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Mitochondrial respiratory dysfunction disturbs neuronal and cardiac lineage commitment of human iPSCs.

ABSTRACT: Mitochondrial diseases are genetically heterogeneous and present a broad clinical spectrum among patients; in most cases, genetic determinants of mitochondrial diseases are heteroplasmic mitochondrial DNA (mtDNA) mutations. However, it is uncertain whether and how heteroplasmic mtDNA mutations affect particular cellular fate-determination processes, which are closely associated with the cell-type-specific pathophysiology of mitochondrial diseases. In this study, we established two isogenic induced pluripotent stem cell (iPSC) lines each carrying different proportions of a heteroplasmic m.3243A>G mutation from the same patient; one exhibited apparently normal and the other showed most likely impaired mitochondrial respiratory function. Low proportions of m.3243A>G exhibited no apparent molecular pathogenic influence on directed differentiation into neurons and cardiomyocytes, whereas high proportions of m.3243A>G showed both induced neuronal cell death and inhibited cardiac lineage commitment. Such neuronal and cardiac maturation defects were also confirmed using another patient-derived iPSC line carrying quite high proportion of m.3243A>G. In conclusion, mitochondrial respiratory dysfunction strongly inhibits maturation and survival of iPSC-derived neurons and cardiomyocytes; our presenting data also suggest that appropriate mitochondrial maturation actually contributes to cellular fate-determination processes during development.

SUBMITTER: Yokota M

PROVIDER: S-EPMC5386384 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Mitochondrial respiratory dysfunction disturbs neuronal and cardiac lineage commitment of human iPSCs.

Yokota Mutsumi M Hatakeyama Hideyuki H Ono Yasuha Y Kanazawa Miyuki M Goto Yu-Ichi YI

Cell death & disease 20170112 1

Mitochondrial diseases are genetically heterogeneous and present a broad clinical spectrum among patients; in most cases, genetic determinants of mitochondrial diseases are heteroplasmic mitochondrial DNA (mtDNA) mutations. However, it is uncertain whether and how heteroplasmic mtDNA mutations affect particular cellular fate-determination processes, which are closely associated with the cell-type-specific pathophysiology of mitochondrial diseases. In this study, we established two isogenic induc ...[more]

PMID: 28079893

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Project description:BACKGROUND:Cardiac differentiation from human pluripotent stem cells provides a unique opportunity to study human heart development in vitro and offers a potential cell source for cardiac regeneration. Compared to the large body of studies investigating cardiac maturation and cardiomyocyte subtype-specific induction, molecular events underlying cardiac lineage commitment from pluripotent stem cells at early stage remain poorly characterized. RESULTS:In order to uncover key molecular events and regulators controlling cardiac lineage commitment from a pluripotent state during differentiation, we performed single-cell RNA-Seq sequencing and obtained high-quality data for 6879 cells collected from 6 stages during cardiac differentiation from human embryonic stem cells and identified multiple cell subpopulations with distinct molecular features. Through constructing developmental trajectory of cardiac differentiation and putative ligand-receptor interactions, we revealed crosstalk between cardiac progenitor cells and endoderm cells, which could potentially provide a cellular microenvironment supporting cardiac lineage commitment at day 5. In addition, computational analyses of single-cell RNA-Seq data unveiled ETS1 (ETS Proto-Oncogene 1) activation as an important downstream event induced by crosstalk between cardiac progenitor cells and endoderm cells. Consistent with the findings from single-cell analysis, chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-Seq) against ETS1 revealed genomic occupancy of ETS1 at cardiac structural genes at day 9 and day 14, whereas ETS1 depletion dramatically compromised cardiac differentiation. CONCLUSION:Together, our study not only characterized the molecular features of different cell types and identified ETS1 as a crucial factor induced by cell-cell crosstalk contributing to cardiac lineage commitment from a pluripotent state, but may also have important implications for understanding human heart development at early embryonic stage, as well as directed manipulation of cardiac differentiation in regenerative medicine.

Project description:Statins, which reduce LDL-cholesterol by inhibition of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, are among the most widely prescribed drugs. Skeletal myopathy is a known statin-induced adverse effect associated with mitochondrial changes. We hypothesized that similar effects would occur in cardiac myocytes in a lipophilicity-dependent manner between 2 common statins: atorvastatin (lipophilic) and pravastatin (hydrophilic). Neonatal cardiac ventricular myocytes were treated with atorvastatin and pravastatin for 48 h. Both statins induced endoplasmic reticular (ER) stress, but only atorvastatin inhibited ERK1/2T202/Y204, AktSer473, and mammalian target of rapamycin signaling; reduced protein abundance of caveolin-1, dystrophin, epidermal growth factor receptor, and insulin receptor-β; decreased Ras homolog gene family member A activation; and induced apoptosis. In cardiomyocyte-equivalent HL-1 cells, atorvastatin, but not pravastatin, reduced mitochondrial oxygen consumption. When male mice underwent atorvastatin and pravastatin administration per os for up to 7 mo, only long-term atorvastatin, but not pravastatin, induced elevated serum creatine kinase; swollen, misaligned, size-variable, and disconnected cardiac mitochondria; alteration of ER structure; repression of mitochondria- and endoplasmic reticulum-related genes; and a 21% increase in mortality in cardiac-specific vinculin-knockout mice during the first 2 months of administration. To our knowledge, we are the first to demonstrate in vivo that long-term atorvastatin administration alters cardiac ultrastructure, a finding with important clinical implications.-Godoy, J. C., Niesman, I. R., Busija, A. R., Kassan, A., Schilling, J. M., Schwarz, A., Alvarez, E. A., Dalton, N. D., Drummond, J. C., Roth, D. M., Kararigas, G., Patel, H. H., Zemljic-Harpf, A. E. Atorvastatin, but not pravastatin, inhibits cardiac Akt/mTOR signaling and disturbs mitochondrial ultrastructure in cardiac myocytes.

Project description:BACKGROUND:Parkinson's disease (PD) is one of the neurodegeneration diseases characterized by the gradual loss of dopaminergic (DA) neurons in the substantia nigra region of the brain. Substantial evidence indicates that at the cellular level mitochondrial dysfunction is a key factor leading to pathological features such as neuronal death and accumulation of misfolded ?-synuclein aggregations. Autologous transplantation of healthy purified mitochondria has shown to attenuate phenotypes in vitro and in vivo models of PD. However, there are significant technical difficulties in obtaining large amounts of purified mitochondria with normal function. In addition, the half-life of mitochondria varies between days to a few weeks. Thus, identifying a continuous source of healthy mitochondria via intercellular mitochondrial transfer is an attractive option for therapeutic purposes. In this study, we asked whether iPSCs derived astrocytes can serve as a donor to provide functional mitochondria and rescue injured DA neurons after rotenone exposure in an in vitro model of PD. METHODS:We generated DA neurons and astrocytes from human iPSCs and hESCs. We established an astroglial-neuronal co-culture system to investigate the intercellular mitochondrial transfer, as well as the neuroprotective effect of mitochondrial transfer. We employed immunocytochemistry and FACS analysis to track mitochondria. RESULTS:We showed evidence that iPSCs-derived astrocytes or astrocytic conditioned media (ACM) can rescue DA neurons degeneration via intercellular mitochondrial transfer in a rotenone induced in vitro PD model. Specifically, we showed that iPSCs-derived astrocytes from health spontaneously release functional mitochondria into the media. Mito-Tracker Green tagged astrocytic mitochondria were detected in the ACM and were shown to be internalized by the injured neurons via a phospho-p38 depended pathway. Transferred mitochondria were able to significantly reverse DA neurodegeneration and axonal pruning following exposure to rotenone. When rotenone injured neurons were cultured in presence of ACM depleted of mitochondria (by ultrafiltration), the neuroprotective effects were abolished. CONCLUSIONS:Our studies provide the proof of principle that iPSCs-derived astrocytes can act as mitochondria donor to the injured DA neurons and attenuate pathology. Using iPSCs derived astrocytes as a donor can provide a novel strategy that can be further developed for cellular therapy for PD.

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Mitochondrial respiratory dysfunction disturbs neuronal and cardiac lineage commitment of human iPSCs.

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Mitochondrial respiratory dysfunction disturbs neuronal and cardiac lineage commitment of human iPSCs.

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