scholarly journals mTOR and autophagy pathways are dysregulated in murine and human models of Schaaf-Yang syndrome

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Emeline Crutcher ◽  
Rituraj Pal ◽  
Fatemeh Naini ◽  
Ping Zhang ◽  
Magdalena Laugsch ◽  
...  
Keyword(s):  
Neurodevelopmental Disorder ◽  
Mammalian Target Of Rapamycin ◽  
Induced Pluripotent Stem Cell ◽  
Fibroblast Cell ◽  
Null Mouse ◽  
Chromosome 15 ◽  
Feeding Difficulties ◽  
Pathogenic Variants ◽  
Induced Pluripotent ◽  
Fibroblast Cell Lines

Abstract MAGEL2 is a maternally imprinted, paternally expressed gene, located in the Prader-Willi region of human chromosome 15. Pathogenic variants in the paternal copy of MAGEL2 cause Schaaf-Yang syndrome (SHFYNG), a neurodevelopmental disorder related to Prader-Willi syndrome (PWS). Patients with SHFYNG, like PWS, manifest neonatal hypotonia, feeding difficulties, hypogonadism, intellectual disability and sleep apnea. However, individuals with SHFYNG have joint contractures, greater cognitive impairment, and higher prevalence of autism than seen in PWS. Additionally, SHFYNG is associated with a lower prevalence of hyperphagia and obesity than PWS. Previous studies have shown that truncating variants in MAGEL2 lead to SHFYNG. However, the molecular pathways involved in manifestation of the SHFYNG disease phenotype are still unknown. Here we show that a Magel2 null mouse model and fibroblast cell lines from individuals with SHFYNG exhibit increased expression of mammalian target of rapamycin (mTOR) and decreased autophagy. Additionally, we show that SHFYNG induced pluripotent stem cell (iPSC)-derived neurons exhibit impaired dendrite formation. Alterations in SHFYNG patient fibroblast lines and iPSC-derived neurons are rescued by treatment with the mTOR inhibitor rapamycin. Collectively, our findings identify mTOR as a potential target for the development of pharmacological treatments for SHFYNG.

Unlocking Personalized Biomedicine and Drug Discovery with Human Induced Pluripotent Stem Cell–Derived Cardiomyocytes: Fit for Purpose or Forever Elusive?

2020 ◽  
Vol 60 (1) ◽  
pp. 529-551 ◽  
Author(s):  
Tessa de Korte ◽  
Puspita A. Katili ◽  
Nurul A.N. Mohd Yusof ◽  
Berend J. van Meer ◽  
Umber Saleem ◽  
...  
Keyword(s):  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
Genome Engineering ◽  
Induced Pluripotent Stem Cell ◽  
Pathogenic Variants ◽  
Development Costs ◽  
Regulatory Guidelines ◽  
Fit For Purpose ◽  
Safety Assessments ◽  
Induced Pluripotent

In recent decades, drug development costs have increased by approximately a hundredfold, and yet about 1 in 7 licensed drugs are withdrawn from the market, often due to cardiotoxicity. This review considers whether technologies using human induced pluripotent stem cell–derived cardiomyocytes (hiPSC-CMs) could complement existing assays to improve discovery and safety while reducing socioeconomic costs and assisting with regulatory guidelines on cardiac safety assessments. We draw on lessons from our own work to suggest a panel of 12 drugs that will be useful in testing the suitability of hiPSC-CM platforms to evaluate contractility. We review issues, including maturity versus complexity, consistency, quality, and cost, while considering a potential need to incorporate auxiliary approaches to compensate for limitations in hiPSC-CM technology. We give examples on how coupling hiPSC-CM technologies with Cas9/CRISPR genome engineering is starting to be used to personalize diagnosis, stratify risk, provide mechanistic insights, and identify new pathogenic variants for cardiovascular disease.

scholarly journals SUN-556 Cardiac Phenotype in Familial Partial Lipodystrophy

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Abdelwahab Jalal Eldin ◽  
Baris Akinci ◽  
Andre Monteiro da Rocha ◽  
Rasimcan Meral ◽  
Ilgin Yildirim Simsir ◽  
...  
Keyword(s):  
Atrial Fibrillation ◽  
Stem Cell ◽  
Action Potential Duration ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Conduction System ◽  
Partial Lipodystrophy ◽  
Pathogenic Variants ◽  
Familial Partial Lipodystrophy ◽  
Induced Pluripotent

Abstract Background Pathogenic variants in Lamin A/C (LMNA) gene are the most common monogenic etiology in Familial Partial Lipodystrophy (FPLD) causing FPLD2. LMNA pathogenic variants have been previously associated with cardiomyopathy, familial arrhythmias or conduction system abnormalities independent of lipodystrophy. We aimed to assess cardiac impacts of FPLD, and to explore the extent of overlap between cardiolaminopathies and FPLD. Methods We conducted a retrospective review of an established cohort of 122 patients (age range: 13-77, M/F 21/101) with FPLD from Michigan (n = 83) and Turkey (n = 39) with an accessible cardiac evaluation. Also, functional syncytia of mature human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from a FLPD2 patient was studied for assessment of autonomous rhythm and action potential duration with optical mapping using a voltage sensitive dye. Results In the whole study cohort, 95 (78%) patients had cardiac alterations (25% ischemic heart disease, 36% arrhythmia, 16% conduction abnormality, 20% prolonged QT interval, 11% cardiomyopathy, and 15% congestive heart failure). The likelihood of having an arrhythmia (OR; 3.95, 95% CI: 1.49-10.49) and conduction disease (OR: 3.324, 95% CI: 1.33-8.31) was significantly higher in patients with LMNA pathogenic variants. Patients with LMNA pathogenic variants were at high risk for atrial fibrillation/flutter (OR: 6.77, 95% CI: 1.27- 39.18). The time to first arrhythmia was significantly shorter in the LMNA group with a higher hazard rate of 3.04 (95% CI: 1.29-7.17, p = 0.032). Non-482 LMNA pathogenic variants were more likely to be associated with cardiac events (vs. 482 LMNA: OR: 4.74, 95% CI: 1.41- 15.98 for arrhythmia; OR: 17.67, 95% CI: 2.44- 127.68 for atrial fibrillation/flutter; OR: 5.71, 95% CI: 1.37- 23.76 for conduction disease. hiPSC-CMs from a FPLD2 patient had higher frequency of autonomous activity, and shorter Fridericia corrected action potential duration at 80% repolarization compared to control cardiomyocytes. Furthermore, FPLD2 functional syncytia of mature hiPSC-CMs presented several rhythm alterations such as early after-depolarizations, spontaneous quiescence and spontaneous tachyarrhythmia; none of those were observed in the control cell lines. Finally, FPLD2 hiPSC-CMs presented significantly slower recovery in chronotropic changes induced by isoproterenol exposure; which indicates disrupted beta-adrenergic response. Conclusion Our results suggest the need for vigilant cardiac monitoring in FPLD, especially in patients with FPLD2 who have an increased risk to develop cardiac arrhythmias and conduction system diseases. In addition, study of human induced pluripotent stem cell-derived cardiomyocytes may prove useful to understand the mechanism of cardiac disease and arrhythmias and to create precision therapy opportunities in the future.

scholarly journals Development of a Cardiac Sarcomere Functional Genomics Platform to Enable Scalable Interrogation of Human TNNT2 Variants

Circulation ◽  
2020 ◽  
Vol 142 (23) ◽  
pp. 2262-2275
Author(s):  
Anthony M. Pettinato ◽  
Feria A. Ladha ◽  
David J. Mellert ◽  
Nicholas Legere ◽  
Rachel Cohn ◽  
...  
Keyword(s):  
Heart Failure ◽  
Stem Cell ◽  
Hypertrophic Cardiomyopathy ◽  
Dilated Cardiomyopathy ◽  
Pluripotent Stem Cell ◽  
Induced Pluripotent Stem Cell ◽  
Pathogenic Variants ◽  
Calcium Affinity ◽  
Uncertain Significance ◽  
Induced Pluripotent

Background: Pathogenic TNNT2 variants are a cause of hypertrophic and dilated cardiomyopathies, which promote heart failure by incompletely understood mechanisms. The precise functional significance for 87% of TNNT2 variants remains undetermined, in part, because of a lack of functional genomics studies. The knowledge of which and how TNNT2 variants cause hypertrophic and dilated cardiomyopathies could improve heart failure risk determination, treatment efficacy, and therapeutic discovery, and provide new insights into cardiomyopathy pathogenesis, as well. Methods: We created a toolkit of human induced pluripotent stem cell models and functional assays using CRISPR/Cas9 to study TNNT2 variant pathogenicity and pathophysiology. Using human induced pluripotent stem cell–derived cardiomyocytes in cardiac microtissue and single-cell assays, we functionally interrogated 51 TNNT2 variants, including 30 pathogenic/likely pathogenic variants and 21 variants of uncertain significance. We used RNA sequencing to determine the transcriptomic consequences of pathogenic TNNT2 variants and adapted CRISPR/Cas9 to engineer a transcriptional reporter assay to assist prediction of TNNT2 variant pathogenicity. We also studied variant-specific pathophysiology using a thin filament–directed calcium reporter to monitor changes in myofilament calcium affinity. Results: Hypertrophic cardiomyopathy–associated TNNT2 variants caused increased cardiac microtissue contraction, whereas dilated cardiomyopathy–associated variants decreased contraction. TNNT2 variant–dependent changes in sarcomere contractile function induced graded regulation of 101 gene transcripts, including MAPK (mitogen-activated protein kinase) signaling targets, HOPX , and NPPB . We distinguished pathogenic TNNT2 variants from wildtype controls using a sarcomere functional reporter engineered by inserting tdTomato into the endogenous NPPB locus. On the basis of a combination of NPPB reporter activity and cardiac microtissue contraction, our study provides experimental support for the reclassification of 2 pathogenic/likely pathogenic variants and 2 variants of uncertain significance. Conclusions: Our study found that hypertrophic cardiomyopathy–associated TNNT2 variants increased cardiac microtissue contraction, whereas dilated cardiomyopathy–associated variants decreased contraction, both of which paralleled changes in myofilament calcium affinity. Transcriptomic changes, including NPPB levels, directly correlated with sarcomere function and can be used to predict TNNT2 variant pathogenicity.

MitoCellPhe reveals mitochondrial morphologies in single fibroblasts and clustered stem cells

2021 ◽  
Author(s):  
Ajibola B. Bakare ◽  
Fibi Meshrkey ◽  
Benjamin Lowe ◽  
Carson Molder ◽  
Raj R Rao ◽  
...  
Keyword(s):  
Stem Cell ◽  
Mitochondrial Dynamics ◽  
Single Cells ◽  
Biological Significance ◽  
Induced Pluripotent Stem Cell ◽  
Fibroblast Cell ◽  
Cell Types ◽  
Health And Disease ◽  
Diseased State ◽  
Induced Pluripotent

Mitochondria are dynamic organelles that differ significantly in their morphologies across cell types, reflecting specific cellular needs and stages in development. Despite the wide biological significance in disease and health, delineating mitochondrial morphologies in complex systems remains challenging. Here, we present the Mitochondrial Cellular Phenotype (MitoCellPhe) tool developed for quantifying mitochondrial morphologies and demonstrate its utility in delineating differences in mitochondrial morphologies in a human fibroblast and human induced pluripotent stem cell (hiPSC) line. MitoCellPhe generates 24 parameters, allowing for a comprehensive analysis of mitochondrial structures and importantly allows for quantification to be performed on mitochondria in images containing single cells or clusters of cells. With this tool, we were able to validate previous findings that show networks of mitochondria in healthy fibroblast cell lines and a more fragmented morphology in hiPSCs. Using images generated from control and diseased fibroblasts and hiPSCs, we also demonstrate the efficacy of the toolset in delineating differences in morphologies between healthy and the diseased state in both stem cell (hiPSC) and differentiated fibroblast cells. Our results demonstrate that MitoCellPhe enables high-throughput, sensitive, detailed and quantitative mitochondrial morphological assessment and thus enables better biological insights into mitochondrial dynamics in health and disease

scholarly journals Metabolomics signatures of a subset of RET variants according to their oncogenic risk level

2019 ◽  
Vol 26 (3) ◽  
pp. 379-389 ◽  
Author(s):  
Charlotte Veyrat-Durebex ◽  
Nathalie Bouzamondo ◽  
Morgane Le Mao ◽  
Juan Manuel Chao de la Barca ◽  
Céline Bris ◽  
...  
Keyword(s):  
Fibroblast Cell ◽  
Risk Level ◽  
Multivariate Statistical ◽  
Risk Levels ◽  
Pathogenic Variants ◽  
Cell Extracts ◽  
Murine Fibroblast ◽  
Metabolomics Study ◽  
Medullary Thyroid Carcinomas ◽  
Fibroblast Cell Lines

Thirty percent of medullary thyroid carcinomas (MTCs) are related to dominant germline pathogenic variants in the RET proto-oncogene. According to their aggressiveness, these pathogenic variants are classified in three risk levels: ‘moderate’, ‘high’ and ‘highest’. The present study compares the metabolomics profiles of five pathogenic variants, whether already classified or not. We have generated six stable murine fibroblast cell lines (NIH3T3) expressing the WT allele or variants of the human RET gene, with different levels of pathogenicity, including the M918V variant that is yet to be accurately classified. We carried out a targeted metabolomics study of the cell extracts with a QTRAP mass spectrometer, using the Biocrates Absolute IDQ p180 kit, which allows the quantification of 188 endogenous molecules. The data were then subjected to multivariate statistical analysis. One hundred seventy three metabolites were accurately measured. The metabolic profiles of the cells expressing the RET variants were found to be correlated with their oncogenic risk. In addition, the statistical model we constructed for predicting the oncogenic risk attributed a moderate risk to the M918V variant. Our results indicate that metabolomics may be useful for characterizing the pathogenicity of the RET gene variants and their levels of aggressiveness.

scholarly journals Establishment and characterization of immortalized Gli-null mouse embryonic fibroblast cell lines

2008 ◽  
Vol 9 (1) ◽  
pp. 49 ◽  
Author(s):  
Robert J Lipinski ◽  
Maarten F Bijlsma ◽  
Jerry J Gipp ◽  
David J Podhaizer ◽  
Wade Bushman
Keyword(s):  
Cell Lines ◽  
Fibroblast Cell ◽  
Mouse Embryonic Fibroblast ◽  
Null Mouse ◽  
Mouse Embryonic Fibroblast Cell ◽  
Embryonic Fibroblast ◽  
Fibroblast Cell Lines

scholarly journals Neural-specific alterations in glycosphingolipid biosynthesis and cell signaling associated with two human ganglioside GM3 Synthase Deficiency variants

2021 ◽  
Author(s):  
Michelle Dookwah ◽  
Shannon K Wagner ◽  
Mayumi Ishihara ◽  
Seok-ho Yu ◽  
Heidi Ulrichs ◽  
...  
Keyword(s):  
Cell Signaling ◽  
Neurodevelopmental Disorder ◽  
Growth Factor Receptor ◽  
Ganglioside Gm3 ◽  
Pathogenic Variants ◽  
Gm3 Synthase ◽  
Terminal Galactose ◽  
Induced Pluripotent ◽  
Induced Apoptosis ◽  
Surface Proteome

GM3 Synthase Deficiency (GM3SD) is a neurodevelopmental disorder resulting from pathogenic variants in the ST3GAL5 gene, which encodes GM3 synthase, a glycosphingolipid (GSL)-specific sialyltransferase. This enzyme adds a single alpha3-linked sialic acid to the terminal galactose of lactosylceramide (LacCer) to produce the monosialylated ganglioside GM3. In turn, GM3 is extended by other glycosyltransferases to generate nearly all the complex gangliosides enriched in neural tissue. Pathogenic mechanisms that account for neural phenotypes associated with GM3SD are not known. To explore how loss of GM3 impacts neural-specific glycolipid glycosylation and cell signaling, GM3SD patient fibroblasts bearing one of two different ST3GAL5 variants were reprogrammed to induced pluripotent stem cells (iPSCs) and then differentiated to neural crest cells (NCCs). GM3 and GM3-derived gangliosides were undetectable in iPSCs and NCCs from both variants, while LacCer precursor levels were elevated compared to wildtype (WT). NCCs of both variants synthesized elevated levels of neutral lacto- and globo-series, as well as minor alternatively sialylated, GSLs compared to WT. Shifts in ceramide profiles associated with iPSC and NCC GSLs were also detected in GM3SD variants. Altered GSL profiles in the GM3SD cells were accompanied by dynamic changes in the cell surface proteome, protein O-GlcNAcylation, and receptor tyrosine kinase abundance. GM3SD cells also exhibited increased apoptosis and sensitivity to erlotnib, an inhibitor of epidermal growth factor receptor signaling. Pharmacologic inhibition of O-GlcNAcase increased protein O-GlcNAcylation and significantly rescued baseline and erlotnib-induced apoptosis. Collectively, these findings indicate broad effects on cell signaling during differentiation of GM3SD patient-derived iPSCs to NCCs. Thus, human GM3SD cells provide a novel platform to investigate structure/function relationships that connect GSL diversity to cell signaling, cell survival, and neural differentiation.

scholarly journals Induced pluripotent stem cells from subjects with Lesch-Nyhan disease

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Diane J. Sutcliffe ◽  
Ashok R. Dinasarapu ◽  
Jasper E. Visser ◽  
Joery den Hoed ◽  
Fatemeh Seifar ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Induced Pluripotent Stem Cell ◽  
Shotgun Proteomics ◽  
Significant Heterogeneity ◽  
Functional Studies ◽  
Pathogenic Variants ◽  
Cell Layers ◽  
Hypoxanthine Guanine Phosphoribosyltransferase ◽  
Induced Pluripotent

AbstractLesch-Nyhan disease (LND) is an inherited disorder caused by pathogenic variants in the HPRT1 gene, which encodes the purine recycling enzyme hypoxanthine–guanine phosphoribosyltransferase (HGprt). We generated 6 induced pluripotent stem cell (iPSC) lines from 3 individuals with LND, along with 6 control lines from 3 normal individuals. All 12 lines had the characteristics of pluripotent stem cells, as assessed by immunostaining for pluripotency markers, expression of pluripotency genes, and differentiation into the 3 primary germ cell layers. Gene expression profiling with RNAseq demonstrated significant heterogeneity among the lines. Despite this heterogeneity, several anticipated abnormalities were readily detectable across all LND lines, including reduced HPRT1 mRNA. Several unexpected abnormalities were also consistently detectable across the LND lines, including decreases in FAR2P1 and increases in RNF39. Shotgun proteomics also demonstrated several expected abnormalities in the LND lines, such as absence of HGprt protein. The proteomics study also revealed several unexpected abnormalities across the LND lines, including increases in GNAO1 decreases in NSE4A. There was a good but partial correlation between abnormalities revealed by the RNAseq and proteomics methods. Finally, functional studies demonstrated LND lines had no HGprt enzyme activity and resistance to the toxic pro-drug 6-thioguanine. Intracellular purines in the LND lines were normal, but they did not recycle hypoxanthine. These cells provide a novel resource to reveal insights into the relevance of heterogeneity among iPSC lines and applications for modeling LND.

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