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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 H1 Oct4-EGFP 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 that include signals from both Oct4-EGFP and 2-NBDG. These images were obtained (immediately after replacing with fresh mTeSR1 medium) by non-saturated time-exposure guided by an autoexposure software (Zeiss Inc.). No 2-NBDG indicates fluorescence background images produced from Oct4-EGFP signal without the use of 2-NBDG. The fluorescence intensity was determined by the signal/noise ratio between cellular fluorescence (signal) and background (noise). The fluorescent background in the 2-NBDG control was due to non-saturated auto exposure using the Zeiss Axiovert imaging system. 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 indicated by arrows in the inset of Fig 5C4. Lower panel: the corresponding phase images of the upper panel. (D) Quantitative analysis of Oct4-EGFP signals without the addition of 2-NBDG. (E) Quantitative analysis of 2-NBDG accumulation in Fig 5B. (F) Quantitative analysis of 2-NBDG retention and glycogen labeling by counting 2-NBDG loci as presented in Fig 5C. Columns represent mean fluorescence intensity measured from at least 4 random colonies and bar standard deviations. Abbreviations (depicted sequentially): BIO, 2 μM GSK3i (BIO); 2i, 2 μM BIO + 1 μM MEKi; 3i, 2i + 1 μM BMP4i; 2iL, 2i + 10 ng/mL LIF; 3iL, 3i + 10 ng/mL LIF; 2-NBDG, (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose), a fluorescent glucose derivative, overlapping with EGFP signals. Scale bars represent 100 μm.
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pone.0142554.g005: 2-NBDG accumulation and retention in H1 Oct4-EGFP 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 that include signals from both Oct4-EGFP and 2-NBDG. These images were obtained (immediately after replacing with fresh mTeSR1 medium) by non-saturated time-exposure guided by an autoexposure software (Zeiss Inc.). No 2-NBDG indicates fluorescence background images produced from Oct4-EGFP signal without the use of 2-NBDG. The fluorescence intensity was determined by the signal/noise ratio between cellular fluorescence (signal) and background (noise). The fluorescent background in the 2-NBDG control was due to non-saturated auto exposure using the Zeiss Axiovert imaging system. 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 indicated by arrows in the inset of Fig 5C4. Lower panel: the corresponding phase images of the upper panel. (D) Quantitative analysis of Oct4-EGFP signals without the addition of 2-NBDG. (E) Quantitative analysis of 2-NBDG accumulation in Fig 5B. (F) Quantitative analysis of 2-NBDG retention and glycogen labeling by counting 2-NBDG loci as presented in Fig 5C. Columns represent mean fluorescence intensity measured from at least 4 random colonies and bar standard deviations. Abbreviations (depicted sequentially): BIO, 2 μM GSK3i (BIO); 2i, 2 μM BIO + 1 μM MEKi; 3i, 2i + 1 μM BMP4i; 2iL, 2i + 10 ng/mL LIF; 3iL, 3i + 10 ng/mL LIF; 2-NBDG, (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose), a fluorescent glucose derivative, overlapping with EGFP signals. Scale bars represent 100 μm.

Mentions: As suggested from the above results, GSK-3 inhibition by GSK3i (CHIR99021) in ROCKi-mediated monolayer culture resulted in increase in glycogen synthesis and elevated Oct-4 expression, suggesting these hPSCs acquired an altered pluripotent state. To verify whether there is also altered glycogen synthesis presented in hPSCs with different pluripotent states, we modified primed hPSCs under our current culture protocols by treating them with the naïve hPSC culture components (3iL) included in a well-established protocol (Fig 5A) [13]. We systematically analyzed glycogen variations in hPSCs under BIO, 2i, 3i, 2iL, and 3iL. As indicated in the phase images of Fig 5B, there were progressive morphological changes after 48-hour treatments of H1 Oct4-EGFP cells with above conditions. These treated cells were morphologically similar to those described naïve cells [13]. Moreover, these cells did not commit to lineage differentiation as monitored by increased Oct4-EGFP expression in these cells (Fig 5B and 5D). There are 1.2-, 1.6-, 1.6-, 1.5-, and 1.7-fold elevations in Oct4-EGFP expression compared to non-treated control, respectively under BIO, 2i, 3i, 2iL, and 3iL culture conditions (Fig 5D). Even a 1.2-fold increase in Oct-EGFP expression in BIO-treated cells (compared to non-treated control) is considered to be significant (Fig 5D: columns 1 and 2, P = 0.008). Thus, these data suggest that H1 Oct4-EGFP cells treated with naïve growth conditions acquire altered pluripotent states, similar to those previously described naïve-like hPSCs [13].


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 H1 Oct4-EGFP 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 that include signals from both Oct4-EGFP and 2-NBDG. These images were obtained (immediately after replacing with fresh mTeSR1 medium) by non-saturated time-exposure guided by an autoexposure software (Zeiss Inc.). No 2-NBDG indicates fluorescence background images produced from Oct4-EGFP signal without the use of 2-NBDG. The fluorescence intensity was determined by the signal/noise ratio between cellular fluorescence (signal) and background (noise). The fluorescent background in the 2-NBDG control was due to non-saturated auto exposure using the Zeiss Axiovert imaging system. 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 indicated by arrows in the inset of Fig 5C4. Lower panel: the corresponding phase images of the upper panel. (D) Quantitative analysis of Oct4-EGFP signals without the addition of 2-NBDG. (E) Quantitative analysis of 2-NBDG accumulation in Fig 5B. (F) Quantitative analysis of 2-NBDG retention and glycogen labeling by counting 2-NBDG loci as presented in Fig 5C. Columns represent mean fluorescence intensity measured from at least 4 random colonies and bar standard deviations. Abbreviations (depicted sequentially): BIO, 2 μM GSK3i (BIO); 2i, 2 μM BIO + 1 μM MEKi; 3i, 2i + 1 μM BMP4i; 2iL, 2i + 10 ng/mL LIF; 3iL, 3i + 10 ng/mL LIF; 2-NBDG, (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose), a fluorescent glucose derivative, overlapping with EGFP signals. Scale bars represent 100 μm.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4643957&req=5

pone.0142554.g005: 2-NBDG accumulation and retention in H1 Oct4-EGFP 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 that include signals from both Oct4-EGFP and 2-NBDG. These images were obtained (immediately after replacing with fresh mTeSR1 medium) by non-saturated time-exposure guided by an autoexposure software (Zeiss Inc.). No 2-NBDG indicates fluorescence background images produced from Oct4-EGFP signal without the use of 2-NBDG. The fluorescence intensity was determined by the signal/noise ratio between cellular fluorescence (signal) and background (noise). The fluorescent background in the 2-NBDG control was due to non-saturated auto exposure using the Zeiss Axiovert imaging system. 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 indicated by arrows in the inset of Fig 5C4. Lower panel: the corresponding phase images of the upper panel. (D) Quantitative analysis of Oct4-EGFP signals without the addition of 2-NBDG. (E) Quantitative analysis of 2-NBDG accumulation in Fig 5B. (F) Quantitative analysis of 2-NBDG retention and glycogen labeling by counting 2-NBDG loci as presented in Fig 5C. Columns represent mean fluorescence intensity measured from at least 4 random colonies and bar standard deviations. Abbreviations (depicted sequentially): BIO, 2 μM GSK3i (BIO); 2i, 2 μM BIO + 1 μM MEKi; 3i, 2i + 1 μM BMP4i; 2iL, 2i + 10 ng/mL LIF; 3iL, 3i + 10 ng/mL LIF; 2-NBDG, (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose), a fluorescent glucose derivative, overlapping with EGFP signals. Scale bars represent 100 μm.
Mentions: As suggested from the above results, GSK-3 inhibition by GSK3i (CHIR99021) in ROCKi-mediated monolayer culture resulted in increase in glycogen synthesis and elevated Oct-4 expression, suggesting these hPSCs acquired an altered pluripotent state. To verify whether there is also altered glycogen synthesis presented in hPSCs with different pluripotent states, we modified primed hPSCs under our current culture protocols by treating them with the naïve hPSC culture components (3iL) included in a well-established protocol (Fig 5A) [13]. We systematically analyzed glycogen variations in hPSCs under BIO, 2i, 3i, 2iL, and 3iL. As indicated in the phase images of Fig 5B, there were progressive morphological changes after 48-hour treatments of H1 Oct4-EGFP cells with above conditions. These treated cells were morphologically similar to those described naïve cells [13]. Moreover, these cells did not commit to lineage differentiation as monitored by increased Oct4-EGFP expression in these cells (Fig 5B and 5D). There are 1.2-, 1.6-, 1.6-, 1.5-, and 1.7-fold elevations in Oct4-EGFP expression compared to non-treated control, respectively under BIO, 2i, 3i, 2iL, and 3iL culture conditions (Fig 5D). Even a 1.2-fold increase in Oct-EGFP expression in BIO-treated cells (compared to non-treated control) is considered to be significant (Fig 5D: columns 1 and 2, P = 0.008). Thus, these data suggest that H1 Oct4-EGFP cells treated with naïve growth conditions acquire altered pluripotent states, similar to those previously described naïve-like hPSCs [13].

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