Comparing the mitochondrial signatures in ESCs and iPSCs and their neural derivations

ABSTRACT Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have distinct origins: ESCs are derived from pre-implanted embryos while iPSCs are reprogrammed somatic cells. Both have their own characteristics and lineage specificity, and both are valuable tools for studying human neurological development and disease. Thus far, few studies have analyzed how differences between stem cell types influence mitochondrial function and mitochondrial DNA (mtDNA) homeostasis during differentiation into neural and glial lineages. In this study, we compared mitochondrial function and mtDNA replication in human ESCs and iPSCs at three different stages − pluripotent, neural progenitor and astrocyte. We found that while ESCs and iPSCs have a similar mitochondrial signature, neural and astrocyte derivations manifested differences. At the neural stem cell (NSC) stage, iPSC-NSCs displayed decreased ATP production and a reduction in mitochondrial respiratory chain (MRC) complex IV expression compared to ESC-NSCs. IPSC-astrocytes showed increased mitochondrial activity including elevated ATP production, MRC complex IV expression, mtDNA copy number and mitochondrial biogenesis relative to those derived from ESCs. These findings show that while ESCs and iPSCs are similar at the pluripotent stage, differences in mitochondrial function may develop during differentiation and must be taken into account when extrapolating results from different cell types. Abbreviation: BSA: Bovine serum albumin; DCFDA: 2′,7′‐dichlorodihydrofluorescein diacetate; DCX: Doublecortin; EAAT-1: Excitatory amino acid transporter 1; ESCs: Embryonic stem cells; GFAP: Glial fibrillary acidic protein; GS: Glutamine synthetase; iPSCs: Induced pluripotent stem cells; LC3B: Microtubule-associated protein 1 light chain 3β; LC-MS: Liquid chromatography-mass spectrometry; mito-ROS: Mitochondrial ROS; MMP: Mitochondrial membrane potential; MRC: Mitochondrial respiratory chain; mtDNA: Mitochondrial DNA; MTDR: MitoTracker Deep Red; MTG: MitoTracker Green; NSCs: Neural stem cells; PDL: Poly-D-lysine; PFA: Paraformaldehyde; PGC-1α: PPAR-γ coactivator-1 alpha; PPAR-γ: Peroxisome proliferator-activated receptor-gamma; p-SIRT1: Phosphorylated sirtuin 1; p-ULK1: Phosphorylated unc-51 like autophagy activating kinase 1; qPCR: Quantitative PCR; RT: Room temperature; RT-qPCR: Quantitative reverse transcription PCR; SEM: Standard error of the mean; TFAM: Mitochondrial transcription factor A; TMRE: Tetramethylrhodamine ethyl ester; TOMM20: Translocase of outer mitochondrial membrane 20 Graphical Abstract

[1]  G. Sullivan,et al.  Nicotinamide Riboside and Metformin Ameliorate Mitophagy Defect in Induced Pluripotent Stem Cell-Derived Astrocytes With POLG Mutations , 2021, Frontiers in Cell and Developmental Biology.

[2]  G. Sullivan,et al.  Stem cell derived astrocytes with POLG mutations and mitochondrial dysfunction including abnormal NAD+ metabolism is toxic for neurons , 2020, bioRxiv.

[3]  G. Sullivan,et al.  N-acetylcysteine amide ameliorates mitochondrial dysfunction and reduces oxidative stress in hiPSC-derived dopaminergic neurons with POLG mutation , 2020, Experimental Neurology.

[4]  G. Sullivan,et al.  A method for differentiating human induced pluripotent stem cells toward functional cardiomyocytes in 96-well microplates , 2020, Scientific Reports.

[5]  G. Sullivan,et al.  Disease‐specific phenotypes in iPSC‐derived neural stem cells with POLG mutations , 2020, EMBO molecular medicine.

[6]  S. Lashen,et al.  Genetically unmatched human iPSC and ESC exhibit equivalent gene expression and neuronal differentiation potential , 2017, Scientific Reports.

[7]  G. Sullivan,et al.  Development of a rapid screen for the endodermal differentiation potential of human pluripotent stem cell lines , 2016, Scientific Reports.

[8]  H. Seo,et al.  Mitochondrial and metabolic remodeling during reprogramming and differentiation of the reprogrammed cells. , 2015, Stem cells and development.

[9]  A. Urbach,et al.  Comparing ESC and iPSC—Based Models for Human Genetic Disorders , 2014, Journal of clinical medicine.

[10]  A. del Sol,et al.  Modelling Fanconi anemia pathogenesis and therapeutics using integration-free patient-derived iPSCs , 2014, Nature Communications.

[11]  Simona Casini,et al.  Isogenic human pluripotent stem cell pairs reveal the role of a KCNH2 mutation in long-QT syndrome , 2013, The EMBO journal.

[12]  M. Mattson,et al.  Involvement of PGC-1α in the formation and maintenance of neuronal dendritic spines , 2012, Nature Communications.

[13]  R. Stewart,et al.  Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells , 2011, Nature.

[14]  Riitta Lahesmaa,et al.  Copy number variation and selection during reprogramming to pluripotency , 2011, Nature.

[15]  Hynek Wichterle,et al.  A functionally characterized test set of human induced pluripotent stem cells , 2011, Nature Biotechnology.

[16]  D. Turner,et al.  Neuronal-Astrocyte Metabolic Interactions: Understanding the Transition Into Abnormal Astrocytoma Metabolism , 2011, Journal of neuropathology and experimental neurology.

[17]  E. Kirkness,et al.  Somatic coding mutations in human induced pluripotent stem cells , 2011, Nature.

[18]  Richard A Young,et al.  Chromatin structure and gene expression programs of human embryonic and induced pluripotent stem cells. , 2010, Cell stem cell.

[19]  Tomohiro Kono,et al.  Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells , 2010, Nature.

[20]  Mike J. Mason,et al.  Induced pluripotent stem cells and embryonic stem cells are distinguished by gene expression signatures. , 2009, Cell stem cell.

[21]  P. Puigserver,et al.  Metabolic adaptations through the PGC‐1α and SIRT1 pathways , 2008 .

[22]  S. Yamanaka,et al.  Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors , 2006, Cell.

[23]  S. Nemoto,et al.  SIRT1 Functionally Interacts with the Metabolic Regulator and Transcriptional Coactivator PGC-1α* , 2005, Journal of Biological Chemistry.

[24]  Wilhelm Haas,et al.  Nutrient control of glucose homeostasis through a complex of PGC-1α and SIRT1 , 2005, Nature.

[25]  P. Puigserver,et al.  Peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1 alpha): transcriptional coactivator and metabolic regulator. , 2003, Endocrine reviews.

[26]  J. Thomson,et al.  Embryonic stem cell lines derived from human blastocysts. , 1998, Science.

[27]  P. Puigserver,et al.  A Cold-Inducible Coactivator of Nuclear Receptors Linked to Adaptive Thermogenesis , 1998, Cell.

[28]  P. Puigserver,et al.  Metabolic adaptations through the PGC-1 alpha and SIRT1 pathways. , 2008, FEBS letters.

[29]  Steven P Gygi,et al.  Nutrient control of glucose homeostasis through a complex of PGC-1alpha and SIRT1. , 2005, Nature.