Immunofluorescence analysis of coexpression of the phospho-glycogen synthase (pGS-Ser641) and GSK-3β in hPSCs.

<p>Coexpression of pGS-Ser641 and GSK-3β in H1 control cells (<b>A</b>), H1 cells treated with 100 ng/mL of BMP-4 (<b>B</b>), and 3 μM GSK3i (CHIR99021) (<b>C</b>) for 48 hours. The cellular genomic DNAs were stained by the Hoechst 33342 dye (Hoechst). The images were collected with a fluorescence microscope (Zeiss). (<b>D</b> and <b>E</b>) Box-and-Whisker plots (with 5–95% percentile) of H1 cells under the indicated treatments: Control 2 (i.e., Cont 2) in both D and E is an additionally untreated control of H1 cells. The plus signs (+) in the plots indicate the location of mean values determined from 116 to 150 individual cells by the ImageJ program. One of two independent experiments is shown. Scale bars in the images represent 50 μm. (<b>F-H</b>) Simple Western experiments: H1 Oct4-EGFP (abbreviated as H1) cells were cultured under the monolayer culture condition for 2 passages and treated with naïve growth conditions as indicated for 48 hours. Simple Western was carried out in H1 Oct4-EGFP cell lysates using specific antibodies against GSK-3β, glycogen synthase (C-terminal), and phospho-glycogen synthase at Ser641 (pGS-Ser641). Protein expression was normalized to GAPDH protein control run in the same capillary (lane). Fold changes (FC) of protein expression related to untreated controls were labeled beneath each lane. The lanes marked by asterisk signs (in Fig 8F) were the results obtained from a separate experiment.</p

Growth, differentiation, and glycogen synthesis in human embryonic stem cells (hESCs).

<p>A hypothetical model is presented to elucidate major signaling pathways that are associated with glycogen synthase kinase 3 (GSK-3) and glycogen synthesis. (<b>A)</b> In this model, glucose transporter-mediated uptake of glucose is activated by an insulin-receptor signaling pathway. (<b>B</b>) Glucose takes part in aerobic glycolysis in the cytoplasm and oxidative phosphorylation in mitochondria to produce energy for hESC proliferation and self-renewal. Presumably, excessive glucose is converted to glycogen by activated glycogen synthase (GSa) upon stress and differentiation signaling to enhance hPSC survival. Glycogen can be decomposed in the presence of phosphorylated glycogen phorsphoylase (pGP) whenever necessary. (<b>C</b>) The insulin signaling pathway also activates the PI3K-AKT pathway, which phosphorylates GSK-3. The GSK-3 phosphorylation leads to its inactivation and subsequently inhibits the phosphorylation of glycogen synthase (GS). Thus, activation of the PI3K-AKT pathway increases glycogen synthesis. (<b>D</b>) The mechanism of BMP-4-induced glycogen body formation is likely through the inhibition of GSK-3 by the putative Smad pathways. (<b>E</b>) The mechanism by which the GSK3i CHIR modulates the synthesis of glycogen is likely through the inhibition of GSK-3 activity, thereby altering glycogen synthase activity. (<b>F</b>) Concomitantly, GSK-3 inhibitors (e.g., CHIR99021 and BIO) may promote hPSC differentiation by activation of the β-catenin-WNT pathway. (<b>G</b>) The function of aggregated glycogen bodies is unclear and may be associated with response to extracellular stress and differentiation signals such as BMP-4. (<b>H</b>) Under sustained Oct-4 expression conditions, GSK3i-mediated glycogen accumulation concomitant with Wnt activation and other naïve growth components enhances the transition from the primed pluripotent to the naïve state in hPSCs. The proposed mechanisms in this model supported by this study are color-highlighted. The “?” symbols indicate inconclusive observations. The abbreviations are: 2iL, the naïve pluripotent growth condition that include GSK3i, MEKi, and LIF; 3iL, the naïve pluripotent growth condition that include GSK3i, MEKi, BMP4i, and LIF; AKT, the serine-threonine protein kinase encoded by v-akt murine thymoma viral oncogene homolog; CHIR, CHIR99021; GPi, dephosphorylated glycogen phosphorylase (inactive form); GSa, dephosphorylated glycogen synthase (active form); GSK-3, glycogen synthase kinase 3; pGPa, phosphorylated glycogen phosphorylase (active form); pGSi, phosphorylated glycogen synthase (inactive form); PI3K, the phosphoinositide 3-kinase; and β-cat, β-catenin.</p

2-NBDG accumulation and retention in NIH-i12 iPSCs under naïve hPSC growth conditions.

<p>(<b>A</b>) Schema of 2-NBDG accumulation and retention (glycogen labeling) experiments. (<b>B</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. (<b>C</b>) 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142554#pone.0142554.g005" target="_blank">Fig 5</a>. (<b>D</b>) Quantitative analysis of mean fluorescence intensity (FI) in Fig 6B. (<b>E</b>, <b>F</b>) 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.</p

Glycogen colorimetric assays of H9 NANOG reporter (H9 hNanog-pGZ) cells under naïve hPSC growth conditions.

<p>The reporter cells (abbreviated as H9 Cont) were treated with GSK3i (BIO), 3i (GSK3i + MEKi + BMP4i), and 3iL (3i + LIF) as described in Materials and Methods. Glycogen colorimetric assays were performed under the same condition as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142554#pone.0142554.g001" target="_blank">Fig 1</a>.</p

Transmission electron micrograph (TEM) analysis of glycogen synthesis in hPSCs.

<p>(<b>A, C</b>) Representative images of untreated H1 and BC1 cells grown as a non-colony type monolayer (mc) on 2.5% Matrigel. (<b>E</b>) Untreated BC1 cells grown as colonies on Matrigel. (B, D, F) Representative images of hPSCs (left panel) treated with 100 ng/mL of BMP-4. The red colored arrowheads indicate stained glycogen aggregates in the cytoplasm of the cell. Abbreviations: G, glycogen particles or aggregates; GB, glycogen bodies; Nu, the nucleus of the cell; Nuo, the nucleolus of in the nucleus. Scale bars represent 1 μm in A, B, C, D, and F and 2 μm in E.</p

2-NBDG accumulation and retention in H1 Oct4-EGFP under naïve hPSC growth conditions.

<p>(<b>A</b>) Schema of 2-NBDG accumulation and retention (glycogen labeling) experiments. (<b>B</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. (<b>C</b>) 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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142554#pone.0142554.g005" target="_blank">Fig 5</a>C4. Lower panel: the corresponding phase images of the upper panel. (<b>D</b>) Quantitative analysis of Oct4-EGFP signals without the addition of 2-NBDG. <b>(E</b>) Quantitative analysis of 2-NBDG accumulation in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142554#pone.0142554.g005" target="_blank">Fig 5B</a>. (<b>F</b>) Quantitative analysis of 2-NBDG retention and glycogen labeling by counting 2-NBDG loci as presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142554#pone.0142554.g005" target="_blank">Fig 5C</a>. 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.</p

Variations in glycogen synthesis in human pluripotent stem cells (hPSCs).

<p>(A) Schema of the glycogen assay based on the protocol from BioVision Inc.: glycogen was hydrolyzed (H) into glucose, then glucose developed (D) into an intermediate and reduce probe (P) to produce color products. (B) Determination of glycogen content in hPSCs grown under various growth conditions. Column 1: MCF7 breast cancer cells used control (Cont) for the assessment of glycogen levels; Column 2: H1 colonies grown on MEF as previously descried in Materials and Methods. The glycogen content was measured at cell passage number 38. Column 3: H1 colonies initially grown on MEF for 35 passages followed by growing on 2.5% BD Matrigel (MG) in mTeSR1 for 5 passages. Column 4: H9 cells were initially grown on MEF for 32 passages followed by passaging on 2.5% BD Matrigel (MG) in mTeSR1 medium for 9 passages. Column 5: BC1 human iPSCs were initially grown on MEF for 50 passages followed by passaging on 2.5% BD Matrigel (MG) in mTeSR1 medium for 26 passages.</p

TEM analysis of glycogen synthesis and the formation of glycogen bodies mediated by BMP-4 and GSK-3 inhibition in hESCs.

<p>(<b>A</b>) Glycogen body formation in untreated H1 cells (control). (<b>B</b>) Glycogen body formation in BMP-4 treated H1 cells. (<b>C</b>) Glycogen body formation in GSK3i (CHIR99021)-treated H1cells. The asterisk sign indicates a glycogen defect region in the glycogen body, which likely resulted from dissociation of glycogen aggregates when the specimens were floating in solution during sample preparation. Additional TEM micrographs are available in Supporting Information <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142554#pone.0142554.s001" target="_blank">S1 Fig</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142554#pone.0142554.s002" target="_blank">S2 Fig</a>. (<b>D</b>) Box-and-Whisker plots of glycogen to cell ratios (with 10–90% percentile) in hESC control (n = 49 cells), BMP-4 treated cells (n = 29), and GSK3i treated cells (n = 22). The statistics included both H1 and H9 cells to increase statistical power. All hESC (i.e., H1 and H9) cells were grown as a non-colony type monolayer (mc) on 2.5% BD Matrigel in meTeSR1 medium and then treated with 100 ng/mL of BMP-4 and 3 μM GSK3i for 48 hours. The red-colored arrowheads indicate the formation of glycogen bodies (GB) in the cytoplasm of the cell. The plus signs (+) in the plots indicate the location of mean values. Abbreviations: GB, glycogen bodies with defined boundaries; Nu, the nucleus of the cells. Scale bars in (A, B) represent 2 μm; and scale bars in (C) represent 1 μm.</p

Immunofluorescence analysis of the expression of the pluripotent marker Oct-4 in human pluripotent stem cells (hPSCs).

<p>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). (<b>D</b> and <b>E</b>) 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.</p

Immunohistochemical staining of ossicles derived from FePro or GFP labeled and unlabeled BMSCs.

<p>A representative ossicle derived from unlabeled (A) and FePro labeled (B) BMSCs at 8 weeks, stained with H & E showing comparable abundant bone formation and abundant hematopoiesis. Immunohistochemistry staining for GFP of a representative ossicle derived from BMSCs labeled with both FePro and lentivirus carrying GFP (C) and control unlabeled BMSCs (D).</p