Mitochondrial DNA sequencing of iPSC, parental cells, and iPSC derived cardiomyocytes
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PROVIDER: EGAS00001005560 | EGA |
REPOSITORIES: EGA
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Project description:Induced pluripotent stem cells (iPSCs) are a valuable resource for neurological disease-modeling and drug discovery, due to their ability to differentiate into neurons reflecting the genetics of the patient from which they are derived. iPSC-derived cultures, however, are highly variable due to differences in culture conditions. We investigated the effect of iPSC passage number on differentiation to optimize the generation of functional, mature sensory neurons (iPSC-dSNs). Three iPSC lines were differentiated into iPSC-dSNs at passage numbers within each of the following ranges: low (LP; 5-10), middle (MP; 20-26), and high (HP; 30-38). Morphology and pluripotency of the parent iPSCs were assessed prior to differentiation at each passage number. iPSC-dSNs were evaluated based on electrophysiological properties and expression of key neuronal markers. All iPSC lines displayed the same morphology and were similarly pluripotent across passage numbers. iPSC-dSNs were also morphologically comparable across passage numbers. However, the expression levels of neuronal markers and an analysis of sodium channel function indicated greater maturity in LP iPSC-dSNs. Our results demonstrate that lower passage numbers may be better suited for differentiation into peripheral sensory neurons. Further studies are warranted to elucidate factors that may contribute to the variability associated with iPSC passage number.
2022-07-19 | GSE193571 | GEO
Project description:Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is often caused by an adenine to guanine variant at m.3243 (m.3243A>G) of the MT-TL1 gene. To understand how this pathogenic variant affects the nervous system, we differentiated human induced pluripotent stem cells (iPSCs) into excitatory neurons with normal (low heteroplasmy) and impaired (high heteroplasmy) mitochondrial function from MELAS patients with the m.3243A>G pathogenic variant. We combined micro-electrode array (MEA) measurements with RNA sequencing (MEA-seq) and found reduced expression of genes involved in mitochondrial respiration and presynaptic function, as well as non-cell autonomous processes in co-cultured astrocytes. Finally, we show that the clinical phase II drug sonlicromanol can improve neuronal network activity when treatment is initiated early in development. This was intricately linked with changes in the neuronal transcriptome. Overall, we provide insight in transcriptomic changes in iPSC-derived neurons with high m.3243A>G heteroplasmy, and show the pathology is partially reversible by sonlicromanol.
2021-07-29 | GSE154825 | GEO
Project description:Mitochondrial heteroplasmy, the presence of more than one mtDNA variant in a cell or individual is not as uncommon as previously thought. It is mostly due to the high mutation rate of the mtDNA and limited repair mechanisms present in the mitochondrion. The phenomenon has been studied mostly in human samples and in medical contexts. Heteroplasmy has also been researched in other species in fields such as forensics or genetic foot printing, but these studies usually focused on contained families within closely related species. Here we describe a large cross-species evaluation of heteroplasmy in mammals. We employed a novel approach to detect mitochondrial heteroplasmy in both novel and previously reported ChIP-sequencing datasets, which include concomitant mitochondrial DNA sequenced in the experiment. Here, we report novel ChIP-seq experiments for H3K4me1 and CEBPA across mammals, as well as some H3K4me3, H3K27ac and total histone H3 experiments. Most of the reported CEBPA experiments are good quality pull-downs, however the quality of many of the other experiments reported here has not been interrogated in detail. Whereas this does not affect the investigation of mitochondrial DNA pollution for the purposes of this study, both H3K4me1 and total histone H3 ChIP-seq datasets were often sequenced to relatively low depth and showed low ChIP enrichment compared to the other antibodies.
2016-05-13 | E-MTAB-3933 | biostudies-arrayexpress
Project description:Mitochondria generate signals of adaptation that regulate nuclear genes expression via retrograde signaling. But this phenomenon is complexified when qualitatively different mitochondria and mitochondrial DNA (mtDNA) coexist within cells. Although this cellular state of heteroplasmy leads to divergent phenotypes clinically, its consequences on cellular function and the cellular transcriptome are unknown. To interrogate this phenomenon, we generated somatic cell cybrids harboring increasing levels of a common mtDNA mutation (tRNALeu(UUR) 3243A>G) and mapped the resulting cellular phenotypes and transcriptional profiles across the complete range of heteroplasmy. Small increases in mutant mtDNAs caused relatively modest defect in mitochondrial oxidative capacity, but resulted in sharp transitions in mitochondrial ultrastructure and in the nuclear and mitochondrial transcriptomes, with the critical functional threshold corresponding to the induction of epigenetic regulatory systems. Principal component analysis underscores how each heteroplasmy level occupies a different "transcriptional space", with low levels heteroplasmy (20-30%) producing a dose-response linear progression in one direction, and mutationload of 50, 60 and 90% producing changes in the opposite direction. Hence, subtle changes in mitochondrial energetics can act through the epigenome to generate the phenotypes of the common “complex” diseases. Cells were generated by transferring the wildtype (3243A) and mutant (3243G) mtDNAs from a heteroplasmic 3243A>G patient’s lymphoblastoid cell line into 143B(TK-) mtDNA-deficient (ρo) cells and selected for transmitochondrial cybrids. Subsequent mtDNA depletion, reamplification, and cloning (Wiseman and Attardi, 1978) resulted in a series of stable cybrids harboring approximately 0, 20, 30, 50, 60, 90, and 100% 3243G mutant mtDNAs. Total RNA extracted from each cell line was then extracted, depleted of rRNA, and measured in sequenced in triplicates.
2014-09-02 | E-GEOD-56158 | biostudies-arrayexpress
Project description:Affect of iPSC Passage Number on Neuronal Differentiation
| PRJNA796610 | ENA
Project description:Mitochondrial DNA (mtDNA) 3243A>G tRNALeu(UUR) heteroplasmic mutation (m.3243A>G) exhibits clinically heterogeneous phenotypes. While the high mtDNA heteroplasmy exceeding a critical threshold causes mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes (MELAS) syndrome, the low mtDNA heteroplasmy causes maternally inherited diabetes with or without deafness (MIDD) syndrome. How quantitative differences in mtDNA heteroplasmy produces distinct pathological states has remained elusive. Here we show that despite striking similarities in the energy metabolic gene expression signature, the mitochondrial bioenergetics, biogenesis and fuel catabolic functions are distinct in cells harboring low or high levels of the m.3243A>G mutation compared to wild type cells. We further demonstrate that the low heteroplasmic mutant cells exhibit a coordinate induction of transcriptional regulators of the mitochondrial biogenesis, glucose and fatty acid metabolism pathways that lack in near homoplasmic mutant cells compared to wild type cells. Altogether, these results shed new biological insights on the potential mechanisms by which low mtDNA heteroplasmy may progressively cause diabetes mellitus.
2019-04-09 | GSE129091 | GEO
Project description:Hepatocytes generated from human induced pluripotent stem cells (hiPSCs) are unprecedented resources for pharmaceuticals and cell therapy. However, little attention has so far been paid to variations among hiPSC lines in terms of their hepatic differentiation. We developed an improved hepatic differentiation protocol and compared multiple hiPSC lines. This comparison indicated that the hepatic differentiation propensity varies among sibling hiPSC clones derived from the same adult human dermal fibroblasts (aHDFs). In addition, hiPSC clones derived from peripheral blood cells (PB-iPSCs) consistently showed good hepatic differentiation efficiency, whereas many hiPSC clones from adult dermal fibroblasts (aHDF-iPSCs) showed poor hepatic differentiation. However, when we compared hiPSCs from blood and dermal fibroblasts from the same individuals, we found that variations in hepatic differentiation were largely attributable to donor differences, rather than to the types of the original cells. In order to understand the molecular mechanisms underlying the observed variations in hepatic differentiation, we performed microarray analyses of sibling aHDF-iPSC clones, and aHDF- and PB-iPSC clones from the same individuals. sibling aHDF-iPSC clones (201B6 and 201B7; derived from the same aHDFs) (1) undifferentiated state (n=4, biological replicate #1-#4) (2) CXCR4-positive cell populations sorted by flowcytometry after 7 days of endodermal differentiation (n=4, biological replicate #1-#4) (3) CXCR4-negative cell populations sorted by flowcytometry after 7 days of endodermal differentiation (n=4, biological replicate #1-#4)
2012-07-15 | E-GEOD-38152 | biostudies-arrayexpress
Project description:Global microRNA analysis of (a) human embryonic stem cells, (b) low passage episomal stromal-primed myeloid iPSC, (c) intermediate passage fibroblast iPSC, and (d) samples of corresponding donor cells.
2017-12-31 | GSE44423 | GEO
Project description:Global gene expression analysis of (a) human embryonic stem cells, (b) low passage episomal stromal-primed myeloid iPSC, (c) intermediate passage fibroblast iPSC, and (d) samples of corresponding donor cells.
2016-09-13 | GSE44421 | GEO
Project description:Hepatocytes generated from human induced pluripotent stem cells (hiPSCs) are unprecedented resources for pharmaceuticals and cell therapy. However, little attention has so far been paid to variations among hiPSC lines in terms of their hepatic differentiation. We developed an improved hepatic differentiation protocol and compared multiple hiPSC lines. This comparison indicated that the hepatic differentiation propensity varies among sibling hiPSC clones derived from the same adult human dermal fibroblasts (aHDFs). In addition, hiPSC clones derived from peripheral blood cells (PB-iPSCs) consistently showed good hepatic differentiation efficiency, whereas many hiPSC clones from adult dermal fibroblasts (aHDF-iPSCs) showed poor hepatic differentiation. However, when we compared hiPSCs from blood and dermal fibroblasts from the same individuals, we found that variations in hepatic differentiation were largely attributable to donor differences, rather than to the types of the original cells. In order to understand the molecular mechanisms underlying the observed variations in hepatic differentiation, we performed microarray analyses of sibling aHDF-iPSC clones, and aHDF- and PB-iPSC clones from the same individuals. Undifferentiated aHDF- and PB-iPSCs from the same individuals (two Parkinson’s disease patients (PD #1 and PD #2) and one adult healthy donor (donor91))
2012-07-15 | E-GEOD-38153 | biostudies-arrayexpress