Human inducible pluripotent stem cell-derived sensory neurons express multiple functional ion channels and GPCRs

AbstractAimsAim of the study was to characterize functional ion channel and GPCR responses by using selective pharmacological tools and intracellular calcium imaging from human inducible pluripotent stem cell-derived sensory neurons.MethodsSensory neurons were generated from human keratinocytes that were reprogrammed to inducible pluripotent stem cells by using standard Yamanaka factors. Inducible pluripotent stem cells were differentiated into sensory neurons by using 2 differentiation protocols (small molecule and PA6 co-culture). Sensory neurons were loaded with intracellular calcium dye Fluo-4. Single-cell calcium imaging was performed with Photometrics Evolve EM-CCD camera at physiological temperature. Cells were perfused with a Ringer solution at 2–3 ml/min into which pharmacological compounds were dissolved. Data was analyzed with Till Photonics Offline Analysis program.ResultsMost of the results were obtained from PA6 differentiated neurons. 50 s application of 50 mM KCl solution was used as diagnostic tool to activate voltage-gated calcium channels and thereby evoke intracellular calcium elevation. Functional ASIC, NMDA, kainate and TRPA1 ion channels were present in a subset of sensory neurons. Majority of sensory neurons showed robust responses to purinergic stimulation with ATP and histaminergic stimulation with histamine, but not to subtype selective histamine H1, H2 or H4 stimulation suggesting the presence of H3 receptor subtype All cells responded strongly to protease-activated receptor stimulation with a low dose of trypsin. Interestingly, at single-cell level notable heterogeneity of ion channel and GPCR responses was observed.ConclusionsOur results suggest that iPS-derived sensory neurons will be valuable in further pharmacological studies as well as sensory neuropathy disease modeling.

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A selective cytotoxic adenovirus vector for concentration of pluripotent stem cells in human pluripotent stem cell-derived neural progenitor cells

Scientific Reports ◽

10.1038/s41598-021-90928-7 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Takamasa Hirai ◽

Ken Kono ◽

Rumi Sawada ◽

Takuya Kuroda ◽

Satoshi Yasuda ◽

...

Keyword(s):

Stem Cells ◽

Flow Cytometry ◽

Stem Cell ◽

Progenitor Cells ◽

Pluripotent Stem Cells ◽

Pluripotent Stem Cell ◽

Neural Progenitor Cells ◽

Neural Progenitor ◽

Sensitive Detection ◽

Quality And Safety

AbstractHighly sensitive detection of residual undifferentiated pluripotent stem cells is essential for the quality and safety of cell-processed therapeutic products derived from human induced pluripotent stem cells (hiPSCs). We previously reported the generation of an adenovirus (Ad) vector and adeno-associated virus vectors that possess a suicide gene, inducible Caspase 9 (iCasp9), which makes it possible to sensitively detect undifferentiated hiPSCs in cultures of hiPSC-derived cardiomyocytes. In this study, we investigated whether these vectors also allow for detection of undifferentiated hiPSCs in preparations of hiPSC-derived neural progenitor cells (hiPSC-NPCs), which have been expected to treat neurological disorders. To detect undifferentiated hiPSCs, the expression of pluripotent stem cell markers was determined by immunostaining and flow cytometry. Using immortalized NPCs as a model, the Ad vector was identified to be the most efficient among the vectors tested in detecting undifferentiated hiPSCs. Moreover, we found that the Ad vector killed most hiPSC-NPCs in an iCasp9-dependent manner, enabling flow cytometry to detect undifferentiated hiPSCs intermingled at a lower concentration (0.002%) than reported previously (0.1%). These data indicate that the Ad vector selectively eliminates hiPSC-NPCs, thus allowing for sensitive detection of hiPSCs. This cytotoxic viral vector could contribute to ensuring the quality and safety of hiPSCs-NPCs for therapeutic use.

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Application of the Pluripotent Stem Cells and Genomics in Cardiovascular Research—What We Have Learnt and Not Learnt until Now

Cells ◽

10.3390/cells10113112 ◽

2021 ◽

Vol 10 (11) ◽

pp. 3112

Author(s):

Michael Simeon ◽

Seema Dangwal ◽

Agapios Sachinidis ◽

Michael Xavier Doss

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Stem Cell Research ◽

Pluripotent Stem Cells ◽

Pluripotent Stem Cell ◽

Genome Engineering ◽

Tourism Industry ◽

Full Potential ◽

Cardiovascular Research ◽

Global Pandemic

Personalized regenerative medicine and biomedical research have been galvanized and revolutionized by human pluripotent stem cells in combination with recent advances in genomics, artificial intelligence, and genome engineering. More recently, we have witnessed the unprecedented breakthrough life-saving translation of mRNA-based vaccines for COVID-19 to contain the global pandemic and the investment in billions of US dollars in space exploration projects and the blooming space-tourism industry fueled by the latest reusable space vessels. Now, it is time to examine where the translation of pluripotent stem cell research stands currently, which has been touted for more than the last two decades to cure and treat millions of patients with severe debilitating degenerative diseases and tissue injuries. This review attempts to highlight the accomplishments of pluripotent stem cell research together with cutting-edge genomics and genome editing tools and, also, the promises that have still not been transformed into clinical applications, with cardiovascular research as a case example. This review also brings to our attention the scientific and socioeconomic challenges that need to be effectively addressed to see the full potential of pluripotent stem cells at the clinical bedside.

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GENERATION OF RABBIT PLURIPOTENT STEM CELL LINES

Reproduction Fertility and Development ◽

10.1071/rdv24n1ab246 ◽

2012 ◽

Vol 24 (1) ◽

pp. 286

Author(s):

A. Dinnyes ◽

M. K. Pirity ◽

E. Gocza ◽

P. Osteil ◽

N. Daniel ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Pluripotent Stem Cells ◽

Pluripotent Stem Cell ◽

Pluripotent Cells ◽

Domestic Species ◽

Isolation And Characterization ◽

Induced Pluripotent ◽

Pluripotency Genes

Pluripotent stem cells have the capacity to divide indefinitely and to differentiate to all the somatic tissues. They can be genetically manipulated in vitro by knocking in and out genes, therefore they serve as an excellent tool for gene-function studies and for the generation of models for human diseases. Since 1981, when the first mouse embryonic stem cell (ESC) line was generated, several attempts have been made to generate pluripotent stem cells from other species as it would help us to understand the differences and similarities of signaling pathways involved in pluripotency and differentiation, and would reveal whether the fundamental mechanism controlling self-renewal of pluripotent cells is conserved among different species. This review gives an overlook of embryonic and induced pluripotent stem cell (iPSCs) research in the rabbit which is one of the most relevant non-rodent species for animal models. To date, several lines of putative ESCs and iPSCs have been described in the rabbit. All expressed stem cell-associated markers and exhibited longevity and pluripotency in vitro, but none have been proven to exhibit full pluripotency in vivo. Moreover, similarly to several domestic species, markers used to characterize the putative ESCs are not fully adequate because studies in domestic species have revealed that they are not specific to the pluripotent inner cell mass. Future validation of rabbit pluripotent stem cells would benefit greatly from a reliable panel of molecular markers specific to pluripotent cells of the developing rabbit embryo. The status of isolation and characterization of the putative pluripotency genes in rabbit will be discussed. Using rabbit specific pluripotency genes we might be able to reprogram somatic cells and generate induced pluripotent stem cells more efficiently thus overcome some of the challenges towards harnessing the potential of this technology. This study was financed by EU FP7 (PartnErS, PIAP-GA-2008-218205; InduHeart, PEOPLE-IRG-2008-234390; InduVir, PEOPLE-IRG-2009-245808; RabPstem, PERG07-GA-2010-268422; PluriSys, HEALTH-2007-B-223485; AniStem, PIAP-GA-2011-286264), NKTH-OTKA-EU-7KP HUMAN-MB08-C-80-205; Plurabbit, OMFB-00130-00131/2010 ANR-NKTH/09-GENM-010-01.

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Challenges and Solutions for Commercial Scale Manufacturing of Allogeneic Pluripotent Stem Cell Products

Bioengineering ◽

10.3390/bioengineering7020031 ◽

2020 ◽

Vol 7 (2) ◽

pp. 31

Author(s):

Brian Lee ◽

Breanna S. Borys ◽

Michael S. Kallos ◽

Carlos A. V. Rodrigues ◽

Teresa P. Silva ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Therapy ◽

Pluripotent Stem Cells ◽

Pluripotent Stem Cell ◽

Large Scale ◽

Process Development ◽

High Quality ◽

Commercial Scale ◽

Allogeneic Cell Therapy

Allogeneic cell therapy products, such as therapeutic cells derived from pluripotent stem cells (PSCs), have amazing potential to treat a wide variety of diseases and vast numbers of patients globally. However, there are various challenges related to the manufacturing of PSCs in large enough quantities to meet commercial needs. This manuscript addresses the challenges for the process development of PSCs production in a bioreactor, and also presents a scalable bioreactor technology that can be a possible solution to remove the bottleneck for the large-scale manufacturing of high-quality therapeutic cells derived from PSCs.

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In Situ Expansion, Differentiation, and Electromechanical Coupling of Human Cardiac Muscle in a 3D Bioprinted, Chambered Organoid

Circulation Research ◽

10.1161/circresaha.119.316155 ◽

2020 ◽

Vol 127 (2) ◽

pp. 207-224 ◽

Cited By ~ 10

Author(s):

Molly E. Kupfer ◽

Wei-Han Lin ◽

Vasanth Ravikumar ◽

Kaiyan Qiu ◽

Lu Wang ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Induced Pluripotent Stem Cells ◽

Cardiac Muscle ◽

Pluripotent Stem Cells ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

Pump Function ◽

Induced Pluripotent

Rationale: One goal of cardiac tissue engineering is the generation of a living, human pump in vitro that could replace animal models and eventually serve as an in vivo therapeutic. Models that replicate the geometrically complex structure of the heart, harboring chambers and large vessels with soft biomaterials, can be achieved using 3-dimensional bioprinting. Yet, inclusion of contiguous, living muscle to support pump function has not been achieved. This is largely due to the challenge of attaining high densities of cardiomyocytes—a notoriously nonproliferative cell type. An alternative strategy is to print with human induced pluripotent stem cells, which can proliferate to high densities and fill tissue spaces, and subsequently differentiate them into cardiomyocytes in situ. Objective: To develop a bioink capable of promoting human induced pluripotent stem cell proliferation and cardiomyocyte differentiation to 3-dimensionally print electromechanically functional, chambered organoids composed of contiguous cardiac muscle. Methods and Results: We optimized a photo-crosslinkable formulation of native ECM (extracellular matrix) proteins and used this bioink to 3-dimensionally print human induced pluripotent stem cell–laden structures with 2 chambers and a vessel inlet and outlet. After human induced pluripotent stem cells proliferated to a sufficient density, we differentiated the cells within the structure and demonstrated function of the resultant human chambered muscle pump. Human chambered muscle pumps demonstrated macroscale beating and continuous action potential propagation with responsiveness to drugs and pacing. The connected chambers allowed for perfusion and enabled replication of pressure/volume relationships fundamental to the study of heart function and remodeling with health and disease. Conclusions: This advance represents a critical step toward generating macroscale tissues, akin to aggregate-based organoids, but with the critical advantage of harboring geometric structures essential to the pump function of cardiac muscle. Looking forward, human chambered organoids of this type might also serve as a test bed for cardiac medical devices and eventually lead to therapeutic tissue grafting.

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Towards consistent generation of pancreatic lineage progenitors from human pluripotent stem cells

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2014.0365 ◽

2015 ◽

Vol 370 (1680) ◽

pp. 20140365 ◽

Cited By ~ 19

Author(s):

Maria Rostovskaya ◽

Nicholas Bredenkamp ◽

Austin Smith

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Lines ◽

Pluripotent Stem Cells ◽

Pluripotent Stem Cell ◽

Human Pluripotent Stem Cells ◽

Type I ◽

Pancreatic Progenitors ◽

Stem Cell Lines

Human pluripotent stem cells can in principle be used as a source of any differentiated cell type for disease modelling, drug screening, toxicology testing or cell replacement therapy. Type I diabetes is considered a major target for stem cell applications due to the shortage of primary human beta cells. Several protocols have been reported for generating pancreatic progenitors by in vitro differentiation of human pluripotent stem cells. Here we first assessed one of these protocols on a panel of pluripotent stem cell lines for capacity to engender glucose sensitive insulin-producing cells after engraftment in immunocompromised mice. We observed variable outcomes with only one cell line showing a low level of glucose response. We, therefore, undertook a systematic comparison of different methods for inducing definitive endoderm and subsequently pancreatic differentiation. Of several protocols tested, we identified a combined approach that robustly generated pancreatic progenitors in vitro from both embryo-derived and induced pluripotent stem cells. These findings suggest that, although there are intrinsic differences in lineage specification propensity between pluripotent stem cell lines, optimal differentiation procedures may consistently direct a substantial fraction of cells into pancreatic specification.

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Young at Heart: Pioneering Approaches to Model Nonischaemic Cardiomyopathy with Induced Pluripotent Stem Cells

Stem Cells International ◽

10.1155/2016/4287158 ◽

2016 ◽

Vol 2016 ◽

pp. 1-15 ◽

Cited By ~ 3

Author(s):

Aoife Gowran ◽

Marco Rasponi ◽

Roberta Visone ◽

Patrizia Nigro ◽

Gianluca L. Perrucci ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Induced Pluripotent Stem Cells ◽

Drug Screening ◽

Pluripotent Stem Cells ◽

Pluripotent Stem Cell ◽

Human Fibroblasts ◽

Induced Pluripotent Stem Cell ◽

Induced Pluripotent

A mere 9 years have passed since the revolutionary report describing the derivation of induced pluripotent stem cells from human fibroblasts and the first in-patient translational use of cells obtained from these stem cells has already been achieved. From the perspectives of clinicians and researchers alike, the promise of induced pluripotent stem cells is alluring if somewhat beguiling. It is now evident that this technology is nascent and many areas for refinement have been identified and need to be considered before induced pluripotent stem cells can be routinely used to stratify, treat and cure patients, and to faithfully model diseases for drug screening purposes. This review specifically addresses the pioneering approaches to improve induced pluripotent stem cell based models of nonischaemic cardiomyopathy.

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Paracrine Effects of the Pluripotent Stem Cell-Derived Cardiac Myocytes Salvage the Injured Myocardium

Circulation Research ◽

10.1161/circresaha.117.310803 ◽

2017 ◽

Vol 121 (6) ◽

Cited By ~ 65

Author(s):

Atsushi Tachibana ◽

Michelle R. Santoso ◽

Morteza Mahmoudi ◽

Praveen Shukla ◽

Lei Wang ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Left Ventricular Ejection Fraction ◽

Cardiac Myocytes ◽

Pluripotent Stem Cells ◽

Pluripotent Stem Cell ◽

Left Ventricular ◽

Left Ventricular Ejection ◽

Ventricular Ejection Fraction ◽

Paracrine Effects

Rationale: Cardiac myocytes derived from pluripotent stem cells have demonstrated the potential to mitigate damage of the infarcted myocardium and improve left ventricular ejection fraction. However, the mechanism underlying the functional benefit is unclear. Objective: To evaluate whether the transplantation of cardiac-lineage differentiated derivatives enhance myocardial viability and restore left ventricular ejection fraction more effectively than undifferentiated pluripotent stem cells after a myocardial injury. Herein, we utilize novel multimodality evaluation of human embryonic stem cells (hESCs), hESC-derived cardiac myocytes (hCMs), human induced pluripotent stem cells (iPSCs), and iPSC-derived cardiac myocytes (iCMs) in a murine myocardial injury model. Methods and Results: Permanent ligation of the left anterior descending coronary artery was induced in immunosuppressed mice. Intramyocardial injection was performed with (1) hESCs (n=9), (2) iPSCs (n=8), (3) hCMs (n=9), (4) iCMs (n=14), and (5) PBS control (n=10). Left ventricular ejection fraction and myocardial viability, measured by cardiac magnetic resonance imaging and manganese-enhanced magnetic resonance imaging, respectively, was significantly improved in hCM- and iCM-treated mice compared with pluripotent stem cell- or control-treated mice. Bioluminescence imaging revealed limited cell engraftment in all treated groups, suggesting that the cell secretions may underlie the repair mechanism. To determine the paracrine effects of the transplanted cells, cytokines from supernatants from all groups were assessed in vitro. Gene expression and immunohistochemistry analyses of the murine myocardium demonstrated significant upregulation of the promigratory, proangiogenic, and antiapoptotic targets in groups treated with cardiac lineage cells compared with pluripotent stem cell and control groups. Conclusions: This study demonstrates that the cardiac phenotype of hCMs and iCMs salvages the injured myocardium effectively than undifferentiated stem cells through their differential paracrine effects.

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Ion Channel Expression and Characterization in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

Stem Cells International ◽

10.1155/2018/6067096 ◽

2018 ◽

Vol 2018 ◽

pp. 1-14 ◽

Cited By ~ 15

Author(s):

Zhihan Zhao ◽

Huan Lan ◽

Ibrahim El-Battrawy ◽

Xin Li ◽

Fanis Buljubasic ◽

...

Keyword(s):

Stem Cell ◽

Ion Channels ◽

Ion Channel ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

Biological Study ◽

Adrenergic Stimulation ◽

Cell Therapies ◽

Channel Expression ◽

Induced Pluripotent

Background. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are providing new possibilities for the biological study, cell therapies, and drug discovery. However, the ion channel expression and functions as well as regulations in hiPSC-CMs still need to be fully characterized. Methods. Cardiomyocytes were derived from hiPS cells that were generated from two healthy donors. qPCR and patch clamp techniques were used for the study. Results. In addition to the reported ion channels, INa, ICa-L, ICa-T, If, INCX, IK1, Ito, IKr, IKs IKATP, IK-pH, ISK1–3, and ISK4, we detected both the expression and currents of ACh-activated (KACh) and Na+-activated (KNa) K+, volume-regulated and calcium-activated (Cl-Ca) Cl−, and TRPV channels. All the detected ion currents except IK1, IKACh, ISK, IKNa, and TRPV1 currents contribute to AP duration. Isoprenaline increased ICa-L, If, and IKs but reduced INa and INCX, without an effect on Ito, IK1, ISK1–3, IKATP, IKr, ISK4, IKNa, ICl-Ca, and ITRPV1. Carbachol alone showed no effect on the tested ion channel currents. Conclusion. Our data demonstrate that most ion channels, which are present in healthy or diseased cardiomyocytes, exist in hiPSC-CMs. Some of them contribute to action potential performance and are regulated by adrenergic stimulation.

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Human pluripotent stem cell-derived cartilaginous organoids promote scaffold-free healing of critical size long bone defects

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02580-7 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Wai Long Tam ◽

Luís Freitas Mendes ◽

Xike Chen ◽

Raphaëlle Lesage ◽

Inge Van Hoven ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Pluripotent Stem Cells ◽

Pluripotent Stem Cell ◽

Critical Size ◽

Bone Defects ◽

Long Bone ◽

Tissue Formation ◽

Induced Pluripotent

Abstract Background Bones have a remarkable capacity to heal upon fracture. Yet, in large defects or compromised conditions healing processes become impaired, resulting in delayed or non-union. Current therapeutic approaches often utilize autologous or allogeneic bone grafts for bone augmentation. However, limited availability of these tissues and lack of predictive biological response result in limitations for clinical demands. Tissue engineering using viable cell-based implants is a strategic approach to address these unmet medical needs. Methods Herein, the in vitro and in vivo cartilage and bone tissue formation potencies of human pluripotent stem cells were investigated. The induced pluripotent stem cells were specified towards the mesodermal lineage and differentiated towards chondrocytes, which subsequently self-assembled into cartilaginous organoids. The tissue formation capacity of these organoids was then challenged in an ectopic and orthotopic bone formation model. Results The derived chondrocytes expressed similar levels of collagen type II as primary human articular chondrocytes and produced stable cartilage when implanted ectopically in vivo. Upon targeted promotion towards hypertrophy and priming with a proinflammatory mediator, the organoids mediated successful bridging of critical size long bone defects in immunocompromised mice. Conclusions These results highlight the promise of induced pluripotent stem cell technology for the creation of functional cartilage tissue intermediates that can be explored for novel bone healing strategies.

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