RAS Regulates the Transition from Naive to Primed Pluripotent Stem Cells

Altshuler, Anna; Verbuk, Mila; Bhattacharya, Swarnabh; Abramovich, Ifat; Haklai, Roni; Hanna, Jacob; Kloog, Yoel; Gottlieb, Eyal; Shalom-Feuerstein, Ruby

doi:10.1016/j.stemcr.2018.01.004

Cited by 27 publications

(21 citation statements)

References 44 publications

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“…However, in this study, the decrease in resting pH i and the downregulation of the acid extrusion mechanism were demonstrated during the early loss of pluripotency in hiPSCs either in HEPES-buffered conditions or in 5% CO 2 /HCO 3 - -buffered conditions. These contradictory results may be due to the different pluripotent states between mESCs and hiPSCs, i.e ., naïve and primed pluripotency, respectively[54,55]. As expected, the cells in the preprimed (naïve) and primed states significantly increased the pH i at 48 and 72 hours during early differentiation in mESCs.…”

Section: Discussionmentioning

confidence: 65%

Functional and molecular mechanism of intracellular pH regulation in human inducible pluripotent stem cells

Chao

Huang

et al. 2018

WJSC

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AIMTo establish a functional and molecular model of the intracellular pH (pHi) regulatory mechanism in human induced pluripotent stem cells (hiPSCs).METHODShiPSCs (HPS0077) were kindly provided by Dr. Dai from the Tri-Service General Hospital (IRB No. B-106-09). Changes in the pHi were detected either by microspectrofluorimetry or by a multimode reader with a pH-sensitive fluorescent probe, BCECF, and the fluorescent ratio was calibrated by the high K+/nigericin method. NH4Cl and Na-acetate prepulse techniques were used to induce rapid intracellular acidosis and alkalization, respectively. The buffering power (β) was calculated from the ΔpHi induced by perfusing different concentrations of (NH4)2SO4. Western blot techniques and immunocytochemistry staining were used to detect the protein expression of pHi regulators and pluripotency markers.RESULTSIn this study, our results indicated that (1) the steady-state pHi value was found to be 7.5 ± 0.01 (n = 20) and 7.68 ± 0.01 (n =20) in HEPES and 5% CO2/HCO3--buffered systems, respectively, which were much greater than that in normal adult cells (7.2); (2) in a CO2/HCO3--buffered system, the values of total intracellular buffering power (β) can be described by the following equation: βtot = 107.79 (pHi)2 - 1522.2 (pHi) + 5396.9 (correlation coefficient R2 = 0.85), in the estimated pHi range of 7.1-8.0; (3) the Na+/H+ exchanger (NHE) and the Na+/HCO3- cotransporter (NBC) were found to be functionally activated for acid extrusion for pHi values less than 7.5 and 7.68, respectively; (4) V-ATPase and some other unknown Na+-independent acid extruder(s) could only be functionally detected for pHi values less than 7.1; (5) the Cl-/ OH- exchanger (CHE) and the Cl-/HCO3- anion exchanger (AE) were found to be responsible for the weakening of intracellular proton loading; (6) besides the CHE and the AE, a Cl--independent acid loading mechanism was functionally identified; and (7) in hiPSCs, a strong positive correlation was observed between the loss of pluripotency and the weakening of the intracellular acid extrusion mechanism, which included a decrease in the steady-state pHi value and diminished the functional activity and protein expression of the NHE and the NBC.CONCLUSIONFor the first time, we established a functional and molecular model of a pHi regulatory mechanism and demonstrated its strong positive correlation with hiPSC pluripotency.

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Section: Discussionmentioning

confidence: 65%

Functional and molecular mechanism of intracellular pH regulation in human inducible pluripotent stem cells

Chao

Huang

et al. 2018

WJSC

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“…Signaling pathways regulating ESC fate differ between mESCs and hESCs, and our study provides another aspect of this difference in signal transduction. Recently, Altshuler and colleagues have shown that all three RAS paralogs regulate the transition from naïve to primed state in mESCs and HRAS, KRAS and NRAS display a similar pattern of activation and overlapping roles in mESC differentiation [ 22 ]. However, we clearly observed the difference between the patterns of these paralogs in regulating the downstream pathways, which are involved in maintaining pluripotency.…”

Section: Discussionmentioning

confidence: 99%

“…However, not much is known about the role of RAS proteins in regulating pluripotency or differentiation of hPSCs, especially hiPSCs. It has been shown that RAS proteins regulate the transition from naïve to primed ESCs in mice [ 22 ] and RAS nullyzygosity reduces the proliferation of mouse (m) ESCs and prohibits their differentiation [ 23 ].…”

Section: Introductionmentioning

confidence: 99%

bFGF-mediated pluripotency maintenance in human induced pluripotent stem cells is associated with NRAS-MAPK signaling

et al. 2018

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BackgroundHuman pluripotent stem cells (PSCs) open new windows for basic research and regenerative medicine due to their remarkable properties, i.e. their ability to self-renew indefinitely and being pluripotent. There are different, conflicting data related to the role of basic fibroblast growth factor (bFGF) in intracellular signal transduction and the regulation of pluripotency of PSCs. Here, we investigated the effect of bFGF and its downstream pathways in pluripotent vs. differentiated human induced (hi) PSCs.MethodsbFGF downstream signaling pathways were investigated in long-term culture of hiPSCs from pluripotent to differentiated state (withdrawing bFGF) using immunoblotting, immunocytochemistry and qPCR. Subcellular distribution of signaling components were investigated by simple fractionation and immunoblotting upon bFGF stimulation. Finally, RAS activity and RAS isoforms were studied using RAS assays both after short- and long-term culture in response to bFGF stimulation.ResultsOur results revealed that hiPSCs were differentiated into the ectoderm lineage upon withdrawing bFGF as an essential pluripotency mediator. Pluripotency markers OCT4, SOX2 and NANOG were downregulated, following a drastic decrease in MAPK pathway activity levels. Notably, a remarkable increase in phosphorylation levels of p38 and JAK/STAT3 was observed in differentiated hiPSCs, while the PI3K/AKT and JNK pathways remained active during differentiation. Our data further indicate that among the RAS paralogs, NRAS predominantly activates the MAPK pathway in hiPSCs.ConclusionCollectively, the MAPK pathway appears to be the prime signaling pathway downstream of bFGF for maintaining pluripotency in hiPSCs and among the MAPK pathways, the activity of NRAS-RAF-MEK-ERK is decreased during differentiation, whereas p38 is activated and JNK remains constant.Electronic supplementary materialThe online version of this article (10.1186/s12964-018-0307-1) contains supplementary material, which is available to authorized users.

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“…More recently Ras has been implicated in controlling a number of events that are critical for the transition from naïve to primed pluripotency, including stimulation of glycolysis, epithelial-mesenchymal transition (marked by an increase in N-Cadherin) and an increase in H3K27me3. Interestingly, blocking glycolysis reversed the effect of Ras on N-Cadherin and H3K27me3, suggesting these effects are downstream of the metabolic remodeling (Altshuler et al, 2018). Recent work has demonstrated that downregulation of SIRT2 and upregulation of SIRT1 are a molecular signature of primed hPSCs, and that these NAD-dependent deacetylases regulate primed pluripotency through distinct mechanisms (Cha et al, 2017).…”

Section: Pluripotent Stem Cell Metabolism Glycolysismentioning

confidence: 99%

Energy Metabolism Regulates Stem Cell Pluripotency

Tsogtbaatar

Landin

Minter-Dykhouse

et al. 2020

Front. Cell Dev. Biol.

173

138

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Pluripotent stem cells (PSCs) are characterized by their unique capacity for both unlimited self-renewal and their potential to differentiate to all cell lineages contained within the three primary germ layers. While once considered a distinct cellular state, it is becoming clear that pluripotency is in fact a continuum of cellular states, all capable of self-renewal and differentiation, yet with distinct metabolic, mitochondrial and epigenetic features dependent on gestational stage. In this review we focus on two of the most clearly defined states: "naïve" and "primed" PSCs. Like other rapidly dividing cells, PSCs have a high demand for anabolic precursors necessary to replicate their genome, cytoplasm and organelles, while concurrently consuming energy in the form of ATP. This requirement for both anabolic and catabolic processes sufficient to supply a highly adapted cell cycle in the context of reduced oxygen availability, distinguishes PSCs from their differentiated progeny. During early embryogenesis PSCs adapt their substrate preference to match the bioenergetic requirements of each specific developmental stage. This is reflected in different mitochondrial morphologies, membrane potentials, electron transport chain (ETC) compositions, and utilization of glycolysis. Additionally, metabolites produced in PSCs can directly influence epigenetic and transcriptional programs, which in turn can affect self-renewal characteristics. Thus, our understanding of the role of metabolism in PSC fate has expanded from anabolism and catabolism to include governance of the pluripotent epigenetic landscape. Understanding the roles of metabolism and the factors influencing metabolic pathways in naïve and primed pluripotent states provide a platform for understanding the drivers of cell fate during development. This review highlights the roles of the major metabolic pathways in the acquisition and maintenance of the different states of pluripotency.

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RAS Regulates the Transition from Naive to Primed Pluripotent Stem Cells

Cited by 27 publications

References 44 publications

Functional and molecular mechanism of intracellular pH regulation in human inducible pluripotent stem cells

Functional and molecular mechanism of intracellular pH regulation in human inducible pluripotent stem cells

bFGF-mediated pluripotency maintenance in human induced pluripotent stem cells is associated with NRAS-MAPK signaling

Energy Metabolism Regulates Stem Cell Pluripotency

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