47 results on '"hiPSC"'
Search Results
2. Generation of two induced pluripotent stem cell lines from hereditary amyloidosis patients with polyneuropathy carrying heterozygous transthyretin (TTR) mutation
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Juan Melesio, Bernardo Bonilauri, Audrey Li, Paul D. Pang, Ronglih Liao, Ronald M. Witteles, Joseph C. Wu, and Karim Sallam
- Subjects
hiPSC ,Stem cells ,Pluripotency ,TTR ,Transthyretin ,Polyneuropathy ,Biology (General) ,QH301-705.5 - Abstract
Hereditary transthyretin amyloidosis with polyneuropathy (ATTR-PN) results from specific TTR gene mutations. In this study, we generated two induced pluripotent stem cell (iPSC) lines derived from ATTR-PN patients with heterozygous TTR gene mutations (Ala97Ser and Phe64Leu). These iPSC lines exhibited normal morphology, karyotype, high pluripotency marker expression, and differentiation into cells representing all germ layers. The generation of these iPSC lines serve as a valuable tool for investigating the mechanisms of ATTR-PN across various cell types and facilitating patient-specific in vitro amyloidosis modeling.
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- 2024
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3. Isogenic hiPSC-derived liver-on-chip platforms: A valuable tool for modeling metabolic liver diseases
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Kehinde Oluwasegun Aina
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Liver ,hiPSC ,Organ-on-chip ,Inflammation ,Fibrosis ,Medicine - Abstract
The liver is a complex organ with vital functions in metabolism, detoxification, and immunity. The anatomy, physiology, and cellular composition of the liver are crucial for comprehending its spatial heterogeneity and regulation of homeostasis. Hepatocytes, liver sinusoidal endothelial cells (LSECs), hepatic stellate cells (HSCs), and liver macrophages play pivotal roles in liver function and pathology. Liver diseases such as NAFLD, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), and Liver fibrosis are prevalent and pose significant health challenges. Studying liver development provides insights into liver model evolution, including differentiation protocols for human hepatocyte-like cells (HLCs), hiPSC -derived endothelial cells, stellate cells and macrophages.Currently, the research landscape of liver tissue models encompasses in vivo and in vitro approaches, including 2D and 3D liver cell culture methods, on-chip systems, and patient-derived hiPSC-based liver disease models. The integration of hiPSCs with micro-physiological systems holds promise for recapitulating liver function and disease in vitro. However, challenges remain in achieving the physiological relevance and scalability of liver models. Advances in the research landscape of liver tissue models are discussed, providing insights into identifying individual patient groups, the current status quo, and prospects of liver research at the interface of developmental biology, tissue engineering, and disease modeling which will allow conclusions to be drawn about the molecular mechanisms of liver diseases and ultimately the targeted use of suitable therapeutics.
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- 2023
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4. Characterization and AAV-mediated CRB gene augmentation in human-derived CRB1KO and CRB1KOCRB2+/− retinal organoids
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Nanda Boon, Xuefei Lu, Charlotte A. Andriessen, Michaela Orlovà, Peter M.J. Quinn, Camiel J.F. Boon, and Jan Wijnholds
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CRB1 ,CRB2 ,AAV ,hiPSC ,retinal organoids ,gene therapy ,Genetics ,QH426-470 ,Cytology ,QH573-671 - Abstract
The majority of patients with mutations in CRB1 develop either early-onset retinitis pigmentosa as young children or Leber congenital amaurosis as newborns. The cause for the phenotypic variability in CRB1-associated retinopathies is unknown, but might be linked to differences in CRB1 and CRB2 protein levels in Müller glial cells and photoreceptor cells. Here, CRB1KO and CRB1KOCRB2+/− differentiation day 210 retinal organoids showed a significant decrease in the number of photoreceptor nuclei in a row and a significant increase in the number of photoreceptor cell nuclei above the outer limiting membrane. This phenotype with outer retinal abnormalities is similar to CRB1 patient-derived retinal organoids and Crb1 or Crb2 mutant mouse retinal disease models. The CRB1KO and CRB1KOCRB2+/− retinal organoids develop an additional inner retinal phenotype due to the complete loss of CRB1 from Müller glial cells, suggesting an essential role for CRB1 in proper localization of neuronal cell types. Adeno-associated viral (AAV) transduction was explored at early and late stages of organoid development. Moreover, AAV-mediated gene augmentation therapy with AAV.hCRB2 improved the outer retinal phenotype in CRB1KO retinal organoids. Altogether, these data provide essential information for future gene therapy approaches for patients with CRB1-associated retinal dystrophies.
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- 2023
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5. hiPSC-derived GRN-deficient astrocytes delay spiking activity of developing neurons
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Christopher Lee, Jonathan Frew, Nicholas L. Weilinger, Stefan Wendt, Wenji Cai, Stefano Sorrentino, Xiujuan Wu, Brian A. MacVicar, Stephanie M. Willerth, and Haakon B. Nygaard
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Frontotemporal dementia ,Progranulin ,hiPSC ,Astrocytes ,Neurons ,Multielectrode array ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
Frontotemporal dementia (FTD) refers to a group of neurodegenerative disorders that are characterized by pathology predominantly localized to the frontal and temporal lobes. Approximately 40% of FTD cases are familial, and up to 20% of these are caused by heterozygous loss of function mutations in the gene encoding for progranulin (PGRN), GRN. The mechanisms by which loss of PGRN leads to FTD remain incompletely understood. While astrocytes and microglia have long been linked to the neuropathology of FTD due to mutations in GRN (FTD-GRN), a primary mechanistic role of these supporting cells have not been thoroughly addressed. In contrast, mutations in MAPT, another leading cause of familial FTD, greatly alters astrocyte gene expression leading to subsequent non-cell autonomous effects on neurons, suggesting similar mechanisms may be present in FTD-GRN. Here, we utilized human induced pluripotent stem cell (hiPSC)-derived neural tissue carrying a homozygous GRN R493X−/− knock-in mutation to investigate in vitro whether GRN mutant astrocytes have a non-cell autonomous effect on neurons. Using microelectrode array (MEA) analysis, we demonstrate that the development of spiking activity of neurons cultured with GRN R493X−/− astrocytes was significantly delayed compared to cultures with WT astrocytes. Histological analysis of synaptic markers in these cultures showed an increase in GABAergic synaptic markers and a decrease in glutamatergic synaptic markers during this period when activity was delayed. We also demonstrate that this effect may be due in-part to soluble factors. Overall, this work represents one of the first studies investigating astrocyte-induced neuronal pathology in GRN mutant hiPSCs, and supports the hypothesis of astrocyte involvement in the early pathophysiology of FTD.
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- 2023
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6. Generation of miR-15a/16-1 cluster-deficient human induced pluripotent stem cell line (DMBi001-A-2) using CRISPR/Cas9 gene editing
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Jacek Stępniewski, Mateusz Jeż, and Józef Dulak
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hiPSC ,microRNA-15a ,microRNA-16-1 ,CRISPR/Cas9 ,Cardiomyocytes ,Cardiac hypertrophy ,Biology (General) ,QH301-705.5 - Abstract
miR-15a/16-1 cluster, composed of MIR15A and MIR16-1 genes located in close proximity on chromosome 13 was described to regulate post-natal cell cycle withdrawal of cardiomyocytes in mice. In humans, on the other hand, the level of miR-15a-5p and miR-16-p was negatively associated with the severity of cardiac hypertrophy. Therefore, to better understand the role of these microRNAs in human cardiomyocytes in regard to their proliferative potential and hypertrophic growth, we generated hiPSC line with complete deletion of miR-15a/16-1 cluster using CRISPR/Cas9 gene editing. Obtained cells demonstrate expression of pluripotency markers, differentiation capacity into all three germ layers and normal karyotype.
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- 2023
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7. Validating human induced pluripotent stem cell-specific quality control tests for the release of an intermediate drug product in a Good Manufacturing Practice quality system.
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Novoa J, Westra I, Steeneveld E, Neves NF, Daleman L, Asensio AB, Davis RP, Carlotti F, Freund C, Rabelink T, Meij P, and Wieles B
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- Humans, Reproducibility of Results, Genetic Vectors, Induced Pluripotent Stem Cells cytology, Quality Control, Cell Differentiation
- Abstract
One of the challenges in Good Manufacturing Practice (GMP)-compliant human induced pluripotent stem cell (hiPSC) production is the validation of quality control (QC) tests specific for hiPSCs, which are required for GMP batch release. This study presents a comprehensive description of the validation process for hiPSC-specific GMP-compliant QC assays; more specifically, the validation of assays to assess the potential presence of residual episomal vectors (REVs), the expression of markers of the undifferentiated state and the directed differentiation potential of hiPSCs. Critical aspects and specific acceptance criteria were formulated in a validation plan prior to assay validation. Assay specificity, sensitivity and reproducibility were tested, and the equipment used for each assay was subjected to performance qualification. A minimum input of 20 000 cells (120 ng of genomic DNA) was defined for accurate determination of the presence of REVs. Furthermore, since vector loss in hiPSC lines is a passage-dependent process, we advocate screening for REVs between passages eight and 10, as testing at earlier passages might lead to unnecessary rejection of hiPSC lines. The cutoff value for assessment of markers of the undifferentiated state was set to the expression of at least three individual markers on at least 75% of the cells. When multi-color flow cytometry panels are used, a fluorescence minus one control is advised to ensure the control for fluorescent spread. For the assay to assess the directed differentiation potential, the detection limit was set to two of three positive lineage-specific markers for each of the three individual germ layers. All of our assays proved to be reproducible and specific. Our data demonstrate that our implemented analytical procedures are suitable as QC assays for the batch release of GMP-compliant hiPSCs., Competing Interests: Declaration of Competing Interest The authors have no commercial, proprietary or financial interest in the products or companies described in this article., (Copyright © 2024 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)
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- 2024
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8. The generation of human induced pluripotent stem cell lines from individuals of Black African ancestry in South Africa.
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Naidoo J, Hurrell T, and Scholefield J
- Abstract
The lack of equitable representation of African diversity in scientific resources, such as genome-wide association studies and human induced pluripotent stem cell (hiPSC) repositories, has perpetuated inequalities in the advancement of health research. HiPSCs could be transformative in regenerative and precision medicine, therefore, the generation of diverse lines is critical in the establishment of African-relevant preclinical cellular models. HiPSC lines were derived from two healthy donors of Black African ancestry using Sendai virus reprogramming of dermal fibroblasts, and characterised to confirm stemness markers, trilineage differentiation, and genetic integrity. These hiPSCs represent a valuable resource for modelling African relevant disease biology., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)
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- 2024
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9. Altered action potential waveform and shorter axonal initial segment in hiPSC-derived motor neurons with mutations in VRK1
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Rémi Bos, Khalil Rihan, Patrice Quintana, Lara El-Bazzal, Nathalie Bernard-Marissal, Nathalie Da Silva, Rosette Jabbour, André Mégarbané, Marc Bartoli, Frédéric Brocard, and Valérie Delague
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Motoneuron ,Action potential waveform ,VRK1 ,hiPSC ,Inherited peripheral neuropathy ,Motor neuron disease ,Neurosciences. Biological psychiatry. Neuropsychiatry ,RC321-571 - Abstract
We recently described new pathogenic variants in VRK1, in patients affected with distal Hereditary Motor Neuropathy associated with upper motor neurons signs. Specifically, we provided evidences that hiPSC-derived Motor Neurons (hiPSC-MN) from these patients display Cajal Bodies (CBs) disassembly and defects in neurite outgrowth and branching. We here focused on the Axonal Initial Segment (AIS) and the related firing properties of hiPSC-MNs from these patients. We found that the patient's Action Potential (AP) was smaller in amplitude, larger in duration, and displayed a more depolarized threshold while the firing patterns were not altered. These alterations were accompanied by a decrease in the AIS length measured in patients' hiPSC-MNs. These data indicate that mutations in VRK1 impact the AP waveform and the AIS organization in MNs and may ultimately lead to the related motor neuron disease.
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- 2022
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10. Validation of an automated cell counting method for cGMP manufacturing of human induced pluripotent stem cells
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Paolo Manzini, Valeria Peli, Araceli Rivera-Ordaz, Silvia Budelli, Mario Barilani, and Lorenza Lazzari
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Cell counting ,hiPSC ,Validation ,Analytical method ,Cell manufacturing ,Cell therapy ,Biotechnology ,TP248.13-248.65 - Abstract
Human induced pluripotent stem cells (hiPSCs) must be manufactured as advanced therapy medicinal products (ATMPs) for innovative tissue replacement clinical applications. Yet, production of hiPSCs under current Good Manufacturing Practice (cGMP) presents many hurdles, such as the large-scale cell expansion needed to reach therapeutically-relevant hiPSC doses. For the monitoring of this phase, a fast and reliable cell counting method should be used. Conventional manual cell counting by the hemocytometer method is dependent on the operator's expertise and is time-consuming. Therefore, automation of sample preparation and analysis is needed to improve precision and rapidity of hiPSC cell counting. We investigated whether an automated cell counting method could be validated for use with hiPSCs, in comparison with a reference cell counting method included in the European Pharmacopeia, 10th edition. The proposed method was the fluorescence imaging-based NucleoCounter NC-100 system, whereas the reference method was manual cell counting using a Bürker hemocytometer. The validation strategy complied with EudraLex cGMP regulations for ATMP manufacturing and ICH Q2(R1) indications for validation of analytical methods. The use of the NucleoCounter NC-100 system for automated cell counting was validated, focusing on accuracy, specificity, intra- and inter-operator reproducibility, range and linearity, showing higher precision than the manual method. The automated method can be used more effectively than the manual one for hiPSC cell counting. Thus, this piece of work paves the way for all cGMP facilities that want to pursue hiPSC manufacturing for clinical use.
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- 2022
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11. Reliable generation of glial enriched progenitors from human fibroblast-derived iPSCs
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Irene L. Llorente, Emily A. Hatanaka, Michael E. Meadow, Yuan Xie, William E. Lowry, and S. Thomas Carmichael
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hiPSC ,Astrocytes ,White matter ,Stroke ,Manufacturing ,Assays ,Biology (General) ,QH301-705.5 - Abstract
White matter stroke (WMS) occurs as small infarcts in deep penetrating blood vessels in the brain and affects the regions of the brain that carry connections, termed the subcortical white matter. WMS progresses over years and has devastating clinical consequences. Unlike large grey matter strokes, WMS disrupts the axonal architecture of the brain and depletes astrocytes, oligodendrocyte lineage cells, axons and myelinating cells, resulting in abnormalities of gait and executive function. An astrocytic cell-based therapy is positioned as a strong therapeutic candidate after WMS. In this study we report, the reliable generation of a novel stem cell-based therapeutic product, glial enriched progenitors (GEPs) derived from human induced pluripotent stem cells (hiPSCs). By transient treatment of hiPSC derived neural progenitors (hiPSC-NPCs) with the small molecule deferoxamine, a prolyl hydroxylase inhibitor, for three days hiPSC-NPCs become permanently biased towards an astrocytic fate, producing hiPSC-GEPs. In preparation for clinical application, we have developed qualification assays to ensure identity, safety, purity, and viability of the cells prior to manufacture. Using tailored q-RT-PCR-based assays, we have demonstrated the lack of pluripotency in our final therapeutic candidate cells (hiPSC-GEPs) and we have identified the unique genetic profile of hiPSC-GEPs that is clearly distinct from the parent lines, hiPSCs and iPSC-NPCs. After completion of the viability assay, we have stablished the therapeutic window of use for hiPSC-GEPs in future clinical applications (7 h). Lastly, we were able to reliably and consistently produce a safe therapeutic final product negative for contamination by any human or murine viral pathogens, selected bacteria, common laboratory mycoplasmas, growth of any aerobes, anaerobes, yeast, or fungi and 100 times less endotoxin levels than the maximum acceptable value. This study demonstrates the reliable and safe generation of patient derived hiPSC-GEPs that are clinically ready as a cell-based therapeutic approach for WMS.
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- 2021
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12. In vitro modeling of cranial placode differentiation: Recent advances, challenges, and perspectives.
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Griffin C and Saint-Jeannet JP
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- Animals, Humans, Vertebrates, Cell Differentiation, Signal Transduction, Gene Expression Regulation, Developmental, Skull, Ectoderm
- Abstract
Cranial placodes are transient ectodermal thickenings that contribute to a diverse array of organs in the vertebrate head. They develop from a common territory, the pre-placodal region that over time segregates along the antero-posterior axis into individual placodal domains: the adenohypophyseal, olfactory, lens, trigeminal, otic, and epibranchial placodes. These placodes terminally differentiate into the anterior pituitary, the lens, and contribute to sensory organs including the olfactory epithelium, and inner ear, as well as several cranial ganglia. To study cranial placodes and their derivatives and generate cells for therapeutic purposes, several groups have turned to in vitro derivation of placodal cells from human embryonic stem cells (hESCs) or induced pluripotent stem cells (hiPSCs). In this review, we summarize the signaling cues and mechanisms involved in cranial placode induction, specification, and differentiation in vivo, and discuss how this knowledge has informed protocols to derive cranial placodes in vitro. We also discuss the benefits and limitations of these protocols, and the potential of in vitro cranial placode modeling in regenerative medicine to treat cranial placode-related pathologies., Competing Interests: Declaration of competing interest The authors report no competing interests. The authors alone are responsible for the content and writing of this article., (Copyright © 2023 Elsevier Inc. All rights reserved.)
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- 2024
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13. Generation of two induced pluripotent stem cell lines from hereditary amyloidosis patients with polyneuropathy carrying heterozygous transthyretin (TTR) mutation.
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Melesio J, Bonilauri B, Li A, Pang PD, Liao R, Witteles RM, Wu JC, and Sallam K
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- Humans, Prealbumin genetics, Prealbumin metabolism, Mutation genetics, Induced Pluripotent Stem Cells metabolism, Amyloid Neuropathies, Familial genetics, Amyloid Neuropathies, Familial metabolism, Polyneuropathies genetics, Polyneuropathies metabolism
- Abstract
Hereditary transthyretin amyloidosis with polyneuropathy (ATTR-PN) results from specific TTR gene mutations. In this study, we generated two induced pluripotent stem cell (iPSC) lines derived from ATTR-PN patients with heterozygous TTR gene mutations (Ala97Ser and Phe64Leu). These iPSC lines exhibited normal morphology, karyotype, high pluripotency marker expression, and differentiation into cells representing all germ layers. The generation of these iPSC lines serve as a valuable tool for investigating the mechanisms of ATTR-PN across various cell types and facilitating patient-specific in vitro amyloidosis modeling., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Joseph C. Wu reports a relationship with Greenstone Biosciences that includes: co-founder & scientific advisory board member. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)
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- 2024
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14. A circular RNA map for human induced pluripotent stem cells of foetal origin
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Mario Barilani, Alessandro Cherubini, Valeria Peli, Francesca Polveraccio, Valentina Bollati, Federica Guffanti, Alessandro Del Gobbo, Cristiana Lavazza, Silvia Giovanelli, Nicola Elvassore, and Lorenza Lazzari
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Cord blood ,Multipotent mesenchymal stromal cells ,hiPSC ,miRNA ,circRNA ,PGRN ,Medicine ,Medicine (General) ,R5-920 - Abstract
Background: Adult skin fibroblasts represent the most common starting cell type used to generate human induced pluripotent stem cells (F-hiPSC) for clinical studies. Yet, a foetal source would offer unique advantages, primarily the absence of accumulated somatic mutations. Herein, we generated hiPSC from cord blood multipotent mesenchymal stromal cells (MSC-hiPSC) and compared them with F-hiPSC. Assessment of the full activation of the pluripotency gene regulatory network (PGRN) focused on circular RNA (circRNA), recently proposed to participate in the control of pluripotency. Methods: Reprogramming was achieved by a footprint-free strategy. Self-renewal and pluripotency of cord blood MSC-hiPSC were investigated in vitro and in vivo, compared to parental MSC, to embryonic stem cells and to F-hiPSC. High-throughput array-based approaches and bioinformatics analyses were applied to address the PGRN. Findings: Cord blood MSC-hiPSC successfully acquired a complete pluripotent identity. Functional comparison with F-hiPSC showed no differences in terms of i) generation of mesenchymal-like derivatives, ii) their subsequent adipogenic, osteogenic and chondrogenic commitment, and iii) their hematopoietic support ability. At the transcriptional level, specific subsets of mRNA, miRNA and circRNA (n = 4,429) were evidenced, casting a further layer of complexity on the PGRN regulatory crosstalk. Interpretation: A circRNA map of transcripts associated to naïve and primed pluripotency is provided for hiPSC of clinical-grade foetal origin, offering insights on still unreported regulatory circuits of the PGRN to consider for the optimization and development of efficient differentiation protocols for clinical translation. Funding: This research was funded by Ricerca Corrente 2012–2018 by the Italian Ministry of Health.
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- 2020
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15. Comparison of 2D and 3D neural induction methods for the generation of neural progenitor cells from human induced pluripotent stem cells
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Abinaya Chandrasekaran, Hasan X. Avci, Anna Ochalek, Lone N. Rösingh, Kinga Molnár, Lajos László, Tamás Bellák, Annamária Téglási, Krisztina Pesti, Arpad Mike, Phetcharat Phanthong, Orsolya Bíró, Vanessa Hall, Narisorn Kitiyanant, Karl-Heinz Krause, Julianna Kobolák, and András Dinnyés
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Neural induction ,Neural progenitor cells ,hiPSC ,Electron microscopy ,Patch clamp ,2D-3D neural induction ,Biology (General) ,QH301-705.5 - Abstract
Neural progenitor cells (NPCs) from human induced pluripotent stem cells (hiPSCs) are frequently induced using 3D culture methodologies however, it is unknown whether spheroid-based (3D) neural induction is actually superior to monolayer (2D) neural induction. Our aim was to compare the efficiency of 2D induction with 3D induction method in their ability to generate NPCs, and subsequently neurons and astrocytes. Neural differentiation was analysed at the protein level qualitatively by immunocytochemistry and quantitatively by flow cytometry for NPC (SOX1, PAX6, NESTIN), neuronal (MAP2, TUBB3), cortical layer (TBR1, CUX1) and glial markers (SOX9, GFAP, AQP4). Electron microscopy demonstrated that both methods resulted in morphologically similar neural rosettes. However, quantification of NPCs derived from 3D neural induction exhibited an increase in the number of PAX6/NESTIN double positive cells and the derived neurons exhibited longer neurites. In contrast, 2D neural induction resulted in more SOX1 positive cells. While 2D monolayer induction resulted in slightly less mature neurons, at an early stage of differentiation, the patch clamp analysis failed to reveal any significant differences between the electrophysiological properties between the two induction methods. In conclusion, 3D neural induction increases the yield of PAX6+/NESTIN+ cells and gives rise to neurons with longer neurites, which might be an advantage for the production of forebrain cortical neurons, highlighting the potential of 3D neural induction, independent of iPSCs' genetic background.
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- 2017
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16. CXCR4 and CXCR7 play distinct roles in cardiac lineage specification and pharmacologic β-adrenergic response
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Delaine K. Ceholski, Irene C. Turnbull, Venu Pothula, Laura Lecce, Andrew A. Jarrah, Changwon Kho, Ahyoung Lee, Lahouaria Hadri, Kevin D. Costa, Roger J. Hajjar, and Sima T. Tarzami
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CXCR4 ,CXCR7 ,Cardiogenesis ,hiPSC ,Engineered tissue ,Biology (General) ,QH301-705.5 - Abstract
CXCR4 and CXCR7 are prominent G protein-coupled receptors (GPCRs) for chemokine stromal cell-derived factor-1 (SDF-1/CXCL12). This study demonstrates that CXCR4 and CXCR7 induce differential effects during cardiac lineage differentiation and β-adrenergic response in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Using lentiviral vectors to ablate CXCR4 and/or CXCR7 expression, hiPSC-CMs were tested for phenotypic and functional properties due to gene knockdown. Gene expression and flow cytometry confirmed the pluripotent and cardiomyocyte phenotype of undifferentiated and differentiated hiPSCs, respectively. Although reduction of CXCR4 and CXCR7 expression resulted in a delayed cardiac phenotype, only knockdown of CXCR4 delayed the spontaneous beating of hiPSC-CMs. Knockdown of CXCR4 and CXCR7 differentially altered calcium transients and β-adrenergic response in hiPSC-CMs. In engineered cardiac tissues, depletion of CXCR4 or CXCR7 had opposing effects on developed force and chronotropic response to β-agonists. This work demonstrates distinct roles for the SDF-1/CXCR4 or CXCR7 network in hiPSC-derived ventricular cardiomyocyte specification, maturation and function.
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- 2017
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17. Early Development of Definitive Erythroblasts from Human Pluripotent Stem Cells Defined by Expression of Glycophorin A/CD235a, CD34, and CD36
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Bin Mao, Shu Huang, Xulin Lu, Wencui Sun, Ya Zhou, Xu Pan, Jinfeng Yu, Mowen Lai, Bo Chen, Qiongxiu Zhou, Song Mao, Guohui Bian, Jiaxi Zhou, Tatsutoshi Nakahata, and Feng Ma
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hESC ,hiPSC ,hematopoiesis ,development ,erythroblasts ,erythropoiesis ,endothelial cells ,GPA ,CD36 ,CD34 ,Medicine (General) ,R5-920 ,Biology (General) ,QH301-705.5 - Abstract
The development of human erythroid cells has been mostly examined in models of adult hematopoiesis, while their early derivation during embryonic and fetal stages is largely unknown. We observed the development and maturation of erythroblasts derived from human pluripotent stem cells (hPSCs) by an efficient co-culture system. These hPSC-derived early erythroblasts initially showed definitive characteristics with a glycophorin A+ (GPA+) CD34lowCD36− phenotype and were distinct from adult CD34+ cell-derived ones. After losing CD34 expression, early GPA+CD36− erythroblasts matured into GPA+CD36low/+ stage as the latter expressed higher levels of β-globin along with a gradual loss of mesodermal and endothelial properties, and terminally suppressed CD36. We establish a unique in vitro model to trace the early development of hPSC-derived erythroblasts by serial expression of CD34, GPA, and CD36. Our findings may provide insight into the understanding of human early erythropoiesis and, ultimately, therapeutic potential.
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- 2016
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18. Development of cartilage tissue using a stirred bioreactor and human iPSC-derived limb bud mesenchymal cells.
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Fujisawa Y, Takao T, Yamada D, and Takarada T
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- Humans, Rats, Animals, Limb Buds, Chondrocytes metabolism, Hyaline Cartilage, Cell Differentiation, Tissue Engineering methods, Aggrecans metabolism, Bioreactors, Chondrogenesis, Induced Pluripotent Stem Cells metabolism
- Abstract
Production of cartilaginous particles for regenerative medicine requires a large supply of chondrocytes and development of suitable production techniques. Previously, we successfully produced human induced pluripotent stem cell (hiPSC)-derived limb bud mesenchymal cells (ExpLBM cells) with a high chondrogenic differentiation potential that stably proliferate. It may be possible to use these cells in combination with a stirred bioreactor to develop a tissue-engineered cell culture technology with potential for scale-up to facilitate production of large amounts of cartilaginous particles. ExpLBM cells derived from 414C2 and Ff-I 14s04 (human leukocyte antigen homozygous) hiPSCs were seeded into a stirred bioreactor containing cartilage induction medium. To characterize the cartilaginous particles produced, we performed real-time quantitative reverse transcription-polymerase chain reaction and histological analyses. Additionally, we transplanted the cartilage tissue into osteochondral defects of immunocompromised rats to assess its functionality, and evaluated engraftment of the grafted tissue. We successfully produced large amounts of cartilaginous particles via cartilage induction culture in a stirred bioreactor. This tissue exhibited significantly increased expression levels of type II collagen (COL2), aggrecan (ACAN), and SRY-box transcription factor 9 (SOX9), as well as positive Safranin O and Toluidine blue staining, indicating that it possesses characteristics of hyaline cartilage. Furthermore, engrafted tissues in osteochondral knee defects of immunodeficient rats were positively stained for human vimentin, COL2, and ACAN as well as with Safranin O. In this study, we successfully generated large amounts of hiPSC-derived cartilaginous particles using a combination of tissue engineering techniques. This method is promising as a cartilage regeneration technology with potential for scale-up., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)
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- 2023
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19. Comparative assessment of Ca 2+ oscillations in 2- and 3-dimensional hiPSC derived and isolated cortical neuronal networks.
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Imredy JP, Roussignol G, Clouse H, Salvagiotto G, and Mazelin-Winum L
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- Humans, Mice, Animals, Cells, Cultured, Reproducibility of Results, Neurons, Drug Discovery, Cell Differentiation, Induced Pluripotent Stem Cells
- Abstract
Human induced Pluripotent Stem Cell (hiPSC) derived neural cells offer great potential for modelling neurological diseases and toxicities and have found application in drug discovery and toxicology. As part of the European Innovative Medicines Initiative (IMI2) NeuroDeRisk (Neurotoxicity De-Risking in Preclinical Drug Discovery), we here explore the Ca
2+ oscillation responses of 2D and 3D hiPSC derived neuronal networks of mixed Glutamatergic/GABAergic activity with a compound set encompassing both clinically as well as experimentally determined seizurogenic compounds. Both types of networks are scored against Ca2+ responses of a primary mouse cortical neuronal 2D network model serving as an established comparator assay. Parameters of frequency and amplitude of spontaneous global network Ca2+ oscillations and the drug-dependent directional changes to these were assessed, and predictivity of seizurogenicity scored using contingency table analysis. In addition, responses between models were compared between both 2D models as well as between 2D and 3D models. Concordance of parameter responses was best between the hiPSC neurospheroid and the mouse primary cortical neuron model (77% for frequency and 65% for amplitude). Decreases in spontaneous Ca2+ oscillation frequency and amplitude were found to be the most basic shared determinants of risk of seizurogenicity between the mouse and the neurospheroid model based on testing of clinical compounds with documented seizurogenic activity. Increases in spontaneous Ca2+ oscillation frequency were primarily observed with the 2D hIPSC model, though the specificity of this effect to seizurogenic clinical compounds was low (33%), while decreases to spike amplitude in this model were more predictive of seizurogenicity. Overall predictivities of the models were similar, with sensitivity of the assays typically exceeding specificity due to high false positive rates. Higher concordance of the hiPSC 3D model over the 2D model when compared to mouse cortical 2D responses may be the result of both a longer maturation time of the neurospheroid (84-87 days for 3D vs. 22-24 days for 2D maturation) as well as the 3-dimensional nature of network connections established. The simplicity and reproducibility of spontaneous Ca2+ oscillation readouts support further investigation of hiPSC derived neuronal sources and their 2- and 3-dimensional networks for neuropharmacological safety screening., Competing Interests: Declaration of Competing Interest The authors are employees of their respective companies and confirm that there are no known conflicts of interest associated with this publication and there has been no significant financial support for this work that could have influenced its outcome. Mention of trade names or commercial products does not constitute endorsement or recommendation for use., (Copyright © 2023. Published by Elsevier Inc.)- Published
- 2023
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20. Comparative study for the IMI2-NeuroDeRisk project on microelectrode arrays to derisk drug-induced seizure liability.
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Zhai J, Traebert M, Zimmermann K, Delaunois A, Royer L, Salvagiotto G, Carlson C, and Lagrutta A
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- Rats, Humans, Animals, Microelectrodes, Cells, Cultured, Seizures chemically induced, Neurons, Induced Pluripotent Stem Cells
- Abstract
Introduction: In the framework of the IMI2-NeuroDeRisk consortium, three in vitro electrophysiology assays were compared to improve preclinical prediction of seizure-inducing liabilities., Methods: Two cell models, primary rat cortical neurons and human induced pluripotent stem cell (hiPSC)-derived glutamatergic neurons co-cultured with hiPSC-derived astrocytes were tested on two different microelectrode array (MEA) platforms, Maestro Pro (Axion Biosystems) and Multiwell-MEA-System (Multi Channel Systems), in three separate laboratories. Pentylenetetrazole (PTZ) and/or picrotoxin (PTX) were included in each plate as positive (n = 3-6 wells) and ≤0.2% DMSO was used as negative controls (n = 3-12 wells). In general, concentrations in a range of 0.1-30 μM were tested, anchored, when possible, on clinically relevant exposures (unbound C
max ) were tested. Activity thresholds for drug-induced changes were set at 20%. To evaluate sensitivity, specificity and predictivity of the cell models, seizurogenic responses were defined as changes in 4 or more endpoints. Concentration dependence trends were also considered., Results: Neuronal activity of 33 compounds categorized as positive tool drugs, seizure-positive or seizure-negative compounds was evaluated. Acute drug effects (<60 min) were compared to baseline recordings. Time points < 15 min exhibited stronger, less variable responses to many of the test agents. For many compounds a reduction and cessation of neuronal activity was detected at higher test concentrations. There was not a single pattern of seizurogenic activity detected, even among tool compounds, likely due to different mechanisms of actions and/or off-target profiles. A post-hoc analysis focusing on changes indicative of neuronal excitation is presented., Conclusion: All cell models showed good sensitivity, ranging from 70 to 86%. Specificity ranged from 40 to 70%. Compared to more conventional measurements of evoked activity in hippocampal slices, these plate-based models provide higher throughput and the potential to study subacute responses. Yet, they may be limited by the random, spontaneous nature of their network activity., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest. All co-authors are employed by pharmaceuticals or biotechnology companies interested in developing novel in vitro assays to improve human health and in assessing methodological improvements to this end, without bias. This research was conducted in the context of the IMI2: NeuroDeRisk non-competitive industry-academic consortium funded by the European Medicines Agency. Mention of trade names or commercial products does not constitute endorsement or recommendation for use., (Copyright © 2023 Merck Sharp & Dohme LLC., a subsidiary of Merck & Co., Inc., Rahway, NJ, USA, The Author(s). Published by Elsevier Inc. All rights reserved.)- Published
- 2023
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21. Biomaterial-driven kidney organoid maturation
- Subjects
Biomaterials ,Kidney organoids ,hiPSC - Abstract
Differentiation of human-induced pluripotent stem cells (hiPSCs) toward kidney organoids is known to suffer from batch-to-batch differences, off-target populations, and skewered cellular compositions. Application of synthetic hydrogels as a tool for hiPSC-differentiation may provide additional control over this variable process. This review discusses important material properties that affect kidney organoid generation. We summarize cellular adhesive cues for synthetic materials, that allow transduction of the mechanical forces to the cell, and how these signals directly affect Hippo-Canonical Wnt signaling and morphogenic events. In addition, chemical strategies are discussed that allow spatiotemporal presentation of biochemical agents to the cell in a material-dependent approach.
- Published
- 2022
22. Epithelial folding of alveolar cells derived from human induced pluripotent stem cells on artificial basement membrane.
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Rofaani E, He Y, Peng J, and Chen Y
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- Humans, Alveolar Epithelial Cells metabolism, Membranes, Artificial, Basement Membrane, Laminin pharmacology, Laminin metabolism, Epithelial Cells metabolism, Morphogenesis, Induced Pluripotent Stem Cells metabolism
- Abstract
Epithelial folding depends on mechanical properties of both epithelial cells and underlying basement membrane (BM). While folding is essential for tissue morphogenesis and functions, it is difficult to recapitulate features of a growing epithelial monolayer for in vitro modeling due to lack of in vivo like BM. Herein, we report a method to overcome this difficulty by culturing on an artificial basement membrane (ABM) the primordial lung progenitors (PLPs) from human induced pluripotent stem cells (hiPSCs). The ABM was achieved by self-assembling collagen IV and laminin, the two principal natural BM proteins, in the pores of a monolayer of crosslinked gelatin nanofibers deposited on a honeycomb micro-frame. The hiPSC-PLPs were seeded on the ABM for alveolar differentiation under submerged and air-liquid interface culture conditions. As results, the forces generated by the growing epithelial monolayer led to a geometry-dependent folding. Analysis of strain distribution in a clamped membrane provided instrumental insights into some of the observed phenomena. Moreover, the forces generated by the growing epithelial layer led to a high-level expression of surfactant protein C and a high percentage of aquaporin 5 positive cells compared with the results obtained with a nanofiber-covered bulk substrate. Thus, this work demonstrated the importance of recapitulating natural BM for advanced epithelial modeling. STATEMENT OF SIGNIFICANCE: The effort to develop in vitro epithelial models has not been entirely successful to date, due to lack of in vivo like basement membrane (BM). This lack has been overcome by using a microfabricated dense thin and pliable sheet like structure made of natural BM proteins. With such an artificial BM, alveolar epithelial deformation and folding could be studied and date could be correlated to numerical analyses of a plate theory. This method is simple and effective, enabling further developments in epithelial tissue modeling., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Ltd.)
- Published
- 2023
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23. Generation of miR-15a/16-1 cluster-deficient human induced pluripotent stem cell line (DMBi001-A-2) using CRISPR/Cas9 gene editing.
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Stępniewski J, Jeż M, and Dulak J
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- Humans, Animals, Mice, Gene Editing, CRISPR-Cas Systems genetics, Cardiomegaly, Induced Pluripotent Stem Cells metabolism, MicroRNAs genetics, MicroRNAs metabolism
- Abstract
miR-15a/16-1 cluster, composed of MIR15A and MIR16-1 genes located in close proximity on chromosome 13 was described to regulate post-natal cell cycle withdrawal of cardiomyocytes in mice. In humans, on the other hand, the level of miR-15a-5p and miR-16-p was negatively associated with the severity of cardiac hypertrophy. Therefore, to better understand the role of these microRNAs in human cardiomyocytes in regard to their proliferative potential and hypertrophic growth, we generated hiPSC line with complete deletion of miR-15a/16-1 cluster using CRISPR/Cas9 gene editing. Obtained cells demonstrate expression of pluripotency markers, differentiation capacity into all three germ layers and normal karyotype., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)
- Published
- 2023
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24. Vitamin C transport in neurons and epithelia is regulated by secretory carrier-associated membrane protein-2 (SCAMP2).
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Rashid MA, Lin-Moshier Y, Gunaratne GS, Subramanian S, Marchant JS, and Subramanian VS
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- Humans, HEK293 Cells, Cell Membrane metabolism, Ascorbic Acid pharmacology, Ascorbic Acid metabolism, Neurons metabolism, Protein Transport, Carrier Proteins metabolism, Membrane Proteins genetics, Membrane Proteins metabolism, Proteomics, Sodium-Coupled Vitamin C Transporters genetics, Sodium-Coupled Vitamin C Transporters metabolism
- Abstract
The human sodium-dependent vitamin C transporter-1 (hSVCT1) is localized at the apical membrane domain of polarized intestinal and renal epithelial cells to mediate ascorbic acid (AA) uptake. Currently, little is known about the array of interacting proteins that aid hSVCT1 trafficking and functional expression at the cell surface. Here we used an affinity tagging ('One-STrEP') and proteomic approach to identify hSVCT1 interacting proteins, which resolved secretory carrier-associated membrane protein-2 (SCAMP2) as a novel accessary protein partner. SCAMP2 was validated as an accessory protein by co-immunoprecipitation with hSVCT1. Co-expression of hSVCT1 and SCAMP2 in HEK-293 cells revealed both proteins co-localized in intracellular structures and at the plasma membrane. Functionally, over-expression of SCAMP2 potentiated
14 C-AA uptake, and reciprocally silencing endogenous SCAMP2 decreased14 C-AA uptake. Finally, knockdown of endogenous hSVCT1 or SCAMP2 impaired differentiation of human-induced pluripotent stem cells (hiPSCs) toward a neuronal fate. These results establish SCAMP2 as a novel hSVCT1 accessary protein partner that regulates AA uptake in absorptive epithelia and during neurogenesis., Competing Interests: Declaration of competing interest No potential conflict of interest was reported by the author(s)., (Copyright © 2023 Elsevier B.V. All rights reserved.)- Published
- 2023
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25. Genome-engineering technologies for modeling and treatment of cystic fibrosis.
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Dębczyński M, Mojsak D, Minarowski Ł, Maciejewska M, Lisowski P, and Mróz RM
- Subjects
- Humans, Gene Editing, Technology, Cystic Fibrosis genetics, Cystic Fibrosis therapy
- Abstract
Cystic fibrosis (CF) is an autosomal recessive disease caused by defects in the CF transmembrane conductance regulator (CFTR) protein. Due to the genetic nature of the disease, interventions in the genome can target any underlying alterations and potentially provide permanent disease resolution. The current development of gene-editing tools, such as designer nuclease technology capable of genome correction, holds great promise for both CF and other genetic diseases. In recent years, Cas9-based technologies have enabled the generation of genetically defined human stem cell and disease models based on induced pluripotent stem cells (iPSC). In this article, we outline the potential and possibilities of using CRISPR/Cas9-based gene-editing technology in CF modeling., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Medical University of Bialystok. Published by Elsevier B.V. All rights reserved.)
- Published
- 2023
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26. Generation of an induced pluripotent stem cell line (CSS012-A (7672)) carrying the p.G376D heterozygous mutation in the TARDBP protein
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Jessica Rosati, Daniela Ferrari, Angela D'Anzi, Elisa Perciballi, Mario Sabatelli, Filomena Altieri, Angelo L. Vescovi, Laura Bernardini, Barbara Torres, Serena Lattante, D'Anzi, A, Altieri, F, Perciballi, E, Ferrari, D, Torres, B, Bernardini, L, Lattante, S, Sabatelli, M, Vescovi, A, and Rosati, J
- Subjects
0301 basic medicine ,QH301-705.5 ,Cellular differentiation ,Induced Pluripotent Stem Cells ,TARDBP, hiPSC, familial ALS ,Germ layer ,Biology ,medicine.disease_cause ,Settore MED/03 - GENETICA MEDICA ,TARDBP ,Induced Pluripotent Stem Cell ,hiPSC ,Cell Line ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Humans ,Biology (General) ,Induced pluripotent stem cell ,Mutation ,Genetic heterogeneity ,Amyotrophic Lateral Sclerosis ,Cell Differentiation ,Cell Biology ,General Medicine ,Fibroblasts ,Phenotype ,Cell biology ,030104 developmental biology ,Cell culture ,Fibroblast ,familial ALS ,030217 neurology & neurosurgery ,Developmental Biology ,Human ,Amyotrophic Lateral Sclerosi - Abstract
Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative condition with phenotypic and genetic heterogeneity. It is characterized by the selective vulnerability and the progressive loss of the neural population. Here, an induced pluripotent stem cell (iPSC) line was generated from dermal fibroblasts of an individual carrying the p.G376D mutation in the TDP-43 protein. Fibroblasts were reprogrammed using non-integrating episomal plasmids. There were no karyotype abnormalities, and iPSCs successfully differentiated into all three germ layers. This cell line may prove useful in the study of the pathogenic mechanisms that underpin ALS syndrome.
- Published
- 2021
27. Core Transcription Factors Promote Induction of PAX3-Positive Skeletal Muscle Stem Cells
- Author
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Takahiko Sato, Naoki Goshima, Morio Ueno, Takuya Yamamoto, Koki Higashioka, Chie Sotozono, and Hidetoshi Sakurai
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0301 basic medicine ,medicine.medical_treatment ,MyoD ,Muscle Development ,Biochemistry ,hiPSC ,Cell therapy ,Dystrophin ,Mesoderm ,Mice ,0302 clinical medicine ,Mice, Inbred NOD ,Basic Helix-Loop-Helix Transcription Factors ,Muscular dystrophy ,muscle stem cell ,lcsh:QH301-705.5 ,Mice, Knockout ,lcsh:R5-920 ,Stem Cells ,Cell Differentiation ,Stem-cell therapy ,musculoskeletal system ,Cell biology ,KLF4 ,embryonic structures ,Stem cell ,lcsh:Medicine (General) ,Reprogramming ,muscular dystrophy ,Induced Pluripotent Stem Cells ,Kruppel-Like Transcription Factors ,Biology ,Article ,03 medical and health sciences ,Kruppel-Like Factor 4 ,Genetics ,medicine ,Animals ,Humans ,Progenitor cell ,Muscle, Skeletal ,PAX3 Transcription Factor ,MyoD Protein ,Pax3 ,reprogramming ,Cell Biology ,Fibroblasts ,medicine.disease ,Muscular Dystrophy, Duchenne ,Repressor Proteins ,Disease Models, Animal ,030104 developmental biology ,lcsh:Biology (General) ,030217 neurology & neurosurgery ,Developmental Biology ,Stem Cell Transplantation - Abstract
Summary The use of adult skeletal muscle stem cells (MuSCs) for cell therapy has been attempted for decades, but still encounters considerable difficulties. MuSCs derived from human induced pluripotent stem cells (hiPSCs) are promising candidates for stem cell therapy to treat Duchenne muscular dystrophy (DMD). Here we report that four transcription factors, HEYL, KLF4, MYOD, and PAX3, selected by comprehensive screening of different MuSC populations, enhance the derivation of PAX3-positive myogenic progenitors from fibroblasts and hiPSCs, using medium that promotes the formation of presomitic mesoderm. These induced PAX3-positive cells contribute efficiently to the repair of DMD-damaged myofibers and also reconstitute the MuSC population. These studies demonstrate how a combination of core transcription factors can fine-tune the derivation of MuSCs capable of contributing to the repair of adult skeletal muscle., Highlights • Persistent single MyoD can induce myogenic cells, not muscle stem cells • The combination of Heyl, Klf4, Pax3, and transient MyoD can induce muscle stem cells • Induced PAX3+ cells revealed incorporation into regenerating myofibers of DMD mice, In this article, Sato and colleagues show that four transcription factors, Heyl, Klf4, MyoD, and Pax3, selected by comprehensive screening of mouse muscle stem cells, enhance the derivation of PAX3-positive myogenic progenitors from fibroblasts and hiPSCs. These induced PAX3-positive cells contribute efficiently to the repair of DMD-damaged myofibers and also reconstitute stem cell population.
- Published
- 2019
28. Perspectives on the Use of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in Biomedical Research
- Author
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Christine L. Mummery
- Subjects
0301 basic medicine ,Biomedical Research ,Induced Pluripotent Stem Cells ,cardiomyocytes ,Disease ,Biology ,Bioinformatics ,medicine.disease_cause ,Biochemistry ,hiPSC ,Sudden cardiac death ,03 medical and health sciences ,Drug Discovery ,Genetics ,medicine ,Humans ,Myocytes, Cardiac ,Precision Medicine ,Induced pluripotent stem cell ,lcsh:QH301-705.5 ,cardiovascular cells ,Mutation ,lcsh:R5-920 ,disease models ,microtissues ,Human heart ,Heart ,Cell Biology ,medicine.disease ,cardiomyocyte maturation ,3. Good health ,030104 developmental biology ,lcsh:Biology (General) ,Heart failure ,Healthy individuals ,Perspective ,lcsh:Medicine (General) ,Developmental Biology - Abstract
Cardiovascular disease is still a major cause of ill-health and mortality, heart failure and arrhythmia being among the causes of sudden cardiac death. There are few drugs available for treatment or prevention and it remains difficult to predict who will develop these conditions, even when disease-causing mutations or associated gene variants are identified in individuals or families. This is in part because widely used rodent models may not fully capture the physiology of the human heart. The advent of pluripotent stem cell technology that allows cardiovascular cells to be derived from patients and healthy individuals, in some cases genetically matched through mutation repair, is leading to paradigm shifts in how cardiovascular diseases are studied in humans. However, these cells are often only partially mature imposing some limitations in use. This Perspective reviews aspects of recent advances but also remaining challenges., Christine Mummery provides a Perspective on applications of human iPSC-cardiomyocytes for disease modeling and drug testing, including recently developed advanced 3D-engineered cultures and multicellular formats enhancing the maturity of cardiomyocytes to rapidly expand their utility in drug discovery and cardiotoxicity screens beyond effects on ion channels.
- Published
- 2018
29. Generation of an induced pluripotent stem cell line, CSSi011-A (6534), from an Amyotrophic lateral sclerosis patient with heterozygous L145F mutation in SOD1 gene
- Author
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Marina Goldoni, Sandra D'Alfonso, Fabiola De Marchi, Letizia Mazzini, Elisa Perciballi, Daniela Ferrari, Angela D'Anzi, Alice Di Pierro, Maurizio Gelati, Angelo Luigi Vescovi, Filomena Altieri, Jessica Rosati, Laura Bernardini, D'Anzi, A, Altieri, F, Perciballi, E, Ferrari, D, Bernardini, L, Goldoni, M, Mazzini, L, De Marchi, F, Di Pierro, A, D'Alfonso, S, Gelati, M, Vescovi, A, and Rosati, J
- Subjects
0301 basic medicine ,Somatic cell ,SOD1 ,Biology ,medicine.disease_cause ,hiPSC ,familial ALS ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Missense mutation ,Amyotrophic lateral sclerosis ,Induced pluripotent stem cell ,Gene ,lcsh:QH301-705.5 ,Mutation ,nutritional and metabolic diseases ,Cell Biology ,General Medicine ,Motor neuron ,medicine.disease ,nervous system diseases ,030104 developmental biology ,medicine.anatomical_structure ,nervous system ,lcsh:Biology (General) ,CSSi011-A, ALS, human induced pluripotent stem cell ,Cancer research ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
Among the known causative genes of familial ALS, SOD1 mutation is one of the most common. It encodes for the ubiquitous detoxifying copper/zinc binding SOD1 enzyme, whose mutations selectively cause motor neuron death, although the mechanisms are not as yet clear. What is known is that mutant-mediated toxicity is not caused by loss of its detoxifying activity but by a gain-of-function. In order to better understand the pathogenic mechanisms of SOD1 mutation, a human induced pluripotent stem cell (hiPSC) line was generated from the somatic cells of a female patient carrying a missense variation in SOD1 (L145F).
- Published
- 2020
30. Biodegradable scaffolds facilitate epiretinal transplantation of hiPSC-Derived retinal neurons in nonhuman primates.
- Author
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Luo Z, Xian B, Li K, Li K, Yang R, Chen M, Xu C, Tang M, Rong H, Hu D, Ye M, Yang S, Lu S, Zhang H, and Ge J
- Subjects
- Animals, Axons, Optic Nerve, Retina, Retinal Ganglion Cells, Induced Pluripotent Stem Cells
- Abstract
Transplantation of stem cell-derived retinal neurons is a promising regenerative therapy for optic neuropathy. However, significant anatomic differences compromise its efficacy in large animal models. The present study describes the procedure and outcomes of human-induced pluripotent stem cell (hiPSC)-derived retinal sheet transplantation in primate models using biodegradable materials. Stem cell-derived retinal organoids were seeded on polylactic-coglycolic acid (PLGA) scaffolds and directed toward a retinal ganglion cell (RGC) fate. The seeded tissues showed active proliferation, typical neuronal morphology, and electrical excitability. The cellular scaffolds were then epiretinally transplanted onto the inner surface of rhesus monkey retinas. With sufficient graft-host contact provided by the scaffold, the transplanted tissues survived for up to 1 year without tumorigenesis. Histological examinations indicated survival, further maturation, and migration. Moreover, green fluorescent protein-labeled axonal projections toward the host optic nerve were observed. Cryopreserved organoids were also able to survive and migrate after transplantation. Our results suggest the potential efficacy of RGC replacement therapy in the repair of optic neuropathy for the restoration of visual function. STATEMENT OF SIGNIFICANCE: In the present study, we generated a human retinal sheet by seeding hiPSC-retinal organoid-derived RGCs on a biodegradable PLGA scaffold. We transplanted this retinal sheet onto the inner surface of the rhesus monkey retina. With scaffold support, donor cells survive, migrate and project their axons into the host optic nerve. Furthermore, an effective cryopreservation strategy for retinal organoids was developed, and the thawed organoids were also observed to survive and show cell migration after transplantation., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Ltd.)
- Published
- 2021
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31. Reliable generation of glial enriched progenitors from human fibroblast-derived iPSCs.
- Author
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Llorente IL, Hatanaka EA, Meadow ME, Xie Y, Lowry WE, and Carmichael ST
- Subjects
- Animals, Astrocytes, Cell Differentiation, Fibroblasts, Humans, Mice, Oligodendroglia, Induced Pluripotent Stem Cells
- Abstract
White matter stroke (WMS) occurs as small infarcts in deep penetrating blood vessels in the brain and affects the regions of the brain that carry connections, termed the subcortical white matter. WMS progresses over years and has devastating clinical consequences. Unlike large grey matter strokes, WMS disrupts the axonal architecture of the brain and depletes astrocytes, oligodendrocyte lineage cells, axons and myelinating cells, resulting in abnormalities of gait and executive function. An astrocytic cell-based therapy is positioned as a strong therapeutic candidate after WMS. In this study we report, the reliable generation of a novel stem cell-based therapeutic product, glial enriched progenitors (GEPs) derived from human induced pluripotent stem cells (hiPSCs). By transient treatment of hiPSC derived neural progenitors (hiPSC-NPCs) with the small molecule deferoxamine, a prolyl hydroxylase inhibitor, for three days hiPSC-NPCs become permanently biased towards an astrocytic fate, producing hiPSC-GEPs. In preparation for clinical application, we have developed qualification assays to ensure identity, safety, purity, and viability of the cells prior to manufacture. Using tailored q-RT-PCR-based assays, we have demonstrated the lack of pluripotency in our final therapeutic candidate cells (hiPSC-GEPs) and we have identified the unique genetic profile of hiPSC-GEPs that is clearly distinct from the parent lines, hiPSCs and iPSC-NPCs. After completion of the viability assay, we have stablished the therapeutic window of use for hiPSC-GEPs in future clinical applications (7 h). Lastly, we were able to reliably and consistently produce a safe therapeutic final product negative for contamination by any human or murine viral pathogens, selected bacteria, common laboratory mycoplasmas, growth of any aerobes, anaerobes, yeast, or fungi and 100 times less endotoxin levels than the maximum acceptable value. This study demonstrates the reliable and safe generation of patient derived hiPSC-GEPs that are clinically ready as a cell-based therapeutic approach for WMS., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)
- Published
- 2021
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32. 2D graphene oxide particles induce unwanted loss in pluripotency and trigger early differentiation in human pluripotent stem cells.
- Author
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Heo J, Choi J, Kim JY, Jeong H, Choi D, Han U, Park JH, Park HH, and Hong J
- Subjects
- Cell Differentiation, Humans, Graphite toxicity, Induced Pluripotent Stem Cells, Pluripotent Stem Cells
- Abstract
The potential health hazards of particulates, such as micro/nano-sized plastics and carbon materials have recently received extensive attention. However, their toxicological properties in association with stem cell differentiation is still relatively unexplored. In this study, we elucidated the cytotoxic effects of 2D graphene oxide (GO), in relation to differentiation of human induced pluripotent stem cells (hiPSCs). Supplementation of GO to hiPSCs demonstrated uptake of GO through the plasma membrane and intracellular accumulation was observed. Increasing the concentration of GO led to reduced viability and increased likelihood of hiPSC colony detachment. Moreover, treatment of GO resulted in significant loss in pluripotency markers, OCT-4 and NANOG. In particular, when hiPSCs were cultured with GO in cardiomyocyte induction medium, upregulation of cardiomyocyte marker, NKX2.5, along with observation of early triggering of differentiation were observed. Taken together, our results highlight the risk in the uptake and accumulation of GO on the stem cell development by unwanted loss in pluripotency and accelerated initiation of differentiation., (Copyright © 2021 Elsevier B.V. All rights reserved.)
- Published
- 2021
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33. Loss of sarcomeric proteins via upregulation of JAK/STAT signaling underlies interferon-γ-induced contractile deficit in engineered human myocardium.
- Author
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Zhan RZ, Rao L, Chen Z, Strash N, and Bursac N
- Subjects
- Humans, Interferon-gamma pharmacology, Myocardium, SARS-CoV-2, Signal Transduction, Up-Regulation, COVID-19, Influenza A Virus, H1N1 Subtype
- Abstract
The level of circulating interferon-γ (IFNγ) is elevated in various clinical conditions including autoimmune and inflammatory diseases, sepsis, acute coronary syndrome, and viral infections. As these conditions are associated with high risk of myocardial dysfunction, we investigated the effects of IFNγ on 3D fibrin-based engineered human cardiac tissues ("cardiobundles"). Cardiobundles were fabricated from human pluripotent stem cell-derived cardiomyocytes, exposed to 0-20 ng/ml of IFNγ on culture days 7-14, and assessed for changes in tissue structure, viability, contractile force and calcium transient generation, action potential propagation, cytokine secretion, and expression of select genes and proteins. We found that application of IFNγ induced a dose-dependent reduction in contractile force generation, deterioration of sarcomeric organization, and cardiomyocyte disarray, without significantly altering cell viability, action potential propagation, or calcium transient amplitude. At molecular level, the IFNγ-induced structural and functional deficits could be attributed to altered balance of pro- and anti-inflammatory cytokines, upregulation of JAK/STAT signaling pathway (JAK1, JAK2, and STAT1), and reduced expression of myosin heavy chain, myosin light chain-2v, and sarcomeric α-actinin. Application of clinically used JAK/STAT inhibitors, tofacitinib and baricitinib, fully prevented IFNγ-induced cardiomyopathy, confirming the critical roles of this signaling pathway in inflammatory cardiac disease. Taken together, our in vitro studies in engineered myocardial tissues reveal direct adverse effects of pro-inflammatory cytokine IFNγ on human cardiomyocytes and establish the foundation for a potential use of cardiobundle platform in modeling of inflammatory myocardial disease and therapy. STATEMENT OF SIGNIFICANCE: Various inflammatory and autoimmune diseases including rheumatoid arthritis, sepsis, lupus erythematosus, Chagas disease, and others, as well as viral infections including H1N1 influenza and COVID-19 show increased systemic levels of a pro-inflammatory cytokine interferon-γ (IFNγ) and are associated with high risk of heart disease. Here we explored for the first time if chronically elevated levels of IFNγ can negatively affect structure and function of engineered human heart tissues in vitro. Our studies revealed IFNγ-induced deterioration of myofibrillar organization and contractile force production in human cardiomyocytes, attributed to decreased expression of multiple sarcomeric proteins and upregulation of JAK/STAT signaling pathway. FDA-approved JAK inhibitors fully blocked the adverse effects of IFNγ, suggesting a potentially effective strategy against human inflammatory cardiomyopathy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)
- Published
- 2021
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34. Characterization of iCell cardiomyocytes using single-cell RNA-sequencing methods.
- Author
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Schmid C, Wohnhaas CT, Hildebrandt T, Baum P, and Rast G
- Subjects
- Biomarkers analysis, Cell Cycle genetics, Cell Differentiation genetics, Cell Line, Cell Separation, Drug Evaluation, Preclinical methods, Fibroblasts physiology, Humans, Induced Pluripotent Stem Cells physiology, Transcriptome physiology, Myocytes, Cardiac physiology, RNA-Seq methods, Single-Cell Analysis methods
- Abstract
Introduction: Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes are being evaluated for their use in pharmacological and toxicological testing, particularly for electrophysiological side effects. However, little is known about the composition of the commercially available iCell cardiomyocyte (Fuijifilm Cellular Dynamics) cultures and the transcriptomic phenotype of individual cells., Methods: We characterized iCell cardiomyocytes (assumed to be a mixture of nodal-, atrial-, and ventricular-like cardiomyocytes together with potential residual non-myocytes) using bulk RNA-sequencing, followed by investigation of cellular heterogeneity using two different single-cell RNA-sequencing platforms., Results: Bulk RNA-sequencing identified key cardiac markers (TNNT2, MYL7) as well as fibroblast associated genes (P4HB, VIM), and cardiac ion channels in the iCell cardiomyocyte culture. High-resolution single cell RNA-sequencing demonstrated that both, cardiac and fibroblast-related genes were co-expressed throughout the cell population. This approach resolved two cell clusters within iCell cardiomyocytes. Interestingly, these clusters could not be associated with known cardiac subtypes. However, transcripts of ion channels potentially useful as functional markers for cardiac subtypes were below the detection limits of the single-cell approaches used. Instead, one cluster (10.8% of the cells) is defined by co-expression of cardiac and cell cycle-related genes (e.g. TOP2A). Incorporation of bromodeoxyuridine further confirmed the capability of iCell cardiomyocytes to enter cell cycle., Discussion: The co-expression of cardiac related genes with cell cycle or fibroblast related genes may be interpreted either as aberrant or as an immature feature. However, this excludes the presence of a non-cardiomyocyte sub-population and indicates that some cardiomyocytes themselves enter cell cycle., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2020
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35. Formulation Stability of Amphiphilic Poly(γ-Glutamic Acid) Nanoparticle and Evaluation of Cardiotoxicity of NPs With Human iPSC-Derived 3D-Cardiomyocyte Tissues.
- Author
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Ikeda M, Akagi T, Nagao M, and Akashi M
- Subjects
- Cardiotoxicity, Glutamic Acid, Humans, Myocytes, Cardiac, Polyglutamic Acid analogs & derivatives, Polyglutamic Acid toxicity, Prospective Studies, Induced Pluripotent Stem Cells, Nanoparticles toxicity
- Abstract
We conducted a stability study of biodegradable and amphiphilic nanoparticles (NPs) consisting of phenylalanine-attached poly(γ-glutamic acid) for drug delivery to find the optimal formulation and define the optimal storage conditions using novel quantitative analytical methods. The stability of NP suspension and lyophilized NP powder manufactured by a dimethyl sulfoxide-based and an ethanol-based process was assessed under 5°C, 25°C/60% relative humidity and 40°C/75% relative humidity. The content of phenylalanine-attached poly(γ-glutamic acid), impurities, absolute molecular weight, appearance, clarity of solution, particle size, zeta potential, particle matter, osmolality, water content, and pH were evaluated as parameters of NP stability. Lyophilized NPs with trehalose showed better stability. The lyophilized NP formulation could therefore provide a stable and high-quality product for clinical studies and shows promise as an effective drug delivery system carrier. The cardiotoxicity of prospective impurities contained in NPs and reagents used in the manufacturing process with human-induced pluripotent stem cell-derived 3-dimensional cardiomyocyte tissues by centrifugation layer-by-layer technique was also evaluated. As a result, cardiotoxicity for NPs and reagents was not observed, and it was clarified that the potential risk to human safety from NPs is low. The applicability of the cardiotoxicity evaluation approaches with human-induced pluripotent stem cell-derived 3-dimensional cardiomyocyte tissues will be evaluated by centrifugation layer-by-layer technique., (Copyright © 2020. Published by Elsevier Inc.)
- Published
- 2020
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36. Polyhydroxyphenylvalerate/polycaprolactone nanofibers improve the life-span and mechanoresponse of human IPSC-derived cortical neuronal cells.
- Author
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Cerrone F, Pozner T, Siddiqui A, Ceppi P, Winner B, Rajendiran M, Babu R, Ibrahim HS, Rodriguez BJ, Winkler J, Murphy KJ, and O'Connor KE
- Subjects
- Caspase 3 metabolism, Cell Differentiation drug effects, Cell Line, Cell Movement drug effects, Cell Survival drug effects, Humans, Neurites drug effects, Neurites metabolism, Regression Analysis, Stress, Mechanical, Temperature, Cerebral Cortex cytology, Mechanotransduction, Cellular drug effects, Nanofibers chemistry, Neurons cytology, Polyesters pharmacology
- Abstract
The physico-chemical characteristics of the extracellular matrix (ECM) cause mechanical cues that could elicit responses in the survival rate of cortical neuronal cells. Efficient neurite outgrowth in vitro, is critical for successful cultivation of cortical neuronal cells and the potential for attempts at regeneration of the central nervous system (CNS) in vivo. Relatively soft and hydrophilic, microbially synthesized aromatic polyester, polyhydroxyphenylvalerate (PHPV) was blended 50:50 with the stiff and hydrophobic polycaprolactone (PCL) and electrospun in microfibers for use in a 3D (CellCrown™) configuration and in a 2D coverslip coated configuration. This blend allows a 2.3-fold increase in the life-span of human induced pluripotent stem derived cortical neuronal cells (hiPS) compared to pure PCL fibers. HiPS-derived cortical neuronal cells grown on PHPV/PCL fibers show a 3.8-fold higher cumulative neurite elaboration compared to neurites grown on PCL fibers only. 96% of cortical neuronal cells die after 8 days of growth when plated on PCL fibers alone while >83% and 55% are alive on PHPV/PCL fibers on day 8 and day 17, respectively. An increased migration rate of cortical neuronal cells is also promoted by the blend compared to the PCL fibers alone. The critical survival rate improvement of hiPS derived cortical neuronal cells on PHPV/PCL blend holds promise in using these biocompatible nanofibers as implantable materials for regenerative purposes of an active cortical neuronal population after full maturation in vitro., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)
- Published
- 2020
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37. A comparative study of key physiological stem cell parameters between three human trophoblast cell lines.
- Author
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Li Z, Kurosawa O, and Iwata H
- Subjects
- Cell Differentiation drug effects, Cell Differentiation genetics, Cell Differentiation physiology, Cell Line, Tumor, Choriocarcinoma genetics, Choriocarcinoma metabolism, Female, Fluorescent Antibody Technique, Gene Expression Profiling, Humans, Induced Pluripotent Stem Cells metabolism, Induced Pluripotent Stem Cells physiology, Oligonucleotide Array Sequence Analysis, Placenta metabolism, Placenta physiology, Pregnancy, Stem Cells metabolism, Stem Cells physiology, Trophoblasts physiology, Chorionic Gonadotropin metabolism, Induced Pluripotent Stem Cells cytology, Stem Cells cytology, Trophoblasts cytology, Trophoblasts metabolism
- Abstract
Human trophoblast stem cells (TSCs) play a key role in the placenta. These cells are proliferative, undifferentiated, and can differentiate into mature trophoblast cell types. However, primary human TSCs are difficult to obtain. In our previous study, we established TSCs from human induced pluripotent stem cells (TS
hiPSC ). Here, we aimed to characterize the identity of these TShiPSC cells by comparing them with BeWo choriocarcinoma cells and primary TSCs (CT cells). Compared with BeWo cells, CT and TShiPSC cells showed high secretion of human chorionic gonadotrophin (hCG) and syncytiotrophoblast differentiation ability. Global gene microarray analysis results showed that CT and TShiPSC cells, unlike BeWo cells, could be classified in the same group. Compared with BeWo cells, CT and TShiPSC cells showed high expression levels of TSC-specific genes and low expression of cancer adhesion and invasion genes. Analysis of placental barrier integrity showed that TShiPSC cells could form a good barrier. Prospective studies using TShiPSC cells hold great promise for elucidating the pathogenesis of infertility due to trophoblast defects., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)- Published
- 2020
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38. Generation of hepatobiliary organoids from human induced pluripotent stem cells.
- Author
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Wu F, Wu D, Ren Y, Huang Y, Feng B, Zhao N, Zhang T, Chen X, Chen S, and Xu A
- Subjects
- Cell Differentiation, Cells, Cultured, Humans, Organogenesis, Bile Ducts embryology, Induced Pluripotent Stem Cells cytology, Liver embryology, Organoids cytology
- Abstract
Background & Aims: Human induced pluripotent stem cell (hiPSC)-derived liver modeling systems have the potential to overcome the shortage of donors for clinical application and become a model for drug development. Although several strategies are available to generate hepatic micro-tissues, few have succeeded in generating a liver organoid with hepatobiliary structure from hiPSCs., Methods: At differentiation stages I and II (day 1-15), 25% of mTeSR™ culture medium was added to hepatic differentiation medium to induce endodermal and mesodermal commitment and thereafter hepatic and biliary co-differentiation. At stage III (day 15-45), 10% cholesterol
+ MIX was added to the maturation medium to promote the formation and maturation of the hepatobiliary organoids. Phenotypes and functions of organoids were determined by specific markers and multiple functional assays both in vitro and in vivo., Results: In this system, hiPSCs were induced to form 3D hepatobiliary organoids and to some extent recapitulated key aspects of early hepatogenesis in a parallel fashion. The organoids displayed a series of functional attributes. Specifically, the induced hepatocyte-like cells could take up indocyanine green, accumulate lipid and glycogen, and displayed appropriate secretion ability (albumin and urea) and drug metabolic ability (CYP3A4 activity and inducibility); the biliary structures in the system showed gamma glutamyltransferase activity and the ability to efflux rhodamine and store bile acids. Furthermore, after transplantation into the immune-deficient mice, the organoids survived for more than 8 weeks., Conclusion: This is the first time that functional hepatobiliary organoids have been generated from hiPSCs. The organoid model will be useful for in vitro studies of the molecular mechanisms of liver development and has important potential in the therapy of liver diseases., Lay Summary: Herein, we established a system to generate human induced pluripotent stem cell-derived functional hepatobiliary organoids in vitro, without any exogenous cells or genetic manipulation. To some extent this model was able to recapitulate several key aspects of hepatobiliary organogenesis in a parallel fashion, holding great promise for drug development and liver transplantation., (Copyright © 2019 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)- Published
- 2019
- Full Text
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39. Development of trophoblast cystic structures from human induced pluripotent stem cells in limited-area cell culture.
- Author
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Li Z, Kurosawa O, and Iwata H
- Subjects
- Cell Culture Techniques methods, Cell Differentiation genetics, Cells, Cultured, Female, Gene Expression, Humans, Induced Pluripotent Stem Cells cytology, Organoids cytology, Pregnancy, Trophoblasts cytology, Chorionic Gonadotropin metabolism, Induced Pluripotent Stem Cells metabolism, Organoids metabolism, Trophoblasts metabolism
- Abstract
We developed a novel engineering technique to induce differentiation of human induced pluripotent stem cells (hiPSCs) into organoids mimicking the trophectoderm (TE). Here, hiPSCs were cultured on a limited area of 2-4 mm in diameter. After 15-20 days, spherical cysts appeared on the surface of the limited area. Secretion of human chorionic gonadotrophin (hCG) began to increase after ∼ 20 days and remained dramatically elevated over the next 20 days. Limited-area-cultured cysts exhibited expression of hCG, which was a result of epithelial differentiation. Low expression levels of pluripotent genes and high expression levels of trophoblast lineage-specific genes were detected in the cells of spherical cysts. Multinucleated syncytia trophoblast was observed in the reseeded cystic cells. We observed hiPSC-derived cysts that morphologically resembled trophectoderm in vivo. The limited-area cell culture induced a three-dimensional (3D) trophectoderm organoid, which has potential for use in the study of human trophoblast differentiation and placental morphogenesis., (Copyright © 2018 Elsevier Inc. All rights reserved.)
- Published
- 2018
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40. Cross - site comparison of excitation-contraction coupling using impedance and field potential recordings in hiPSC cardiomyocytes.
- Author
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Bot CT, Juhasz K, Haeusermann F, Polonchuk L, Traebert M, and Stoelzle-Feix S
- Subjects
- Action Potentials drug effects, Action Potentials physiology, Cell Line, Cells, Cultured, Disopyramide pharmacology, Drug Evaluation, Preclinical methods, Drug Evaluation, Preclinical standards, Excitation Contraction Coupling physiology, Humans, Induced Pluripotent Stem Cells physiology, Myocytes, Cardiac physiology, Phenethylamines pharmacology, Potassium Channel Blockers pharmacology, Sulfonamides pharmacology, Anti-Arrhythmia Agents pharmacology, Electric Impedance, Excitation Contraction Coupling drug effects, Induced Pluripotent Stem Cells drug effects, Myocytes, Cardiac drug effects
- Abstract
Introduction: Since 2005 the S7B and E14 guidances from ICH and FDA have been in place to assess a potential drug candidate's ability to cause long QT syndrome. To refine these guidelines, the FDA proposed the Comprehensive in vitro Proarrhythmia Assay (CiPA) initiative, where the assessment of drug effects on cardiac repolarization was one subject of investigation. Within the myocyte validation study, effects of pharmaceutical compounds on human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were assessed and this article will focus on the evaluation of the proarrhythmic potential of 23 blinded drugs in four hiPSC-CM cell lines., Methods: Experiments were performed on the CardioExcyte 96 at different sites. A combined readout of contractility (via impedance) and electrophysiology endpoints (field potentials) was performed., Results: Our data demonstrates that hERG blockers such as dofetilide and further high risk categorized compounds prolong the field potential duration. Arrhythmia were detected in both impedance as well as field potential recordings. Intermediate risk compounds induced arrhythmia in almost all cases at the highest dose. In the case of low risk compounds, either a decrease in FPD
max was observed, or not a significant change from pre-addition control values., Discussion: With exceptions, hiPSC-CMs are sensitive and exhibit at least 10% delayed or shortened repolarization from pre-addition values and arrhythmia after drug application and thus can provide predictive cardiac electrophysiology data. The baseline electrophysiological parameters vary between iPS cells from different sources, therefore positive and negative control recordings are recommended., (Copyright © 2018 Elsevier Inc. All rights reserved.)- Published
- 2018
- Full Text
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41. NEK1 loss-of-function mutation induces DNA damage accumulation in ALS patient-derived motoneurons.
- Author
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Higelin J, Catanese A, Semelink-Sedlacek LL, Oeztuerk S, Lutz AK, Bausinger J, Barbi G, Speit G, Andersen PM, Ludolph AC, Demestre M, and Boeckers TM
- Subjects
- Amyotrophic Lateral Sclerosis pathology, Humans, Mutation, Transfection, Amyotrophic Lateral Sclerosis genetics, DNA Damage genetics, Motor Neurons metabolism, NIMA-Related Kinase 1 genetics
- Abstract
Mutations in genes coding for proteins involved in DNA damage response (DDR) and repair, such as C9orf72 and FUS (Fused in Sarcoma), are associated with neurodegenerative diseases and lead to amyotrophic lateral sclerosis (ALS). Heterozygous loss-of-function mutations in NEK1 (NIMA-related kinase 1) have also been recently found to cause ALS. NEK1 codes for a multifunctional protein, crucially involved in mitotic checkpoint control and DDR. To resolve pathological alterations associated with NEK1 mutation, we compared hiPSC-derived motoneurons carrying a NEK1 mutation with mutant C9orf72 and wild type neurons at basal level and after DNA damage induction. Motoneurons carrying a C9orf72 mutation exhibited cell specific signs of increased DNA damage. This phenotype was even more severe in NEK1
c.2434A>T neurons that showed significantly increased DNA damage at basal level and impaired DDR after induction of DNA damage in an maturation-dependent manner. Our results provide first mechanistic insight in pathophysiological alterations induced by NEK1 mutations and point to a converging pathomechanism of different gene mutations causative for ALS. Therefore, our study contributes to the development of novel therapeutic strategies to reduce DNA damage accumulation in neurodegenerative diseases and ALS., (Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.)- Published
- 2018
- Full Text
- View/download PDF
42. Comparison of 2D and 3D neural induction methods for the generation of neural progenitor cells from human induced pluripotent stem cells.
- Author
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Chandrasekaran A, Avci HX, Ochalek A, Rösingh LN, Molnár K, László L, Bellák T, Téglási A, Pesti K, Mike A, Phanthong P, Bíró O, Hall V, Kitiyanant N, Krause KH, Kobolák J, and Dinnyés A
- Subjects
- Cell Differentiation, Cell Line, Humans, Induced Pluripotent Stem Cells metabolism, Nestin genetics, Nestin metabolism, Neural Stem Cells metabolism, PAX6 Transcription Factor genetics, PAX6 Transcription Factor metabolism, SOXB1 Transcription Factors genetics, SOXB1 Transcription Factors metabolism, Cell Culture Techniques methods, Induced Pluripotent Stem Cells cytology, Neural Stem Cells cytology
- Abstract
Neural progenitor cells (NPCs) from human induced pluripotent stem cells (hiPSCs) are frequently induced using 3D culture methodologies however, it is unknown whether spheroid-based (3D) neural induction is actually superior to monolayer (2D) neural induction. Our aim was to compare the efficiency of 2D induction with 3D induction method in their ability to generate NPCs, and subsequently neurons and astrocytes. Neural differentiation was analysed at the protein level qualitatively by immunocytochemistry and quantitatively by flow cytometry for NPC (SOX1, PAX6, NESTIN), neuronal (MAP2, TUBB3), cortical layer (TBR1, CUX1) and glial markers (SOX9, GFAP, AQP4). Electron microscopy demonstrated that both methods resulted in morphologically similar neural rosettes. However, quantification of NPCs derived from 3D neural induction exhibited an increase in the number of PAX6/NESTIN double positive cells and the derived neurons exhibited longer neurites. In contrast, 2D neural induction resulted in more SOX1 positive cells. While 2D monolayer induction resulted in slightly less mature neurons, at an early stage of differentiation, the patch clamp analysis failed to reveal any significant differences between the electrophysiological properties between the two induction methods. In conclusion, 3D neural induction increases the yield of PAX6
+ /NESTIN+ cells and gives rise to neurons with longer neurites, which might be an advantage for the production of forebrain cortical neurons, highlighting the potential of 3D neural induction, independent of iPSCs' genetic background., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)- Published
- 2017
- Full Text
- View/download PDF
43. Action potential-based MEA platform for in vitro screening of drug-induced cardiotoxicity using human iPSCs and rat neonatal myocytes.
- Author
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Jans D, Callewaert G, Krylychkina O, Hoffman L, Gullo F, Prodanov D, and Braeken D
- Subjects
- Action Potentials drug effects, Animals, Animals, Newborn, Dose-Response Relationship, Drug, Drug Evaluation, Preclinical methods, Humans, Induced Pluripotent Stem Cells drug effects, Microelectrodes, Myocytes, Cardiac drug effects, Quinidine pharmacology, Rats, Rats, Wistar, Action Potentials physiology, Anti-Arrhythmia Agents pharmacology, Cardiotoxins pharmacology, Induced Pluripotent Stem Cells physiology, Myocytes, Cardiac physiology, Semiconductors
- Abstract
Drug-induced cardiotoxicity poses a negative impact on public health and drug development. Cardiac safety pharmacology issues urged for the preclinical assessment of drug-induced ventricular arrhythmia leading to the design of several in vitro electrophysiological screening assays. In general, patch clamp systems allow for intracellular recordings, while multi-electrode array (MEA) technology detect extracellular activity. Here, we demonstrate a complementary metal oxide semiconductor (CMOS)-based MEA system as a reliable platform for non-invasive, long-term intracellular recording of cardiac action potentials at high resolution. Quinidine (8 concentrations from 10
-7 to 2.10-5 M) and verapamil (7 concentrations from 10-11 to 10-5 M) were tested for dose-dependent responses in a network of cardiomyocytes. Electrophysiological parameters, such as the action potential duration (APD), rates of depolarization and repolarization and beating frequency were assessed. In hiPSC, quinidine prolonged APD with EC50 of 2.2·10-6 M. Further analysis indicated a multifactorial action potential prolongation by quinidine: (1) decreasing fast repolarization with IC50 of 1.1·10-6 M; (2) reducing maximum upstroke velocity with IC50 of 2.6·10-6 M; and (3) suppressing spontaneous activity with EC50 of 3.8·10-6 M. In rat neonatal cardiomyocytes, verapamil blocked spontaneous activity with EC50 of 5.3·10-8 M and prolonged the APD with EC50 of 2.5·10-8 M. Verapamil reduced rates of fast depolarization and repolarization with IC50 s of 1.8 and 2.2·10-7 M, respectively. In conclusion, the proposed action potential-based MEA platform offers high quality and stable long-term recordings with high information content allowing to characterize multi-ion channel blocking drugs. We anticipate application of the system as a screening platform to efficiently and cost-effectively test drugs for cardiac safety., (Copyright © 2017 Elsevier Inc. All rights reserved.)- Published
- 2017
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44. CXCR4 and CXCR7 play distinct roles in cardiac lineage specification and pharmacologic β-adrenergic response.
- Author
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Ceholski DK, Turnbull IC, Pothula V, Lecce L, Jarrah AA, Kho C, Lee A, Hadri L, Costa KD, Hajjar RJ, and Tarzami ST
- Subjects
- Biomarkers metabolism, Calcium metabolism, Cardiomegaly pathology, Gene Expression Regulation, Gene Knockdown Techniques, HEK293 Cells, Humans, Induced Pluripotent Stem Cells metabolism, Lentivirus metabolism, Myocytes, Cardiac metabolism, Organogenesis, RNA, Small Interfering metabolism, Cell Lineage, Myocardium cytology, Myocardium metabolism, Receptors, Adrenergic, beta metabolism, Receptors, CXCR metabolism, Receptors, CXCR4 metabolism
- Abstract
CXCR4 and CXCR7 are prominent G protein-coupled receptors (GPCRs) for chemokine stromal cell-derived factor-1 (SDF-1/CXCL12). This study demonstrates that CXCR4 and CXCR7 induce differential effects during cardiac lineage differentiation and β-adrenergic response in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Using lentiviral vectors to ablate CXCR4 and/or CXCR7 expression, hiPSC-CMs were tested for phenotypic and functional properties due to gene knockdown. Gene expression and flow cytometry confirmed the pluripotent and cardiomyocyte phenotype of undifferentiated and differentiated hiPSCs, respectively. Although reduction of CXCR4 and CXCR7 expression resulted in a delayed cardiac phenotype, only knockdown of CXCR4 delayed the spontaneous beating of hiPSC-CMs. Knockdown of CXCR4 and CXCR7 differentially altered calcium transients and β-adrenergic response in hiPSC-CMs. In engineered cardiac tissues, depletion of CXCR4 or CXCR7 had opposing effects on developed force and chronotropic response to β-agonists. This work demonstrates distinct roles for the SDF-1/CXCR4 or CXCR7 network in hiPSC-derived ventricular cardiomyocyte specification, maturation and function., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)
- Published
- 2017
- Full Text
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45. Differentiation of functional endothelial cells from human induced pluripotent stem cells: A novel, highly efficient and cost effective method.
- Author
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Liu X, Qi J, Xu X, Zeisberg M, Guan K, and Zeisberg EM
- Subjects
- Antigens, CD metabolism, Cadherins metabolism, Cell Lineage genetics, Cell Proliferation, Humans, Platelet Endothelial Cell Adhesion Molecule-1 metabolism, Pluripotent Stem Cells, Cell Culture Techniques methods, Cell Differentiation genetics, Endothelial Cells cytology, Induced Pluripotent Stem Cells cytology
- Abstract
Endothelial cells derived from human induced pluripotent stem cells (hiPSC- EC) are of significant value for research on human vascular development, in vitro disease models and drug screening. Here we report an alternative, highly efficient and cost-effective simple three step method (mesoderm induction, endothelial cell differentiation and endothelial cell expansion) to differentiate hiPSC directly into endothelial cells. We demonstrate that efficiency of described method to derive CD31+ and VE-Cadherin+ double positive cells is higher than 80% in 12 days. Most notably we established that hiPSC-EC differentiation efficacy depends on optimization of both mesoderm differentiation and endothelial cell differentiation steps., (Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.)
- Published
- 2016
- Full Text
- View/download PDF
46. Neurotrophic activity of jiadifenolide on neuronal precursor cells derived from human induced pluripotent stem cells.
- Author
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Shoji M, Nishioka M, Minato H, Harada K, Kubo M, Fukuyama Y, and Kuzuhara T
- Subjects
- Cell Differentiation, Cell Enlargement drug effects, Cell Proliferation drug effects, Cell Proliferation physiology, Cells, Cultured, Dose-Response Relationship, Drug, Humans, Induced Pluripotent Stem Cells drug effects, Induced Pluripotent Stem Cells physiology, Neural Stem Cells drug effects, Neurogenesis drug effects, Induced Pluripotent Stem Cells cytology, Nerve Growth Factors administration & dosage, Neural Stem Cells cytology, Neural Stem Cells physiology, Neurogenesis physiology, Sesquiterpenes administration & dosage
- Abstract
Although jiadifenolide has been reported to neurotrophin-like activity in primary cultured rat cortical neurons, it is unknown on that of activity in human neurons. Thus, we aimed to assess neurotrophin-like activity by jiadifenolide in human neuronal cells. We analyzed neuronal precursor cells derived from human induced pluripotent stem cells for microtuble-associated-protein-2 expression by immunofluorescence and western blot, following jiadifenolide treatment. Jiadifenolide promoted dendrite outgrowth, facilitated growth, and prevented death in neuronal cells derived from human induced pluripotent stem cells. Interestingly, jiadifenolide also increased postsynaptic density-95 protein expression suggesting that jiadifenolide promotes neuronal maturation and post-synaptic formation. We demonstrate for the first time that jiadifenolide exhibits neurotrophic effects on human neuronal precursor cells., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2016
- Full Text
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47. Myosin light chain 2-based selection of human iPSC-derived early ventricular cardiac myocytes.
- Author
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Bizy A, Guerrero-Serna G, Hu B, Ponce-Balbuena D, Willis BC, Zarzoso M, Ramirez RJ, Sener MF, Mundada LV, Klos M, Devaney EJ, Vikstrom KL, Herron TJ, and Jalife J
- Subjects
- Adenoviridae genetics, Cardiac Myosins genetics, Cell Differentiation, Cell Lineage, Flow Cytometry, Genes, Reporter, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Humans, Induced Pluripotent Stem Cells metabolism, Myocytes, Cardiac metabolism, Myosin Light Chains genetics, Phenotype, Promoter Regions, Genetic, Protein Isoforms genetics, Protein Isoforms metabolism, Cardiac Myosins metabolism, Cell Separation methods, Heart Ventricles cytology, Induced Pluripotent Stem Cells cytology, Myocytes, Cardiac cytology, Myosin Light Chains metabolism
- Abstract
Applications of human induced pluripotent stem cell derived-cardiac myocytes (hiPSC-CMs) would be strengthened by the ability to generate specific cardiac myocyte (CM) lineages. However, purification of lineage-specific hiPSC-CMs is limited by the lack of cell marking techniques. Here, we have developed an iPSC-CM marking system using recombinant adenoviral reporter constructs with atrial- or ventricular-specific myosin light chain-2 (MLC-2) promoters. MLC-2a and MLC-2v selected hiPSC-CMs were purified by fluorescence-activated cell sorting and their biochemical and electrophysiological phenotypes analyzed. We demonstrate that the phenotype of both populations remained stable in culture and they expressed the expected sarcomeric proteins, gap junction proteins and chamber-specific transcription factors. Compared to MLC-2a cells, MLC-2v selected CMs had larger action potential amplitudes and durations. In addition, by immunofluorescence, we showed that MLC-2 isoform expression can be used to enrich hiPSC-CM consistent with early atrial and ventricular myocyte lineages. However, only the ventricular myosin light chain-2 promoter was able to purify a highly homogeneous population of iPSC-CMs. Using this approach, it is now possible to develop ventricular-specific disease models using iPSC-CMs while atrial-specific iPSC-CM cultures may require additional chamber-specific markers., (© 2013.)
- Published
- 2013
- Full Text
- View/download PDF
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