scholarly journals APOE4 disrupts intracellular lipid homeostasis in human iPSC-derived glia

2021 ◽  
Vol 13 (583) ◽  
pp. eaaz4564
Author(s):  
Grzegorz Sienski ◽  
Priyanka Narayan ◽  
Julia Maeve Bonner ◽  
Nora Kory ◽  
Sebastian Boland ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
Disease Risk ◽  
Induced Pluripotent Stem Cell ◽  
Lipid Homeostasis ◽  
Intracellular Lipid ◽  
Apolipoprotein E Gene ◽  
Genome Wide ◽  
Induced Pluripotent ◽  
Human Ipsc ◽  
Altered Lipid

The E4 allele of the apolipoprotein E gene (APOE) has been established as a genetic risk factor for many diseases including cardiovascular diseases and Alzheimer’s disease (AD), yet its mechanism of action remains poorly understood. APOE is a lipid transport protein, and the dysregulation of lipids has recently emerged as a key feature of several neurodegenerative diseases including AD. However, it is unclear how APOE4 perturbs the intracellular lipid state. Here, we report that APOE4, but not APOE3, disrupted the cellular lipidomes of human induced pluripotent stem cell (iPSC)–derived astrocytes generated from fibroblasts of APOE4 or APOE3 carriers, and of yeast expressing human APOE isoforms. We combined lipidomics and unbiased genome-wide screens in yeast with functional and genetic characterization to demonstrate that human APOE4 induced altered lipid homeostasis. These changes resulted in increased unsaturation of fatty acids and accumulation of intracellular lipid droplets both in yeast and in APOE4-expressing human iPSC-derived astrocytes. We then identified genetic and chemical modulators of this lipid disruption. We showed that supplementation of the culture medium with choline (a soluble phospholipid precursor) restored the cellular lipidome to its basal state in APOE4-expressing human iPSC-derived astrocytes and in yeast expressing human APOE4. Our study illuminates key molecular disruptions in lipid metabolism that may contribute to the disease risk linked to the APOE4 genotype. Our study suggests that manipulating lipid metabolism could be a therapeutic approach to help alleviate the consequences of carrying the APOE4 allele.

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

2017 ◽  
Vol 312 (6) ◽  
pp. H1144-H1153 ◽  
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.

scholarly journals Robust Expression of Functional NMDA Receptors in Human Induced Pluripotent Stem Cell-Derived Neuronal Cultures Using an Accelerated Protocol

2021 ◽  
Vol 14 ◽  
Author(s):  
Jacob B. Ruden ◽  
Mrinalini Dixit ◽  
José C. Zepeda ◽  
Brad A. Grueter ◽  
Laura L. Dugan
Keyword(s):  
Nmda Receptors ◽  
Neural Progenitor Cells ◽  
Induced Pluripotent Stem Cell ◽  
Calcium Flux ◽  
Neuronal Cultures ◽  
Mature Neurons ◽  
Health And Disease ◽  
Nervous System Function ◽  
Induced Pluripotent ◽  
Human Ipsc

N-methyl-D-aspartate (NMDA) receptors are critical for higher-order nervous system function, but in previously published protocols to convert human induced pluripotent stem cells (iPSCs) to mature neurons, functional NMDA receptors (NMDARs) are often either not reported or take an extended time to develop. Here, we describe a protocol to convert human iPSC-derived neural progenitor cells (NPCs) to mature neurons in only 37 days. We demonstrate that the mature neurons express functional NMDARs exhibiting ligand-activated calcium flux, and we document the presence of NMDAR-mediated electrically evoked postsynaptic current. In addition to being more rapid than previous procedures, our protocol is straightforward, does not produce organoids which are difficult to image, and does not involve co-culture with rodent astrocytes. This could enhance our ability to study primate/human-specific aspects of NMDAR function and signaling in health and disease.

scholarly journals Genome-wide molecular effects of the neuropsychiatric 16p11 CNVs in an iPSC-to-iN neuronal model

2020 ◽  
Author(s):  
Thomas R. Ward ◽  
Xianglong Zhang ◽  
Louis C. Leung ◽  
Bo Zhou ◽  
Kristin Muench ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Cell Model ◽  
Copy Number Variants ◽  
Induced Pluripotent Stem Cell ◽  
Neuronal Model ◽  
Autism Spectrum ◽  
Genome Wide ◽  
Induced Pluripotent ◽  
Methylation Patterns

AbstractCopy number variants (CNVs), either deletions or duplications, at the 16p11.2 locus in the human genome are known to increase the risk for autism spectrum disorders (ASD), schizophrenia, and for several other developmental conditions. Here, we investigate the global effects on gene expression and DNA methylation using a 16p11.2 CNV patient-derived induced pluripotent stem cell (iPSC) to induced neuron (iN) cell model system. This approach revealed genome-wide and cell-type specific alterations to both gene expression and DNA methylation patterns and also yielded specific leads on genes potentially contributing to some of the known 16p11.2 patient phenotypes. PCSK9 is identified as a possible contributing factor to the symptoms seen in carriers of the 16p11.2 CNVs. The protocadherin (PCDH) gene family is found to have altered DNA methylation patterns in the CNV patient samples. The iPSC lines used for this study are available through a repository as a resource for research into the molecular etiology of the clinical phenotypes of 16p11.2 CNVs and into that of neuropsychiatric and neurodevelopmental disorders in general.

scholarly journals Chromatin compartment dynamics in a haploinsufficient model of cardiac laminopathy

2019 ◽  
Author(s):  
Alessandro Bertero ◽  
Paul A. Fields ◽  
Alec S. T. Smith ◽  
Andrea Leonard ◽  
Kevin Beussman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Large Scale ◽  
Induced Pluripotent Stem Cell ◽  
Lamin A ◽  
Chromosome Conformation ◽  
Human Cardiomyocytes ◽  
Nuclear Lamins ◽  
Genome Wide ◽  
Induced Pluripotent ◽  
Gene Expression Alterations

AbstractPathogenic mutations in A-type nuclear lamins cause dilated cardiomyopathy, which is postulated to result from dysregulated gene expression due to changes in chromatin organization into active and inactive compartments. To test this, we performed genome-wide chromosome conformation analyses (Hi-C) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with a haploinsufficient mutation for lamin A/C. Compared to gene-corrected cells, mutant hiPSC-CMs have marked electrophysiological and contractile alterations, with modest gene expression changes. While large-scale changes in chromosomal topology are evident, differences in chromatin compartmentalization are limited to a few hotspots that escape inactivation during cardiogenesis. These regions exhibit upregulation of multiple non-cardiac genes including CACNA1A, encoding for neuronal P/Q-type calcium channels. Pharmacological inhibition of the resulting current partially mitigates the electrical alterations. On the other hand, A/B compartment changes do not explain most gene expression alterations in mutant hiPSC-CMs. We conclude that global errors in chromosomal compartmentation are not the primary pathogenic mechanism in heart failure due to lamin A/C haploinsufficiency.SummaryBertero et al. observe that lamin A/C haploinsufficiency in human cardiomyocytes markedly alters electrophysiology, contractility, gene expression, and chromosomal topology. Contrary to expectations, however, changes in chromatin compartments involve just few regions, and most dysregulated genes lie outside these hotspots.Condensed titleGenomic effects of lamin A/C haploinsufficiency

scholarly journals Population-scale proteome variation in human induced pluripotent stem cells

2018 ◽  
Author(s):  
Bogdan A Mirauta ◽  
Daniel D Seaton ◽  
Dalila Bensaddek ◽  
Alejandro Brenes ◽  
Marc J Bonder ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Induced Pluripotent Stem Cell ◽  
Protein Abundance ◽  
Protein Protein Interactions ◽  
Genetic Determinants ◽  
Ips Cell ◽  
Transcriptome Variation ◽  
Regulatory Effects ◽  
Induced Pluripotent ◽  
Human Ipsc

AbstractRealising the potential of human induced pluripotent stem cell (iPSC) technology for drug discovery, disease modelling and cell therapy requires an understanding of variability across iPSC lines. While previous studies have characterized iPS cell lines genetically and transcriptionally, little is known about the variability of the iPSC proteome. Here, we present the first comprehensive proteomic iPSC dataset, analysing 202 iPSC lines derived from 151 donors. We characterise the major genetic determinants affecting proteome and transcriptome variation across iPSC lines and identify key regulatory mechanisms affecting variation in protein abundance. Our data identified >700 human iPSC protein quantitative trait loci (pQTLs). We mapped trans regulatory effects, identifying an important role for protein-protein interactions. We discovered that pQTLs show increased enrichment in disease-linked GWAS variants, compared with RNA-based eQTLs.

scholarly journals Tension-driven multi-scale self-organisation in human iPSC-derived muscle fibers

2021 ◽  
Author(s):  
Qiyan Mao ◽  
Achyuth Acharya ◽  
Alejandra Rodriguez-delaRosa ◽  
Fabio Marchiano ◽  
Benoit Dehapiot ◽  
...  
Keyword(s):  
Induced Pluripotent Stem Cell ◽  
Human Muscle ◽  
Mechanical Tension ◽  
In Vitro System ◽  
Multi Scale ◽  
Self Organisation ◽  
Induced Pluripotent ◽  
Sarcomere Assembly ◽  
Human Ipsc

Human muscle is a hierarchically organised tissue with its contractile cells called myofibers packed into large myofiber bundles. Each myofiber contains periodic myofibrils built by hundreds of contractile sarcomeres that generate large mechanical forces. To better understand the mechanisms that coordinate human muscle morphogenesis from tissue to molecular scales, we adopted a simple in vitro system using induced pluripotent stem cell-derived human myogenic precursors. When grown on an unrestricted two-dimensional substrate, developing myofibers spontaneously align and self-organise into higher-order myofiber bundles, which grow and consolidate to stable sizes. Following a transcriptional boost of sarcomeric components, myofibrils assemble into chains of periodic sarcomeres that emerge across the entire myofiber. By directly probing tension we found that tension build-up precedes sarcomere assembly and increases within each assembling myofibril. Furthermore, we found that myofiber ends stably attach to other myofibers using integrin-based attachments and thus myofiber bundling coincides with stable myofiber bundle attachment in vitro. A failure in stable myofiber attachment results in a collapse followed by a disassembly of the myofibrils. Overall, our results strongly suggest that mechanical tension across sarcomeric components as well as between differentiating myofibers is key to coordinate the multi-scale self-organisation of muscle morphogenesis.

scholarly journals Syncytium cell growth increases Kir2.1 contribution in human iPSC-cardiomyocytes

2020 ◽  
Vol 319 (5) ◽  
pp. H1112-H1122 ◽  
Author(s):  
Weizhen Li ◽  
Julie L. Han ◽  
Emilia Entcheva
Keyword(s):  
Ion Channel ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Cell Contact ◽  
Isolated Cells ◽  
Protein Levels ◽  
Ion Channel Properties ◽  
Induced Pluripotent ◽  
Human Ipsc ◽  
Channel Properties

We identify cell culture density and cell-cell contact as an important factor in determining the expression of a key ion channel at the transcriptional and the protein levels, KCNJ2/Kir2.1, and its contribution to the electrophysiology of human induced pluripotent stem cell-derived cardiomyocytes. Our results indicate that studies on isolated cells, out of tissue context, may underestimate the cellular ion channel properties being characterized.

scholarly journals Human Induced Pluripotent Stem-Cell-Derived Cardiomyocytes as Models for Genetic Cardiomyopathies

2019 ◽  
Vol 20 (18) ◽  
pp. 4381 ◽  
Author(s):  
Andreas Brodehl ◽  
Hans Ebbinghaus ◽  
Marcus-André Deutsch ◽  
Jan Gummert ◽  
Anna Gärtner ◽  
...  
Keyword(s):  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Patient Specific ◽  
Promising Alternative ◽  
Human Ipscs ◽  
Genetic Landscape ◽  
Inherited Cardiomyopathies ◽  
Induced Pluripotent ◽  
Human Ipsc

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.

Abstract 14104: Replacement of Infarct Myocardium by Large Scale-Expanded Human Induced Pluripotent Stem Cell-Derived Cardiac Cell-Sheet in a Porcine Chronic Myocardial Infarction Model

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Keitaro Domae ◽  
Shigeru Miyagawa ◽  
Satsuki Fukushima ◽  
Atsuhiro Saito ◽  
Yukiko Imanishi ◽  
...  
Keyword(s):  
Functional Recovery ◽  
Large Scale ◽  
Induced Pluripotent Stem Cell ◽  
Cardiac Cells ◽  
Cell Group ◽  
Small Scale ◽  
Cardiac Cell ◽  
Cell Sheets ◽  
Induced Pluripotent ◽  
Human Ipsc

Introduction: It has been shown that transplanted induced pluripotent stem cell (iPSC)-derived cardiac cells in the myocardial infarction (MI) heart synchronously contract with native myocardium to mechanically contribute to functional recovery in rodent models. We herein hypothesized that large scale cardiac cell-sheets generated by human iPSCs may induce a greater functional recovery than small scale ones after transplantation in chronic MI heart. Methods: Bioreactor-based three-dimensional suspension culture system was used for generating large scale-expanded human iPSC-derived cardiomyocytes, of which cardiac troponin T positivity was constantly 75-85%. Scaffold-free cell-sheets containing several cell number (1.0х10^6, 10^7, 10^8) were transplanted over the cardiac surface in porcine chronic MI heart (n=5 each). Tacrolimus and prednisolone were daily given in all pigs against xeno-transplantation-inducing immune reaction. Results: Echocardiographically, left ventricular systolic and diastolic dimensions were significantly decreasing and ejection fraction was significantly increasing in the 10^8 cell group. In addition, global myocardial structure was better preserved in the 10^8 cell group with presence of the graft in the infarct area macroscopically (Figure). Moreover, there were significantly less accumulation of interstitial fibrosis in the infarct-remote area and greater vascular density and expression of VEGF, bFGF, and SDF-1 in the infarct-border area in the 10^8 cell group than the other groups at 3 months after the transplantation. Conclusions: Large scale human iPSC-derived cardiac cells were engrafted in the infarct myocardium, showing substantial functional recovery in a porcine chronic MI heart, indicating that artificial cell-based myocardial replacement therapy may be achieved. In contrast, small scale cardiac cells induced modest functional recovery, suggesting paracrine mechanisms of this treatment.

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