Metabolic switching and cell fate decisions: implications for pluripotency, reprogramming and development

Cliff, Tim S; Dalton, Stephen

doi:10.1016/j.gde.2017.06.008

Cited by 79 publications

(58 citation statements)

References 56 publications

(75 reference statements)

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“…We found that genes with novel transcripts are enriched in metabolic processes and specific neural functions related to neurogenesis and oligodendroglial lineage. This is remarkable because both the narrow control of metabolic programming and the expression of genes involved in cell identity are key players in differentiation courses (Cliff and Dalton 2017), and the finding that most novel transcripts concentrate in these categories suggests that important untapped transcript/regulatory diversity could be revealed by long-read sequencing technologies.…”

Section: Sqanti As a Critical Tool To Analyze Whole Transcriptome Quamentioning

confidence: 99%

SQANTI: extensive characterization of long-read transcript sequences for quality control in full-length transcriptome identification and quantification

et al. 2018

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High-throughput sequencing of full-length transcripts using long reads has paved the way for the discovery of thousands of novel transcripts, even in well-annotated mammalian species. The advances in sequencing technology have created a need for studies and tools that can characterize these novel variants. Here, we present SQANTI, an automated pipeline for the classification of long-read transcripts that can assess the quality of data and the preprocessing pipeline using 47 unique descriptors. We apply SQANTI to a neuronal mouse transcriptome using Pacific Biosciences (PacBio) long reads and illustrate how the tool is effective in characterizing and describing the composition of the full-length transcriptome. We perform extensive evaluation of ToFU PacBio transcripts by PCR to reveal that an important number of the novel transcripts are technical artifacts of the sequencing approach and that SQANTI quality descriptors can be used to engineer a filtering strategy to remove them. Most novel transcripts in this curated transcriptome are novel combinations of existing splice sites, resulting more frequently in novel ORFs than novel UTRs, and are enriched in both general metabolic and neural-specific functions. We show that these new transcripts have a major impact in the correct quantification of transcript levels by state-of-the-art short-read-based quantification algorithms. By comparing our iso-transcriptome with public proteomics databases, we find that alternative isoforms are elusive to proteogenomics detection. SQANTI allows the user to maximize the analytical outcome of long-read technologies by providing the tools to deliver quality-evaluated and curated full-length transcriptomes.

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Section: Sqanti As a Critical Tool To Analyze Whole Transcriptome Quamentioning

confidence: 99%

SQANTI: extensive characterization of long-read transcript sequences for quality control in full-length transcriptome identification and quantification

et al. 2018

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“…Deregulated metabolic switching has also been implicated in obesity and type-II diabetes (Vander Heiden et al, 2009), raising the possibility that metabolic switching is important not only for developmental decisions but also for disease pathogenesis. Numerous reports have also described the requirement for a glycolytic mode of metabolism to reprogram fibroblasts to a pluripotent state (Cliff and Dalton, 2017; Folmes et al, 2011; Kida et al, 2015; Ryall et al, 2015a). This metabolic need underscores observations described in this report and elsewhere (Gu et al, 2016; Moussaieff et al, 2015b) that glycolytic flux is linked, but not limited, to maintenance and establishment of the pluripotent state.…”

Section: Discussionmentioning

confidence: 99%

MYC Controls Human Pluripotent Stem Cell Fate Decisions through Regulation of Metabolic Flux

et al. 2017

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SUMMARY As human pluripotent stem cells (hPSCs) exit pluripotency they are thought to switch from a glycolytic mode of energy generation to one more dependent on oxidative phosphorylation. Here we show that although metabolic switching occurs during early mesoderm and endoderm differentiation, high glycolytic flux is maintained and in fact essential during early ectoderm specification. The elevated glycolysis observed in hPSCs requires elevated MYC/MYCN activity. Metabolic switching during endodermal and mesodermal differentiations coincides with a reduction in MYC/MYCN, and can be reversed by ectopically restoring MYC activity. During early ectodermal differentiation, sustained MYCN activity maintains the transcription of 'switch' genes that are rate-limiting for metabolic activity and lineage commitment. Our work, therefore, shows that metabolic switching is lineage-specific and not a required step for exit of pluripotency in hPSCs, and identifies MYC and MYCN as developmental regulators that couple metabolism to pluripotency and cell fate determination.

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“…It is to be noted that the TGFβ pathway is important for pluripotency due to its direct modulation of expression of Nanog (Xu et al, 2008). The increased expression of genes related to the glycolytic pathway in ARPE-19 cells, upon SPIR treatment, reflects the pluripotent state of these cells under these conditions, since aerobic glycolysis supports the maintenance of the pluripotent state by maintaining histone acetylation and the associated open chromatin structure of PSCs (Moussaieff et al, 2015;Gu et al, 2016;Folmes et al, 2011;Guda et al, 2018;Cliff et al, 2017, Dahan et al, 2019).…”

Section: Discussionmentioning

confidence: 99%

Sustained exposure to trypsin causes cells to transition into a state of reversible stemness that is amenable to transdifferentiation

Sharma

Kumar

Sharma

et al. 2019

Preprint

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During cell culture, trypsin, a serine protease, is applied to cells for 5-10 minutes to separate them from each other and from the underlying substratum so that they can be transferred to a different vessel, for re-plating, after growth medium containing 10 % serum has been added to the cells, in a well-known technique known as 'passaging'. The serum in the growth medium contains alpha-1 antitrypsin, which is a potent inhibitor of trypsin, elastase and other serine proteases.Although what is used is bovine serum in which levels of proteins could be different from levels seen in humans, normal human serum contains A1AT (> 1 mg/ml; > ~18 µmol/L) as well as trypsin itself (< 460 ng/ml, or ~0.02 µmol/L), with the former in a ~900-fold molar excess over the latter. Thus, it may be assumed there is also enough A1AT in the bovine serum added during passaging, to neutralize the trypsin (~100 μM) present in the small volume of trypsin-EDTA solution used to separate cells. What are the consequences of not adding serum, when growth medium is added, or of maintaining cells for a few tens of hours in the presence of trypsin, in a serum-free growth medium? What does such sustained exposure to trypsin during cell culture do to cells? More generally, what are the responses of cells within an organism to the balance of trypsin and A1AT in the serum that bathes them constantly? We know that excesses and deficiencies in the levels of either trypsin or A1AT are associated with disease. We know that cellular metabolism can be influenced through signaling involving protease activated membrane GPCR receptors (PAR1-4). In particular, we know of a receptor called PAR2, which is specifically activated by trypsin, expressed by cells at baseline levels, and upregulated through some feedback involving trypsin-activation. We also know that cells at sites of injury or inflammation produce and secrete trypsin, and that this trypsin can act locally upon cells in a variety of ways, all of which have probably not yet been elucidated. Here, we show that sustained exposure to trypsin induces cells to de-differentiate into a stem-like state. We show that if serum is either not added at all, or added and then washed away (after confluency is attained), during cell culture, all cells exposed to exogenously-added trypsin undergo changes in morphology, transcriptome, secretome, and developmental potential, and transition into a state of stemness, in minimal essential medium (MEM). Regardless of their origins, i.e., independent of whether they are derived from primary cultures, cell lines or cancer cell lines, and regardless of the original cell type used, exposure to trypsin (~10 µM; ~250 µg/ml) at a concentration 10-fold lower than that used to separate cells during passaging (~100 μM), over a period of 24-48 hours, causes cells to (1) become rounded, (2) cluster together, (3) get arrested in the G0/G1 stage of the cell cycle, (4) display increased presence of 5-hydroxymethyl cytosine in their nuclei (indicative of reprogramming), (5) display increased...

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Metabolic switching and cell fate decisions: implications for pluripotency, reprogramming and development

Cited by 79 publications

References 56 publications

SQANTI: extensive characterization of long-read transcript sequences for quality control in full-length transcriptome identification and quantification

SQANTI: extensive characterization of long-read transcript sequences for quality control in full-length transcriptome identification and quantification

MYC Controls Human Pluripotent Stem Cell Fate Decisions through Regulation of Metabolic Flux

Sustained exposure to trypsin causes cells to transition into a state of reversible stemness that is amenable to transdifferentiation

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