bims-mireme 2021-09-05 papers

Nat Commun. 2021 Sep 02. 12(1): 5241

Cell reprogramming shapes the mitochondrial DNA landscape.

Wei Wei, Daniel J Gaffney, Patrick F Chinnery.

Individual induced pluripotent stem cells (iPSCs) show considerable phenotypic heterogeneity, but the reasons for this are not fully understood. Comprehensively analysing the mitochondrial genome (mtDNA) in 146 iPSC and fibroblast lines from 151 donors, we show that most age-related fibroblast mtDNA mutations are lost during reprogramming. However, iPSC-specific mutations are seen in 76.6% (108/141) of iPSC lines at a mutation rate of 8.62 × 10-5/base pair. The mutations observed in iPSC lines affect a higher proportion of mtDNA molecules, favouring non-synonymous protein-coding and tRNA variants, including known disease-causing mutations. Analysing 11,538 single cells shows stable heteroplasmy in sub-clones derived from the original donor during differentiation, with mtDNA variants influencing the expression of key genes involved in mitochondrial metabolism and epidermal cell differentiation. Thus, the dynamic mtDNA landscape contributes to the heterogeneity of human iPSCs and should be considered when using reprogrammed cells experimentally or as a therapy.

DOI: https://doi.org/10.1038/s41467-021-25482-x

Am J Physiol Cell Physiol. 2021 09 01.

MitoCellPhe reveals mitochondrial morphologies in single fibroblasts and clustered stem cells.

Ajibola B Bakare, Fibi Meshrkey, Benjamin Lowe, Carson Molder, Raj R Rao, Justin Zhan, Shilpa Iyer.

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.

Keywords: mitochondria; morphology; networks; stem cells; structure

DOI: https://doi.org/10.1152/ajpcell.00231.2021

Development. 2021 Sep 02. pii: dev.199477. [Epub ahead of print]

Y705 and S727 are required for mitochondrial import and transcriptional activities of STAT3 and regulate proliferation of embryonic and tissue stem cells.

Margherita Peron, Alberto Dinarello, Giacomo Meneghetti, Laura Martorano, Riccardo M Betto, Nicola Facchinello, Annachiara Tesoriere, Natascia Tiso, Graziano Martello, Francesco Argenton.

The STAT3 transcription factor, acting both in the nucleus and mitochondria, maintains embryonic stem cell pluripotency and promotes their proliferation. In this work, using zebrafish, we determined in vivo that mitochondrial STAT3 regulates mtDNA transcription in embryonic and larval stem cell niches and that this activity affects their proliferation rates. As a result, we demonstrated that STAT3 import inside mitochondria requires Y705 phosphorylation by Jak, while its mitochondrial transcriptional activity, as well as its effect on proliferation, depends on the MAPK target S727. These data were confirmed using mouse embryonic stem cells: while the Y705 mutated STAT3 cannot enter mitochondria, the S727 mutation does not affect the import in the organelle and is responsible for STAT3-dependent mitochondrial transcription. Surprisingly, STAT3-dependent increase of mitochondrial transcription seems independent from STAT3 binding to STAT3 responsive elements. Finally, loss of function experiments, with chemical inhibition of the JAK/STAT3 pathway or genetic ablation of stat3 gene, demonstrated that STAT3 is also required for cell proliferation in the intestine of zebrafish.

Keywords: ESC; STAT3; mitochondria; transcription; zebrafish

DOI: https://doi.org/10.1242/dev.199477

Stem Cells. 2021 Aug 30.

Cell cycle-coupled changes in the level of reactive oxygen species support the proliferation of human pluripotent stem cells.

Ju S Ivanova, N A Pugovkina, I E Neganova, I V Kozhukharova, N N Nikolsky, O G Lyublinskaya.

The study of proliferation regulation in human pluripotent stem cells is crucial to gain insights into understanding the physiology of these cells. However, redox regulation of the pluripotent cell cycle remains largely unexplored. Here, using human embryonic stem cells (hESCs) as well as human induced pluripotent stem cells (hiPSCs), we demonstrate that the level of reactive oxygen species (ROS) in pluripotent cells oscillates in accordance with the cell cycle progression with the peak occurring at transition from S to G2 /M phase of the cycle. A decrease of this level by antioxidants leads to hindered S-phase initiation and progression but does not affect the early-G1 -phase or mitosis. Cells exposed to antioxidants in the early-G1 -phase accumulate the phosphorylated retinoblastoma protein and overcome the restriction point but are unable to accumulate the main regulators of the S phase - CYCLIN A and GEMININ. Based on the previous findings that CYCLIN A stability is affected by redox homeostasis disturbances in somatic cells, we compared the responses to antioxidant treatments in hESCs and in their differentiated fibroblast-like progeny cells (difESCs). In difESCs, similar to hESCs, a decrease in ROS level results in the disruption of S-phase initiation accompanied by a deficiency of the CYCLIN A level. Moreover, in antioxidant-treated cells, we revealed the accumulation of DNA breaks, which was accompanied with activation of the apoptosis program in pluripotent cells. Thus, we conclude that maintaining the physiological ROS level is essential for promotion of proliferation and accurate DNA synthesis in pluripotent cells and their differentiated descendants. © AlphaMed Press 2021 SIGNIFICANCE STATEMENT: Reactive oxygen species are involved in the regulation of almost all intracellular processes. However, the number of studies devoted to redox signaling in pluripotent cells is quite limited. In our study, we found that maintaining the physiological level of reactive oxygen species is essential for the promotion of proliferation and accurate DNA synthesis in embryonic and induced pluripotent stem cells. A decrease in this level leads to the disturbance of S-phase regulation, loss of DNA integrity, and apoptosis. Hence, this article provides new insight into the redox homeostasis of pluripotent cells, which are a powerful tool for research in developmental biology and biomedical applications.

Keywords: antioxidants; human embryonic stem cells; induced pluripotent stem cells; pluripotent cell cycle; reactive oxygen species; redox homeostasis; redox regulation

DOI: https://doi.org/10.1002/stem.3450

Bioelectricity. 2020 Sep 01. 2(3): 238-250

Dual Bioelectrical Assessment of Human Mesenchymal Stem Cells Using Plasma and Mitochondrial Membrane Potentiometric Probes.

Timothy Kamaldinov, Mariah S Hahn.

Background: Bioelectrical properties are known to impact stem cell fate, state, and function. However, assays that measure bioelectrical properties are generally limited to the plasma membrane potential. In this study, we propose an assay to simultaneously assess cell plasma membrane and mitochondrial membrane potentials. Materials and Methods: Mesenchymal stem cell (MSC) plasma and mitochondrial membrane potentials were measured using flow cytometry and a combination of tetramethylrhodamine, methyl ester (TMRM), and bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC) dyes. We investigated the shifts in the bioelectrical phenotype of MSCs due to extended culture in vitro, activation with interferon-gamma (IFN-γ), and aggregate conditions. Results: MSCs subjected to extended culture in vitro acquired plasma and mitochondrial membrane potentials consistent with a hyperpolarized bioelectrical phenotype. Activation with IFN-γ shifted MSCs toward a state associated with increased levels of both DiBAC and TMRM. MSCs in aggregate conditions were associated with a decrease in TMRM levels, indicating mitochondrial depolarization. Conclusions: Our proposed assay described distinct MSC bioelectrical transitions due to extended in vitro culture, exposure to an inflammatory cytokine, and culture under aggregate conditions. Overall, our assay enables a more complete characterization of MSC bioelectrical properties within a single experiment, and its relative simplicity enables researchers to apply it in variety of settings.

Keywords: mesenchymal stem cells; mitochondrial membrane potential; plasma membrane potential

DOI: https://doi.org/10.1089/bioe.2020.0006

STAR Protoc. 2021 Sep 17. 2(3): 100740

Protocol for intracellular and extracellular metabolite detection in human embryonic stem cells.

Faxiang Xu, Chengcheng Song, Weiwei Liu, Guokai Chen.

Metabolic homeostasis is critical for cell pluripotency and differentiation in human embryonic stem cells (hESCs). It has been reported that metabolic changes specifically regulate cellular signaling during hESC differentiation. This protocol describes procedures for both cell culture and detection of intracellular and extracellular metabolites in hESCs by liquid chromatography-mass spectrometry. Metabolites in glycolysis, citric acid cycle, pentose phosphate pathway, and other metabolic processes can be detected using this approach. For complete details on the use and execution of this protocol, please refer to Song et al., (2019), Yang et al., (2019), Meng et al., (2018), and Chen et al., (2011b).

Keywords: Cell Differentiation; Cell culture; Flow Cytometry/Mass Cytometry; Metabolism; Metabolomics; Stem Cells

DOI: https://doi.org/10.1016/j.xpro.2021.100740

Sci Rep. 2021 Sep 02. 11(1): 17589

Glutamine metabolism regulates endothelial to hematopoietic transition and hematopoietic lineage specification.

Leal Oburoglu, Els Mansell, Niels-Bjarne Woods.

During hematopoietic development, definitive hematopoietic cells are derived from hemogenic endothelial (HE) cells through a process known as endothelial to hematopoietic transition (EHT). During EHT, transitioning cells proliferate and undergo progressive changes in gene expression culminating in the new cell identity with corresponding changes in function, phenotype and morphology. However, the metabolic pathways fueling this transition remain unclear. We show here that glutamine is a crucial regulator of EHT and a rate limiting metabolite in the hematopoietic differentiation of HE cells. Intriguingly, different hematopoietic lineages require distinct derivatives of glutamine. While both derivatives, α-ketoglutarate and nucleotides, are required for early erythroid differentiation of HE during glutamine deprivation, lymphoid differentiation relies on α-ketoglutarate alone. Furthermore, treatment of HE cells with α-ketoglutarate in glutamine-free conditions pushes their differentiation towards lymphoid lineages both in vitro and in vivo, following transplantation into NSG mice. Thus, we report an essential role for glutamine metabolism during EHT, regulating both the emergence and the specification of hematopoietic cells through its various derivatives.

DOI: https://doi.org/10.1038/s41598-021-97194-7