Selected Ionotropic Receptors and Voltage-Gated Ion Channels: More Functional Competence for Human Induced Pluripotent Stem Cell (iPSC)-Derived Nociceptors

Preclinical research using different rodent model systems has largely contributed to the scientific progress in the pain field, however, it suffers from interspecies differences, limited access to human models, and ethical concerns. Human induced pluripotent stem cells (iPSCs) offer major advantages over animal models, i.e., they retain the genome of the donor (patient), and thus allow donor-specific and cell-type specific research. Consequently, human iPSC-derived nociceptors (iDNs) offer intriguingly new possibilities for patient-specific, animal-free research. In the present study, we characterized iDNs based on the expression of well described nociceptive markers and ion channels, and we conducted a side-by-side comparison of iDNs with mouse sensory neurons. Specifically, immunofluorescence (IF) analyses with selected markers including early somatosensory transcription factors (BRN3A/ISL1/RUNX1), the low-affinity nerve growth factor receptor (p75), hyperpolarization-activated cyclic nucleotide-gated channels (HCN), as well as high voltage-gated calcium channels (VGCC) of the CaV2 type, calcium permeable TRPV1 channels, and ionotropic GABAA receptors, were used to address the characteristics of the iDN phenotype. We further combined IF analyses with microfluorimetric Ca2+ measurements to address the functionality of these ion channels in iDNs. Thus, we provide a detailed morphological and functional characterization of iDNs, thereby, underpinning their enormous potential as an animal-free alternative for human specific research in the pain field for unveiling pathophysiological mechanisms and for unbiased, disease-specific personalized drug development.

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Decoding Genetics of Congenital Heart Disease Using Patient-Derived Induced Pluripotent Stem Cells (iPSCs)

Frontiers in Cell and Developmental Biology ◽

10.3389/fcell.2021.630069 ◽

2021 ◽

Vol 9 ◽

Author(s):

Hui Lin ◽

Kim L. McBride ◽

Vidu Garg ◽

Ming-Tao Zhao

Keyword(s):

Stem Cells ◽

Congenital Heart Disease ◽

Heart Disease ◽

Induced Pluripotent Stem Cells ◽

Genome Sequencing ◽

Pluripotent Stem Cells ◽

Congenital Heart ◽

Model Systems ◽

Patient Specific ◽

Induced Pluripotent

Congenital heart disease (CHD) is the most common cause of infant death associated with birth defects. Recent next-generation genome sequencing has uncovered novel genetic etiologies of CHD, from inherited and de novo variants to non-coding genetic variants. The next phase of understanding the genetic contributors of CHD will be the functional illustration and validation of this genome sequencing data in cellular and animal model systems. Human induced pluripotent stem cells (iPSCs) have opened up new horizons to investigate genetic mechanisms of CHD using clinically relevant and patient-specific cardiac cells such as cardiomyocytes, endothelial/endocardial cells, cardiac fibroblasts and vascular smooth muscle cells. Using cutting-edge CRISPR/Cas9 genome editing tools, a given genetic variant can be corrected in diseased iPSCs and introduced to healthy iPSCs to define the pathogenicity of the variant and molecular basis of CHD. In this review, we discuss the recent progress in genetics of CHD deciphered by large-scale genome sequencing and explore how genome-edited patient iPSCs are poised to decode the genetic etiologies of CHD by coupling with single-cell genomics and organoid technologies.

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Characterization of Functional Human Skeletal Myotubes and Neuromuscular Junction Derived—From the Same Induced Pluripotent Stem Cell Source

Bioengineering ◽

10.3390/bioengineering7040133 ◽

2020 ◽

Vol 7 (4) ◽

pp. 133

Author(s):

Xiufang Guo ◽

Agnes Badu-Mensah ◽

Michael C. Thomas ◽

Christopher W. McAleer ◽

James J. Hickman

Keyword(s):

Stem Cell ◽

Pluripotent Stem Cell ◽

Disease Modeling ◽

Neuromuscular Junctions ◽

Functional Characterization ◽

Induced Pluripotent Stem Cell ◽

Patient Specific ◽

Type I ◽

Induced Pluripotent

In vitro generation of functional neuromuscular junctions (NMJs) utilizing the same induced pluripotent stem cell (iPSC) source for muscle and motoneurons would be of great value for disease modeling and tissue engineering. Although, differentiation and characterization of iPSC-derived motoneurons are well established, and iPSC-derived skeletal muscle (iPSC-SKM) has been reported, there is a general lack of systemic and functional characterization of the iPSC-SKM. This study performed a systematic characterization of iPSC-SKM differentiated using a serum-free, small molecule-directed protocol. Morphologically, the iPSC-SKM demonstrated the expression and appropriate distribution of acetylcholine, ryanodine and dihydropyridine receptors. Fiber type analysis revealed a mixture of human fast (Type IIX, IIA) and slow (Type I) muscle types and the absence of animal Type IIB fibers. Functionally, the iPSC-SKMs contracted synchronously upon electrical stimulation, with the contraction force comparable to myofibers derived from primary myoblasts. Most importantly, when co-cultured with human iPSC-derived motoneurons from the same iPSC source, the myofibers contracted in response to motoneuron stimulation indicating the formation of functional NMJs. By demonstrating comparable structural and functional capacity to primary myoblast-derived myofibers, this defined, iPSC-SKM system, as well as the personal NMJ system, has applications for patient-specific drug testing and investigation of muscle physiology and disease.

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Abstract P482: Elucidating And Characterizing The Molecular Mechanistic Role Of Phospholamban L39 Stop In The Pathophysiology Of Cardiomyopathy Using Patient-derived Human Induced Pluripotent Stem Cells And Humanized Knock-in Mouse Model Systems

Circulation Research ◽

10.1161/res.129.suppl_1.p482 ◽

2021 ◽

Vol 129 (Suppl_1) ◽

Author(s):

Anichavezhi Devendran ◽

Rasheed Bailey ◽

Sumanta Kar ◽

Francesca Stillitano ◽

Irene Turnbull ◽

...

Keyword(s):

Stem Cells ◽

Mouse Model ◽

Mononuclear Cells ◽

Embryonic Stem ◽

Model Systems ◽

Patient Specific ◽

Induced Pluripotent

Background: Heart failure (HF) is a complex clinical condition associated with substantial morbidity and mortality worldwide. The contractile dysfunction and arrhythmogenesis related to HF has been linked to the remodelling of calcium (Ca ++ ) handling. Phospholamban (PLN) has emerged as a key regulator of intracellular Ca ++ concentration. Of the PLN mutations, L39X is intriguing as it has not been fully characterized. This mutation is believed to be functionally equivalent to PLN null (KO) but contrary to PLN KO mice, L39X carriers develop a lethal cardiomyopathy (CMP). Our study aims at using induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) from homozygous L39X carriers to elucidate the role of L39X in human pathophysiology. Our plan also involves the characterization of humanized L39X knock-in mice (KM), which we hypothesize will develop a CMP from mis-localization of PLN and disruption of Ca ++ signalling. Methodology and Results: Mononuclear cells from Hom L39X carriers were obtained to generate 11 integration-free patient-specific iPSC clones. The iPSC-CMs were derived using established protocols. Compared to the WT iPSC-CMs, the Hom L39X derived-CMs PLN had an abnormal cytoplasmic distribution and formed intracellular aggregates, with the loss of perinuclear localization. There was also a 70% and 50% reduction of mRNA and protein expression of PLN respectively in L39X compared to WT iPSC-CMs. These findings indicated that L39X PLN is both under-expressed and mis-localized within the cell. To validate this observation in-vivo, we genetically modified FVB mice to harbour the human L39X. Following electroporation, positively transfected mouse embryonic stem cells were injected into host blastocysts to make humanized KM that were subsequently used to generate either a protamine-Cre (endogenous PLN driven expression) or a cardiac TNT mouse (i.e., CMP specific). Conclusion: Our data confirm an abnormal intracellular distribution of PLN, with the loss of perinuclear accumulation and mis-localization, suggestive of ineffective targeting to or retention of L39X. The mouse model will be critically important to validate the in-vitro observations and provides an ideal platform for future studies centred on the development of novel therapeutic strategies including virally delivered CRISPR/Cas9 for in-vivo gene editing and testing of biochemical signalling pathways.

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Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Afford New Opportunities in Inherited Cardiovascular Disease Modeling

Cardiology Research and Practice ◽

10.1155/2016/3582380 ◽

2016 ◽

Vol 2016 ◽

pp. 1-17 ◽

Cited By ~ 6

Author(s):

Daniel R. Bayzigitov ◽

Sergey P. Medvedev ◽

Elena V. Dementyeva ◽

Sevda A. Bayramova ◽

Evgeny A. Pokushalov ◽

...

Keyword(s):

Cardiovascular Disease ◽

Stem Cell ◽

Pluripotent Stem Cell ◽

Molecular Mechanisms ◽

Disease Modeling ◽

Induced Pluripotent Stem Cell ◽

Model Systems ◽

Laboratory Animals ◽

Patient Specific ◽

Induced Pluripotent

Fundamental studies of molecular and cellular mechanisms of cardiovascular disease pathogenesis are required to create more effective and safer methods of their therapy. The studies can be carried out only when model systems that fully recapitulate pathological phenotype seen in patients are used. Application of laboratory animals for cardiovascular disease modeling is limited because of physiological differences with humans. Since discovery of induced pluripotency generating induced pluripotent stem cells has become a breakthrough technology in human disease modeling. In this review, we discuss a progress that has been made in modeling inherited arrhythmias and cardiomyopathies, studying molecular mechanisms of the diseases, and searching for and testing drug compounds using patient-specific induced pluripotent stem cell-derived cardiomyocytes.

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Polyunsaturated fatty acid analogues differentially affect cardiac NaV, CaV, and KV channels through unique mechanisms

eLife ◽

10.7554/elife.51453 ◽

2020 ◽

Vol 9 ◽

Cited By ~ 3

Author(s):

Briana M Bohannon ◽

Alicia de la Cruz ◽

Xiaoan Wu ◽

Jessica J Jowais ◽

Marta E Perez ◽

...

Keyword(s):

Ion Channels ◽

Action Potential ◽

Induced Pluripotent Stem Cell ◽

Voltage Gated ◽

Kv Channels ◽

Cardiac Ion Channels ◽

Induced Pluripotent ◽

Voltage Gated Ion Channels ◽

Ventricular Action Potential ◽

Gated Ion Channels

The cardiac ventricular action potential depends on several voltage-gated ion channels, including NaV, CaV, and KV channels. Mutations in these channels can cause Long QT Syndrome (LQTS) which increases the risk for ventricular fibrillation and sudden cardiac death. Polyunsaturated fatty acids (PUFAs) have emerged as potential therapeutics for LQTS because they are modulators of voltage-gated ion channels. Here we demonstrate that PUFA analogues vary in their selectivity for human voltage-gated ion channels involved in the ventricular action potential. The effects of specific PUFA analogues range from selective for a specific ion channel to broadly modulating cardiac ion channels from all three families (NaV, CaV, and KV). In addition, a PUFA analogue selective for the cardiac IKs channel (Kv7.1/KCNE1) is effective in shortening the cardiac action potential in human-induced pluripotent stem cell-derived cardiomyocytes. Our data suggest that PUFA analogues could potentially be developed as therapeutics for LQTS and cardiac arrhythmia.

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Model systems for studying cellular mechanisms of SCN1A-related epilepsy

Journal of Neurophysiology ◽

10.1152/jn.00824.2015 ◽

2016 ◽

Vol 115 (4) ◽

pp. 1755-1766 ◽

Cited By ~ 27

Author(s):

Soleil S. Schutte ◽

Ryan J. Schutte ◽

Eden V. Barragan ◽

Diane K. O'Dowd

Keyword(s):

Induced Pluripotent Stem Cell ◽

Febrile Seizures ◽

Model Systems ◽

Dravet Syndrome ◽

Response To Treatment ◽

Patient Specific ◽

Cellular Mechanisms ◽

Gene Encoding ◽

Induced Pluripotent ◽

Febrile Seizures Plus

Mutations in SCN1A, the gene encoding voltage-gated sodium channel NaV1.1, cause a spectrum of epilepsy disorders that range from genetic epilepsy with febrile seizures plus to catastrophic disorders such as Dravet syndrome. To date, more than 1,250 mutations in SCN1A have been linked to epilepsy. Distinct effects of individual SCN1A mutations on neuronal function are likely to contribute to variation in disease severity and response to treatment in patients. Several model systems have been used to explore seizure genesis in SCN1A epilepsies. In this article we review what has been learned about cellular mechanisms and potential new therapies from these model systems, with a particular emphasis on the novel model system of knockin Drosophila and a look toward the future with expanded use of patient-specific induced pluripotent stem cell-derived neurons.

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Voltage-gated ion channels mediate the electrotaxis of glioblastoma cells in a hybrid PMMA/PDMS microdevice

10.1101/2020.02.14.948638 ◽

2020 ◽

Author(s):

Hsieh-Fu Tsai ◽

Camilo IJspeert ◽

Amy Q. Shen

Keyword(s):

Machine Learning ◽

Ion Channels ◽

Single Cell ◽

Cell Tracking ◽

Cancer Metastasis ◽

Single Cell Analysis ◽

Single Cells ◽

Patient Specific ◽

Glioblastoma Cells ◽

Voltage Gated

Transformed astrocytes in the most aggressive form cause glioblastoma, the most common cancer in central nervous system with high mortality. The physiological electric field by neuronal local field potentials and tissue polarity may guide the infiltration of glioblastoma cells through the electrotaxis process. However, microenvironments with multiplex gradients are difficult to create. In this work, we have developed a hybrid microfluidic platform to study glioblastoma electrotaxis in controlled microenvironments with high through-put quantitative analysis by a machine learning-powered single cell tracking software. By equalizing the hydrostatic pressure difference between inlets and outlets of the microchannel, uniform single cells can be seeded reliably inside the microdevice. The electrotaxis of two glioblastoma models, T98G and U-251MG, require optimal laminin-containing extracellular matrix and exhibits opposite directional and electro-alignment tendencies. Calcium signaling is a key contributor in glioblastoma pathophysiology but its role in glioblastoma electrotaxis is still an open question. Anodal T98G electrotaxis and cathodal U-251MG electrotaxis require the presence of extracellular calcium cations. U-251MG electrotaxis is dependent on the P/Q-type voltage-gated calcium channel (VGCC) and T98G is dependent on the R-type VGCC. U-251MG and T98G electrotaxis are also mediated by A-type (rapidly inactivating) voltage-gated potassium channels and acid-sensing sodium channels. The involvement of multiple ion channels suggests that the glioblastoma electrotaxis is complex and patient-specific ion channel expression can be critical to develop personalized therapeutics to fight against cancer metastasis. The hybrid microfluidic design and machine learning-powered single cell analysis provide a simple and flexible platform for quantitative investigation of complicated biological systems.

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Challenges and opportunities for modeling monogenic and complex disorders of the human retina via induced pluripotent stem cell technology

Medizinische Genetik ◽

10.1515/medgen-2021-2092 ◽

2021 ◽

Vol 33 (3) ◽

pp. 221-227

Author(s):

Karolina Plössl ◽

Andrea Milenkovic ◽

Bernhard H. F. Weber

Keyword(s):

Stem Cell ◽

Pluripotent Stem Cell ◽

Induced Pluripotent Stem Cell ◽

Model Systems ◽

Patient Specific ◽

Human Retina ◽

Stem Cell Technology ◽

Complex Disorders ◽

Cell Technology ◽

Induced Pluripotent

Abstract The human retina is a highly structured and complex neurosensory tissue central to perceiving and processing visual signals. In a healthy individual, the close interplay between the neuronal retina, the adjacent retinal pigment epithelium and the underlying blood supply, the choriocapillaris, is critical for maintaining eyesight over a lifetime. An impairment of this delicate and metabolically highly active system, caused by genetic alteration, environmental impact or both, results in a multitude of pathological phenotypes of the retina. Understanding and treating these disease processes are motivated by a marked medical need in young as well as in older patients. While naturally occurring or gene-manipulated animal models have been used successfully in ophthalmological research for many years, recent advances in induced pluripotent stem cell technology have opened up new avenues to generate patient-derived retinal model systems. Here, we explore to what extent these cellular models can be useful to mirror human pathologies in vitro ultimately allowing to analyze disease mechanisms and testing treatment options in the cell type of interest on an individual patient-specific genetic background.

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Pluripotency of human embryonic and induced pluripotent stem cells for cardiac and vascular regeneration

Thrombosis and Haemostasis ◽

10.1160/th09-07-0507 ◽

2010 ◽

Vol 104 (07) ◽

pp. 23-29 ◽

Cited By ~ 9

Author(s):

Kenneth R. Boheler

Keyword(s):

Stem Cells ◽

Pluripotent Stem Cells ◽

Ips Cells ◽

Scientific Investigation ◽

Model Systems ◽

Patient Specific ◽

Congenital Defects ◽

Vascular Regeneration ◽

The Creation ◽

Induced Pluripotent

SummaryCardiac and vascular abnormalities and disease syndromes are major causes of death both during human development and with aging. To identify the cause of congenital defects and to combat this epidemic in the aging population, new models must be created for scientific investigation and new therapies must be developed. Recent advances in pluripotent stem cell biology offer renewed hope for tackling these problems. Of particular importance has been the creation of induced pluripotent (iPS) cells from adult tissues and organs through the forced expression of two to four transcription factors. Moreover, iPS cells, which are phenotypically indistinguishable from embryonic stem (ES) cells, can be generated from any patient. This unique capacity when coupled with samples from patients who have congenital and genetic defects of unknown aetiology should permit the creation of new model systems that foment scientific investigation. Moreover, creation of patient-specific cells should overcome many of the immunological limitations that currently impede therapeutic applications associated with other pluripotent stem cells and their derivatives. The aims of this paper will be to discuss cardiac and vascular diseases and show how iPS cells may be employed to overcome some of the most significant scientific and clinical hurdles facing this field.

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Model Systems for Addressing Mechanism of Arrhythmogenesis in Cardiac Repair

Current Cardiology Reports ◽

10.1007/s11886-021-01498-z ◽

2021 ◽

Vol 23 (6) ◽

Author(s):

Xiao-Dong Zhang ◽

Phung N. Thai ◽

Deborah K. Lieu ◽

Nipavan Chiamvimonvat

Keyword(s):

Cardiac Arrhythmias ◽

Cardiac Repair ◽

Mitigation Strategies ◽

Model Systems ◽

Patient Specific ◽

Computational Studies ◽

Cardiac Arrhythmogenesis ◽

Cell Based Therapy ◽

Current Review ◽

Induced Pluripotent

Abstract Purpose of Review Cardiac cell-based therapy represents a promising approach for cardiac repair. However, one of the main challenges is cardiac arrhythmias associated with stem cell transplantation. The current review summarizes the recent progress in model systems for addressing mechanisms of arrhythmogenesis in cardiac repair. Recent Findings Animal models have been extensively developed for mechanistic studies of cardiac arrhythmogenesis. Advances in human induced pluripotent stem cells (hiPSCs), patient-specific disease models, tissue engineering, and gene editing have greatly enhanced our ability to probe the mechanistic bases of cardiac arrhythmias. Additionally, recent development in multiscale computational studies and machine learning provides yet another powerful tool to quantitatively decipher the mechanisms of cardiac arrhythmias. Summary Advancing efforts towards the integrations of experimental and computational studies are critical to gain insights into novel mitigation strategies for cardiac arrhythmias in cell-based therapy.

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