bims-mireme 2021-10-17 papers

J Vis Exp. 2021 Sep 24.

Assessment of Cellular Bioenergetics in Mouse Hematopoietic Stem and Primitive Progenitor Cells using the Extracellular Flux Analyzer.

Surinder Kumar, Morgan Jones, Qing Li, David B Lombard.

Under steady state, hematopoietic stem cells (HSCs) remain largely quiescent and are believed to be predominantly reliant on glycolysis to meet their energetic needs. However, under stress conditions such as infection or blood loss, HSCs become proliferative and rapidly produce downstream progenitor cells, which in turn further differentiate, ultimately producing mature blood cells. During this transition and differentiation process, HSCs exit from quiescence and rapidly undergo a metabolic switch from glycolysis to oxidative phosphorylation (OxPHOS). Various stress conditions, such as aging, cancer, diabetes, and obesity, can negatively impact mitochondrial function and thus can alter the metabolic reprogramming and differentiation of HSCs and progenitors during hematopoiesis. Valuable insights into glycolytic and mitochondrial functions of HSCs and progenitors under normal and stress conditions can be gained through the assessment of their extracellular acidification rate (ECAR) and oxygen consumption rate (OCR), which are indicators of cellular glycolysis and mitochondrial respiration, respectively. Here, a detailed protocol is provided to measure ECAR and OCR in mouse bone marrow-derived lineage-negative cell populations, which include both hematopoietic stem and primitive progenitor cells (HSPCs), using the extracellular flux analyzer. This protocol describes approaches to isolate lineage-negative cells from mouse bone marrow, explains optimization of cell seeding density and concentrations of 2-deoxy-D-glucose (2-DG, a glucose analog that inhibits glycolysis) and various OxPHOS-targeted drugs (oligomycin, FCCP, rotenone, and antimycin A) used in these assays, and describes drug treatment strategies. Key parameters of glycolytic flux, such as glycolysis, glycolytic capacity, and glycolytic reserve, and OxPHOS parameters, such as basal respiration, maximal respiration, proton leak, ATP production, spare respiratory capacity, and coupling efficiency, can be measured in these assays. This protocol allows ECAR and OCR measurements on non-adherent HSPCs and can be generalized to optimize analysis conditions for any type of suspension cells.

DOI: https://doi.org/10.3791/63045

Cell Metab. 2021 Oct 06. pii: S1550-4131(21)00432-0. [Epub ahead of print]

Nutrients in the fate of pluripotent stem cells.

Vivian Lu, Irena J Roy, Michael A Teitell.

Pluripotent stem cells model certain features of early mammalian development ex vivo. Medium-supplied nutrients can influence self-renewal, lineage specification, and earliest differentiation of pluripotent stem cells. However, which specific nutrients support these distinct outcomes, and their mechanisms of action, remain under active investigation. Here, we evaluate the available data on nutrients and their metabolic conversion that influence pluripotent stem cell fates. We also discuss key questions open for investigation in this rapidly expanding area of increasing fundamental and practical importance.

Keywords: development; differentiation; fate; metabolism; nutrients; pluripotent stem cell

DOI: https://doi.org/10.1016/j.cmet.2021.09.013

Mol Biol Rep. 2021 Oct 13.

Intracellular metabolic reprogramming mediated by micro-RNAs in differentiating and proliferating cells under non-diseased conditions.

Varsha Singh.

Intracellular metabolic reprogramming is a critical process the cells carry out to increase biomass, energy fulfillment and genome replication. Cells reprogram their demands from internal catabolic or anabolic activities in coordination with multiple genes and microRNAs which further control the critical processes of differentiation and proliferation. The microRNAs reprogram the metabolism involving mitochondria, the nucleus and the biochemical processes utilizing glucose, amino acids, lipids, and nucleic acids resulting in ATP production. The processes of glycolysis, tricarboxylic acid cycle, or oxidative phosphorylation are also mediated by micro-RNAs maintaining cells and organs in a non-diseased state. Several reports have shown practical applications of metabolic reprogramming for clinical utility to assess various diseases, mostly studying cancer and immune-related disorders. Cells under diseased conditions utilize glycolysis for abnormal growth or proliferation, respectively, affecting mitochondrial paucity and biogenesis. Similar metabolic processes also affect gene expressions and transcriptional regulation for carrying out biochemical reactions. Metabolic reprogramming is equally vital for regulating cell environment to maintain organs and tissues in non-diseased states. This review offers in depth insights and analysis of how miRNAs regulate metabolic reprogramming in four major types of cells undergoing differentiation and proliferation, i.e., immune cells, neuronal cells, skeletal satellite cells, and cardiomyocytes under a non-diseased state. Further, the work systematically summarizes and elaborates regulation of genetic switches by microRNAs through predominantly through cellular reprogramming and metabolic processes for the first time. The observations will lead to a better understanding of disease initiation during the differentiation and proliferation stages of cells, as well as fresh approaches to studying clinical onset of linked metabolic diseases targeting metabolic processes.

Keywords: Cell differentiation; Cell proliferation; Gene regulation; Metabolic reprogramming; MicroRNAs; Signal transduction

DOI: https://doi.org/10.1007/s11033-021-06769-0

Nat Commun. 2021 Oct 13. 12(1): 5977

p107 mediated mitochondrial function controls muscle stem cell proliferative fates.

Debasmita Bhattacharya, Vicky Shah, Oreoluwa Oresajo, Anthony Scimè.

Muscle diseases and aging are associated with impaired myogenic stem cell self-renewal and fewer proliferating progenitors (MPs). Importantly, distinct metabolic states induced by glycolysis or oxidative phosphorylation have been connected to MP proliferation and differentiation. However, how these energy-provisioning mechanisms cooperate remain obscure. Herein, we describe a mechanism by which mitochondrial-localized transcriptional co-repressor p107 regulates MP proliferation. We show p107 directly interacts with the mitochondrial DNA, repressing mitochondrial-encoded gene transcription. This reduces ATP production by limiting electron transport chain complex formation. ATP output, controlled by the mitochondrial function of p107, is directly associated with the cell cycle rate. Sirt1 activity, dependent on the cytoplasmic glycolysis product NAD+, directly interacts with p107, impeding its mitochondrial localization. The metabolic control of MP proliferation, driven by p107 mitochondrial function, establishes a cell cycle paradigm that might extend to other dividing cell types.

DOI: https://doi.org/10.1038/s41467-021-26176-0

STAR Protoc. 2021 Dec 17. 2(4): 100850

Generating stable isolated mitochondrial recipient clones in mammalian cells using MitoPunch mitochondrial transfer.

Alexander J Sercel, Alexander J Napior, Alexander N Patananan, Ting-Hsiang Wu, Pei-Yu Chiou, Michael A Teitell.

This protocol describes the assembly and use of MitoPunch to deliver mitochondria containing mitochondrial DNA (mtDNA) into cells lacking mtDNA (ρ0 cells). MitoPunch generates stable isolated mitochondrial recipient clones with restored mtDNA and recovered respiration, enabling investigation of mtDNA mutations and mtDNA-nuclear DNA interactions in a range of cell types. For complete details on the use and execution of this protocol, please refer to Sercel et al. (2021) and Patananan et al. (2020).

Keywords: Biotechnology and bioengineering; Cell Biology; Cell culture; Cell-based Assays; Metabolism

DOI: https://doi.org/10.1016/j.xpro.2021.100850

FASEB Bioadv. 2021 Oct;3(10): 787-801

Friends and foes: Extracellular vesicles in aging and rejuvenation.

Brian V Lananna, Shin-Ichiro Imai.

Extracellular vesicles (EVs) are released by many different cell types throughout the body and play a role in a diverse range of biological processes. EVs circulating in blood as well as in other body fluids undergo dramatic alterations over an organism's lifespan that are only beginning to be elucidated. The exact nature of these changes is an area of active and intense investigation, but lacks clear consensus due to the substantial heterogeneity in EV subpopulations and insufficiencies in current technologies. Nonetheless, emerging evidence suggests that EVs regulate systemic aging as well as the pathophysiology of age-related diseases. Here, we review the current literature investigating EVs and aging with an emphasis on consequences for the maintenance of human healthspan. Intriguingly, the biological utility of EVs both in vitro and in vivo and across contexts depends on the states of the source cells or tissues. As such, EVs secreted by cells in an aged or pathological state may impose detrimental consequences on recipient cells, while EVs secreted by youthful or healthy cells may promote functional improvement. Thus, it is critical to understand both functions of EVs and tip the balance toward their beneficial effects as an antiaging intervention.

Keywords: NAD+ metabolism; ageing; aging; exosome; extracellular vesicle; longevity

DOI: https://doi.org/10.1096/fba.2021-00077