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From hIPSCs to Adult Cells in a Dish: Promises and Pitfalls

A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Biochemistry".

Deadline for manuscript submissions: closed (30 November 2019) | Viewed by 94751

Special Issue Editors

Dr. Alessandra Rossini

E-Mail Website
Guest Editor
Center for Biomedicine, European Academy of Bolzano/Bozen, Via Galvani 31, I-39100 Bolzano, Italy
Dr. Andrea Barbuti

E-Mail Website
Guest Editor
Department of Biosciences, Università degli Studi di Milano, Milan, Italy
Interests: sinoatrial cells; human-induced pluripotent stem cell (iPSC)
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Human induced-pluripotent stem cells (hIPSCs) represent a source to produce a virtually unlimited number of cells from patients and healthy controls, which are otherwise difficult to obtain, such as, but not limited to, cardiomyocytes and neurons.

Cells derived from hIPSCs can be subsequently applied in preclinical and, possibly in the future, clinical studies such as target validation, drug screening, and tissue regeneration. However, IPSC-derived somatic cells often present significant differences from their adult counterparts in terms of maturity and gene expression.

This Special Issue of IJMS will cover, but will not be not limited to, the following topics:

  • The success and limitations of hIPSC for disease modeling (including cardiac, neurological, liver, skin, blood diseases, and more)
  • How to promote and evaluate the maturation of hiPSC-derived somatic cells
  • hIPSCs to derive tissue and organ models
  • The direct reprogramming of human somatic cells to bypass the generation of iPSC

You are warmly invited to submit original research and review articles related to any of these aspects.

Dr. Alessandra Rossini
Dr. Andrea Barbuti
Guest Editors

Manuscript Submission Information

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

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

Please visit the Instructions for Authors page before submitting a manuscript. There is an Article Processing Charge (APC) for publication in this open access journal. For details about the APC please see here. Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Published Papers (19 papers)

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Research

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15 pages, 2015 KiB  
Article
Functional Comparison of Blood-Derived Human Neural Progenitor Cells
by Eszter Szabó, Flóra Juhász, Edit Hathy, Dóra Reé, László Homolya, Zsuzsa Erdei, János M. Réthelyi and Ágota Apáti
Int. J. Mol. Sci. 2020, 21(23), 9118; https://doi.org/10.3390/ijms21239118 - 30 Nov 2020
Cited by 1 | Viewed by 2522
Abstract
Induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) are promising tools to model complex neurological or psychiatric diseases, including schizophrenia. Multiple studies have compared patient-derived and healthy control NPCs derived from iPSCs in order to investigate cellular phenotypes of this disease, although [...] Read more.
Induced pluripotent stem cell (iPSC)-derived neural progenitor cells (NPCs) are promising tools to model complex neurological or psychiatric diseases, including schizophrenia. Multiple studies have compared patient-derived and healthy control NPCs derived from iPSCs in order to investigate cellular phenotypes of this disease, although the establishment, stabilization, and directed differentiation of iPSC lines are rather expensive and time-demanding. However, interrupted reprogramming by omitting the stabilization of iPSCs may allow for the generation of a plastic stage of the cells and thus provide a shortcut to derive NPSCs directly from tissue samples. Here, we demonstrate a method to generate shortcut NPCs (sNPCs) from blood mononuclear cells and present a detailed comparison of these sNPCs with NPCs obtained from the same blood samples through stable iPSC clones and a subsequent neural differentiation (classical NPCs—cNPCs). Peripheral blood cells were obtained from a schizophrenia patient and his two healthy parents (a case–parent trio), while a further umbilical cord blood sample was obtained from the cord of a healthy new-born. The expression of stage-specific markers in sNPCs and cNPCs were compared both at the protein and RNA levels. We also performed functional tests to investigate Wnt and glutamate signaling and the oxidative stress, as these pathways have been suggested to play important roles in the pathophysiology of schizophrenia. We found similar responses in the two types of NPCs, suggesting that the shortcut procedure provides sNPCs, allowing an efficient screening of disease-related phenotypes. Full article
(This article belongs to the Special Issue From hIPSCs to Adult Cells in a Dish: Promises and Pitfalls)
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15 pages, 2301 KiB  
Article
Targeted Integration of Inducible Caspase-9 in Human iPSCs Allows Efficient in vitro Clearance of iPSCs and iPSC-Macrophages
by Alexandra Lipus, Ewa Janosz, Mania Ackermann, Miriam Hetzel, Julia Dahlke, Theresa Buchegger, Stephanie Wunderlich, Ulrich Martin, Toni Cathomen, Axel Schambach, Thomas Moritz and Nico Lachmann
Int. J. Mol. Sci. 2020, 21(7), 2481; https://doi.org/10.3390/ijms21072481 - 03 Apr 2020
Cited by 10 | Viewed by 3292
Abstract
Induced pluripotent stem cells (iPSCs) offer great promise for the field of regenerative medicine, and iPSC-derived cells have already been applied in clinical practice. However, potential contamination of effector cells with residual pluripotent cells (e.g., teratoma-initiating cells) or effector cell-associated side effects may [...] Read more.
Induced pluripotent stem cells (iPSCs) offer great promise for the field of regenerative medicine, and iPSC-derived cells have already been applied in clinical practice. However, potential contamination of effector cells with residual pluripotent cells (e.g., teratoma-initiating cells) or effector cell-associated side effects may limit this approach. This also holds true for iPSC-derived hematopoietic cells. Given the therapeutic benefit of macrophages in different disease entities and the feasibility to derive macrophages from human iPSCs, we established human iPSCs harboring the inducible Caspase-9 (iCasp9) suicide safety switch utilizing transcription activator-like effector nuclease (TALEN)-based designer nuclease technology. Mono- or bi-allelic integration of the iCasp9 gene cassette into the AAVS1 locus showed no effect on the pluripotency of human iPSCs and did not interfere with their differentiation towards macrophages. In both, iCasp9-mono and iCasp9-bi-allelic clones, concentrations of 0.1 nM AP20187 were sufficient to induce apoptosis in more than 98% of iPSCs and their progeny—macrophages. Thus, here we provide evidence that the introduction of the iCasp9 suicide gene into the AAVS1 locus enables the effective clearance of human iPSCs and thereof derived macrophages. Full article
(This article belongs to the Special Issue From hIPSCs to Adult Cells in a Dish: Promises and Pitfalls)
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13 pages, 4896 KiB  
Article
iPSC-Derived MSCs Versus Originating Jaw Periosteal Cells: Comparison of Resulting Phenotype and Stem Cell Potential
by Felix Umrath, Marbod Weber, Siegmar Reinert, Hans-Peter Wendel, Meltem Avci-Adali and Dorothea Alexander
Int. J. Mol. Sci. 2020, 21(2), 587; https://doi.org/10.3390/ijms21020587 - 16 Jan 2020
Cited by 10 | Viewed by 3099
Abstract
Induced pluripotent stem cell-derived mesenchymal stem cell-like cells (iMSCs) are considered to be a promising source of progenitor cells for approaches in the field of bone regeneration. In a previous study, we described the generation of footprint-free induced pluripotent stem cells (iPSCs) from [...] Read more.
Induced pluripotent stem cell-derived mesenchymal stem cell-like cells (iMSCs) are considered to be a promising source of progenitor cells for approaches in the field of bone regeneration. In a previous study, we described the generation of footprint-free induced pluripotent stem cells (iPSCs) from human jaw periosteal cells (JPCs) by transfection of a self-replicating RNA (srRNA) and subsequent differentiation into functional osteogenic progenitor cells. In order to facilitate the prospective transfer into clinical practice, xeno-free reprogramming and differentiation methods were established. In this study, we compared the properties and stem cell potential of the iMSCs produced from JPC-derived iPSCs with the parental primary JPCs they were generated from. Our results demonstrated, on the one hand, a comparable differentiation potential of iMSCs and JPCs. Additionally, iMSCs showed significantly longer telomere lengths compared to JPCs indicating rejuvenation of the cells during reprogramming. On the other hand, proliferation, mitochondrial activity, and senescence-associated beta-galactosidase (SA-β-gal) activity indicated early senescence of iMSCs. These data demonstrate the requirement of further optimization strategies to improve mesenchymal development of JPC-derived iPSCs in order to take advantage of the best features of reprogrammed and rejuvenated cells. Full article
(This article belongs to the Special Issue From hIPSCs to Adult Cells in a Dish: Promises and Pitfalls)
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17 pages, 5473 KiB  
Article
In Vitro Fabrication of Hybrid Bone/Cartilage Complex Using Mouse Induced Pluripotent Stem Cells
by Phoonsuk Limraksasin, Takeru Kondo, Maolin Zhang, Hiroko Okawa, Thanaphum Osathanon, Prasit Pavasant and Hiroshi Egusa
Int. J. Mol. Sci. 2020, 21(2), 581; https://doi.org/10.3390/ijms21020581 - 16 Jan 2020
Cited by 21 | Viewed by 4332
Abstract
Cell condensation and mechanical stimuli play roles in osteogenesis and chondrogenesis; thus, they are promising for facilitating self-organizing bone/cartilage tissue formation in vitro from induced pluripotent stem cells (iPSCs). Here, single mouse iPSCs were first seeded in micro-space culture plates to form 3-dimensional [...] Read more.
Cell condensation and mechanical stimuli play roles in osteogenesis and chondrogenesis; thus, they are promising for facilitating self-organizing bone/cartilage tissue formation in vitro from induced pluripotent stem cells (iPSCs). Here, single mouse iPSCs were first seeded in micro-space culture plates to form 3-dimensional spheres. At day 12, iPSC spheres were subjected to shaking culture and maintained in osteogenic induction medium for 31 days (Os induction). In another condition, the osteogenic induction medium was replaced by chondrogenic induction medium at day 22 and maintained for a further 21 days (Os-Chon induction). Os induction produced robust mineralization and some cartilage-like tissue, which promoted expression of osteogenic and chondrogenic marker genes. In contrast, Os-Chon induction resulted in partial mineralization and a large area of cartilage tissue, with greatly increased expression of chondrogenic marker genes along with osterix and collagen 1a1. Os-Chon induction enhanced mesodermal lineage commitment with brachyury expression followed by high expression of lateral plate and paraxial mesoderm marker genes. These results suggest that combined use of micro-space culture and mechanical stimuli facilitates hybrid bone/cartilage tissue formation from iPSCs, and that the bone/cartilage tissue ratio in iPSC constructs could be manipulated through the induction protocol. Full article
(This article belongs to the Special Issue From hIPSCs to Adult Cells in a Dish: Promises and Pitfalls)
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25 pages, 2704 KiB  
Article
Characterization of Human Induced Pluripotent Stem Cell-Derived Hepatocytes with Mature Features and Potential for Modeling Metabolic Diseases
by Gustav Holmgren, Benjamin Ulfenborg, Annika Asplund, Karin Toet, Christian X Andersson, Ann Hammarstedt, Roeland Hanemaaijer, Barbara Küppers-Munther and Jane Synnergren
Int. J. Mol. Sci. 2020, 21(2), 469; https://doi.org/10.3390/ijms21020469 - 11 Jan 2020
Cited by 15 | Viewed by 5233
Abstract
There is a strong anticipated future for human induced pluripotent stem cell-derived hepatocytes (hiPS-HEP), but so far, their use has been limited due to insufficient functionality. We investigated the potential of hiPS-HEP as an in vitro model for metabolic diseases by combining transcriptomics [...] Read more.
There is a strong anticipated future for human induced pluripotent stem cell-derived hepatocytes (hiPS-HEP), but so far, their use has been limited due to insufficient functionality. We investigated the potential of hiPS-HEP as an in vitro model for metabolic diseases by combining transcriptomics with multiple functional assays. The transcriptomics analysis revealed that 86% of the genes were expressed at similar levels in hiPS-HEP as in human primary hepatocytes (hphep). Adult characteristics of the hiPS-HEP were confirmed by the presence of important hepatocyte features, e.g., Albumin secretion and expression of major drug metabolizing genes. Normal energy metabolism is crucial for modeling metabolic diseases, and both transcriptomics data and functional assays showed that hiPS-HEP were similar to hphep regarding uptake of glucose, low-density lipoproteins (LDL), and fatty acids. Importantly, the inflammatory state of the hiPS-HEP was low under standard conditions, but in response to lipid accumulation and ER stress the inflammation marker tumor necrosis factor α (TNFα) was upregulated. Furthermore, hiPS-HEP could be co-cultured with primary hepatic stellate cells both in 2D and in 3D spheroids, paving the way for using these co-cultures for modeling non-alcoholic steatohepatitis (NASH). Taken together, hiPS-HEP have the potential to serve as an in vitro model for metabolic diseases. Furthermore, differently expressed genes identified in this study can serve as targets for future improvements of the hiPS-HEP. Full article
(This article belongs to the Special Issue From hIPSCs to Adult Cells in a Dish: Promises and Pitfalls)
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17 pages, 5085 KiB  
Article
Long-Term Stability and Differentiation Potential of Cryopreserved cGMP-Compliant Human Induced Pluripotent Stem Cells
by Mehdi Shafa, Tylor Walsh, Krishna Morgan Panchalingam, Thomas Richardson, Laura Menendez, Xinghui Tian, Sahana Suresh Babu, Saedeh Dadgar, Justin Beller, Fan Yang and Behnam Ahmadian Baghbaderani
Int. J. Mol. Sci. 2020, 21(1), 108; https://doi.org/10.3390/ijms21010108 - 23 Dec 2019
Cited by 11 | Viewed by 6929
Abstract
The clinical effectiveness of human induced pluripotent stem cells (iPSCs) is highly dependent on a few key quality characteristics including the generation of high quality cell bank, long-term genomic stability, post-thaw viability, plating efficiency, retention of pluripotency, directed differentiation, purity, potency, and sterility. [...] Read more.
The clinical effectiveness of human induced pluripotent stem cells (iPSCs) is highly dependent on a few key quality characteristics including the generation of high quality cell bank, long-term genomic stability, post-thaw viability, plating efficiency, retention of pluripotency, directed differentiation, purity, potency, and sterility. We have already reported the establishment of iPSC master cell banks (MCBs) and working cell banks (WCBs) under current good manufacturing procedure (cGMP)-compliant conditions. In this study, we assessed the cellular and genomic stability of the iPSC lines generated and cryopreserved five years ago under cGMP-compliant conditions. iPSC lines were thawed, characterized, and directly differentiated into cells from three germ layers including cardiomyocytes (CMs), neural stem cells (NSCs), and definitive endoderm (DE). The cells were also expanded in 2D and 3D spinner flasks to evaluate their long-term expansion potential in matrix-dependent and feeder-free culture environment. All three lines successfully thawed and attached to the L7TM matrix, and formed typical iPSC colonies that expressed pluripotency markers over 15 passages. iPSCs maintained their differentiation potential as demonstrated with spontaneous and directed differentiation to the three germ layers and corresponding expression of specific markers, respectfully. Furthermore, post-thaw cells showed normal karyotype, negative mycoplasma, and sterility testing. These cells maintained both their 2D and 3D proliferation potential after five years of cryopreservation without acquiring karyotype abnormality, loss of pluripotency, and telomerase activity. These results illustrate the long-term stability of cGMP iPSC lines, which is an important step in establishing a reliable, long-term source of starting materials for clinical and commercial manufacturing of iPSC-derived cell therapy products. Full article
(This article belongs to the Special Issue From hIPSCs to Adult Cells in a Dish: Promises and Pitfalls)
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29 pages, 11471 KiB  
Article
End-to-End Platform for Human Pluripotent Stem Cell Manufacturing
by Puspa R. Pandey, Amarel Tomney, Marites T. Woon, Nicholas Uth, Farjad Shafighi, Igor Ngabo, Haritha Vallabhaneni, Yonatan Levinson, Eytan Abraham and Inbar Friedrich Ben-Nun
Int. J. Mol. Sci. 2020, 21(1), 89; https://doi.org/10.3390/ijms21010089 - 21 Dec 2019
Cited by 25 | Viewed by 7154
Abstract
Industrialization of stem-cell based therapies requires innovative solutions to close the gap between research and commercialization. Scalable cell production platforms are needed to reliably deliver the cell quantities needed during the various stages of development and commercial supply. Human pluripotent stem cells (hPSCs) [...] Read more.
Industrialization of stem-cell based therapies requires innovative solutions to close the gap between research and commercialization. Scalable cell production platforms are needed to reliably deliver the cell quantities needed during the various stages of development and commercial supply. Human pluripotent stem cells (hPSCs) are a key source material for generating therapeutic cell types. We have developed a closed, automated and scalable stirred tank bioreactor platform, capable of sustaining high fold expansion of hPSCs. Such a platform could facilitate the in-process monitoring and integration of online monitoring systems, leading to significantly reduced labor requirements and contamination risk. hPSCs are expanded in a controlled bioreactor using perfused xeno-free media. Cell harvest and concentration are performed in closed steps. The hPSCs can be cryopreserved to generate a bank of cells, or further processed as needed. Cryopreserved cells can be thawed into a two-dimensional (2D) tissue culture platform or a three-dimensional (3D) bioreactor to initiate a new expansion phase, or be differentiated to the clinically relevant cell type. The expanded hPSCs express hPSC-specific markers, have a normal karyotype and the ability to differentiate to the cells of the three germ layers. This end-to-end platform allows a large scale expansion of high quality hPSCs that can support the required cell demand for various clinical indications. Full article
(This article belongs to the Special Issue From hIPSCs to Adult Cells in a Dish: Promises and Pitfalls)
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10 pages, 3267 KiB  
Article
Modeling the Effects of Severe Metabolic Disease by Genome Editing of hPSC-Derived Endothelial Cells Reveals an Inflammatory Phenotype
by Filip Roudnicky, Yanjun Lan, Max Friesen, Gregor Dernick, Jitao David Zhang, Andreas Staempfli, Natalie Bordag, Antje Wagner-Golbs, Klaus Christensen, Martin Ebeling, Martin Graf, Mark Burcin, Claas Aiko Meyer, Chad A Cowan and Christoph Patsch
Int. J. Mol. Sci. 2019, 20(24), 6201; https://doi.org/10.3390/ijms20246201 - 09 Dec 2019
Cited by 3 | Viewed by 2650
Abstract
The kinase AKT2 (PKB) is an important mediator of insulin signaling, for which loss-of-function knockout (KO) mutants lead to early onset diabetes mellitus, and dominant active mutations lead to early development of obesity and endothelial cell (EC) dysfunction. To model EC dysfunction, we [...] Read more.
The kinase AKT2 (PKB) is an important mediator of insulin signaling, for which loss-of-function knockout (KO) mutants lead to early onset diabetes mellitus, and dominant active mutations lead to early development of obesity and endothelial cell (EC) dysfunction. To model EC dysfunction, we used edited human pluripotent stem cells (hPSCs) that carried either a homozygous deletion of AKT2 (AKT2 KO) or a dominant active mutation (AKT2 E17K), which, along with the parental wild type (WT), were differentiated into ECs. Profiling of EC lines indicated an increase in proinflammatory and a reduction in anti-inflammatory fatty acids, an increase in inflammatory chemokines in cell supernatants, increased expression of proinflammatory genes, and increased binding to the EC monolayer in a functional leukocyte adhesion assay for both AKT2 KO and AKT2 E17K. Collectively, these findings suggest that vascular endothelial inflammation that results from dysregulated insulin signaling (homeostasis) may contribute to coronary artery disease, and that either downregulation or upregulation of the insulin pathway may lead to inflammation of endothelial cells. This suggests that the standard of care for patients must be expanded from control of metabolic parameters to include control of inflammation, such that endothelial dysfunction and cardiovascular disorders can ultimately be prevented. Full article
(This article belongs to the Special Issue From hIPSCs to Adult Cells in a Dish: Promises and Pitfalls)
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17 pages, 2670 KiB  
Article
Optical Investigation of Action Potential and Calcium Handling Maturation of hiPSC-Cardiomyocytes on Biomimetic Substrates
by Josè Manuel Pioner, Lorenzo Santini, Chiara Palandri, Daniele Martella, Flavia Lupi, Marianna Langione, Silvia Querceto, Bruno Grandinetti, Valentina Balducci, Patrizia Benzoni, Sara Landi, Andrea Barbuti, Federico Ferrarese Lupi, Luca Boarino, Laura Sartiani, Chiara Tesi, David L. Mack, Michael Regnier, Elisabetta Cerbai, Camilla Parmeggiani, Corrado Poggesi, Cecilia Ferrantini and Raffaele Coppiniadd Show full author list remove Hide full author list
Int. J. Mol. Sci. 2019, 20(15), 3799; https://doi.org/10.3390/ijms20153799 - 03 Aug 2019
Cited by 28 | Viewed by 5478
Abstract
Cardiomyocytes from human induced pluripotent stem cells (hiPSC-CMs) are the most promising human source with preserved genetic background of healthy individuals or patients. This study aimed to establish a systematic procedure for exploring development of hiPSC-CM functional output to predict genetic cardiomyopathy outcomes [...] Read more.
Cardiomyocytes from human induced pluripotent stem cells (hiPSC-CMs) are the most promising human source with preserved genetic background of healthy individuals or patients. This study aimed to establish a systematic procedure for exploring development of hiPSC-CM functional output to predict genetic cardiomyopathy outcomes and identify molecular targets for therapy. Biomimetic substrates with microtopography and physiological stiffness can overcome the immaturity of hiPSC-CM function. We have developed a custom-made apparatus for simultaneous optical measurements of hiPSC-CM action potential and calcium transients to correlate these parameters at specific time points (day 60, 75 and 90 post differentiation) and under inotropic interventions. In later-stages, single hiPSC-CMs revealed prolonged action potential duration, increased calcium transient amplitude and shorter duration that closely resembled those of human adult cardiomyocytes from fresh ventricular tissue of patients. Thus, the major contribution of sarcoplasmic reticulum and positive inotropic response to β-adrenergic stimulation are time-dependent events underlying excitation contraction coupling (ECC) maturation of hiPSC-CM; biomimetic substrates can promote calcium-handling regulation towards adult-like kinetics. Simultaneous optical recordings of long-term cultured hiPSC-CMs on biomimetic substrates favor high-throughput electrophysiological analysis aimed at testing (mechanistic hypothesis on) disease progression and pharmacological interventions in patient-derived hiPSC-CMs. Full article
(This article belongs to the Special Issue From hIPSCs to Adult Cells in a Dish: Promises and Pitfalls)
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17 pages, 9776 KiB  
Article
Increased Cytotoxicity of Herpes Simplex Virus Thymidine Kinase Expression in Human Induced Pluripotent Stem Cells
by Chizuru Iwasawa, Ryota Tamura, Yuki Sugiura, Sadafumi Suzuki, Naoko Kuzumaki, Minoru Narita, Makoto Suematsu, Masaya Nakamura, Kazunari Yoshida, Masahiro Toda, Hideyuki Okano and Hiroyuki Miyoshi
Int. J. Mol. Sci. 2019, 20(4), 810; https://doi.org/10.3390/ijms20040810 - 14 Feb 2019
Cited by 19 | Viewed by 8284
Abstract
Human induced pluripotent stem cells (iPSCs) hold enormous promise for regenerative medicine. The major safety concern is the tumorigenicity of transplanted cells derived from iPSCs. A potential solution would be to introduce a suicide gene into iPSCs as a safety switch. The herpes [...] Read more.
Human induced pluripotent stem cells (iPSCs) hold enormous promise for regenerative medicine. The major safety concern is the tumorigenicity of transplanted cells derived from iPSCs. A potential solution would be to introduce a suicide gene into iPSCs as a safety switch. The herpes simplex virus type 1 thymidine kinase (HSV-TK) gene, in combination with ganciclovir, is the most widely used enzyme/prodrug suicide system from basic research to clinical applications. In the present study, we attempted to establish human iPSCs that stably expressed HSV-TK with either lentiviral vectors or CRISPR/Cas9-mediated genome editing. However, this task was difficult to achieve, because high-level and/or constitutive expression of HSV-TK resulted in the induction of cell death or silencing of HSV-TK expression. A nucleotide metabolism analysis suggested that excessive accumulation of thymidine triphosphate, caused by HSV-TK expression, resulted in an imbalance in the dNTP pools. This unbalanced state led to DNA synthesis inhibition and cell death in a process similar to a “thymidine block”, but more severe. We also demonstrated that the Tet-inducible system was a feasible solution for overcoming the cytotoxicity of HSV-TK expression. Our results provided a warning against using the HSV-TK gene in human iPSCs, particularly in clinical applications. Full article
(This article belongs to the Special Issue From hIPSCs to Adult Cells in a Dish: Promises and Pitfalls)
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10 pages, 1519 KiB  
Article
Enterococcus faecium L-15 Cell-Free Extract Improves the Chondrogenic Differentiation of Human Dental Pulp Stem Cells
by Hyewon Kim, Sangkyu Park, Kichul Kim, Seockmo Ku, Jeongmin Seo and Sangho Roh
Int. J. Mol. Sci. 2019, 20(3), 624; https://doi.org/10.3390/ijms20030624 - 31 Jan 2019
Cited by 9 | Viewed by 3121
Abstract
Hyaline cartilage is a tissue of very low regenerative capacity because of its histology and limited nutrient supply. Cell-based therapies have been spotlighted in the regeneration of damaged cartilage. Dental pulp stem cells (DPSCs) are multipotent and are easily accessible for therapeutic purposes. [...] Read more.
Hyaline cartilage is a tissue of very low regenerative capacity because of its histology and limited nutrient supply. Cell-based therapies have been spotlighted in the regeneration of damaged cartilage. Dental pulp stem cells (DPSCs) are multipotent and are easily accessible for therapeutic purposes. In human gastrointestinal tracts, Enterococcus faecium is a naturally occurring commensal species of lactic acid bacteria. In this work, the human DPSCs were differentiated into chondrocytes using a chondrogenic differentiation medium with or without L-15 extract. We observed that chondrogenic differentiation improved in an E. faecium L-15 extract (L-15)-treated DPSC group via evaluation of chondrogenic-marker mRNA expression levels. In particular, we found that L-15 treatment promoted early-stage DPSC differentiation. Cells treated with L-15 were inhibited at later stages and were less likely to transform into hypertrophic chondrocytes. In L-15-treated groups, the total amount of cartilage extracellular matrix increased during the differentiation process. These results suggest that L-15 promotes chondrogenic differentiation, and that L-15 may be used for cartilage repair or cartilage health supplements. To our knowledge, this is the first report demonstrating the beneficial effect of L-15 treatment on chondrogenic differentiation. Full article
(This article belongs to the Special Issue From hIPSCs to Adult Cells in a Dish: Promises and Pitfalls)
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Review

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52 pages, 1329 KiB  
Review
“Betwixt Mine Eye and Heart a League Is Took”: The Progress of Induced Pluripotent Stem-Cell-Based Models of Dystrophin-Associated Cardiomyopathy
by Davide Rovina, Elisa Castiglioni, Francesco Niro, Sara Mallia, Giulio Pompilio and Aoife Gowran
Int. J. Mol. Sci. 2020, 21(19), 6997; https://doi.org/10.3390/ijms21196997 - 23 Sep 2020
Cited by 5 | Viewed by 3737
Abstract
The ultimate goal of precision disease modeling is to artificially recreate the disease of affected people in a highly controllable and adaptable external environment. This field has rapidly advanced which is evident from the application of patient-specific pluripotent stem-cell-derived precision therapies in numerous [...] Read more.
The ultimate goal of precision disease modeling is to artificially recreate the disease of affected people in a highly controllable and adaptable external environment. This field has rapidly advanced which is evident from the application of patient-specific pluripotent stem-cell-derived precision therapies in numerous clinical trials aimed at a diverse set of diseases such as macular degeneration, heart disease, spinal cord injury, graft-versus-host disease, and muscular dystrophy. Despite the existence of semi-adequate treatments for tempering skeletal muscle degeneration in dystrophic patients, nonischemic cardiomyopathy remains one of the primary causes of death. Therefore, cardiovascular cells derived from muscular dystrophy patients’ induced pluripotent stem cells are well suited to mimic dystrophin-associated cardiomyopathy and hold great promise for the development of future fully effective therapies. The purpose of this article is to convey the realities of employing precision disease models of dystrophin-associated cardiomyopathy. This is achieved by discussing, as suggested in the title echoing William Shakespeare’s words, the settlements (or “leagues”) made by researchers to manage the constraints (“betwixt mine eye and heart”) distancing them from achieving a perfect precision disease model. Full article
(This article belongs to the Special Issue From hIPSCs to Adult Cells in a Dish: Promises and Pitfalls)
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27 pages, 1352 KiB  
Review
A Link between Genetic Disorders and Cellular Impairment, Using Human Induced Pluripotent Stem Cells to Reveal the Functional Consequences of Copy Number Variations in the Central Nervous System—A Close Look at Chromosome 15
by Alessia Casamassa, Daniela Ferrari, Maurizio Gelati, Massimo Carella, Angelo Luigi Vescovi and Jessica Rosati
Int. J. Mol. Sci. 2020, 21(5), 1860; https://doi.org/10.3390/ijms21051860 - 09 Mar 2020
Cited by 3 | Viewed by 4484
Abstract
Recent cutting-edge human genetics technology has allowed us to identify copy number variations (CNVs) and has provided new insights for understanding causative mechanisms of human diseases. A growing number of studies show that CNVs could be associated with physiological mechanisms linked to evolutionary [...] Read more.
Recent cutting-edge human genetics technology has allowed us to identify copy number variations (CNVs) and has provided new insights for understanding causative mechanisms of human diseases. A growing number of studies show that CNVs could be associated with physiological mechanisms linked to evolutionary trigger, as well as to the pathogenesis of various diseases, including cancer, autoimmune disease and mental disorders such as autism spectrum disorders, schizophrenia, intellectual disabilities or attention-deficit/hyperactivity disorder. Their incomplete penetrance and variable expressivity make diagnosis difficult and hinder comprehension of the mechanistic bases of these disorders. Additional elements such as co-presence of other CNVs, genomic background and environmental factors are involved in determining the final phenotype associated with a CNV. Genetically engineered animal models are helpful tools for understanding the behavioral consequences of CNVs. However, the genetic background and the biology of these animal model systems have sometimes led to confusing results. New cellular models obtained through somatic cellular reprogramming technology that produce induced pluripotent stem cells (iPSCs) from human subjects are being used to explore the mechanisms involved in the pathogenic consequences of CNVs. Considering the vast quantity of CNVs found in the human genome, we intend to focus on reviewing the current literature on the use of iPSCs carrying CNVs on chromosome 15, highlighting advantages and limits of this system with respect to mouse model systems. Full article
(This article belongs to the Special Issue From hIPSCs to Adult Cells in a Dish: Promises and Pitfalls)
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16 pages, 313 KiB  
Review
Engineering Prostate Cancer from Induced Pluripotent Stem Cells—New Opportunities to Develop Preclinical Tools in Prostate and Prostate Cancer Studies
by Anastasia C. Hepburn, C. H. Cole Sims, Adriana Buskin and Rakesh Heer
Int. J. Mol. Sci. 2020, 21(3), 905; https://doi.org/10.3390/ijms21030905 - 30 Jan 2020
Cited by 14 | Viewed by 4879
Abstract
One of the key issues hampering the development of effective treatments for prostate cancer is the lack of suitable, tractable, and patient-specific in vitro models that accurately recapitulate this disease. In this review, we address the challenges of using primary cultures and patient-derived [...] Read more.
One of the key issues hampering the development of effective treatments for prostate cancer is the lack of suitable, tractable, and patient-specific in vitro models that accurately recapitulate this disease. In this review, we address the challenges of using primary cultures and patient-derived xenografts to study prostate cancer. We describe emerging approaches using primary prostate epithelial cells and prostate organoids and their genetic manipulation for disease modelling. Furthermore, the use of human prostate-derived induced pluripotent stem cells (iPSCs) is highlighted as a promising complimentary approach. Finally, we discuss the manipulation of iPSCs to generate ‘avatars’ for drug disease testing. Specifically, we describe how a conceptual advance through the creation of living biobanks of “genetically engineered cancers” that contain patient-specific driver mutations hold promise for personalised medicine. Full article
(This article belongs to the Special Issue From hIPSCs to Adult Cells in a Dish: Promises and Pitfalls)
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26 pages, 915 KiB  
Review
The Emergence of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes (hiPSC-CMs) as a Platform to Model Arrhythmogenic Diseases
by Marc Pourrier and David Fedida
Int. J. Mol. Sci. 2020, 21(2), 657; https://doi.org/10.3390/ijms21020657 - 19 Jan 2020
Cited by 27 | Viewed by 5533
Abstract
There is a need for improved in vitro models of inherited cardiac diseases to better understand basic cellular and molecular mechanisms and advance drug development. Most of these diseases are associated with arrhythmias, as a result of mutations in ion channel or ion [...] Read more.
There is a need for improved in vitro models of inherited cardiac diseases to better understand basic cellular and molecular mechanisms and advance drug development. Most of these diseases are associated with arrhythmias, as a result of mutations in ion channel or ion channel-modulatory proteins. Thus far, the electrophysiological phenotype of these mutations has been typically studied using transgenic animal models and heterologous expression systems. Although they have played a major role in advancing the understanding of the pathophysiology of arrhythmogenesis, more physiological and predictive preclinical models are necessary to optimize the treatment strategy for individual patients. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have generated much interest as an alternative tool to model arrhythmogenic diseases. They provide a unique opportunity to recapitulate the native-like environment required for mutated proteins to reproduce the human cellular disease phenotype. However, it is also important to recognize the limitations of this technology, specifically their fetal electrophysiological phenotype, which differentiates them from adult human myocytes. In this review, we provide an overview of the major inherited arrhythmogenic cardiac diseases modeled using hiPSC-CMs and for which the cellular disease phenotype has been somewhat characterized. Full article
(This article belongs to the Special Issue From hIPSCs to Adult Cells in a Dish: Promises and Pitfalls)
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18 pages, 1762 KiB  
Review
Human Induced Pluripotent Stem Cells Derived from a Cardiac Somatic Source: Insights for an In-Vitro Cardiomyocyte Platform
by Alessandra Maria Lodrini, Lucio Barile, Marcella Rocchetti and Claudia Altomare
Int. J. Mol. Sci. 2020, 21(2), 507; https://doi.org/10.3390/ijms21020507 - 13 Jan 2020
Cited by 9 | Viewed by 4554
Abstract
Reprogramming of adult somatic cells into induced pluripotent stem cells (iPSCs) has revolutionized the complex scientific field of disease modelling and personalized therapy. Cardiac differentiation of human iPSCs into cardiomyocytes (hiPSC-CMs) has been used in a wide range of healthy and disease models [...] Read more.
Reprogramming of adult somatic cells into induced pluripotent stem cells (iPSCs) has revolutionized the complex scientific field of disease modelling and personalized therapy. Cardiac differentiation of human iPSCs into cardiomyocytes (hiPSC-CMs) has been used in a wide range of healthy and disease models by deriving CMs from different somatic cells. Unfortunately, hiPSC-CMs have to be improved because existing protocols are not completely able to obtain mature CMs recapitulating physiological properties of human adult cardiac cells. Therefore, improvements and advances able to standardize differentiation conditions are needed. Lately, evidences of an epigenetic memory retained by the somatic cells used for deriving hiPSC-CMs has led to evaluation of different somatic sources in order to obtain more mature hiPSC-derived CMs. Full article
(This article belongs to the Special Issue From hIPSCs to Adult Cells in a Dish: Promises and Pitfalls)
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19 pages, 1225 KiB  
Review
Progress of Induced Pluripotent Stem Cell Technologies to Understand Genetic Epilepsy
by Bruno Sterlini, Floriana Fruscione, Simona Baldassari, Fabio Benfenati, Federico Zara and Anna Corradi
Int. J. Mol. Sci. 2020, 21(2), 482; https://doi.org/10.3390/ijms21020482 - 12 Jan 2020
Cited by 14 | Viewed by 4902
Abstract
The study of the pathomechanisms by which gene mutations lead to neurological diseases has benefit from several cellular and animal models. Recently, induced Pluripotent Stem Cell (iPSC) technologies have made possible the access to human neurons to study nervous system disease-related mechanisms, and [...] Read more.
The study of the pathomechanisms by which gene mutations lead to neurological diseases has benefit from several cellular and animal models. Recently, induced Pluripotent Stem Cell (iPSC) technologies have made possible the access to human neurons to study nervous system disease-related mechanisms, and are at the forefront of the research into neurological diseases. In this review, we will focalize upon genetic epilepsy, and summarize the most recent studies in which iPSC-based technologies were used to gain insight on the molecular bases of epilepsies. Moreover, we discuss the latest advancements in epilepsy cell modeling. At the two dimensional (2D) level, single-cell models of iPSC-derived neurons lead to a mature neuronal phenotype, and now allow a reliable investigation of synaptic transmission and plasticity. In addition, functional characterization of cerebral organoids enlightens neuronal network dynamics in a three-dimensional (3D) structure. Finally, we discuss the use of iPSCs as the cutting-edge technology for cell therapy in epilepsy. Full article
(This article belongs to the Special Issue From hIPSCs to Adult Cells in a Dish: Promises and Pitfalls)
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16 pages, 869 KiB  
Review
The Challenge of Bringing iPSCs to the Patient
by María del Carmen Ortuño-Costela, Victoria Cerrada, Marta García-López and M. Esther Gallardo
Int. J. Mol. Sci. 2019, 20(24), 6305; https://doi.org/10.3390/ijms20246305 - 13 Dec 2019
Cited by 40 | Viewed by 6355
Abstract
The implementation of induced pluripotent stem cells (iPSCs) in biomedical research more than a decade ago, resulted in a huge leap forward in the highly promising area of personalized medicine. Nowadays, we are even closer to the patient than ever. To date, there [...] Read more.
The implementation of induced pluripotent stem cells (iPSCs) in biomedical research more than a decade ago, resulted in a huge leap forward in the highly promising area of personalized medicine. Nowadays, we are even closer to the patient than ever. To date, there are multiple examples of iPSCs applications in clinical trials and drug screening. However, there are still many obstacles to overcome. In this review, we will focus our attention on the advantages of implementing induced pluripotent stem cells technology into the clinics but also commenting on all the current drawbacks that could hinder this promising path towards the patient. Full article
(This article belongs to the Special Issue From hIPSCs to Adult Cells in a Dish: Promises and Pitfalls)
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56 pages, 1308 KiB  
Review
Human Induced Pluripotent Stem-Cell-Derived Cardiomyocytes as Models for Genetic Cardiomyopathies
by Andreas Brodehl, Hans Ebbinghaus, Marcus-André Deutsch, Jan Gummert, Anna Gärtner, Sandra Ratnavadivel and Hendrik Milting
Int. J. Mol. Sci. 2019, 20(18), 4381; https://doi.org/10.3390/ijms20184381 - 06 Sep 2019
Cited by 39 | Viewed by 7213
Abstract
In the last few decades, many pathogenic or likely pathogenic genetic mutations in over hundred different genes have been described for non-ischemic, genetic cardiomyopathies. However, the functional knowledge about most of these mutations is still limited because the generation of adequate animal models [...] Read more.
In the last few decades, many pathogenic or likely pathogenic genetic mutations in over hundred different genes have been described for non-ischemic, genetic cardiomyopathies. However, the functional knowledge about most of these mutations is still limited because the generation of adequate animal models is time-consuming and challenging. Therefore, human induced pluripotent stem cells (iPSCs) carrying specific cardiomyopathy-associated mutations are a promising alternative. Since the original discovery that pluripotency can be artificially induced by the expression of different transcription factors, various patient-specific-induced pluripotent stem cell lines have been generated to model non-ischemic, genetic cardiomyopathies in vitro. In this review, we describe the genetic landscape of non-ischemic, genetic cardiomyopathies and give an overview about different human iPSC lines, which have been developed for the disease modeling of inherited cardiomyopathies. We summarize different methods and protocols for the general differentiation of human iPSCs into cardiomyocytes. In addition, we describe methods and technologies to investigate functionally human iPSC-derived cardiomyocytes. Furthermore, we summarize novel genome editing approaches for the genetic manipulation of human iPSCs. This review provides an overview about the genetic landscape of inherited cardiomyopathies with a focus on iPSC technology, which might be of interest for clinicians and basic scientists interested in genetic cardiomyopathies. Full article
(This article belongs to the Special Issue From hIPSCs to Adult Cells in a Dish: Promises and Pitfalls)
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