Analysis of proteomic profiling of mouse embryonic stem cells derived from fertilized, parthenogenetic and androgenetic blastocysts

DOI: 10.4236/scd.2011.11001   PDF   HTML     4,275 Downloads   10,772 Views   Citations


Embryonic stem cells (ESCs) are derived from the inner cell mass (ICM) of preimplantation embryos. ESCs exhibit true pluripotency, i.e., the ability to differentiate into cells of all three germ layers in the developing embryo. We used 2-DE MALDI-TOF/TOF to identify differentially expressed proteins among three types of mouse embryonic stem cells (ESCs) derived from ferti-lized, parthenogenetic, and androgenetic (fESC, pESC and aESC, respectively) blastocysts. We detected more than 800 proteins on silver- stained gels of whole protein extracts from each type of ESC. Of these, 52 differentially expressed proteins were identified by the MALDI–TOF/TOF analyzer, including 32 (fESCs vs. pESCs), 28 (fESCs vs. aESCs) and 17 (pESCs vs. aESCs) prominent proteins with significantly higher or lower expression relative to the comparison cells. Among the 32 proteins from fESCs, 12 were significantly increased in expression and 20 were reduced in expression in fESCs com-pared with pESCs. Similarly, 10 of 28 and 8 of 17 proteins were more highly expressed in fESCs and pESCs compared with aESCs, respectively. In contrast, 18 of 28 and 9 of 17 proteins were reduced in expression in fESCs and pESCs compared with aESCs, respectively. Of the eight protein candidates in fESCs, four were in-creased and four were decreased in expression relative to both pESCs and aESCs in the 2-DE analysis. Differential expression of these pro-teins were confirmed by mRNA expression analysis using real time RT-PCR. For three pro-teins, ANXA5, CLIC1 and SRM, Western blot analysis corroborated the expression patterns indicated by the 2-DE results. ANXA5 and CLIC1 were increased in expression and SRM was de-creased in expression in fESCs compared with both pESCs and aESCs. The differentially ex-pressed proteins identified in the present study warrant further investigation towards the goal of their potential application in ESC therapy.

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Cui, X. , Shen, X. , Lee, C. , Kang, Y. , Wakayama, T. and Kim, N. (2011) Analysis of proteomic profiling of mouse embryonic stem cells derived from fertilized, parthenogenetic and androgenetic blastocysts. Stem Cell Discovery, 1, 1-15. doi: 10.4236/scd.2011.11001.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Evans, M.J. and Kaufman, M.H. (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature, 292, 154-156. doi:10.1038/292154a0
[2] Thomson, J.A., Itskovitz-Eldor J., Shapiro S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S. and Jones, J.M. (1998) Embryonic stem cell lines derived from human blastocysts. Science, 282, 1145-1147. doi:10.1126/science.282.5391.1145
[3] Allen, N.D., Barton, S.C., Hilton, K., Norris, M.L. and Surani, M.A. (1994) A functional analysis of imprinting in parthenogenetic embryonic stem cells. Development, 120, 1473-1482.
[4] Eckardt, S, Leu, N.A., Bradley, H.L., Kato, H., Bunting, K.D. and McLaughlin, K.J. (2007) Hematopoietic reconstitution with androgenetic and gynogenetic stem cells. Genes Deveplment, 21, 409-419. doi:10.1101/gad.1524207
[5] Li, C., Chen, Z., Liu, Z., Huang, J., Zhang, W., Zhou, L., Keefe, D.L. and Liu, L. (2009) Correlation of expression and methylation of imprinted genes with pluripotency of parthenogenetic embryonic stem cells. Human Molecular Genetics, 18, 2177- 2187. doi:10.1093/hmg/ddp150
[6] Baharvand, H., Fathi, A., Gourabi, H., Mollamohammadi, S. and Salekdeh, G.H. (2008) Identification of mouse embryonic stem cell-associated proteins. Journal of Proteome Research, 7, 412-423. doi:10.1021/pr700560t
[7] Intoh, A., Kurisaki, A., Yamanaka, Y., Hirano, H., Fukuda, H., Sugino, H. and Asashima, M. (2009) Proteomic analysis of membrane proteins expressed specifically in pluripotent murine embryonic stem cells. Proteomics, 9, 126-137. doi:10.1002/pmic.200800496
[8] Lee, C.K., Kim, H.J., Lee, Y.R., So, H.H., Park, H.J., Won, K.J., Park, T., Lee, K.Y., Lee, H.M. and Kim, B. (2007) Analysis of peroxiredoxin decreasing oxidative stress in hypertensive aortic smooth muscle. Biochimica at Biophysica Acta, 1774, 848-855.
[9] Zhang, D.X., Cui, X.S. and Kim, N.H. (2009) Involvement of polyadenylation status on maternal gene expression during in vitro maturation of porcine oocytes. Molecular Reproduction Development, 76, 881-889. doi:10.1002/mrd.21056
[10] Cary. Statisitics. (1985) SAS User's Gudie Version 5, NC, SAS.
[11] Torrie, RGDSa, J.H. (1980) Principles and Procedures of Statistics. New York, McGraw Hill Book Companies.
[12] Hikichi, T., Wakayama, S., Mizutani, E., Takashima, Y., Kishigami, S., Van Thuan, N., Ohta, H., Thuy Bui, H., Nishikawa, S. and Wakayama, T. (2007) Differentiation potential of parthenogenetic embryonic stem cells is improved by nuclear transfer. Stem Cells, 25, 46-53. doi:10.1634/stemcells.2006-0439
[13] Hikichi, T., Ohta, H., Wakayama, S. and Wakayama, T. (2010) Functional full-term placentas formed from parthenogenetic embryos using serial nuclear transfer. Development, 137, 2841-2847. doi:10.1242/dev.051375
[14] Wakayama, S., Ohta, H., Kishigami, S., Thuan, N.V., Hikichi, T., Mizutani, E., Miyake, M. and Wakayama, T. (2005) Establishment of male and female nuclear transfer embryonic stem cell lines from different mouse strains and tissues. Biology of Reproduction, 72, 932-936. doi:10.1095/biolreprod.104.035105
[15] Wakayama, T., Tabar, V., Rodriguez, I., Perry, A.C., Studer, L. and Mombaerts, P. (2001) Differentiation of embryonic stem cell lines generated from adult somatic cells by nuclear transfer. Science, 292, 740-743. doi:10.1126/science.1059399
[16] Wakayama, S., Jakt, M.L., Suzuki, M., Araki, R., Hikichi, T., Kishigami, S., Ohta, H., Van Thuan, N., Mizutani, E., Sakaide, Y., Senda, S., Tanaka, S., Okada, M., Miyake, M., Abe, M., Nishikawa, S., Shiota, K. and Wakayama, T. (2006) Equivalency of nuclear transfer-derived embryonic stem cells to those derived from fertilized mouse blastocysts. Stem Cells, 24, 2023-2033. doi:10.1634/stemcells.2005-0537
[17] Niwa, H., Miyazaki, J. and Smith, A.G. (2000) Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nature Genetics, 24, 372-376. doi:10.1038/74199
[18] Mitsui, K., Tokuzawa, Y., Itoh, H., Segawa, K., Murakami, M., Takahashi, K., Maruyama M, Maeda M and Yamanaka S. (2003) The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell, 113, 631-642. doi:10.1016/S0092-8674(03)00393-3
[19] Kashyap, V., Rezende, N.C., Scotland, K.B., Shaffer, S.M., Persson, J.L., Gudas, L.J. and Mongan, N.P. (2009) Regulation of stem cell pluripotency and differentiation involves a mutual regulatory circuit of the NANOG, OCT4, and SOX2 pluripotency transcription factors with polycomb repressive complexes and stem cell microRNAs. Stem Cells Development, 18, 1093-1108. doi:10.1089/scd.2009.0113
[20] Dinger, T.C., Eckardt, S., Choi, S.W., Camarero, G., Kurosaka, S., Hornich, V., McLaughlin, K.J. and Muller, A.M. (2008) Androgenetic embryonic stem cells form neural progenitor cells in vivo and in vitro. Stem Cells, 26, 1474-1483. doi:10.1634/stemcells.2007-0877
[21] Teramura, T., Onodera, Y., Murakami, H., Ito, S., Mihara, T., Takehara, T., Kato, H., Mitani,T., Anzai, M., Matsumoto, K., Saeki, K., Fukuda, K., Sagawa, N. and Osoi, Y. (2009) Mouse androgenetic embryonic stem cells differentiated to multiple cell lineages in three embryonic germ layers in vitro. Journal of Reproduction and Development, 55, 283-292. doi:10.1262/jrd.20146
[22] Dedman BASaJR. Annexins. Biometals (1998) 11, 399-404. doi:10.1023/A:1009205925714
[23] Russo-Marie, F. and Annexin, V. (1999) Phospholipid metabolism. Clinical Chemistry and Laboratory Medicine, 37, 287-291. doi:10.1515/CCLM.1999.050
[24] Ozerova, S.G. and Minin, A.A. (2008) A study of proteins of annexin group in early fish development. IV. Identification of calcium-binding proteins in zebrafish egg by mass spectrometry. Ontogenez, 39, 222-226.
[25] Avalos-Rodriguez, A., Ortiz-Muniz, A.R., Ortega-Camarillo, C., Vergara-Onofre, M., Rosado-Garcia, A. and Rosales-Torres, A.M. (2004) Fluorometric study of rabbit sperm head membrane phospholipid asymmetry during capacitation and acrosome reaction using Annexin-V FITC. Archives of Anthology, 50, 273-285. doi:10.1080/01485010490448741
[26] Cromer, B.A., Morton, C.J., Board, P.G. and Parker, M.W. (2002) From glutathione transferase to pore in a CLIC. European Biophysics Journal, 31, 356-364. doi:10.1007/s00249-002-0219-1
[27] Ashley, R.H. (2003) Challenging accepted ion channel biology: P64 and the CLIC family of putative intracellular anion channel proteins (Review). Molecular Membrane Biology, 20, 1-11. doi:10.1080/09687680210042746
[28] Harrop, S.J., DeMaere, M.Z., Fairlie, W.D., Reztsova, T., Valenzuela, S.M., Mazzanti, M., Tonini, R., Qiu, M.R., Jankova, L., Warton, K., Bauskin, A.R., Wu, W.M., Pankhurst, S., Campbell, T.J., Breit, S.N. and Curmi, P.M. (2001) Crystal structure of a soluble form of the intracellular chloride ion channel CLIC1 (NCC27) at 1.4-A resolution. The Journal of Biological Chemistry, 276, 44993-45000. doi:10.1074/jbc.M107804200
[29] Yang, J.Y., Jung, J.Y., Cho, S.W., Choi, H.J., Kim, S.W., Kim, S.Y, Kim, H.J., Jang, C.H., Lee, M.G., Han, J. and Shin, C.S. (2009) Chloride intracellular channel 1 regulates osteoblast differentiation. Bone, 45, 1175-1185. doi:10.1016/j.bone.2009.08.012
[30] Edwards, J.C. (2006) The CLIC1 chloride channel is regulated by the cystic fibrosis transmembrane conductance regulator when expressed in Xenopus oocytes. Journal of Membrane Biology, 213, 39-46. doi:10.1007/s00232-006-0059-5
[31] Lee, H.Y., Cui, X.S., Lee, K.A. and Kim, N.H. (2006) Annealing control primer system identifies differentially expressed genes in blastocyst-stage porcine parthenotes. Zygote, 14, 71-80. doi:10.1017/S0967199406003571
[32] Cui, X.S. and Kim, N.H. (2005) Polyamines inhibit apoptosis in porcine parthenotes developing in vitro. Molecular Reproduction and Development, 70, 471-477. doi:10.1002/mrd.20228
[33] Pegg, A.E. (1988) Polyamine metabolism and its importance in neoplastic growth and a target for chemotherapy. Cancer Research, 48, 759-774.
[34] Stefanelli C, P.C., Tantini, B., Fattori, M., Stanic, I., Mackintosh, C.A., Flamigni, F., Guarnieri, C., Caldarera, C.M. and Pegg, A.E. (2001) Effect of polyamine depletion on caspase activation: A study with spermine synthase-deficient cells. Biochemical Journal, 355, 199-206. doi:10.1042/0264-6021:3550199
[35] Imai, A., Matsuyama, T., Hanzawa, Y., Akiyama, T., Tamaoki, M., Saji, H., Shirano, Y., Kato, T., Hayashi, H., Shibata, D., Tabata, S., Komeda, Y. and Takahashi, T. (2004) Spermidine synthase genes are essential for survival of Arabidopsis. Plant Physiology, 135, 1565-1573. doi:10.1104/pp.104.041699
[36] Kasukabe, Y., He, L., Nada, K., Misawa, S., Ihara, I. and Tachibana, S. (2004) Overexpression of spermidine synthase enhances tolerance to multiple environmental stresses and up-regulates the expression of various stress-regulated genes in transgenic Arabidopsis thaliana. Plant Cell Physiology, 45, 712-722. doi:10.1093/pcp/pch083
[37] Hardy, K. (1997) Cell death in the mammalian blastocyst. Molecular Human Reproduction, 3, 919-925. doi:10.1093/molehr/3.10.919

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