Advanced
Ganglioside GM1 influences the proliferation rate of mouse induced pluripotent stem cells
Ganglioside GM1 influences the proliferation rate of mouse induced pluripotent stem cells
BMB Reports. 2012. Dec, 45(12): 713-718
Copyright © 2012, Korean Society for Biochemistry and Molecular Biology
This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
  • Received : July 02, 2012
  • Accepted : September 03, 2012
  • Published : December 30, 2012
Download
PDF
e-PUB
PubReader
PPT
Export by style
Share
Article
Author
Metrics
Cited by
TagCloud
About the Authors
Jae-Sung Ryu
Department of Biological Science, College of Natural Sciences, Wonkwang University, Iksan 570-749, Korea
Kyu-Tae Chang
National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), O-chang 363-883, Korea
Ju-Taek Lee
Department of Biological Science, College of Natural Sciences, Wonkwang University, Iksan 570-749, Korea
Malg-Um Lim
Department of Biological Science, College of Natural Sciences, Wonkwang University, Iksan 570-749, Korea
Hyun-Ki Min
Department of Biological Science, College of Natural Sciences, Wonkwang University, Iksan 570-749, Korea
Yoon-Ju Na
Department of Biological Science, College of Natural Sciences, Wonkwang University, Iksan 570-749, Korea
Su-Bin Lee
Department of Biological Science, College of Natural Sciences, Wonkwang University, Iksan 570-749, Korea
Gislain Moussavou
Department of Biological Science, College of Natural Sciences, Wonkwang University, Iksan 570-749, Korea
Sun-Uk Kim
National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), O-chang 363-883, Korea
Ji-Su Kim
National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), O-chang 363-883, Korea
Kinarm Ko
Center for Stem Cell Research, Institute of Biomedical Sciences and Technology, Konkuk University, Seoul 143-701, Korea
Kisung Ko
Department of Medicine, College of Medicine, Chung-Ang University, Seoul 156-756, Korea
Kyung-A Hwang
Department of Agrofood Resources, National Academy of Agricultural Science, RDA, Suwon 441-853, Korea
Eun-Jeong Jeong
Center for Development and Differentiation, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Korea
Jeong-Woong Lee
Center for Development and Differentiation, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Daejeon 305-806, Korea
Young-Kug Choo
Department of Biological Science, College of Natural Sciences, Wonkwang University, Iksan 570-749, Korea

Abstract
Gangliosides play important roles in the control of several biological processes, including proliferation and transmembrane signaling. In this study, we demonstrate the effect of ganglioside GM1 on the proliferation of mouse induced pluripotent stem cells (miPSCs). The proliferation rate of miPSCs was lower than in mouse embryonic stem cells (mESCs). Fluorescence activated cell sorting analysis showed that the percentage of cells in the G2/M phase in miPSCs was lower than that in mESCs. GM1 was expressed in mESCs, but not miPSCs. To confirm the role of GM1 in miPSC proliferation, miPSCs were treated with GM1. GM1-treated miPSCs exhibited increased cell proliferation and a larger number of cells in the G2/M phase. Furthermore, phosphorylation of mitogen-activated protein kinases was increased in GM1-treated miPSCs. [BMB Reports 2012; 45(12): 713-718]
Keywords
INTRODUCTION
Induced pluripotent stem cells (iPSCs) have been generated from mouse fibroblasts by retroviral introduction of four defined transcription factors: Oct4 , Sox2 , Klf4 , and c-Myc (1) . iPSCs are indistinguishable from embryonic stem cells (ESCs) in terms of morphology, self-renewal, and expression of ESC markers (2 , 3) . In some recent studies, iPSCs were generated from various cell types, including neural stem cells, liver cells, stomach cells, and pancreatic beta cells, and can give rise to chimeric mice that are competent for germline transmission (4 , 5) . However, other studies found differences between ESCs and iPSCs, including different proliferation rates (2 , 6) .
Gangliosides are complex glycosphingolipids that contain one or more sialic acids, and which are ubiquitously expressed in the cell plasma membrane (7) . The biosynthesis of gangliosides from ceramide involves the stepwise regulation of glycosyltransferases in the endoplasmic reticulum and Golgi apparatus, where diverse forms of gangliosides, including GM3, GD3, and GT3 of the a-, b-, and c-series are assembled (Supplementary Fig. 1) (8 , 9) . These gangliosides are thought to be involved in the control of several biological processes, including apoptosis, mouse embryonic development, cell proliferation, cell surface interactions, cell differentiation, and transmembrane signaling (10 , 11) . GM3 is mostly expressed during embryogenesis in mice (12 , 13) , but its expression is decreased during brain development (14) . GD1a is also expressed during late embryogenesis (E9-E16), and increased expression of GD1a is observed during mouse brain development (12 , 14) . In particular, GM1 has been shown to possess antineurotoxic, neuroprotective, and neurorestorative capabilities, as well as the ability to facilitate neuronal commitment (15 , 16) . Moreover, GM1 appears to increase neural stem cell, smooth muscle cell, and rat PC-12 cell proliferation (17 - 19) .
Gangliosides regulate mitogen-activated protein (MAP) kinase signaling pathways in fundamental cellular processes (20 , 21) . In particular, the extracellular signal-regulated kinase (ERK) 1/2 MAP kinase has been reported to regulate cell proliferation. Specifically, GM1 induces vascular smooth muscle cell proliferation via ERK 1/2 (18) , and activated ERK 1/2 triggers an increase in DNA synthesis in human glioma cells (22) .
In this study, we compared the proliferation rate and ganglioside expression pattern in cultured mouse induced pluripotent stem cells (miPSCs) and mouse embryonic stem cells (mESCs). We also analyzed the effect of GM1 on the cell proliferation rate and MAP kinase-signaling pathway in miPSCs.
RESULTS
- Comparison of miPSC and mESC proliferation and cell cycle
miPSCs and mESCs exhibited the same characteristics, such as expressions of alkaline phosphatase, stage-specific embryonic antigen-1, and pluripotency markers (Supplementary Fig. 2). However, a difference was apparent in the proliferation rate between miPSCs and mESCs ( Fig. 1 A). The time-dependent cell viability was investigated over 4 days. Although the proliferation rate of miPSCs and mESCs did not differ significantly during the first 2 days, the proliferation rate of miPSCs was lower than that of mESCs on day 3.
PPT Slide
Lager Image
Comparison of cell proliferation rates and the cell cycle in miPSCs and mESCs. (A) miPSCs and mESCs were seeded at a density of 5 × 103 cells/well in 24-well plates, and the cell proliferation rates were analyzed over 4 days by the MTT assay (**P < 0.01, ***P < 0.001). (B) Cell cycle distribution profiles determined by fluorescence-activated cell sorting (FACS)/flow cytometry at 4 days (96 h) of culture of miPSCs and mESCs. Data are presented as mean ± S.D. (C) Expression of G2/M phaseand S phase-related proteins in miPSCs and mESCs. The expression of cyclin B, Cdc2, cyclin E, cyclin A, and Cdk2 proteins was measured by western blotting using specific antibodies.
To determine whether cell cycle arrest or apoptosis had been induced, the distribution of cells in each cell cycle phase was analyzed by flow cytometry. As shown in Fig. 1 B, the percentage of mESCs and miPSCs in the G1 phase was 20.19 ± 3.3% and 32.25 ± 3.2%, respectively, at 4 days of culture. The percentage of cells in the S and G2/M phases was also higher in mESCs than in miPSCs. Although the expression level of cell cycle-related proteins increased over 4 days in both cell types, the rate of this increase was higher in mESCs compared with miPSCs. Western blot analysis revealed that the expression of cell cycle progression (S and G2/M phase)-related cyclins (cyclin E, cyclin A, cyclin B, and cdc2) and cyclin-dependent kinase 2 (cdk2) in mESCs was higher than miPSCs ( Fig. 1 C), whereas the expression of G1 phase-related cyclins and cdks was higher in mESCs (Supplementary Fig. 3).
- Differential expression of gangliosides in miPSCs
The expression patterns of gangliosides in miPSCs and mESCs were different, and the GM3 and GD1a expression levels in mESCs were significantly higher than miPSCs ( Fig. 2 A). Of particular interest, GM1 was expressed in mESCs but not in miPSCs. In addition, immunofluorescence showed the expression of gangliosides in miPSCs and mESCs ( Fig. 2 B, Supplementary Fig. 4).
PPT Slide
Lager Image
Expression of gangliosides in miPSCs and mESCs. (A) Differential expression of gangliosides in miPSCs and mESCs. Lanes 1 and 2, ganglioside standard markers; lane 3, mEFs; lane 4, miPSCs; lane 5, mESCs. (B) Immunofluorescence staining of gangliosides. GM3 and GD1a expression (Cy5, red) and EGFP (green) were detected in miPSCs and mESCs. GM1 expression (Cy5, red) was observed in mESCs but not in miPSCs.
- Effect of GM1 on the proliferation of miPSCs
To determine whether GM1 increased the proliferation of miPSCs, cells were treated with various concentrations of GM1 over 4 days. The expression of pluripotent markers and the mRNA level were unaltered by the GM1 treatment (Supplementary Fig. 5). Immunofluorescence with antibody against GM1 resulted in the detection of GM1 in GM1-treated miPSCs (Supplementary Fig. 5).
The proliferation rate of miPSCs treated with GM1 was higher than that of untreated miPSCs. In particular, the proliferation rate of cells treated with 5 and 10 μM GM1 over 4 days was higher than that of untreated cells ( Fig. 3 A).
PPT Slide
Lager Image
Effect of GM1 on the proliferation rate and the cell cycle of miPSCs. (A) miPSCs were seeded at a density of 5 × 103 cells/well in 24-well plates with various concentrations of GM1 (0, 1, 2, 5, and 10 μM) for 4 days, and the cell proliferation was then analyzed by the MTT assay (*P < 0.05, **P < 0.01, ***P < 0.001). (B) Cell cycle distribution profiles determined by FACS of a 4-day (96-h) culture of miPSCs and mESCs. Data are presented as mean ± S.D. (C) Expression of G2/M phase and S phase-related proteins in GM1-treated miPSCs. The expression of cyclin B, Cdc2, cyclin E, cyclin A, and Cdk2 was measured by Western blotting using specific antibodies.
To elucidate the molecular mechanism underlying the proliferation rate of the treated miPSCs, the cell cycle stage was analyzed. As shown in Fig. 3 B, the percentage of GM1-treated miPSCs in the G1 phase was lower than untreated miPSCs. However, the percentage of cells in the S and G2/M phases was greater in GM1-treated miPSCs than in untreated miPSCs. In particular, miPSCs treated with 5 and 10 μM GM1 showed a further increased percentage of cells in the S and G2/M phases. In addition, Western blot analysis also revealed the presence of S, G2/M, and G1 phase-related proteins ( Fig. 3 C, Supplementary Fig. 6).
- Increased MAP kinase signaling by GM1
The phosphorylation of ERK1/2 MAP kinase controls cell proliferation and differentiation (23 , 24) . To determine whether the GM1 treatment increased proliferation rate via the ERK1/2 MAP kinase pathway, activation of this pathway was examined by Western blot analysis of GM1-treated miPSCs over 4 days. Western blotting using antibodies directed against phospho-c-Raf, -MEK1/2, and -ERK1/2 MAP kinases showed that these proteins were phosphorylated in the cells during this time ( Fig. 4 ). GM1-treated miPSCs exhibited increased phosphorylation of c-Raf, MEK1/2, and ERK1/2 MAP kinase.
DISCUSSION
Stem cells are undifferentiated cells endowed with a high potential for proliferation and the capacity for self-renewal. Stem cells are very important from the biological and clinical perspectives. In basic biological studies and clinical use, stem cell surface markers are essential for identification and isolation of stem cells. Gangliosides localized at the cell surface can serve as markers (25) . Many gangliosides expressed in pluripotent stem cells, multipotent stem cells, and cancer stem cells have been identified by biochemical and immunological analyses. Some gangliosides are excellent biomarkers of stem cells (26) . miPSCs, which are pluripotent cells artificially generated from somatic cells such as fibroblasts by introducing Oct4 , Sox2 , c-Myc , and Klf4 , express SSEA-1 (1) . Presently, miPSCs expressed stem cell markers including AP, SSEA-1, Oct4 , Sox2 , and Nanog . Bhave et al . investigated the difference in the proliferation between iPSCs and ESCs (6) . Our results revealed a difference in the proliferation rates of these cells. We demonstrated that miPSCs have similar characteristics to mESCs, but different proliferation rates as compared with mESCs.
PPT Slide
Lager Image
Effect of GM1 on MAP kinase activation in miPSCs. The activation of MAP kinases, including c-Raf, MEK1/2, and ERK1/2, was analyzed by Western blotting of miPSCs treated with various concentrations of GM1 (0, 1, 2, 5, and 10 μM).
High performance thin layer chromatography (HPTLC) analyses have revealed the expression of GM3, GM1, and GD1a in E14 mESCs and GM3, GM1, and GD3 in J1 mESCs (27 - 29) . Presently, GM1 was expressed in mESCs, but not in miPSCs. Moreover, our results showed a difference in proliferation rate between miPSCs and mESCs. Therefore, we hypothesize that ganglioside GM1 increases the proliferation rate of miPSCs. GM1 has been implicated in neuronal development and differentiation (30 , 31) . In addition, GM1 has been shown to activate the high affinity nerve growth factor receptor tyrosine kinase (TrkA) in PC12 cells (32) . Exogenous GM1 increases the proliferation of neural stem cells, vascular smooth muscle cells, and brain cells (18 , 33 - 35) , and over-expression of GM1 enhances PC12 cell proliferation (19) . Our results show that exogenous GM1 enhanced the proliferation rate of miPSCs. Furthermore, on the basis of cell cycle analysis, the number of GM1-treated miPSCs in the G2/M phase was higher than the number of untreated miPSCs in the G2/M phase. These results demonstrate that GM1 induces an increased proliferation rate.
Gangliosides regulate the MAP kinase signaling pathways that are important in fundamental cellular processes (20 , 21) . In particular, ERK1/2 MAP kinase pathway has been reported to regulate cell proliferation. Treatment with GM1 activates MAP kinase signaling and increases DNA synthesis in human glioma cells (22) . GM1 activates growth factor receptor tyrosine kinase, and this activation inhibits apoptosis (36) . GM1 treatment of murine neuroblastoma cells increases the ERK1/2 MAP kinase activity (37) . Depletion of glycosphingolipids following treatment with a glycosphingolipid synthesis inhibitor, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), represses the activation of the MAP kinase pathway and retards the proliferation of mouse neuroepithelial cells (21) . Similarly, in mESCs, knock-down of glucosylceramide synthase using small hairpin RNAs inhibits the Ras-MAP kinase pathway and thereby decreases cell proliferation (38) . In addition, dysfunction of the cerebellum and peripheral nerves and alteration of the Ras-MAP kinase pathway have been shown in glycosylceramide synthase-conditional knockout mice (39) .
In this study, we showed the correlation between the proliferation rate and ganglioside expression in miPSCs and mESCs. We demonstrated that GM1 induced the miPSCs proliferation rate. Thus, our results indicate that the treatment with GM1 enhances the proliferation rate of miPSCs via activation of the ERK1/2 MAP kinase pathway.
MATERIALS AND METHODS
- miPSCs and mESCs culture
miPSCs derived from OG2 (Oct-4GFP) mEFs by retrovirus-mediated delivery and expression Oct-4, Sox-2, Klf-4, and c-Myc, and mESCs derived from OG2 mice were generous gifts from Dr. Jeong Tae Do (CHA Stem Cell Institute and CHA Biotech, CHA University, Seoul, Korea). These cells were maintained on mitomycin C-treated mEF feeder plates in standard mESC medium containing DMEM supplemented with 15% fetal bovine serum (Hyclone, Logan, UT), non-essential amino acids, 2 mM L -glutamine, 0.1 mM β-mercaptoethanol, penicillin (100 U/ml), streptomycin (100 μg/ml), and leukemia inhibitory factor (1,000 U/ml; Millipore, Billerica, MA) at 37℃ in 5% CO 2 .
- Viability assay
Cells were seeded at a density of 1 × 10 4 cells/well in 24-well plates. The cells were then treated with GM1, after which cell proliferation was determined by the established 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based assay. Each well was incubated with MTT solution (Sigma-Aldrich, St. Louis, MO) for 4 h, and the absorbance of each well was measured at 590 nm using a spectrophotometer.
- FACS analysis
Cells were trypsinized and harvested in phosphate-buffered saline (PBS) followed by 2 washes with PBS. The cells were then resuspended in 70% ethanol overnight at 4℃. The fixed cells were collected by centrifugation, resuspended in RNaseA solution (0.2 mg/ml RNaseA in PBS), and incubated at 37℃ for 30 min. The cells were then pelleted and incubated in propidium iodide (Sigma-Aldrich) staining solution (20 mg/ml propidium iodide in PBS) overnight at 4℃. The cells were analyzed using a FACSCalibur flow cytometer (BD Biosciences, Bedford, MA) and Cell Quest software (BD Biosciences) was used for data acquisition and analysis.
- Western blot analysis
Cells were homogenized in RIPA buffer (Sigma-Aldrich) and centrifuged at 15,000× g for 30 min. The protein concentration was measured using the Bradford method. Equal amounts of protein (30 μg) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a Hybond ECL nitrocellulose membrane (Amersham Pharmacia, Piscataway, NJ). Blots were blocked for 2 h at room temperature with 5% (w/v) non-fat dried milk in Tris-buffered saline containing 0.05% Tween 20. Each membrane was then incubated for 16 h at 4℃ with specific antibody against cyclin B, Cdc2, cyclin D, Cdk4, Cdk6, cyclin A, cyclin E, Cdk2, c-Raf, phospho-c-Raf, MEK1/2, phospho-MEK1/2, ERK1/2, phospho-ERK1/2, or β-actin (each 1:500; Santa Cruz Biotechnology, Santa Cruz, CA) as specified by the manufacturer. Each blot was then incubated with the corresponding horseradish peroxidase-conjugated anti-mouse, anti-rabbit secondary antibody (Santa Cruz Biotechnology) and proteins were visualized by the enhanced chemiluminescence system (Pierce, Rockford, IL).
- HPTLC analysis
HPTLC analysis of the gangliosides was performed using a 10 × 10 cm HPTLC 5651 plate (Merck, Darmstadt, Germany) as previously described (29) . Purified gangliosides were applied to the HPTLC plates, which were subsequently developed in chloroform/methanol/0.25% CaCl 2 ㆍH 2 O (50:40:10, v/v/v). The gangliosides were visualized with 0.2% resorcinol. Bovine brain gangliosides were used as markers for individual ganglioside species (Matreya LLC; Pleasant Gap, PA).
- Immunocytochemistry
Cells were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature and permeabilized in 0.25% Triton X-100 for 10 min at 37℃. The fixed, permeabilized cells were blocked for 20 min in 5% bovine serum albumin (BSA)/PBS, followed by incubation with primary antibodies against SSEA-1 (Chemicon, Temecula, CA) and gangliosides GM3, GM2, GM1, and GD1a (Seikagaku, Tokyo, Japan) in 5% BSA/PBS overnight at 4℃. Next, the cells were washed with 1% BSA/PBS. A fluorescent secondary antibody, goat anti-mouse IgM-Cy5 for detecting SSEA-1 and gangliosides (Chemicon), was then applied at a dilution of 1:500. Hoechst 33342 reagent (Sigma-Aldrich) was used to stain the nuclei. The stained cells were observed under a model FV300 confocal scanning laser fluorescence microscope (Olympus, Tokyo, Japan).
- Statistical analysis
All data are presented as mean ± S.D. Comparisons of multiple groups were performed by one-way analysis of variance (ANOVA), followed by pairwise comparisons with a Bonferroni post hoc test. All data were analyzed using GraphPad Prism version 4.00 software (GraphPad Software, La Jolla, CA).
Acknowledgements
This research was supported by a grant (Code# PJ0074922012) from the Korean Rural Development Administration, and has been supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education Science and Technology (2010-0022316 and KCG5401011), and the KRIBB/KRCF Research Initiative Program (NAP), Korea.
References
Takahashi K. , Yamanaka S. (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126 663 - 676    DOI : 10.1016/j.cell.2006.07.024
Okita K. , Ichisaka T. , Yamanaka S. (2007) Generation of germline-competent induced pluripotent stem cells. Nature 448 313 - 317    DOI : 10.1038/nature05934
Maherali N. , Sridharan R. , Xie W. , Utikal J. , Eminli S. , Arnold K. , Stadtfeld M. , Yachechko R. , Tchieu J. , Jaenisch R. , Plath K. , Hochedlinger K. (2007) Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution. Cell. Stem. Cell 1 55 - 70    DOI : 10.1016/j.stem.2007.05.014
Aoi T. , Yae K. , Nakagawa M. , Ichisaka T. , Okita K. , Takahashi K. , Chiba T. , Yamanaka S. (2008) Generation of pluripotent stem cells from adult mouse liver and stomach cells. Science 321 699 - 702    DOI : 10.1126/science.1154884
Stadtfeld M. , Brennand K. , Hochedlinger K. (2008) Reprogramming of pancreatic beta cells into induced pluripotent stem cells. Curr. Biol. 18 890 - 894    DOI : 10.1016/j.cub.2008.05.010
Bhave V. S. , Paranjpe S. , Bowen W. C. , Donthamsetty S. , Bell A. W. , Khillan J. S. , Michalopoulos G. K. (2011) Genes inducing iPS phenotype play a role in hepatocyte survival and proliferation in vitro and liver regeneration in vivo. Hepatology 54 1360 - 1370    DOI : 10.1002/hep.24507
Hakomori S. (1990) Bifunctional role of glycosphingolipids. Modulators for transmembrane signaling and mediators for cellular interactions. J. Biol. Chem. 265 18713 - 18716
van Meer G. (1993) Transport and sorting of membrane lipids. Curr. Opin. Cell Biol. 5 661 - 673    DOI : 10.1016/0955-0674(93)90137-F
Tettamanti G. (2004) Ganglioside/glycosphingolipid turnover: new concepts. Glycoconj. J. 20 301 - 317    DOI : 10.1023/B:GLYC.0000033627.02765.cc
Hakomori S. , Yamamura S. , Handa A. K. (1998) Signal transduction through glyco(sphingo)lipids. Introduction and recent studies on glyco(sphingo)lipid-enriched microdomains. Ann. N. Y. Acad. Sci. 845 1 - 10    DOI : 10.1111/j.1749-6632.1998.tb09657.x
Kwak D. H. , Jin J. W. , Ryu J. S. , Ko K. , Lee S. D. , Lee J. W. , Kim J. S. , Jung K. Y. , Ma J. Y. , Hwang K. A. , Chang K. T. , Choo Y. K. (2011) Regulatory roles of ganglioside GQ1b in neuronal cell differentiation of mouse embryonic stem cells. BMB Rep. 44 799 - 804    DOI : 10.5483/BMBRep.2011.44.12.799
Ji M. Y. , Lee Y. C. , Do S. 2nd , Nam S. Y. , Jung K. Y. , Kim H. M. , Park L. K. , Choo Y. K. (2000) Developmental patterns of mST3GalV mRNA expression in the mouse: in situ hybridization using DIG-labeled RNA probes. Arch. Pharm. Res. 23 525 - 530    DOI : 10.1007/BF02976584
Kim B. H. , Jung J. U. , Ko K. , Kim W. S. , Kim S. M. , Ryu J. S. , Jin J. W. , Yang H. J. , Kim J. S. , Kwon H. C. , Nam S. Y. , Kwak D. H. , Park Y. I. , Koo D. B. , Choo Y. K. (2008) Expression of ganglioside GT1b in mouse embryos at different developmental stages after cryopreservation. Arch. Pharm. Res. 31 88 - 95    DOI : 10.1007/s12272-008-1125-6
Ngamukote S. , Yanagisawa M. , Ariga T. , Ando S. , Yu R. K. (2007) Developmental changes of glycosphingolipids and expression of glycogenes in mouse brains. J. Neurochem. 103 2327 - 2341    DOI : 10.1111/j.1471-4159.2007.04910.x
Goettl V. M. , Zhang H. , Burrows A. C. , Wemlinger T. A. , Neff N. H. , Hadjiconstantinou M. (2003) GM1 enhances dopaminergic markers in the brain of aged rats. Exp. Neurol. 183 665 - 672    DOI : 10.1016/S0014-4886(03)00223-1
Goettl V. M. , Wemlinger T. A. , Duchemin A. M. , Neff N. H. , Hadjiconstantinou M. (1999) GM1 ganglioside restores dopaminergic neurochemical and morphological markers in aged rats. Neuroscience 92 991 - 1000    DOI : 10.1016/S0306-4522(98)00756-8
Duan J. G. , Xiang T. , Chen H. , Liu M. (2007) Role of extrinsic ganglioside GM1 in proliferation and differentiation of neural stem cells. Sichuan Da Xue Xue Bao Yi Xue Ban 38 260 - 263
Gouni-Berthold I. , Seul C. , Ko Y. , Hescheler J. , Sachinidis A. (2001) Gangliosides GM1 and GM2 induce vascular smooth muscle cell proliferation via extracellular signal-regulated kinase 1/2 pathway. Hypertension 38 1030 - 1037    DOI : 10.1161/hy1101.093104
Nishio M. , Tajima O. , Furukawa K. , Urano T. (2005) Over-expression of GM1 enhances cell proliferation with epidermal growth factor without affecting the receptor localization in the microdomain in PC12 cells. Int. J. Oncol. 26 191 - 199
Hashiramoto A. , Mizukami H. , Yamashita T. (2006) Ganglioside GM3 promotes cell migration by regulating MAPK and c-Fos/AP-1. Oncogene 25 3948 - 3955    DOI : 10.1038/sj.onc.1209416
Yanagisawa M. , Nakamura K. , Taga T. (2005) Glycosphingolipid synthesis inhibitor represses cytokine-induced activation of the Ras-MAPK pathway in embryonic neural precursor cells. J. Biochem. 138 285 - 291    DOI : 10.1093/jb/mvi129
Van Brocklyn J. R. , Vandenheede J. R. , Fertel R. , Yates A. J. , Rampersaud A. A. (1997) Ganglioside GM1 activates the mitogen-activated protein kinase Erk2 and p70 S6 kinase in U-1242 MG human glioma cells. J. Neurochem. 69 116 - 125    DOI : 10.1046/j.1471-4159.1997.69010116.x
Backer J. M. , Myers M. G. Jr. , Shoelson S. E. , Chin D. J. , Sun X. J. , Miralpeix M. , Hu P. , Margolis B. , Skolnik E. Y. , Schlessinger J. , White M. F. (1992) Phosphatidylinositol 3'-kinase is activated by association with IRS-1 during insulin stimulation. EMBO J. 11 3469 - 3479
Ihle J. N. , Witthuhn B. A. , Quelle F. W. , Silvennoinen O. , Tang B. , Yi T. (1994) Protein tyrosine phosphorylation in the regulation of hematopoiesis by receptors of the cytokine-receptor superfamily. Blood Cells 20 65 - 80
Yanagisawa M. , Yu R. K. (2007) The expression and functions of glycoconjugates in neural stem cells. Glycobiology 17 57R - 74R    DOI : 10.1093/glycob/cwm018
Yanagisawa M. (2011) Stem cell glycolipids. Neurochem Res. 36 1623 - 1635    DOI : 10.1007/s11064-010-0358-1
Kimber S. J. , Brown D. G. , Pahlsson P. , Nilsson B. (1993) Carbohydrate antigen expression in murine embryonic stem cells and embryos. II. Sialylated antigens and glycolipid analysis. Histochem J. 25 628 - 641    DOI : 10.1007/BF00157877
Kwak D. H. , Yu K. , Kim S. M. , Lee D. H. , Jung J. U. , Seo J. W. , Kim N. , Lee S. , Jung K. Y. , You H. K. , Kim H. A. , Choo Y. K. (2006) Dynamic changes of gangliosides expression during the differentiation of embryonic and mesenchymal stem cells into neural cells. Exp. Mol. Med. 38 668 - 676    DOI : 10.1038/emm.2006.79
Lee D. H. , Koo D. B. , Ko K. , Kim S. M. , Jung J. U. , Ryu J. S. , Jin J. W. , Yang H. J. , Do S. I. , Jung K. Y. , Choo Y. K. (2007) Effects of daunorubicin on ganglioside expression and neuronal differentiation of mouse embryonic stem cells. Biochem. Biophys. Res. Commun. 362 313 - 318    DOI : 10.1016/j.bbrc.2007.07.142
Ledeen R. W. (1984) Biology of gangliosides: neuritogenic and neuronotrophic properties. J. Neurosci. Res. 12 147 - 159    DOI : 10.1002/jnr.490120204
Leon A. , Dal Toso R. , Presti D. , Benvegnu D. , Facci L. , Kirschner G. , Tettamanti G. , Toffano G. (1988) Development and survival of neurons in dissociated fetal mesencephalic serum-free cell cultures: II. Modulatory effects of gangliosides. J. Neurosci. 8 746 - 753
Mutoh T. , Tokuda A. , Miyadai T. , Hamaguchi M. , Fujiki N. (1995) Ganglioside GM1 binds to the Trk protein and regulates receptor function. Proc. Natl. Acad. Sci. U.S.A. 92 5087 - 5091    DOI : 10.1073/pnas.92.11.5087
Wang Q. , Wang Z. P. , Xu Q. , Bao N. (2009) Effects of ganglioside 1 and nerve growth factor on the proliferation of neural stem cells in vitro. Zhongguo Dang Dai Er Ke Za Zhi 11 841 - 845
Duan J. G. , Xiang T. , Chen H. , Liu M. (2007) Role of extrinsic ganglioside GM1 in proliferation and differentiation of neural stem cells. Sichuan Da Xue Xue Bao Yi Xue Ban 38 260 - 263
Duchemin A. M. , Ren Q. , Mo L. , Neff N. H. , Hadjiconstantinou M. (2002) GM1 ganglioside induces phosphorylation and activation of Trk and Erk in brain. J. Neurochem. 81 696 - 707    DOI : 10.1046/j.1471-4159.2002.00831.x
Ferrari G. , Greene L. A. (1998) Promotion of neuronal survival by GM1 ganglioside. Phenomenology and mechanism of action. Ann. N. Y. Acad. Sci. 845 263 - 273    DOI : 10.1111/j.1749-6632.1998.tb09679.x
Singleton D. W. , Lu C. L. , Colella R. , Roisen F. J. (2000) Promotion of neurite outgrowth by protein kinase inhibitors and ganglioside GM1 in neuroblastoma cells involves MAP kinase ERK1/2. Int. J. Dev. Neurosci. 18 797 - 805    DOI : 10.1016/S0736-5748(00)00047-2
Jung J. U. , Ko K. , Lee D. H. , Chang K. T. , Choo Y. K. (2009) The roles of glycosphingolipids in the proliferation and neural differentiation of mouse embryonic stem cells. Exp. Mol. Med. 41 935 - 945    DOI : 10.3858/emm.2009.41.12.099
Jennemann R. , Sandhoff R. , Wang S. , Kiss E. , Gretz N. , Zuliani C. , Martin-Villalba A. , Jager R. , Schorle H. , Kenzelmann M. , Bonrouhi M. , Wiegandt H. , Grone H. J. (2005) Cell-specific deletion of glucosylceramide synthase in brain leads to severe neural defects after birth. Proc. Natl. Acad. Sci. U.S.A. 102 12459 - 12464    DOI : 10.1073/pnas.0500893102