bims-mireme 2021-05-09 papers

bims-mireme

Biomed News

on Mitochondria in regenerative medicine

Issue of 2021–05–09
nine papers selected by
Brian Spurlock, University of Alabama at Birmingham

Retinoic acid induces NELFA-mediated 2C-like state of mouse embryonic stem cells associates with epigenetic modifications and metabolic processes in chemically defined media.
ASCs derived from burn patients are more prone to increased oxidative metabolism and reactive oxygen species upon passaging.
Monitoring mammalian mitochondrial translation with MitoRiboSeq.
Preconditioning of mesenchymal stem cells with ghrelin exerts superior cardioprotection in aged heart through boosting mitochondrial function and autophagy flux.
Hypoxia-inducible factor-1α-mediated upregulation of CD99 promotes the proliferation of placental mesenchymal stem cells by regulating ERK1/2.
Fatty acid oxidation is required for embryonic stem cell survival during metabolic stress.
PINK1 contained in huMSC-derived exosomes prevents cardiomyocyte mitochondrial calcium overload in sepsis via recovery of mitochondrial Ca2+ efflux.
Metformin perturbs pancreatic differentiation from human embryonic stem cells.
Bivalve molluscs as model systems for studying mitochondrial biology.

Cell Prolif. 2021 May 07. e13049

Retinoic acid induces NELFA-mediated 2C-like state of mouse embryonic stem cells associates with epigenetic modifications and metabolic processes in chemically defined media.

Yanqiu Wang, Qin Na, Xihe Li, Wee-Wei Tee, Baojiang Wu, Siqin Bao.

   OBJECTIVES: Mouse embryonic stem cells (ESCs) are derived from the inner cell mass of blastocyst-stage embryos and cultured in different culture media with varied pluripotency. Sporadically, a small population of ESCs exhibit 2-cell stage embryonic features in serum containing medium. However, whether ESCs can transit into 2-cell embryo-like (2C-like) cells in the chemically defined media remains largely unknown.
MATERIALS AND METHODS: We established a robust in vitro induction system, based on retinoic acid (RA) containing chemically defined media, which can efficiently increase the subpopulation of 2C-like cells. Further test the pluripotency and 2C features of ESCs cultured in RA. 2C reporter-positive cells were selected by FACS; the level of protein was detected via immunofluorescence staining and western blot; the level gene expressions were measured by RNA-seq.
RESULTS: Retinoic acid drives a NELFA (negative elongation factor A)-mediated 2C-like state in mouse ESCs, characterized with 2C-specific transcriptional networks and the ability to contribute trophectoderm (TE) when injected into developing embryos. In addition, RA treatment triggers DNA hypomethylation, active histone modification, suppressed glycolysis metabolism and reduced protein synthesis activity of ESCs.
CONCLUSIONS: We showed that RA has a broader role in 2C-like cells state, not only is one of the upstream regulators of the 2C-like state in chemically defined media but also illuminates genetic and epigenetic regulations that govern ESCs to 2C-like transition.

Keywords:  2C-like cells; NELFA; embryonic stem cell; epigenetic; mouse; retinoic acid

DOI:  https://doi.org/10.1111/cpr.13049
Stem Cell Res Ther. 2021 May 06. 12(1): 270

ASCs derived from burn patients are more prone to increased oxidative metabolism and reactive oxygen species upon passaging.

David M Burmeister, Grace Chu-Yuan Chu, Tony Chao, Tiffany C Heard, Belinda I Gómez, Linda E Sousse, Shanmugasundaram Natesan, Robert J Christy.

   BACKGROUND: Patients with severe burn injury (over 20% of the total body surface area) experience profound hypermetabolism which significantly prolongs wound healing. Adipose-derived stem cells (ASCs) have been proposed as an attractive solution for treating burn wounds, including the potential for autologous ASC expansion. While subcutaneous adipocytes display an altered metabolic profile post-burn, it is not known if this is the case with the stem cells associated with the adipose tissue.
METHODS: ASCs were isolated from discarded burn skin of severely injured human subjects (BH, n = 6) and unburned subcutaneous adipose tissue of patients undergoing elective abdominoplasty (UH, n = 6) and were analyzed at passages 2, 4, and 6. Flow cytometry was used to quantify ASC cell surface markers CD90, CD105, and CD73. Mitochondrial abundance and reactive oxygen species (ROS) production were determined with MitoTracker Green and MitoSOX Red, respectively, while JC-10 Mitochondrial Membrane Potential Assays were also performed. Mitochondrial respiration and glycolysis were analyzed with a high-resolution respirometer (Seahorse XFe24 Analyzer).
RESULTS: There was no difference in age between BH and UH (34 ± 6 and 41 ± 4 years, respectively, P = 0.49). While passage 2 ASCs had lower ASC marker expression than subsequent passages, there were no significant differences in the expression between BH and UH ASCs. Similarly, no differences in mitochondrial abundance or membrane potential were found amongst passages or groups. Two-way ANOVA showed a significant effect (P < 0.01) of passaging on mitochondrial ROS production, with increased ROS in BH ASCs at later passages. Oxidative phosphorylation capacities (leak and maximal respiration) increased significantly in BH ASCs (P = 0.035) but not UH ASCs. On the contrary, basal glycolysis significantly decreased in BH ASCs (P = 0.011) with subsequent passaging, but not UH ASCs.
CONCLUSIONS: In conclusion, ASCs from burned individuals become increasingly oxidative and less glycolytic upon passaging when compared to ASCs from unburned patients. This increase in oxidative capacities was associated with ROS production in later passages. While the autologous expansion of ASCs holds great promise for treating burned patients with limited donor sites, the potential negative consequences of using them require further investigation.

Keywords:  Adipose stem cells; Burn; Glycolysis; Mitochondria; Oxidative phosphorylation; ROS; Respirometry

DOI:  https://doi.org/10.1186/s13287-021-02327-4
Nat Protoc. 2021 May 05.

Monitoring mammalian mitochondrial translation with MitoRiboSeq.

Sophia Hsin-Jung Li, Michel Nofal, Lance R Parsons, Joshua D Rabinowitz, Zemer Gitai.

Several essential components of the electron transport chain, the major producer of ATP in mammalian cells, are encoded in the mitochondrial genome. These 13 proteins are translated within mitochondria by 'mitoribosomes'. Defective mitochondrial translation underlies multiple inborn errors of metabolism and has been implicated in pathologies such as aging, metabolic syndrome and cancer. Here, we provide a detailed ribosome profiling protocol optimized to interrogate mitochondrial translation in mammalian cells (MitoRiboSeq), wherein mitoribosome footprints are generated with micrococcal nuclease and mitoribosomes are separated from cytosolic ribosomes and other RNAs by ultracentrifugation in a single straightforward step. We highlight critical steps during library preparation and provide a step-by-step guide to data analysis accompanied by open-source bioinformatic code. Our method outputs mitoribosome footprints at single-codon resolution. Codons with high footprint densities are sites of mitoribosome stalling. We recently applied this approach to demonstrate that defects in mitochondrial serine catabolism or in mitochondrial tRNA methylation cause stalling of mitoribosomes at specific codons. Our method can be applied to study basic mitochondrial biology or to characterize abnormalities in mitochondrial translation in patients with mitochondrial disorders.

DOI: https://doi.org/10.1038/s41596-021-00517-1
Eur J Pharmacol. 2021 May 02. pii: S0014-2999(21)00295-8. [Epub ahead of print] 174142

Preconditioning of mesenchymal stem cells with ghrelin exerts superior cardioprotection in aged heart through boosting mitochondrial function and autophagy flux.

Liqiang Sun, Wenlong Zhang.

  Application of mesenchymal stem cells (MSCs) is considered as a promising cell-based therapy to induce cardioprotection against ischemia-reperfusion (IR) injury. Preconditioning of MSCs is the key strategy to improve MSCs functions in vitro and their efficacy in vivo, especially in elderly subjects in whom cardioprotection is lost. This study investigated the effects of preconditioning of human umbilical cord-derived MSCs with ghrelin and their combination with nicotinamide-mononucleotide (NMN) on cardioprotection, and the role of autophagy flux and mitochondrial function in aged hearts subjected to IR injury. Aged Sprague Dawley rats (20-22 months old) were subjected to LAD occlusion-induced myocardial IR injury and treated with ghrelin-preconditioned or unconditioned-MSCs at early reperfusion. NMN (500 mg/kg, i.p) was also administered at early reperfusion and repeated 12 h later. Intra-myocardial injection of ghrelin-preconditioned MSCs reduced infarct size and cardiotroponin release of aged myocardium, and improved cardiac function following IR injury. MSCs preconditioning with ghrelin restored IR-induced mitochondrial reactive oxygen species and membrane potential depolarization and enhanced ATP production. To reveal possible mechanism, preconditioned-MSCs increased autophagy flux by downregulating the overexpression of Beclin-1 and P62 proteins and increasing the LC3-II expression and LC3-II/LC3-I ratio. Moreover, combining NMN to ghrelin-preconditioned MSCs synergistically augmented its protective effects on infarct size and mitochondrial function. All above effects were abolished by autophagy flux inhibitor, chloroquine. Thus, ghrelin may serve as a promising candidate to improve the cardioprotective efficacy of MSC-based therapy via autophagy/mitochondrial pathway and that NMN serves as a good booster in combination therapy in aged hearts.

Keywords:  Autophagy; Cardioprotection; Ghrelin; Mesenchymal stem cell; Mitochondria

DOI:  https://doi.org/10.1016/j.ejphar.2021.174142
World J Stem Cells. 2021 Apr 26. 13(4): 317-330

Hypoxia-inducible factor-1α-mediated upregulation of CD99 promotes the proliferation of placental mesenchymal stem cells by regulating ERK1/2.

Xu-Dong Feng, Jia-Qi Zhu, Jia-Hang Zhou, Fei-Yan Lin, Bing Feng, Xiao-Wei Shi, Qiao-Ling Pan, Jiong Yu, Lan-Juan Li, Hong-Cui Cao.

   BACKGROUND: As human placenta-derived mesenchymal stem cells (hP-MSCs) exist in a physiologically hypoxic microenvironment, various studies have focused on the influence of hypoxia. However, the underlying mechanisms remain to be further explored.
AIM: The aim was to reveal the possible mechanisms by which hypoxia enhances the proliferation of hP-MSCs.
METHODS: A hypoxic cell incubator (2.5% O2) was used to mimic a hypoxic microenvironment. Cell counting kit-8 and 5-ethynyl-20-deoxyuridine incorporation assays were used to assay the proliferation of hP-MSCs. The cell cycle was profiled by flow cytometry. Transcriptome profiling of hP-MSCs under hypoxia was performed by RNA sequencing. CD99 mRNA expression was assayed by reverse transcription-polymerase chain reaction. Small interfering RNA-mediated hypoxia-inducible factor 1α (HIF-1α) or CD99 knockdown of hP-MSCs, luciferase reporter assays, and the ERK1/2 signaling inhibitor PD98059 were used in the mechanistic analysis. Protein expression was assayed by western blotting; immunofluorescence assays were conducted to evaluate changes in expression levels.
RESULTS: Hypoxia enhanced hP-MSC proliferation, increased the expression of cyclin E1, cyclin-dependent kinase 2, and cyclin A2, and decreased the expression of p21. Under hypoxia, CD99 expression was increased by HIF-1α. CD99-specific small interfering RNA or the ERK1/2 signaling inhibitor PD98059 abrogated the hypoxia-induced increase in cell proliferation.
CONCLUSION: Hypoxia promoted hP-MSCs proliferation in a manner dependent on CD99 regulation of the MAPK/ERK signaling pathway in vitro.

Keywords:  CD99; Hypoxia; Hypoxia-inducible factor 1α; MAPK/ERK signaling pathway; Mesenchymal stem cells; Proliferation; RNA sequencing assay

DOI:  https://doi.org/10.4252/wjsc.v13.i4.317
EMBO Rep. 2021 May 05. e52122

Fatty acid oxidation is required for embryonic stem cell survival during metabolic stress.

Hualong Yan, Navdeep Malik, Young-Im Kim, Yunlong He, Mangmang Li, Wendy Dubois, Huaitian Liu, Tyler J Peat, Joe T Nguyen, Yu-Chou Tseng, Gamze Ayaz, Waseem Alzamzami, King Chan, Thorkell Andresson, Lino Tessarollo, Beverly A Mock, Maxwell P Lee, Jing Huang.

  Metabolic regulation is critical for the maintenance of pluripotency and the survival of embryonic stem cells (ESCs). The transcription factor Tfcp2l1 has emerged as a key factor for the naïve pluripotency of ESCs. Here, we report an unexpected role of Tfcp2l1 in metabolic regulation in ESCs-promoting the survival of ESCs through regulating fatty acid oxidation (FAO) under metabolic stress. Tfcp2l1 directly activates many metabolic genes in ESCs. Deletion of Tfcp2l1 leads to an FAO defect associated with upregulation of glucose uptake, the TCA cycle, and glutamine catabolism. Mechanistically, Tfcp2l1 activates FAO by inducing Cpt1a, a rate-limiting enzyme transporting free fatty acids into the mitochondria. ESCs with defective FAO are sensitive to cell death induced by glycolysis inhibition and glutamine deprivation. Moreover, the Tfcp2l1-Cpt1a-FAO axis promotes the survival of quiescent ESCs and diapause-like blastocysts induced by mTOR inhibition. Thus, our results reveal how ESCs orchestrate pluripotent and metabolic programs to ensure their survival in response to metabolic stress.

Keywords:  Tfcp2l1; diapause; embryonic stem cell; fatty acid oxidation; metabolism

DOI:  https://doi.org/10.15252/embr.202052122
Stem Cell Res Ther. 2021 May 06. 12(1): 269

PINK1 contained in huMSC-derived exosomes prevents cardiomyocyte mitochondrial calcium overload in sepsis via recovery of mitochondrial Ca2+ efflux.

Qin Zhou, Min Xie, Jing Zhu, Qin Yi, Bin Tan, Yasha Li, Liang Ye, Xinyuan Zhang, Ying Zhang, Jie Tian, Hao Xu.

   BACKGROUND: Sepsis is a systemic inflammatory response to a local severe infection that may lead to multiple organ failure and death. Previous studies have shown that 40-50% of patients with sepsis have diverse myocardial injuries and 70 to 90% mortality rates compared to 20% mortality in patients with sepsis without myocardial injury. Therefore, uncovering the mechanism of sepsis-induced myocardial injury and finding a target-based treatment are immensely important.
OBJECTIVE: The present study elucidated the mechanism of sepsis-induced myocardial injury and examined the value of human umbilical cord mesenchymal stem cells (huMSCs) for protecting cardiac function in sepsis.
METHODS: We used cecal ligation and puncture (CLP) to induce sepsis in mice and detect myocardial injury and cardiac function using serological markers and echocardiography. Cardiomyocyte apoptosis and heart tissue ultrastructure were detected using TdT-mediated dUTP Nick-End Labeling (TUNEL) and transmission electron microscopy (TEM), respectively. Fura-2 AM was used to monitor Ca2+ uptake and efflux in mitochondria. FQ-PCR and Western blotting detected expression of mitochondrial Ca2+ distribution regulators and PTEN-induced putative kinase 1 (PINK1). JC-1 was used to detect the mitochondrial membrane potential (Δψm) of cardiomyocytes.
RESULTS: We found that expression of PINK1 decreased in mouse hearts during sepsis, which caused cardiomyocyte mitochondrial Ca2+ efflux disorder, mitochondrial calcium overload, and cardiomyocyte injury. In contrast, we found that exosomes isolated from huMSCs (huMSC-exo) carried Pink1 mRNA, which could be transferred to recipient cardiomyocytes to increase PINK1 expression. The reduction in cardiomyocyte mitochondrial calcium efflux was reversed, and cardiomyocytes recovered from injury. We confirmed the effect of the PINK1-PKA-NCLX axis on mitochondrial calcium homeostasis in cardiomyocytes during sepsis.
CONCLUSION: The PINK1-PKA-NCLX axis plays an important role in mitochondrial calcium efflux in cardiomyocytes. Therefore, PINK1 may be a therapeutic target to protect cardiomyocyte mitochondria, and the application of huMSC-exo is a promising strategy against sepsis-induced heart dysfunction.

Keywords:  Calcium overload; Cardiac dysfunction; Mitochondrial Ca2+ efflux; PINK1; Sepsis

DOI:  https://doi.org/10.1186/s13287-021-02325-6
Diabetes. 2021 May 06. pii: db200722. [Epub ahead of print]

Metformin perturbs pancreatic differentiation from human embryonic stem cells.

Linh Nguyen, Lillian Yuxian Lim, Shirley Suet Lee Ding, Nur Shabrina Amirruddin, Shawn Hoon, Shiao-Yng Chan, Adrian Kee Keong Teo.

Metformin is becoming a popular treatment before and during pregnancy but current literature on in utero exposure to metformin lacks long-term clinical trials and mechanistic studies. Current literature on the effects of metformin on mature pancreatic β cells highlighted its dual, opposing, protective or inhibitory, effects depending on metabolic environments. However, the impact of metformin on developing human pancreatic β cells remains unknown. Here, we investigated the potential effects of metformin exposure on human pancreatic β cell development and function in vitro In the absence of metabolic challenges such as high levels of glucose and fatty acids, metformin exposure impaired the development and function of pancreatic β cells, with downregulation of pancreatic genes and dysfunctional mitochondrial respiration. It also affected the insulin secretion function of pancreatic β cells. These findings call for further in-depth evaluation of the exposure of human embryonic and fetal tissue during pregnancy to metformin, and its implications on long-term offspring health.

DOI: https://doi.org/10.2337/db20-0722
Integr Comp Biol. 2021 May 04. pii: icab057. [Epub ahead of print]

Bivalve molluscs as model systems for studying mitochondrial biology.

Fabrizio Ghiselli, Mariangela Iannello, Giovanni Piccinini, Liliana Milani.

  The class Bivalvia is a highly successful and ancient taxon including ∼25,000 living species. During their long evolutionary history bivalves adapted to a wide range of physicochemical conditions, habitats, biological interactions, and feeding habits. Bivalves can have strikingly different size, and despite their apparently simple body plan, they evolved very different shell shapes, and complex anatomic structures. One of the most striking features of this class of animals is their peculiar mitochondrial biology: some bivalves have facultatively anaerobic mitochondria that allow them to survive prolonged periods of anoxia/hypoxia. Moreover, more than 100 species have now been reported showing the only known evolutionarily stable exception to the strictly maternal inheritance of mitochondria in animals, named doubly uniparental inheritance. Mitochondrial activity is fundamental to eukaryotic life, and thanks to their diversity and uncommon features, bivalves represent a great model system to expand our knowledge about mitochondrial biology, so far limited to a few species. We highlight recent works studying mitochondrial biology in bivalves at either genomic or physiological level. A link between these two approaches is still missing, and we believe that an integrated approach and collaborative relationships are the only possible ways to be successful in such endeavour.

Keywords:  Doubly Uniparental Inheritance; heteroplasmy; mitochondrial bottleneck; mitochondrial genomics; mitochondrial inheritance; physiological adaptation

DOI:  https://doi.org/10.1093/icb/icab057