Scaffold-Mediated Developmental Effects on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes Are Preserved After External Support Removal

Human induced pluripotent stem (hiPS) cells have been used as a cell source for regenerative therapy and disease modeling. The purity of hiPS-cardiomyocytes (hiPS-CMs) has markedly improved with advancements in cell culture and differentiation protocols. However, the morphological features and molecular properties of the relatively immature cells are still unclear, which has hampered their clinical application. The aim of the present study was to investigate the extent to which topographic substrates actively influence hiPS-CMs. hiPS-CMs were seeded on randomized oriented fiber substrate (random), anisotropic aligned fiber substrate (align), and flat non-scaffold substrate (flat). After culturing for one week, the hiPS-CMs on the aligned patterns showed more mature-like properties, including elongated rod shape, shorter duration of action potential, accelerated conduction velocity, and elevated cardiac gene expression. Subsequently, to determine whether this development was irreversible or was altered after withdrawal of the structural support, the hiPS-CMs were harvested from the three different patterns and reseeded on the non-scaffold (flat) pattern. After culturing for one more week, the improvements in morphological and functional properties diminished, although hiPS-CMs pre-cultured on the aligned pattern retained the molecular features of development, which were even more significant as compared to that observed during the pre-culture stage. Our results suggested that the anisotropic fiber substrate can induce the formation of geometrical mimic-oriented heart tissue in a short time. Although the morphological and electrophysiological properties of hiPS-CMs obtained via facilitated maturation somehow rely on the existence of an exterior scaffold, the molecular developmental features were preserved even in the absence of the external support, which might persist throughout hiPS-CM development.

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Dynamic loading of human engineered heart tissue enhances contractile function and drives a desmosome-linked disease phenotype

Science Translational Medicine ◽

10.1126/scitranslmed.abd1817 ◽

2021 ◽

Vol 13 (603) ◽

pp. eabd1817

Author(s):

Jacqueline M. Bliley ◽

Mathilde C. S. C. Vermeer ◽

Rebecca M. Duffy ◽

Ivan Batalov ◽

Duco Kramer ◽

...

Keyword(s):

Dynamic Loading ◽

Disease Modeling ◽

Contractile Function ◽

Three Dimensional ◽

Induced Pluripotent Stem Cell ◽

Heart Tissue ◽

Disease Phenotype ◽

Heart Formation ◽

Induced Pluripotent ◽

And Function

The role that mechanical forces play in shaping the structure and function of the heart is critical to understanding heart formation and the etiology of disease but is challenging to study in patients. Engineered heart tissues (EHTs) incorporating human induced pluripotent stem cell (hiPSC)–derived cardiomyocytes have the potential to provide insight into these adaptive and maladaptive changes. However, most EHT systems cannot model both preload (stretch during chamber filling) and afterload (pressure the heart must work against to eject blood). Here, we have developed a new dynamic EHT (dyn-EHT) model that enables us to tune preload and have unconstrained contractile shortening of >10%. To do this, three-dimensional (3D) EHTs were integrated with an elastic polydimethylsiloxane strip providing mechanical preload and afterload in addition to enabling contractile force measurements based on strip bending. Our results demonstrated that dynamic loading improves the function of wild-type EHTs on the basis of the magnitude of the applied force, leading to improved alignment, conduction velocity, and contractility. For disease modeling, we used hiPSC-derived cardiomyocytes from a patient with arrhythmogenic cardiomyopathy due to mutations in the desmoplakin gene. We demonstrated that manifestation of this desmosome-linked disease state required dyn-EHT conditioning and that it could not be induced using 2D or standard 3D EHT approaches. Thus, a dynamic loading strategy is necessary to provoke the disease phenotype of diastolic lengthening, reduction of desmosome counts, and reduced contractility, which are related to primary end points of clinical disease, such as chamber thinning and reduced cardiac output.

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Mutation-specific differences in arrhythmias and drug responses in CPVT patients: simultaneous patch clamp and video imaging of iPSC derived cardiomyocytes

Molecular Biology Reports ◽

10.1007/s11033-019-05201-y ◽

2019 ◽

Vol 47 (2) ◽

pp. 1067-1077 ◽

Cited By ~ 2

Author(s):

R. P. Pölönen ◽

H. Swan ◽

K. Aalto-Setälä

Keyword(s):

Disease Modeling ◽

Induced Pluripotent Stem Cell ◽

Polymorphic Ventricular Tachycardia ◽

Patient Specific ◽

Electrophysiological Properties ◽

Electrophysiological Measurements ◽

Skin Biopsies ◽

Induced Pluripotent ◽

Almost All ◽

And Control

AbstractCatecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac disease characterized by arrhythmias under adrenergic stress. Mutations in the cardiac ryanodine receptor (RYR2) are the leading cause for CPVT. We characterized electrophysiological properties of CPVT patient-specific induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) carrying different mutations in RYR2 and evaluated effects of carvedilol and flecainide on action potential (AP) and contractile properties of hiPSC-CMs. iPSC-CMs were generated from skin biopsies of CPVT patients carrying exon 3 deletion (E3D) and L4115F mutation in RYR2. APs and contractile movement were recorded simultaneously from the same hiPSC-CMs. Differences in AP properties of ventricular like CMs were seen in CPVT and control CMs: APD90 of both E3D (n = 20) and L4115F (n = 25) CPVT CMs was shorter than in control CMs (n = 15). E3D-CPVT CMs had shortest AP duration, lowest AP amplitude, upstroke velocity and more depolarized diastolic potential than controls. Adrenaline had positive and carvedilol and flecainide negative chronotropic effect in all hiPSC CMs. CPVT CMs had increased amount of delayed after depolarizations (DADs) and early after depolarizations (EADs) after adrenaline exposure. E3D CPVT CMs had the most DADs, EADs, and tachyarrhythmia. Discordant negatively coupled alternans was seen in L4115F CPVT CMs. Carvedilol cured almost all arrhythmias in L4115F CPVT CMs. Both drugs decreased contraction amplitude in all hiPSC CMs. E3D CPVT CMs have electrophysiological properties, which render them more prone to arrhythmias. iPSC-CMs provide a unique platform for disease modeling and drug screening for CPVT. Combining electrophysiological measurements, we can gain deeper insight into mechanisms of arrhythmias.

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Dynamic Loading of Human Engineered Heart Tissue Enhances Contractile Function and Drives Desmosome-linked Disease Phenotype

10.1101/2020.05.25.111690 ◽

2020 ◽

Author(s):

Jacqueline M. Bliley ◽

Mathilde C.S.C Vermeer ◽

Rebecca M. Duffy ◽

Ivan Batalov ◽

Duco Kramer ◽

...

Keyword(s):

Dynamic Loading ◽

Disease Modeling ◽

Contractile Function ◽

Induced Pluripotent Stem Cell ◽

Heart Tissue ◽

Disease Phenotype ◽

Heart Formation ◽

Induced Pluripotent ◽

And Function ◽

Engineered Heart Tissues

ABSTRACTThe role mechanical forces play in shaping the structure and function of the heart is critical to understanding heart formation and the etiology of disease but is challenging to study in patients. Engineered heart tissues (EHTs) incorporating human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have the potential to provide insight into these adaptive and maladaptive changes in the heart. However, most EHT systems are unable to model both preload (stretch during chamber filling) and afterload (pressure the heart must work against to eject blood). Here, we have developed a new dynamic EHT (dyn-EHT) model that enables us to tune preload and have unconstrained fractional shortening of >10%. To do this, 3D EHTs are integrated with an elastic polydimethylsiloxane (PDMS) strip that provides mechanical pre- and afterload to the tissue in addition to enabling contractile force measurements based on strip bending. Our results demonstrate in wild-type EHTs that dynamic loading is beneficial based on the magnitude of the forces, leading to improved alignment, conduction velocity, and contractility. For disease modeling, we use hiPSC–derived cardiomyocytes from a patient with arrhythmogenic cardiomyopathy (ACM) due to mutations in desmoplakin. We demonstrate that manifestation of this desmosome-linked disease state requires the dyn-EHT conditioning and that it cannot be induced using 2D or standard 3D EHT approaches. Thus, dynamic loading strategy is necessary to provoke a disease phenotype (diastolic lengthening, reduction of desmosome counts, and reduced contractility), which are akin to primary endpoints of clinical disease, such as chamber thinning and reduced cardiac output.Single Sentence SummaryDevelopment of a dynamic mechanical loading platform to improve contractile function of engineered heart tissues and study cardiac disease progression.

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Emerging hiPSC Models for Drug Discovery in Neurodegenerative Diseases

International Journal of Molecular Sciences ◽

10.3390/ijms22158196 ◽

2021 ◽

Vol 22 (15) ◽

pp. 8196

Author(s):

Dorit Trudler ◽

Swagata Ghatak ◽

Stuart A. Lipton

Keyword(s):

Drug Discovery ◽

Animal Models ◽

Neurodegenerative Diseases ◽

Disease Modeling ◽

Induced Pluripotent Stem Cell ◽

Neural Function ◽

Neural Cells ◽

Human Samples ◽

Healthy Donors ◽

Induced Pluripotent

Neurodegenerative diseases affect millions of people worldwide and are characterized by the chronic and progressive deterioration of neural function. Neurodegenerative diseases, such as Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington’s disease (HD), represent a huge social and economic burden due to increasing prevalence in our aging society, severity of symptoms, and lack of effective disease-modifying therapies. This lack of effective treatments is partly due to a lack of reliable models. Modeling neurodegenerative diseases is difficult because of poor access to human samples (restricted in general to postmortem tissue) and limited knowledge of disease mechanisms in a human context. Animal models play an instrumental role in understanding these diseases but fail to comprehensively represent the full extent of disease due to critical differences between humans and other mammals. The advent of human-induced pluripotent stem cell (hiPSC) technology presents an advantageous system that complements animal models of neurodegenerative diseases. Coupled with advances in gene-editing technologies, hiPSC-derived neural cells from patients and healthy donors now allow disease modeling using human samples that can be used for drug discovery.

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NRF2 is required for structural and metabolic maturation of human induced pluripotent stem cell-derived ardiomyocytes

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02264-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Xinyuan Zhang ◽

Liang Ye ◽

Hao Xu ◽

Qin Zhou ◽

Bin Tan ◽

...

Keyword(s):

Stem Cell ◽

Oxidative Phosphorylation ◽

Pluripotent Stem Cell ◽

Disease Modeling ◽

Induced Pluripotent Stem Cell ◽

Metabolic Energy ◽

Cell Maturation ◽

Great Promise ◽

Functional Changes ◽

Induced Pluripotent

Abstract Background Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) hold great promise for regenerative medicine and in drugs screening. Despite displaying key cardiomyocyte phenotypic characteristics, they more closely resemble fetal/neonatal cardiomyocytes and are still immature; these cells mainly rely on glucose as a substrate for metabolic energy, while mature cardiomyocytes mainly employ oxidative phosphorylation of fatty acids. Studies showed that the alteration of metabolism pattern from glycolysis to oxidative phosphorylation improve the maturity of hiPSC-CMs. As a transcription factor, accumulating evidences showed the important role of NRF2 in the regulation of energy metabolism, which directly regulates the expression of mitochondrial respiratory complexes. Therefore, we hypothesized that NRF2 is involved in the maturation of hiPSC-CMs. Methods The morphological and functional changes related to mitochondria and cell maturation were analyzed by knock-down and activation of NRF2. Results The results showed that the inhibition of NRF2 led to the retardation of cell maturation. The activation of NRF2 leads to a more mature hiPSC-CMs phenotype, as indicated by the increase of cardiac maturation markers, sarcomere length, calcium transient dynamics, the number and fusion events of mitochondria, and mitochondrial respiration. Bioinformatics analysis showed that in addition to metabolism-related genes, NRF2 also activates the expression of myocardial ion channels. Conclusions These findings indicated that NRF2 plays an important role in the maturation of hiPSC-CMs. The present work provides greater insights into the molecular regulation of hiPSC-CMs metabolism and theoretical basis in drug screening, disease modeling, and alternative treatment.

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A Temperature-Controlled Patch Clamp Platform Demonstrated on Jurkat T Lymphocytes and Human Induced Pluripotent Stem Cell-Derived Neurons

Bioengineering ◽

10.3390/bioengineering7020046 ◽

2020 ◽

Vol 7 (2) ◽

pp. 46 ◽

Cited By ~ 1

Author(s):

Jann Harberts ◽

Max Kusch ◽

John O’Sullivan ◽

Robert Zierold ◽

Robert H. Blick

Keyword(s):

Stem Cell ◽

T Lymphocytes ◽

Patch Clamp ◽

Pluripotent Stem Cell ◽

Room Temperature ◽

Standard Procedure ◽

Induced Pluripotent Stem Cell ◽

Electrophysiological Properties ◽

Temperature Controlled ◽

Induced Pluripotent

Though patch clamping at room temperature is a widely disseminated standard procedure in the electrophysiological community, it does not represent the biological system in mammals at around 37 °C. In order to better mimic the natural environment in electrophysiological studies, we present a custom-built, temperature-controlled patch clamp platform for upright microscopes, which can easily be adapted to any upright patch clamp setup independently, whether commercially available or home built. Our setup can both cool and heat the platform having only small temperature variations of less than 0.5 °C. We demonstrate our setup with patch clamp measurements at 36 °C on Jurkat T lymphocytes and human induced pluripotent stem cell-derived neurons. Passive membrane parameters and characteristic electrophysiological properties, such as the gating properties of voltage-gated ion channels and the firing of action potentials, are compared to measurements at room temperature. We observe that many processes that are not explicitly considered as temperature dependent show changes with temperature. Thus, we believe in the need of a temperature control in patch clamp measurements if improved physiological conditions are required. Furthermore, we advise researchers to only compare electrophysiological results directly that have been measured at similar temperatures since small variations in cellular properties might be caused by temperature alterations.

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Disease Modeling and Disease Gene Discovery in Cardiomyopathies: A Molecular Study of Induced Pluripotent Stem Cell Generated Cardiomyocytes

International Journal of Molecular Sciences ◽

10.3390/ijms22073311 ◽

2021 ◽

Vol 22 (7) ◽

pp. 3311

Author(s):

Satish Kumar ◽

Joanne E. Curran ◽

Kashish Kumar ◽

Erica DeLeon ◽

Ana C. Leandro ◽

...

Keyword(s):

Stem Cell ◽

Pluripotent Stem Cell ◽

Disease Gene ◽

Disease Modeling ◽

Gene Discovery ◽

Induced Pluripotent Stem Cell ◽

P Value ◽

Disease Gene Discovery ◽

Induced Pluripotent

The in vitro modeling of cardiac development and cardiomyopathies in human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs) provides opportunities to aid the discovery of genetic, molecular, and developmental changes that are causal to, or influence, cardiomyopathies and related diseases. To better understand the functional and disease modeling potential of iPSC-differentiated CMs and to provide a proof of principle for large, epidemiological-scale disease gene discovery approaches into cardiomyopathies, well-characterized CMs, generated from validated iPSCs of 12 individuals who belong to four sibships, and one of whom reported a major adverse cardiac event (MACE), were analyzed by genome-wide mRNA sequencing. The generated CMs expressed CM-specific genes and were highly concordant in their total expressed transcriptome across the 12 samples (correlation coefficient at 95% CI =0.92 ± 0.02). The functional annotation and enrichment analysis of the 2116 genes that were significantly upregulated in CMs suggest that generated CMs have a transcriptomic and functional profile of immature atrial-like CMs; however, the CMs-upregulated transcriptome also showed high overlap and significant enrichment in primary cardiomyocyte (p-value = 4.36 × 10−9), primary heart tissue (p-value = 1.37 × 10−41) and cardiomyopathy (p-value = 1.13 × 10−21) associated gene sets. Modeling the effect of MACE in the generated CMs-upregulated transcriptome identified gene expression phenotypes consistent with the predisposition of the MACE-affected sibship to arrhythmia, prothrombotic, and atherosclerosis risk.

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Machine Learning Approach to Automated Quality Identification of Human Induced Pluripotent Stem Cell Colony Images

Computational and Mathematical Methods in Medicine ◽

10.1155/2016/3091039 ◽

2016 ◽

Vol 2016 ◽

pp. 1-15 ◽

Cited By ~ 13

Author(s):

Henry Joutsijoki ◽

Markus Haponen ◽

Jyrki Rasku ◽

Katriina Aalto-Setälä ◽

Martti Juhola

Keyword(s):

Machine Learning ◽

Stem Cell ◽

Pluripotent Stem Cell ◽

Disease Modeling ◽

Induced Pluripotent Stem Cell ◽

Patient Specific ◽

Support Vector ◽

Ips Cell ◽

Cell Technology ◽

Induced Pluripotent

The focus of this research is on automated identification of the quality of human induced pluripotent stem cell (iPSC) colony images. iPS cell technology is a contemporary method by which the patient’s cells are reprogrammed back to stem cells and are differentiated to any cell type wanted. iPS cell technology will be used in future to patient specific drug screening, disease modeling, and tissue repairing, for instance. However, there are technical challenges before iPS cell technology can be used in practice and one of them is quality control of growing iPSC colonies which is currently done manually but is unfeasible solution in large-scale cultures. The monitoring problem returns to image analysis and classification problem. In this paper, we tackle this problem using machine learning methods such as multiclass Support Vector Machines and several baseline methods together with Scaled Invariant Feature Transformation based features. We perform over 80 test arrangements and do a thorough parameter value search. The best accuracy (62.4%) for classification was obtained by using ak-NN classifier showing improved accuracy compared to earlier studies.

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Contribution of two-pore K+ channels to cardiac ventricular action potential revealed using human iPSC-derived cardiomyocytes

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00107.2017 ◽

2017 ◽

Vol 312 (6) ◽

pp. H1144-H1153 ◽

Cited By ~ 13

Author(s):

Sam Chai ◽

Xiaoping Wan ◽

Drew M. Nassal ◽

Haiyan Liu ◽

Christine S. Moravec ◽

...

Keyword(s):

Action Potential ◽

Expression Profile ◽

Cardiac Electrophysiology ◽

Human Heart ◽

Induced Pluripotent Stem Cell ◽

Heart Tissue ◽

Induced Pluripotent ◽

Interfering Rna ◽

Human Ipsc ◽

Physiological Systems

Two-pore K+ (K2p) channels have been described in modulating background conductance as leak channels in different physiological systems. In the heart, the expression of K2p channels is heterogeneous with equivocation regarding their functional role. Our objective was to determine the K2p expression profile and their physiological and pathophysiological contribution to cardiac electrophysiology. Induced pluripotent stem cells (iPSCs) generated from humans were differentiated into cardiomyocytes (iPSC-CMs). mRNA was isolated from these cells, commercial iPSC-CM (iCells), control human heart ventricular tissue (cHVT), and ischemic (iHF) and nonischemic heart failure tissues (niHF). We detected 10 K2p channels in the heart. Comparing quantitative PCR expression of K2p channels between human heart tissue and iPSC-CMs revealed K2p1.1, K2p2.1, K2p5.1, and K2p17.1 to be higher expressed in cHVT, whereas K2p3.1 and K2p13.1 were higher in iPSC-CMs. Notably, K2p17.1 was significantly lower in niHF tissues compared with cHVT. Action potential recordings in iCells after K2p small interfering RNA knockdown revealed prolongations in action potential depolarization at 90% repolarization for K2p2.1, K2p3.1, K2p6.1, and K2p17.1. Here, we report the expression level of 10 human K2p channels in iPSC-CMs and how they compared with cHVT. Importantly, our functional electrophysiological data in human iPSC-CMs revealed a prominent role in cardiac ventricular repolarization for four of these channels. Finally, we also identified K2p17.1 as significantly reduced in niHF tissues and K2p4.1 as reduced in niHF compared with iHF. Thus, we advance the notion that K2p channels are emerging as novel players in cardiac ventricular electrophysiology that could also be remodeled in cardiac pathology and therefore contribute to arrhythmias. NEW & NOTEWORTHY Two-pore K+ (K2p) channels are traditionally regarded as merely background leak channels in myriad physiological systems. Here, we describe the expression profile of K2p channels in human-induced pluripotent stem cell-derived cardiomyocytes and outline a salient role in cardiac repolarization and pathology for multiple K2p channels.

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Generation of 2.5D lung bud organoid from human induced pluripotent stem cell

Clinical Hemorheology and Microcirculation ◽

10.3233/ch-219111 ◽

2021 ◽

pp. 1-14

Author(s):

Xun Xu ◽

Yan Nie ◽

Weiwei Wang ◽

Imran Ullah ◽

Wing Tai Tung ◽

...

Keyword(s):

Single Cell ◽

Disease Modeling ◽

3D Culture ◽

Induced Pluripotent Stem Cell ◽

Patient Specific ◽

Homogeneous Distribution ◽

Cell Seeding ◽

Differentiation Potential ◽

Induced Pluripotent ◽

Defined Culture

Human induced pluripotent stem cells (hiPSCs) are a promising cell source to generate the patient-specific lung organoid given their superior differentiation potential. However, the current 3D cell culture approach is tedious and time-consuming with a low success rate and high batch-to-batch variability. Here, we explored the establishment of lung bud organoids by systematically adjusting the initial confluence levels and homogeneity of cell distribution. The efficiency of single cell seeding and clump seeding was compared. Instead of the traditional 3D culture, we established a 2.5D organoid culture to enable the direct monitoring of the internal structure via microscopy. It was found that the cell confluence and distribution prior to induction were two key parameters, which strongly affected hiPSC differentiation trajectories. Lung bud organoids with positive expression of NKX 2.1, in a single-cell seeding group with homogeneously distributed hiPSCs at 70%confluence (SC_70%_hom) or a clump seeding group with heterogeneously distributed cells at 90%confluence (CL_90%_het), can be observed as early as 9 days post induction. These results suggest that a successful lung bud organoid formation with single-cell seeding of hiPSCs requires a moderate confluence and homogeneous distribution of cells, while high confluence would be a prominent factor to promote the lung organoid formation when seeding hiPSCs as clumps. 2.5D organoids generated with defined culture conditions could become a simple, efficient, and valuable tool facilitating drug screening, disease modeling and personalized medicine.

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