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Variations in Glycogen Synthesis in Human Pluripotent Stem Cells with Altered Pluripotent States.

ABSTRACT: Human pluripotent stem cells (hPSCs) represent very promising resources for cell-based regenerative medicine. It is essential to determine the biological implications of some fundamental physiological processes (such as glycogen metabolism) in these stem cells. In this report, we employ electron, immunofluorescence microscopy, and biochemical methods to study glycogen synthesis in hPSCs. Our results indicate that there is a high level of glycogen synthesis (0.28 to 0.62 μg/μg proteins) in undifferentiated human embryonic stem cells (hESCs) compared with the glycogen levels (0 to 0.25 μg/μg proteins) reported in human cancer cell lines. Moreover, we found that glycogen synthesis was regulated by bone morphogenetic protein 4 (BMP-4) and the glycogen synthase kinase 3 (GSK-3) pathway. Our observation of glycogen bodies and sustained expression of the pluripotent factor Oct-4 mediated by the potent GSK-3 inhibitor CHIR-99021 reveals an altered pluripotent state in hPSC culture. We further confirmed glycogen variations under different naïve pluripotent cell growth conditions based on the addition of the GSK-3 inhibitor BIO. Our data suggest that primed hPSCs treated with naïve growth conditions acquire altered pluripotent states, similar to those naïve-like hPSCs, with increased glycogen synthesis. Furthermore, we found that suppression of phosphorylated glycogen synthase was an underlying mechanism responsible for altered glycogen synthesis. Thus, our novel findings regarding the dynamic changes in glycogen metabolism provide new markers to assess the energetic and various pluripotent states in hPSCs. The components of glycogen metabolic pathways offer new assays to delineate previously unrecognized properties of hPSCs under different growth conditions.

SUBMITTER: Chen RJ

PROVIDER: S-EPMC4643957 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundFragile X syndrome (FXS), a neurodevelopmental disorder, is a leading monogenetic cause of intellectual disability and autism spectrum disorder. Notwithstanding the extensive studies using rodent and other pre-clinical models of FXS, which have provided detailed mechanistic insights into the pathophysiology of this disorder, it is only relatively recently that human stem cell-derived neurons have been employed as a model system to further our understanding of the pathophysiological events that may underlie FXS. Our study assesses the physiological properties of human pluripotent stem cell-derived cortical neurons lacking fragile X mental retardation protein (FMRP).MethodsElectrophysiological whole-cell voltage- and current-clamp recordings were performed on two control and three FXS patient lines of human cortical neurons derived from induced pluripotent stem cells. In addition, we also describe the properties of an isogenic pair of lines in one of which FMR1 gene expression has been silenced.ResultsNeurons lacking FMRP displayed bursts of spontaneous action potential firing that were more frequent but shorter in duration compared to those recorded from neurons expressing FMRP. Inhibition of large conductance Ca2+-activated K+ currents and the persistent Na+ current in control neurons phenocopies action potential bursting observed in neurons lacking FMRP, while in neurons lacking FMRP pharmacological potentiation of voltage-dependent Na+ channels phenocopies action potential bursting observed in control neurons. Notwithstanding the changes in spontaneous action potential firing, we did not observe any differences in the intrinsic properties of neurons in any of the lines examined. Moreover, we did not detect any differences in the properties of miniature excitatory postsynaptic currents in any of the lines.ConclusionsPharmacological manipulations can alter the action potential burst profiles in both control and FMRP-null human cortical neurons, making them appear like their genetic counterpart. Our studies indicate that FMRP targets that have been found in rodent models of FXS are also potential targets in a human-based model system, and we suggest potential mechanisms by which activity is altered.

Project description:BACKGROUND: Human iPS cells (hiPSCs) have attracted considerable attention for applications to drug screening and analyses of disease mechanisms, and even as next generation materials for regenerative medicine. Genetic reprogramming of human somatic cells to a pluripotent state was first achieved by the ectopic expression of four factors (Sox2, Oct4, Klf4 and c-Myc), using a retrovirus. Subsequently, this method was applied to various human cells, using different combinations of defined factors. However, the transcription factor-induced acquisition of replication competence and pluripotency raises the question as to how exogenous factors induce changes in the inner and outer cellular states. RESULTS: We analyzed both the RNA profile, to reveal changes in gene expression, and the glycan profile, to identify changes in glycan structures, between 51 cell samples of four parental somatic cell (SC) lines from amniotic mesodermal, placental artery endothelial, and uterine endometrium sources, fetal lung fibroblast (MRC-5) cells, and nine hiPSC lines that were originally established. The analysis of this information by standard statistical techniques combined with a network approach, named network screening, detected significant expression differences between the iPSCs and the SCs. Subsequent network analysis of the gene expression and glycan signatures revealed that the glycan transfer network is associated with known epitopes for differentiation, e.g., the SSEA epitope family in the glycan biosynthesis pathway, based on the characteristic changes in the cellular surface states of the hiPSCs. CONCLUSIONS: The present study is the first to reveal the relationships between gene expression patterns and cell surface changes in hiPSCs, and reinforces the importance of the cell surface to identify established iPSCs from SCs. In addition, given the variability of iPSCs, which is related to the characteristics of the parental SCs, a glycosyltransferase expression assay might be established to define hiPSCs more precisely and thus facilitate their standardization, which are important steps towards the eventual therapeutic applications of hiPSCs.

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Variations in Glycogen Synthesis in Human Pluripotent Stem Cells with Altered Pluripotent States.

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