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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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Immunofluorescence analysis of the expression of the pluripotent marker Oct-4 in human pluripotent stem cells (hPSCs).Oct-4 expression in H1 control cells (A), H1 cells treated with 100 ng/mL of BMP-4 (B), and H1 cells treated with 3 μM GSK3i (CHIR99021) for 48 hours (C). The cellular genomic DNAs were stained by the Hoechst 33342 dye (Hoechst). The images were collected with a fluorescence microscope (Zeiss). (D and E) Box-and-Whisker plots of Oct-4 expression (with 5–95% percentile) in both H1 and BC1 cells under the indicated treatments. Control 2 (Cont2) in D is an additionally untreated control of H1 cells. The plus signs (+) in the plots indicate the location of the mean values determined from 116 to 150 individual cells by the ImageJ program. One of two independent experiments is shown. Scale bars represent 50 μm.
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pone.0142554.g004: Immunofluorescence analysis of the expression of the pluripotent marker Oct-4 in human pluripotent stem cells (hPSCs).Oct-4 expression in H1 control cells (A), H1 cells treated with 100 ng/mL of BMP-4 (B), and H1 cells treated with 3 μM GSK3i (CHIR99021) for 48 hours (C). The cellular genomic DNAs were stained by the Hoechst 33342 dye (Hoechst). The images were collected with a fluorescence microscope (Zeiss). (D and E) Box-and-Whisker plots of Oct-4 expression (with 5–95% percentile) in both H1 and BC1 cells under the indicated treatments. Control 2 (Cont2) in D is an additionally untreated control of H1 cells. The plus signs (+) in the plots indicate the location of the mean values determined from 116 to 150 individual cells by the ImageJ program. One of two independent experiments is shown. Scale bars represent 50 μm.

Mentions: To further verify the relationship between glycogen utilization and the pluripotent state of hPSCs, we monitored the expression of the pluripotent factor Oct-4 (Fig 4). Our data indicate that the average Oct-4 immunofluorescence intensity in H1 control cells (i.e., mean ± s.e.m. = 50.2 ± 1.0; n = 150 cells) was significantly reduced in BMP-4-treated cells (i.e., mean ± s.e.m. = 35.5 ± 1.3; n = 150 cells) (Fig 4D: columns 1 and 3; unpaired t-test, two-tailed, P < 0.0001). Similar Oct-4 expression patterns were also found in BC1 cells treated with BMP-4 (Fig 4D: columns 1 and 2). These data suggest that BMP-4 inhibits Oct-4 expression, consistent with BMP-4 functioning as a differentiation factor under stem cell culture conditions [34].


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)

Immunofluorescence analysis of the expression of the pluripotent marker Oct-4 in human pluripotent stem cells (hPSCs).Oct-4 expression in H1 control cells (A), H1 cells treated with 100 ng/mL of BMP-4 (B), and H1 cells treated with 3 μM GSK3i (CHIR99021) for 48 hours (C). The cellular genomic DNAs were stained by the Hoechst 33342 dye (Hoechst). The images were collected with a fluorescence microscope (Zeiss). (D and E) Box-and-Whisker plots of Oct-4 expression (with 5–95% percentile) in both H1 and BC1 cells under the indicated treatments. Control 2 (Cont2) in D is an additionally untreated control of H1 cells. The plus signs (+) in the plots indicate the location of the mean values determined from 116 to 150 individual cells by the ImageJ program. One of two independent experiments is shown. Scale bars represent 50 μm.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0142554.g004: Immunofluorescence analysis of the expression of the pluripotent marker Oct-4 in human pluripotent stem cells (hPSCs).Oct-4 expression in H1 control cells (A), H1 cells treated with 100 ng/mL of BMP-4 (B), and H1 cells treated with 3 μM GSK3i (CHIR99021) for 48 hours (C). The cellular genomic DNAs were stained by the Hoechst 33342 dye (Hoechst). The images were collected with a fluorescence microscope (Zeiss). (D and E) Box-and-Whisker plots of Oct-4 expression (with 5–95% percentile) in both H1 and BC1 cells under the indicated treatments. Control 2 (Cont2) in D is an additionally untreated control of H1 cells. The plus signs (+) in the plots indicate the location of the mean values determined from 116 to 150 individual cells by the ImageJ program. One of two independent experiments is shown. Scale bars represent 50 μm.
Mentions: To further verify the relationship between glycogen utilization and the pluripotent state of hPSCs, we monitored the expression of the pluripotent factor Oct-4 (Fig 4). Our data indicate that the average Oct-4 immunofluorescence intensity in H1 control cells (i.e., mean ± s.e.m. = 50.2 ± 1.0; n = 150 cells) was significantly reduced in BMP-4-treated cells (i.e., mean ± s.e.m. = 35.5 ± 1.3; n = 150 cells) (Fig 4D: columns 1 and 3; unpaired t-test, two-tailed, P < 0.0001). Similar Oct-4 expression patterns were also found in BC1 cells treated with BMP-4 (Fig 4D: columns 1 and 2). These data suggest that BMP-4 inhibits Oct-4 expression, consistent with BMP-4 functioning as a differentiation factor under stem cell culture conditions [34].

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