose M. de Hoyos-Vega, Jong Hoon Choi, Caden D. Duffy, Alan M. Gonzalez-Suarez, Yuji Ishida, Kianna M. Nguyen, Kihak Gwon, Quinn P. Peterson, Takeshi Saito, Gulnaz Stybayeva and Alexander Revzin
Cells 2023, 12(15), 1982; https://doi.org/10.3390/cells12151982 - 01 Aug 2023
Cited by 2 | Viewed by 1290
Abstract
Human pluripotent stem cells (hPSCs) are capable of unlimited proliferation and can undergo differentiation to give rise to cells and tissues of the three primary germ layers. While directing lineage selection of hPSCs has been an active area of research, improving the efficiency [...] Read more.
Human pluripotent stem cells (hPSCs) are capable of unlimited proliferation and can undergo differentiation to give rise to cells and tissues of the three primary germ layers. While directing lineage selection of hPSCs has been an active area of research, improving the efficiency of differentiation remains an important objective. In this study, we describe a two-compartment microfluidic device for co-cultivation of adult human hepatocytes and stem cells. Both cell types were cultured in a 3D or spheroid format. Adult hepatocytes remained highly functional in the microfluidic device over the course of 4 weeks and served as a source of instructive paracrine cues to drive hepatic differentiation of stem cells cultured in the neighboring compartment. The differentiation of stem cells was more pronounced in microfluidic co-cultures compared to a standard hepatic differentiation protocol. In addition to improving stem cell differentiation outcomes, the microfluidic co-culture system described here may be used for parsing signals and mechanisms controlling hepatic cell fate. Full article
(This article belongs to the Special Issue Advances in iPSC-Based Disease Modeling: From Two Dimensional Monolayers to Organoids)
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21 pages, 5113 KiB  
Article
A Shared Pathogenic Mechanism for Valproic Acid and SHROOM3 Knockout in a Brain Organoid Model of Neural Tube Defects
by Taylor N. Takla, Jinghui Luo, Roksolana Sudyk, Joy Huang, John Clayton Walker, Neeta L. Vora, Jonathan Z. Sexton, Jack M. Parent and Andrew M. Tidball
Cells 2023, 12(13), 1697; https://doi.org/10.3390/cells12131697 - 23 Jun 2023
Cited by 6 | Viewed by 1950
Abstract
Neural tube defects (NTDs), including anencephaly and spina bifida, are common major malformations of fetal development resulting from incomplete closure of the neural tube. These conditions lead to either universal death (anencephaly) or severe lifelong complications (spina bifida). Despite hundreds of genetic mouse [...] Read more.
Neural tube defects (NTDs), including anencephaly and spina bifida, are common major malformations of fetal development resulting from incomplete closure of the neural tube. These conditions lead to either universal death (anencephaly) or severe lifelong complications (spina bifida). Despite hundreds of genetic mouse models of neural tube defect phenotypes, the genetics of human NTDs are poorly understood. Furthermore, pharmaceuticals, such as antiseizure medications, have been found clinically to increase the risk of NTDs when administered during pregnancy. Therefore, a model that recapitulates human neurodevelopment would be of immense benefit to understand the genetics underlying NTDs and identify teratogenic mechanisms. Using our self-organizing single rosette cortical organoid (SOSR-COs) system, we have developed a high-throughput image analysis pipeline for evaluating the SOSR-CO structure for NTD-like phenotypes. Similar to small molecule inhibition of apical constriction, the antiseizure medication valproic acid (VPA), a known cause of NTDs, increases the apical lumen size and apical cell surface area in a dose-responsive manner. GSK3β and HDAC inhibitors caused similar lumen expansion; however, RNA sequencing suggests VPA does not inhibit GSK3β at these concentrations. The knockout of SHROOM3, a well-known NTD-related gene, also caused expansion of the lumen, as well as reduced f-actin polarization. The increased lumen sizes were caused by reduced cell apical constriction, suggesting that impingement of this process is a shared mechanism for VPA treatment and SHROOM3-KO, two well-known causes of NTDs. Our system allows the rapid identification of NTD-like phenotypes for both compounds and genetic variants and should prove useful for understanding specific NTD mechanisms and predicting drug teratogenicity. Full article
(This article belongs to the Special Issue Advances in iPSC-Based Disease Modeling: From Two Dimensional Monolayers to Organoids)
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19 pages, 4963 KiB  
Article
Preclinical Studies with Glioblastoma Brain Organoid Co-Cultures Show Efficient 5-ALA Photodynamic Therapy
by Leire Pedrosa, Carmen Bedia, Diouldé Diao, Alejandra Mosteiro, Abel Ferrés, Elisabetta Stanzani, Fina Martínez-Soler, Avelina Tortosa, Estela Pineda, Iban Aldecoa, Marc Centellas, Marta Muñoz-Tudurí, Ana Sevilla, Àngels Sierra and José Juan González Sánchez
Cells 2023, 12(8), 1125; https://doi.org/10.3390/cells12081125 - 10 Apr 2023
Cited by 4 | Viewed by 3090
Abstract
Background: The high recurrence of glioblastoma (GB) that occurs adjacent to the resection cavity within two years of diagnosis urges an improvement of therapies oriented to GB local control. Photodynamic therapy (PDT) has been proposed to cleanse infiltrating tumor cells from parenchyma to [...] Read more.
Background: The high recurrence of glioblastoma (GB) that occurs adjacent to the resection cavity within two years of diagnosis urges an improvement of therapies oriented to GB local control. Photodynamic therapy (PDT) has been proposed to cleanse infiltrating tumor cells from parenchyma to ameliorate short long-term progression-free survival. We examined 5-aminolevulinic acid (5-ALA)-mediated PDT effects as therapeutical treatment and determined optimal conditions for PDT efficacy without causing phototoxic injury to the normal brain tissue. Methods: We used a platform of Glioma Initiation Cells (GICs) infiltrating cerebral organoids with two different glioblastoma cells, GIC7 and PG88. We measured GICs-5-ALA uptake and PDT/5-ALA activity in dose-response curves and the efficacy of the treatment by measuring proliferative activity and apoptosis. Results: 5-ALA (50 and 100 µg/mL) was applied, and the release of protoporphyrin IX (PpIX) fluorescence measures demonstrated that the emission of PpIX increases progressively until its stabilization at 24 h. Moreover, decreased proliferation and increased apoptosis corroborated the effect of 5-ALA/PDT on cancer cells without altering normal cells. Conclusions: We provide evidence about the effectiveness of PDT to treat high proliferative GB cells in a complex in vitro system, which combines normal and cancer cells and is a useful tool to standardize new strategic therapies. Full article
(This article belongs to the Special Issue Advances in iPSC-Based Disease Modeling: From Two Dimensional Monolayers to Organoids)
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18 pages, 1323 KiB  
Article
Gene-Edited Human-Induced Pluripotent Stem Cell Lines to Elucidate DAND5 Function throughout Cardiac Differentiation
by José M. Inácio, Mafalda M. Nunes, Micael Almeida, Fernando Cristo, Rui Anjos and José A. Belo
Cells 2023, 12(4), 520; https://doi.org/10.3390/cells12040520 - 05 Feb 2023
Viewed by 2108
Abstract
(1) Background: The contribution of gene-specific variants for congenital heart disease, one of the most common congenital disabilities, is still far from our complete understanding. Here, we applied a disease model using human-induced pluripotent stem cells (hiPSCs) to evaluate the function of DAND5 [...] Read more.
(1) Background: The contribution of gene-specific variants for congenital heart disease, one of the most common congenital disabilities, is still far from our complete understanding. Here, we applied a disease model using human-induced pluripotent stem cells (hiPSCs) to evaluate the function of DAND5 on human cardiomyocyte (CM) differentiation and proliferation. (2) Methods: Taking advantage of our DAND5 patient-derived iPSC line, we used CRISPR-Cas9 gene-editing to generate a set of isogenic hiPSCs (DAND5-corrected and DAND5 full-mutant). The hiPSCs were differentiated into CMs, and RT-qPCR and immunofluorescence profiled the expression of cardiac markers. Cardiomyocyte proliferation was analysed by flow cytometry. Furthermore, we used a multi-electrode array (MEA) to study the functional electrophysiology of DAND5 hiPSC-CMs. (3) Results: The results indicated that hiPSC-CM proliferation is affected by DAND5 levels. Cardiomyocytes derived from a DAND5 full-mutant hiPSC line are more proliferative when compared with gene-corrected hiPSC-CMs. Moreover, parallel cardiac differentiations showed a differential cardiac gene expression profile, with upregulated cardiac progenitor markers in DAND5-KO hiPSC-CMs. Microelectrode array (MEA) measurements demonstrated that DAND5-KO hiPSC-CMs showed prolonged field potential duration and increased spontaneous beating rates. In addition, conduction velocity is reduced in the monolayers of hiPSC-CMs with full-mutant genotype. (4) Conclusions: The absence of DAND5 sustains the proliferation of hiPSC-CMs, which alters their electrophysiological maturation properties. These results using DAND5 hiPSC-CMs consolidate the findings of the in vitro and in vivo mouse models, now in a translational perspective. Altogether, the data will help elucidate the molecular mechanism underlying this human heart disease and potentiates new therapies for treating adult CHD. Full article
(This article belongs to the Special Issue Advances in iPSC-Based Disease Modeling: From Two Dimensional Monolayers to Organoids)
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17 pages, 3212 KiB  
Article
Impact of Neurons on Patient-Derived Cardiomyocytes Using Organ-On-A-Chip and iPSC Biotechnologies
by Albin A. Bernardin, Sarah Colombani, Antoine Rousselot, Virginie Andry, Yannick Goumon, Hélène Delanoë-Ayari, Côme Pasqualin, Bernard Brugg, Etienne D. Jacotot, Jean-Luc Pasquié, Alain Lacampagne and Albano C. Meli
Cells 2022, 11(23), 3764; https://doi.org/10.3390/cells11233764 - 25 Nov 2022
Cited by 5 | Viewed by 2904
Abstract
In the heart, cardiac function is regulated by the autonomic nervous system (ANS) that extends through the myocardium and establishes junctions at the sinus node and ventricular levels. Thus, an increase or decrease in neuronal activity acutely affects myocardial function and chronically affects [...] Read more.
In the heart, cardiac function is regulated by the autonomic nervous system (ANS) that extends through the myocardium and establishes junctions at the sinus node and ventricular levels. Thus, an increase or decrease in neuronal activity acutely affects myocardial function and chronically affects its structure through remodeling processes. The neuro–cardiac junction (NCJ), which is the major structure of this system, is poorly understood and only a few cell models allow us to study it. Here, we present an innovant neuro–cardiac organ-on-chip model to study this structure to better understand the mechanisms involved in the establishment of NCJ. To create such a system, we used microfluidic devices composed of two separate cell culture compartments interconnected by asymmetric microchannels. Rat PC12 cells were differentiated to recapitulate the characteristics of sympathetic neurons, and cultivated with cardiomyocytes derived from human induced pluripotent stem cells (hiPSC). We confirmed the presence of a specialized structure between the two cell types that allows neuromodulation and observed that the neuronal stimulation impacts the excitation–contraction coupling properties including the intracellular calcium handling. Finally, we also co-cultivated human neurons (hiPSC-NRs) with human cardiomyocytes (hiPSC-CMs), both obtained from the same hiPSC line. Hence, we have developed a neuro–cardiac compartmentalized in vitro model system that allows us to recapitulate the structural and functional properties of the neuro–cardiac junction and that can also be used to better understand the interaction between the heart and brain in humans, as well as to evaluate the impact of drugs on a reconstructed human neuro–cardiac system. Full article
(This article belongs to the Special Issue Advances in iPSC-Based Disease Modeling: From Two Dimensional Monolayers to Organoids)
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17 pages, 3764 KiB  
Article
Hyperglycemia Negatively Affects IPSC-Derived Myoblast Proliferation and Skeletal Muscle Regeneration and Function
by Agnes Badu-Mensah, Paola Valinski, Hemant Parsaud, James J. Hickman and Xiufang Guo
Cells 2022, 11(22), 3674; https://doi.org/10.3390/cells11223674 - 18 Nov 2022
Cited by 4 | Viewed by 1763
Abstract
Diabetic myopathy is a co-morbidity diagnosed in most diabetes mellitus patients, yet its pathogenesis is still understudied, which hinders the development of effective therapies. This project aimed to investigate the effect of hyperglycemia on human myoblast physiology, devoid of other complicating factors, by [...] Read more.
Diabetic myopathy is a co-morbidity diagnosed in most diabetes mellitus patients, yet its pathogenesis is still understudied, which hinders the development of effective therapies. This project aimed to investigate the effect of hyperglycemia on human myoblast physiology, devoid of other complicating factors, by utilizing human myoblasts derived from induced pluripotent stem cells (iPSCs), in a defined in vitro system. IPSC-derived myoblasts were expanded under three glucose conditions: low (5 mM), medium (17.5 mM) or high (25 mM). While hyperglycemic myoblasts demonstrated upregulation of Glut4 relative to the euglycemic control, myoblast proliferation demonstrated a glucose dose-dependent impedance. Further cellular analysis revealed a retarded cell cycle progression trapped at the S phase and G2/M phase and an impaired mitochondrial function in hyperglycemic myoblasts. Terminal differentiation of these hyperglycemic myoblasts resulted in significantly hypertrophic and highly branched myotubes with disturbed myosin heavy chain arrangement. Lastly, functional assessment of these myofibers derived from hyperglycemic myoblasts demonstrated comparatively increased fatigability. Collectively, the hyperglycemic myoblasts demonstrated deficient muscle regeneration capability and functionality, which falls in line with the sarcopenia symptoms observed in diabetic myopathy patients. This human-based iPSC-derived skeletal muscle hyperglycemic model provides a valuable platform for mechanistic investigation of diabetic myopathy and therapeutic development. Full article
(This article belongs to the Special Issue Advances in iPSC-Based Disease Modeling: From Two Dimensional Monolayers to Organoids)
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17 pages, 2906 KiB  
Article
The Impaired Neurodevelopment of Human Neural Rosettes in HSV-1-Infected Early Brain Organoids
by Leonardo D’Aiuto, Jill K. Caldwell, Callen T. Wallace, Tristan R. Grams, Maribeth A. Wesesky, Joel A. Wood, Simon C. Watkins, Paul R. Kinchington, David C. Bloom and Vishwajit L. Nimgaonkar
Cells 2022, 11(22), 3539; https://doi.org/10.3390/cells11223539 - 09 Nov 2022
Cited by 3 | Viewed by 1770
Abstract
Intrauterine infections during pregnancy by herpes simplex virus (HSV) can cause significant neurodevelopmental deficits in the unborn/newborn, but clinical studies of pathogenesis are challenging, and while animal models can model some aspects of disease, in vitro studies of human neural cells provide a [...] Read more.
Intrauterine infections during pregnancy by herpes simplex virus (HSV) can cause significant neurodevelopmental deficits in the unborn/newborn, but clinical studies of pathogenesis are challenging, and while animal models can model some aspects of disease, in vitro studies of human neural cells provide a critical platform for more mechanistic studies. We utilized a reductionist approach to model neurodevelopmental outcomes of HSV-1 infection of neural rosettes, which represent the in vitro equivalent of differentiating neural tubes. Specifically, we employed early-stage brain organoids (ES-organoids) composed of human induced pluripotent stem cells (hiPSCs)-derived neural rosettes to investigate aspects of the potential neuropathological effects induced by the HSV-1 infections on neurodevelopment. To allow for the long-term differentiation of ES-organoids, viral infections were performed in the presence of the antiviral drug acyclovir (ACV). Despite the antiviral treatment, HSV-1 infection caused organizational changes in neural rosettes, loss of structural integrity of infected ES-organoids, and neuronal alterations. The inability of ACV to prevent neurodegeneration was associated with the generation of ACV-resistant mutants during the interaction of HSV-1 with differentiating neural precursor cells (NPCs). This study models the effects of HSV-1 infection on the neuronal differentiation of NPCs and suggests that this environment may allow for accelerated development of ACV-resistance. Full article
(This article belongs to the Special Issue Advances in iPSC-Based Disease Modeling: From Two Dimensional Monolayers to Organoids)
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18 pages, 16451 KiB  
Article
Two Different Therapeutic Approaches for SARS-CoV-2 in hiPSCs-Derived Lung Organoids
by Paola Spitalieri, Federica Centofanti, Michela Murdocca, Maria Giovanna Scioli, Andrea Latini, Silvia Di Cesare, Gennaro Citro, Antonio Rossi, Augusto Orlandi, Shane Miersch, Sachdev S. Sidhu, Pier Paolo Pandolfi, Annalisa Botta, Federica Sangiuolo and Giuseppe Novelli
Cells 2022, 11(7), 1235; https://doi.org/10.3390/cells11071235 - 05 Apr 2022
Cited by 19 | Viewed by 3982
Abstract
The global health emergency for SARS-CoV-2 (COVID-19) created an urgent need to develop new treatments and therapeutic drugs. In this study, we tested, for the first time on human cells, a new tetravalent neutralizing antibody (15033-7) targeting Spike protein and a synthetic peptide [...] Read more.
The global health emergency for SARS-CoV-2 (COVID-19) created an urgent need to develop new treatments and therapeutic drugs. In this study, we tested, for the first time on human cells, a new tetravalent neutralizing antibody (15033-7) targeting Spike protein and a synthetic peptide homologous to dipeptidyl peptidase-4 (DPP4) receptor on host cells. Both could represent powerful immunotherapeutic candidates for COVID-19 treatment. The infection begins in the proximal airways, namely the alveolar type 2 (AT2) cells of the distal lung, which express both ACE2 and DPP4 receptors. Thus, to evaluate the efficacy of both approaches, we developed three-dimensional (3D) complex lung organoid structures (hLORGs) derived from human-induced pluripotent stem cells (iPSCs) and resembling the in vivo organ. Afterward, hLORGs were infected by different SARS-CoV-2 S pseudovirus variants and treated by the Ab15033-7 or DPP4 peptide. Using both approaches, we observed a significant reduction of viral entry and a modulation of the expression of genes implicated in innate immunity and inflammatory response. These data demonstrate the efficacy of such approaches in strongly reducing the infection efficiency in vitro and, importantly, provide proof-of-principle evidence that hiPSC-derived hLORGs represent an ideal in vitro system for testing both therapeutic and preventive modalities against COVID-19. Full article
(This article belongs to the Special Issue Advances in iPSC-Based Disease Modeling: From Two Dimensional Monolayers to Organoids)
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Review

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28 pages, 1278 KiB  
Review
Revolutionizing Disease Modeling: The Emergence of Organoids in Cellular Systems
by Rita Silva-Pedrosa, António José Salgado and Pedro Eduardo Ferreira
Cells 2023, 12(6), 930; https://doi.org/10.3390/cells12060930 - 18 Mar 2023
Cited by 11 | Viewed by 4786
Abstract
Cellular models have created opportunities to explore the characteristics of human diseases through well-established protocols, while avoiding the ethical restrictions associated with post-mortem studies and the costs associated with researching animal models. The capability of cell reprogramming, such as induced pluripotent stem cells [...] Read more.
Cellular models have created opportunities to explore the characteristics of human diseases through well-established protocols, while avoiding the ethical restrictions associated with post-mortem studies and the costs associated with researching animal models. The capability of cell reprogramming, such as induced pluripotent stem cells (iPSCs) technology, solved the complications associated with human embryonic stem cells (hESC) usage. Moreover, iPSCs made significant contributions for human medicine, such as in diagnosis, therapeutic and regenerative medicine. The two-dimensional (2D) models allowed for monolayer cellular culture in vitro; however, they were surpassed by the three-dimensional (3D) cell culture system. The 3D cell culture provides higher cell–cell contact and a multi-layered cell culture, which more closely respects cellular morphology and polarity. It is more tightly able to resemble conditions in vivo and a closer approach to the architecture of human tissues, such as human organoids. Organoids are 3D cellular structures that mimic the architecture and function of native tissues. They are generated in vitro from stem cells or differentiated cells, such as epithelial or neural cells, and are used to study organ development, disease modeling, and drug discovery. Organoids have become a powerful tool for understanding the cellular and molecular mechanisms underlying human physiology, providing new insights into the pathogenesis of cancer, metabolic diseases, and brain disorders. Although organoid technology is up-and-coming, it also has some limitations that require improvements. Full article
(This article belongs to the Special Issue Advances in iPSC-Based Disease Modeling: From Two Dimensional Monolayers to Organoids)
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25 pages, 862 KiB  
Review
Modeling Movement Disorders via Generation of hiPSC-Derived Motor Neurons
by Masuma Akter and Baojin Ding
Cells 2022, 11(23), 3796; https://doi.org/10.3390/cells11233796 - 27 Nov 2022
Cited by 4 | Viewed by 2143
Abstract
Generation of motor neurons (MNs) from human-induced pluripotent stem cells (hiPSCs) overcomes the limited access to human brain tissues and provides an unprecedent approach for modeling MN-related diseases. In this review, we discuss the recent progression in understanding the regulatory mechanisms of MN [...] Read more.
Generation of motor neurons (MNs) from human-induced pluripotent stem cells (hiPSCs) overcomes the limited access to human brain tissues and provides an unprecedent approach for modeling MN-related diseases. In this review, we discuss the recent progression in understanding the regulatory mechanisms of MN differentiation and their applications in the generation of MNs from hiPSCs, with a particular focus on two approaches: induction by small molecules and induction by lentiviral delivery of transcription factors. At each induction stage, different culture media and supplements, typical growth conditions and cellular morphology, and specific markers for validation of cell identity and quality control are specifically discussed. Both approaches can generate functional MNs. Currently, the major challenges in modeling neurological diseases using iPSC-derived neurons are: obtaining neurons with high purity and yield; long-term neuron culture to reach full maturation; and how to culture neurons more physiologically to maximize relevance to in vivo conditions. Full article
(This article belongs to the Special Issue Advances in iPSC-Based Disease Modeling: From Two Dimensional Monolayers to Organoids)
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12 pages, 4144 KiB  
Review
Human Pluripotent Stem Cell-Based Models for Hirschsprung Disease: From 2-D Cell to 3-D Organoid Model
by Kathy Nga-Chu Lui and Elly Sau-Wai NGAN
Cells 2022, 11(21), 3428; https://doi.org/10.3390/cells11213428 - 29 Oct 2022
Cited by 4 | Viewed by 2524
Abstract
Hirschsprung disease (HSCR) is a complex congenital disorder caused by defects in the development of the enteric nervous system (ENS). It is attributed to failures of the enteric neural crest stem cells (ENCCs) to proliferate, differentiate and/or migrate, leading to the absence of [...] Read more.
Hirschsprung disease (HSCR) is a complex congenital disorder caused by defects in the development of the enteric nervous system (ENS). It is attributed to failures of the enteric neural crest stem cells (ENCCs) to proliferate, differentiate and/or migrate, leading to the absence of enteric neurons in the distal colon, resulting in colonic motility dysfunction. Due to the oligogenic nature of the disease, some HSCR conditions could not be phenocopied in animal models. Building the patient-based disease model using human induced pluripotent stem cells (hPSC) has opened up a new opportunity to untangle the unknowns of the disease. The expanding armamentarium of hPSC-based therapies provides needed new tools for developing cell-replacement therapy for HSCR. Here we summarize the recent studies of hPSC-based models of ENS in 2-D and 3-D culture systems. These studies have highlighted how hPSC-based models complement the population-based genetic screens and bioinformatic approaches for the discovery of new HSCR susceptibility genes and provide a human model for the close-to-physiological functional studies. We will also discuss the potential applications of these hPSC-based models in translational medicines and their advantages and limitations. The use of these hPSC-based models for drug discovery or cell replacement therapy likely leads to new treatment strategies for HSCR in the future. Further improvements in incorporating hPSC-based models with the human-mouse chimera model and organ-on-a-chip system for establishing a better disease model of HSCR and for drug discovery will further propel us to success in the development of an efficacious treatment for HSCR. Full article
(This article belongs to the Special Issue Advances in iPSC-Based Disease Modeling: From Two Dimensional Monolayers to Organoids)
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14 pages, 1238 KiB  
Review
Lung Organoids as Model to Study SARS-CoV-2 Infection
by Li Peng, Li Gao, Xinya Wu, Yuxin Fan, Meixiao Liu, Jingjing Chen, Jieqin Song, Jing Kong, Yan Dong, Bingxue Li, Aihua Liu and Fukai Bao
Cells 2022, 11(17), 2758; https://doi.org/10.3390/cells11172758 - 04 Sep 2022
Cited by 7 | Viewed by 2483
Abstract
Coronavirus disease-2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic and has severely affected socio-economic conditions and people’s life. The lung is the major target organ infected and (seriously) damaged by SARS-CoV-2, so a comprehensive understanding [...] Read more.
Coronavirus disease-2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic and has severely affected socio-economic conditions and people’s life. The lung is the major target organ infected and (seriously) damaged by SARS-CoV-2, so a comprehensive understanding of the virus and the mechanism of infection are the first choices to overcome COVID-19. Recent studies have demonstrated the enormous value of human organoids as platforms for virological research, making them an ideal tool for researching host–pathogen interactions. In this study, the various existing lung organoids and their identification biomarkers and applications are summarized. At the same time, the seven coronaviruses currently capable of infecting humans are outlined. Finally, a detailed summary of existing studies on SARS-CoV-2 using lung organoids is provided and includes pathogenesis, drug development, and precision treatment. This review highlights the value of lung organoids in studying SARS-CoV-2 infection, bringing hope that research will alleviate COVID-19-associated lung infections. Full article
(This article belongs to the Special Issue Advances in iPSC-Based Disease Modeling: From Two Dimensional Monolayers to Organoids)
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16 pages, 2480 KiB  
Review
RNA-Binding Proteins: Emerging Therapeutics for Vascular Dysfunction
by Victoria A. Cornelius, Hojjat Naderi-Meshkin, Sophia Kelaini and Andriana Margariti
Cells 2022, 11(16), 2494; https://doi.org/10.3390/cells11162494 - 11 Aug 2022
Cited by 8 | Viewed by 2151
Abstract
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 [...] Read more.
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. Full article
(This article belongs to the Special Issue Advances in iPSC-Based Disease Modeling: From Two Dimensional Monolayers to Organoids)
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25 pages, 2596 KiB  
Protocol
An Optimized Workflow to Generate and Characterize iPSC-Derived Motor Neuron (MN) Spheroids
by María José Castellanos-Montiel, Mathilde Chaineau, Anna Kristyna Franco-Flores, Ghazal Haghi, Dulce Carrillo-Valenzuela, Wolfgang E. Reintsch, Carol X.-Q. Chen and Thomas M. Durcan
Cells 2023, 12(4), 545; https://doi.org/10.3390/cells12040545 - 08 Feb 2023
Cited by 4 | Viewed by 3029
Abstract
A multitude of in vitro models based on induced pluripotent stem cell (iPSC)-derived motor neurons (MNs) have been developed to investigate the underlying causes of selective MN degeneration in motor neuron diseases (MNDs). For instance, spheroids are simple 3D models that have the [...] Read more.
A multitude of in vitro models based on induced pluripotent stem cell (iPSC)-derived motor neurons (MNs) have been developed to investigate the underlying causes of selective MN degeneration in motor neuron diseases (MNDs). For instance, spheroids are simple 3D models that have the potential to be generated in large numbers that can be used across different assays. In this study, we generated MN spheroids and developed a workflow to analyze them. To start, the morphological profiling of the spheroids was achieved by developing a pipeline to obtain measurements of their size and shape. Next, we confirmed the expression of different MN markers at the transcript and protein levels by qPCR and immunocytochemistry of tissue-cleared samples, respectively. Finally, we assessed the capacity of the MN spheroids to display functional activity in the form of action potentials and bursts using a microelectrode array approach. Although most of the cells displayed an MN identity, we also characterized the presence of other cell types, namely interneurons and oligodendrocytes, which share the same neural progenitor pool with MNs. In summary, we successfully developed an MN 3D model, and we optimized a workflow that can be applied to perform its morphological, gene expression, protein, and functional profiling over time. Full article
(This article belongs to the Special Issue Advances in iPSC-Based Disease Modeling: From Two Dimensional Monolayers to Organoids)
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