Continuous hypoxic culturing maintains activation of Notch and allows long‐term propagation of human embryonic stem cells without spontaneous differentiation

Prasad, S. M.; Czepiel, Marcin; Cetinkaya, Cihan; Smigielska, K.; Weli, Simon Chioma; Lysdahl, Helle; Gabrielsen, Anette; Petersen, K.; Ehlers, Niels; Fink, Trine; Minger, SL; Zachar, Vladimír

doi:10.1111/j.1365-2184.2008.00571.x

Cited by 98 publications

(92 citation statements)

References 33 publications

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“…Embryo culture in atmospheric oxygen (20%), compared to physiological concentrations, is associated with perturbed gene expression (Harvey et al 2004, Rinaudo et al 2006 changes to the proteome (Katz-Jaffe et al 2005), alterations in metabolic activity (Lane & Gardner 2005, Wale & Gardner 2012, Wale & Gardner 2013) and retarded development in several species, including the human (Thompson et al 1990, Meintjes et al 2009, Waldenströ m et al 2009). In previous reports, hES cell lines showed changes in metabolism (Forristal et al 2013, Harvey et al 2014, epigenetics (Petruzzelli et al 2014), transcription (Forsyth et al 2008, Westfall et al 2008, self-renewal capacity (Prasad et al 2009), clonal recovery , Hewitt et al 2006 and pluripotency (Ezashi et al 2005) in response to changes in environmental oxygen concentration. For example, culture of hES cells in physiological oxygen (w5%) results in increased glucose consumption and increased lactate production when compared to cells cultured in 20% oxygen (Forristal et al 2013, Harvey et al 2014, Turner et al 2014.…”

Section: Introductionmentioning

confidence: 85%

Distinct profiles of human embryonic stem cell metabolism and mitochondria identified by oxygen

Lees¹,

Rathjen²,

Sheedy³

et al. 2015

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Oxygen is a powerful regulator of cell function and embryonic development. It has previously been determined that oxygen regulates human embryonic stem (hES) cell glycolytic and amino acid metabolism, but the effects on mitochondria are as yet unknown. Two hES cell lines (MEL1, MEL2) were analyzed to determine the role of 5% (physiological) and 20% (atmospheric) oxygen in regulating mitochondrial activity. In response to extended physiological oxygen culture, MEL2 hES cells displayed reduced mtDNA content, mitochondrial mass and expression of metabolic genes TFAM, NRF1, PPARa and MT-ND4. Furthermore, MEL2 hES cell glucose consumption, lactate production and amino acid turnover were elevated under physiological oxygen. In stark contrast, MEL1 hES cell amino acid and carbohydrate use and mitochondrial function were relatively unaltered in response to oxygen. Furthermore, differentiation kinetics were delayed in the MEL1 hES cell line following BMP4 treatment. Here we report the first incidence of metabolic dysfunction in a hES cell population, defined as a failure to respond to oxygen concentration through the modulation of metabolism, demonstrating that hES cells can be perturbed during culture despite exhibiting the defining characteristics of pluripotent cells. Collectively, these data reveal a central role for oxygen in the regulation of hES cell metabolism and mitochondrial function, whereby physiological oxygen promotes glucose flux and suppresses mitochondrial biogenesis and gene expression.

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Section: Introductionmentioning

confidence: 85%

Distinct profiles of human embryonic stem cell metabolism and mitochondria identified by oxygen

Lees¹,

Rathjen²,

Sheedy³

et al. 2015

Reproduction

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“…Also, culturing human ESCs by constant hypoxic conditions may maintain pluripotency by sustaining Notch activation (67). Recent research data also show that using hypoxic conditions (5% O 2 ) increases the efficiency of generation of iPSCs from mouse MEFs using OCT3/4, Sox2, and Klf4 retroviral transduction, as well as with nonviral vectors, such as plasmid expression vectors or piggyback transposition system (99).…”

Section: Role Of Hypoxia In Pluripotent Stem Cellsmentioning

confidence: 99%

Roles of Reactive Oxygen Species in the Fate of Stem Cells

Chaudhari

Jang³

2014

Antioxidants & Redox Signaling

119

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Significance: Stem cells are characterized by the properties of self-renewal and the ability to differentiate into multiple cell types, and thus maintain tissue homeostasis. Reactive oxygen species (ROS) are a natural byproduct of aerobic metabolism and have roles in cell signaling. Regulation of ROS has a vital role in maintaining ''stemness'' and differentiation of the stem cells, as well as in progression of stem-cell-associated diseases. Recent Advances: As of late, much research has been done on the adverse effects of ROS in stem cells. However, recently it has become apparent that in some cases redox status of the stem cell does have a role in maintaining its identity as such. Both pluripotent and multipotent stem cell types have been reported to possess enzymatic and nonenzymatic mechanisms for detoxification of ROS and to correct oxidative damage to the genome as well as the proteome. Critical Issues: Although context dependent and somewhat varied among different stem cell types, the correlation seems to exist between antioxidant defense level and stem cell fate change (i.e., proliferation, differentiation, and death). Changes in stem cell redox regulation may affect the pathogenesis of various human diseases. Future Directions: Dissecting the defined roles of ROS in distinct stem cell types will greatly enhance their basic and translational applications. Here, we discuss the various roles of ROS in adult, embryonic, and induced pluripotent stem cells.

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“…It has been demonstrated that NICD-dependent transcriptional activity, and thus the stem cell pluripotency, may be regulated by oxygen partial tension (11). It is interesting to note that several previous studies confirmed that pericellular oxygen concentration has an effect on hESCs self-renewal and differentiation (12,13). In the case of hematopoietic stem cells that reside in a relatively hypoxic niche, it has previously been shown that activation of Notch blocked differentiation and resulted in T cell acute lymphoblastic leukemia (14).…”

Section: Introductionmentioning

confidence: 95%

Notch and Hedgehog Signaling Cooperate to Maintain Self-Renewal of Human Embryonic Stem Cells Exposed to Low Oxygen Concentration

et al. 2010

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Background and Objectives: Expansion and maintenance of human embryonic stem cells (hESCs) in undifferentiated state is influenced by complex signals in the microenvironment, including those contingent upon oxygen availability. Responses mediated by Notch and Hedgehog (Hh) have essential role in the growth and maintenance of hESCs, therefore this study examined their effect on the self-renewal of hESCs exposed to low oxygen. Methods and Results: Using potent antagonists γ-secretase inhibitor and cyclopamine, we inhibited Notch and Hh pathways, respectively, in the CLS1 hESC line expanded continuously in a hypoxic atmosphere of 5% oxygen. Immunohistochemical staining and protein assays revealed loss of Oct4 and gain of stage-specific embryonic antigen 1 (SSEA1) markers in the inhibited cells. Semiquantitative real-time RT-PCR, and bromodeoxyuridine and thymidine incorporation assays demonstrated low Oct4 and Nanog mRNA expression, and decreased DNA synthesis, respectively, resulting from the block of each of the pathways. The loss increased significantly with co-inhibition of both pathways. Importantly, Notch and Hh downstream targets, including Hes1, Hey1, GIi1, and Ptc1, were surprisingly suppressed not only by the pathway-specific but also the unrelated inhibitor.Conclusions: These findings demonstrate complementary effect of Notch and Hh signaling in hypoxia enhanced maintenance of hESCs.

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Continuous hypoxic culturing maintains activation of Notch and allows long‐term propagation of human embryonic stem cells without spontaneous differentiation

Abstract: Our data, thus, indicate that hypoxic exposure has the capacity to sustain long-term self-renewal of hESCs and that this effect is mediated through activation of Notch.

Cited by 98 publications

References 33 publications

Distinct profiles of human embryonic stem cell metabolism and mitochondria identified by oxygen

Distinct profiles of human embryonic stem cell metabolism and mitochondria identified by oxygen

Roles of Reactive Oxygen Species in the Fate of Stem Cells

Notch and Hedgehog Signaling Cooperate to Maintain Self-Renewal of Human Embryonic Stem Cells Exposed to Low Oxygen Concentration

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