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

Chen RJ, Zhang G, Garfield SH, Shi YJ, Chen KG, Robey PG, Leapman RD - PLoS ONE (2015)

Bottom Line: Moreover, we found that glycogen synthesis was regulated by bone morphogenetic protein 4 (BMP-4) and the glycogen synthase kinase 3 (GSK-3) pathway.Furthermore, we found that suppression of phosphorylated glycogen synthase was an underlying mechanism responsible for altered glycogen synthesis.The components of glycogen metabolic pathways offer new assays to delineate previously unrecognized properties of hPSCs under different growth conditions.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD, 20892, United States of America.

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.

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2-NBDG accumulation and retention in NIH-i12 iPSCs under naïve hPSC growth conditions.(A) Schema of 2-NBDG accumulation and retention (glycogen labeling) experiments. (B) 2-hour 2-NBDG accumulation in the presence of 10 mM D-glucose. Upper panel: green fluorescence intensity (Fluor) images from 2-NBDG alone. These images were obtained (immediately after replacing with fresh mTeSR1 medium) by non-saturated time-exposure guided by an autoexposure software (Zeiss Inc.). Lower panel: the corresponding phase images of the upper panel. Only brightness was adjusted in phase images (presented in both B and C) to enhance the image presentation in this figure. (C) 2-NBDG retention and glycogen labeling carried out in the presence of 10 mM D-glucose and absence of 2-NBDG. Upper panel: unique fluorescence loci (dots) were derived from 2-NBDG signals as detailed in Fig 5. (D) Quantitative analysis of mean fluorescence intensity (FI) in Fig 6B. (E, F) Quantitative analysis of 2-NBDG retention and glycogen labeling by measuring mean fluorescence intensity (FI, arbitrary units) from at least 4 random colonies (E) and by counting 2-NBDG loci (F). Columns represent mean fluorescence intensity measured from at least 4 random colonies and bar standard deviations. Scale bars represent 100 μm.
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pone.0142554.g006: 2-NBDG accumulation and retention in NIH-i12 iPSCs under naïve hPSC growth conditions.(A) Schema of 2-NBDG accumulation and retention (glycogen labeling) experiments. (B) 2-hour 2-NBDG accumulation in the presence of 10 mM D-glucose. Upper panel: green fluorescence intensity (Fluor) images from 2-NBDG alone. These images were obtained (immediately after replacing with fresh mTeSR1 medium) by non-saturated time-exposure guided by an autoexposure software (Zeiss Inc.). Lower panel: the corresponding phase images of the upper panel. Only brightness was adjusted in phase images (presented in both B and C) to enhance the image presentation in this figure. (C) 2-NBDG retention and glycogen labeling carried out in the presence of 10 mM D-glucose and absence of 2-NBDG. Upper panel: unique fluorescence loci (dots) were derived from 2-NBDG signals as detailed in Fig 5. (D) Quantitative analysis of mean fluorescence intensity (FI) in Fig 6B. (E, F) Quantitative analysis of 2-NBDG retention and glycogen labeling by measuring mean fluorescence intensity (FI, arbitrary units) from at least 4 random colonies (E) and by counting 2-NBDG loci (F). Columns represent mean fluorescence intensity measured from at least 4 random colonies and bar standard deviations. Scale bars represent 100 μm.

Mentions: To reproduce the effects of naïve growth conditions on 2-NBDG retention in iPSCs, we performed the same experiment in NIH-i12, a well-characterized iPSC line reported in the NIH StemCellDB [6]. As revealed in Fig 6D, NIH-i12 cells showed similar accumulation and retention patterns to those of H1 Oct4-EGFP cells presented in Fig 5. One interesting difference between the two cell lines is that BIO had a pronounced effect on 2-NBDG accumulation (Fig 6D: columns 1 and 2), with 1.5 to 1.9-fold elevation in 2-NBDG fluorescence retention (Fig 6E: columns 2 to 5, P < 0.05), and 78-fold increase in 2-NBDG retention based on 2-NBDG fluorescence loci counts (Fig 6F: columns 1 and 2, P = 0.004).


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

Chen RJ, Zhang G, Garfield SH, Shi YJ, Chen KG, Robey PG, Leapman RD - PLoS ONE (2015)

2-NBDG accumulation and retention in NIH-i12 iPSCs under naïve hPSC growth conditions.(A) Schema of 2-NBDG accumulation and retention (glycogen labeling) experiments. (B) 2-hour 2-NBDG accumulation in the presence of 10 mM D-glucose. Upper panel: green fluorescence intensity (Fluor) images from 2-NBDG alone. These images were obtained (immediately after replacing with fresh mTeSR1 medium) by non-saturated time-exposure guided by an autoexposure software (Zeiss Inc.). Lower panel: the corresponding phase images of the upper panel. Only brightness was adjusted in phase images (presented in both B and C) to enhance the image presentation in this figure. (C) 2-NBDG retention and glycogen labeling carried out in the presence of 10 mM D-glucose and absence of 2-NBDG. Upper panel: unique fluorescence loci (dots) were derived from 2-NBDG signals as detailed in Fig 5. (D) Quantitative analysis of mean fluorescence intensity (FI) in Fig 6B. (E, F) Quantitative analysis of 2-NBDG retention and glycogen labeling by measuring mean fluorescence intensity (FI, arbitrary units) from at least 4 random colonies (E) and by counting 2-NBDG loci (F). Columns represent mean fluorescence intensity measured from at least 4 random colonies and bar standard deviations. Scale bars represent 100 μm.
© Copyright Policy
Related In: Results  -  Collection

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pone.0142554.g006: 2-NBDG accumulation and retention in NIH-i12 iPSCs under naïve hPSC growth conditions.(A) Schema of 2-NBDG accumulation and retention (glycogen labeling) experiments. (B) 2-hour 2-NBDG accumulation in the presence of 10 mM D-glucose. Upper panel: green fluorescence intensity (Fluor) images from 2-NBDG alone. These images were obtained (immediately after replacing with fresh mTeSR1 medium) by non-saturated time-exposure guided by an autoexposure software (Zeiss Inc.). Lower panel: the corresponding phase images of the upper panel. Only brightness was adjusted in phase images (presented in both B and C) to enhance the image presentation in this figure. (C) 2-NBDG retention and glycogen labeling carried out in the presence of 10 mM D-glucose and absence of 2-NBDG. Upper panel: unique fluorescence loci (dots) were derived from 2-NBDG signals as detailed in Fig 5. (D) Quantitative analysis of mean fluorescence intensity (FI) in Fig 6B. (E, F) Quantitative analysis of 2-NBDG retention and glycogen labeling by measuring mean fluorescence intensity (FI, arbitrary units) from at least 4 random colonies (E) and by counting 2-NBDG loci (F). Columns represent mean fluorescence intensity measured from at least 4 random colonies and bar standard deviations. Scale bars represent 100 μm.
Mentions: To reproduce the effects of naïve growth conditions on 2-NBDG retention in iPSCs, we performed the same experiment in NIH-i12, a well-characterized iPSC line reported in the NIH StemCellDB [6]. As revealed in Fig 6D, NIH-i12 cells showed similar accumulation and retention patterns to those of H1 Oct4-EGFP cells presented in Fig 5. One interesting difference between the two cell lines is that BIO had a pronounced effect on 2-NBDG accumulation (Fig 6D: columns 1 and 2), with 1.5 to 1.9-fold elevation in 2-NBDG fluorescence retention (Fig 6E: columns 2 to 5, P < 0.05), and 78-fold increase in 2-NBDG retention based on 2-NBDG fluorescence loci counts (Fig 6F: columns 1 and 2, P = 0.004).

Bottom Line: Moreover, we found that glycogen synthesis was regulated by bone morphogenetic protein 4 (BMP-4) and the glycogen synthase kinase 3 (GSK-3) pathway.Furthermore, we found that suppression of phosphorylated glycogen synthase was an underlying mechanism responsible for altered glycogen synthesis.The components of glycogen metabolic pathways offer new assays to delineate previously unrecognized properties of hPSCs under different growth conditions.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, MD, 20892, United States of America.

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.

Show MeSH
Related in: MedlinePlus