Differentiation of neuronal cells using a murine embryonic stem cell-based method

DOI: 10.4236/abb.2013.49113   PDF   HTML     3,609 Downloads   5,383 Views  


The differentiation and screening methodology proposed here is an efficient in vitro system to screen and study effects of small molecules and bioagents and is an alternative to studies that use live animals and embryos. The method is based on engineering a stable murine embryonic stem (ES) cell line expressing lineage-specific promoters that drive selection and reporter genes. Additionally, uniform embryoid bodies (EBs) are used for differentiation studies that allow synchronous differentiation. The reporter and selection marker genes are expressed only in lineages where the promoter is functional. The differentiated cell type can be identified by reporter gene expression and the selection marker can be used for selective enrichment of that particular cell population. The method described here is useful in screening small molecules or bioagents that can differentiate stem cells into particular lineages or cell types. Identified compounds are useful in areas such as stem cell-based regenerative medicine and therapeutics. The method described here has been applied to neuronal cell differentiation.

Share and Cite:

Ezekiel, U. (2013) Differentiation of neuronal cells using a murine embryonic stem cell-based method. Advances in Bioscience and Biotechnology, 4, 853-859. doi: 10.4236/abb.2013.49113.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Xian, H.Q. and Gottlieb, D.I. (2001) Peering into early neurogenesis with embryonic stem cells. Trends in Neurosciences, 24, 685-686. doi:10.1016/S0166-2236(00)01966-4
[2] Leahy, A., Xiong, J.W., Kuhnert, F. and Stuhlmann, H. (1999) Use of developmental marker genes to define temporal and spatial patterns of differentiation during embryoid body formation. Journal of Experimental Zoology, 284, 67-81. doi:10.1002/(SICI)1097-010X(19990615)284:1<67::AID-JEZ10>3.0.CO;2-O
[3] Bain, G. and Gottlieb, D.I. (1998) Neural cells derived by in vitro differentiation of P19 and embryonic stem cells. Perspectives on Developmental Neurobiology, 5, 175-178.
[4] Okabe, S., Forsberg-Nilsson, K., Spiro, A.C., Segal, M. and McKay, R.D. (1996) Development of neuronal precursor cells and functional postmitotic neurons from embryonic stem cells in vitro. Mechanisms of Development, 59, 89-102. doi:10.1016/0925-4773(96)00572-2
[5] Wichterle, H. and Peljto, M. (2008) Differentiation of mouse embryonic stem cells to spinal motor neurons. Current Protocols in Stem Cell Biology, Chapter 1: Unit-1H.
[6] Ng, E.S., Davis, R.P., Azzola, L., Stanley, E.G. and Elefanty, A.G. (2005) Forced aggregation of defined numbers of human embryonic stem cells into embryoid bodies fosters robust, reproducible hematopoietic differentiation. Blood, 106, 1601-1603. doi:10.1182/blood-2005-03-0987
[7] Ezekiel, U.R., Muthuchamy, M., Ryerse, J.S. and Heuertz, R. (2006) Single embryoid body formation in a multiwell plate. Electronic Journal of Biotechnology, 10, 328-335.
[8] Bain, G., Ray, W.J., Yao, M. and Gottlieb, D.I. (1996) Retinoic acid promotes neural and represses mesodermal gene expression in mouse embryonic stem cells in culture. Biochemical and Biophysical Research Communications, 223, 691-694. doi:10.1006/bbrc.1996.0957
[9] Chaerkady, R., Kerr, C.L., Marimuthu, A., Kelkar, D.S., Kashyap, M.K., Gucek, M., Gearhart, J.D. and Pandey, A. (2009) Temporal analysis of neural differentiation using quantitative proteomics. Journal of Proteome Research, 8, 1315-1326. doi:10.1021/pr8006667
[10] Miloso, M., Bertelli, A.A., Nicolini, G. and Tredici, G. (1999) Resveratrol-induced activation of the mitogen-activated protein kinases, ERK1 and ERK2, in human neuronblastoma SH-SY5Y cells. Neuroscience Letters, 264, 141-144. doi:10.1016/S0304-3940(99)00194-9
[11] Ahlemeyer, B. and Krieglstein, J. (2003) Neuroprotective effects of Ginkgo biloba extract. Cellular and Molecular Life Sciences, 60, 1779-1792. doi:10.1007/s00018-003-3080-1
[12] Yoo, D.Y., Nam, Y., Kim, W., Yoo, K.Y., Park, J., Lee, C.H., Choi, J.H., Yoon, Y.S., Kim, D.W., Won, M.H., et al. (2011) Effects of Ginkgo biloba extract on promotion of neurogenesis in the hippocampal dentate gyrus in C57BL/6 mice. Journal of Veterinary Medical Science, 73, 71-76. doi:10.1292/jvms.10-0294
[13] Mahonen, A., Jaaskelainen, T. and Maenpaa, P.H. (1996) A novel vitamin D analog with two double bonds in its side chain. A potent inducer of osteoblastic cell differentiation. Biochemical Pharmacology, 51, 887-892. doi:10.1016/0006-2952(95)02242-2
[14] Watanabe, K., Ueno, M., Kamiya, D., Nishiyama, A., Matsumura, M., Wataya, T., Takahashi, J.B., Nishikawa, S., Nishikawa, S., Muguruma, K., et al. (2007) A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nature Biotechnology, 25, 681-686. doi:10.1038/nbt1310
[15] Li, Z., Theus, M.H. and Wei, L. (2006) Role of ERK 1/2 signaling in neuronal differentiation of cultured embryonic stem cells. Development, Growth & Differentiation, 48, 513-523. doi:10.1111/j.1440-169X.2006.00889.x
[16] Uetsuki, T., Takagi, K., Sugiura, H., and Yoshikawa, K. (1996) Structure and expression of the mouse necdin gene. Identification of a postmitotic neuron-restrictive core promoter. Journal of Biological Chemistry, 271, 918-924. doi:10.1074/jbc.271.2.918
[17] Kuo, C.H., Uetsuki, T., Kim, C.H., Tanaka, H., Li, B.S., Taira, E., Higuchi, H., Okamoto, H., Yoshikawa, K. and Miki, N. (1995) Determination of a necdin cis-acting element required for neuron specific expression by using zebra fish. Biochemical and Biophysical Research Communications, 211, 438-446. doi:10.1006/bbrc.1995.1833
[18] Maruyama, E. (1996) Biochemical characterization of mouse brain necdin. Biochemical Journal, 314, 895-901.
[19] Tucker, K.L., Wang, Y., Dausman, J. and Jaenisch, R. (1997) A transgenic mouse strain expressing four drugselectable marker genes. Nucleic Acids Research, 25, 3745-3746. doi:10.1093/nar/25.18.3745
[20] Brook, F.A. and Gardner, R.L. (1997) The origin and efficient derivation of embryonic stem cells in the mouse. Proceedings of the National Academy of Sciences of the United States of America, 94, 5709-5712. doi:10.1073/pnas.94.11.5709
[21] Martin, C.S., Wight, P.A., Dobretsova, A. and Bronstein, I. (1996) Dual luminescence-based reporter gene assay for luciferase and beta-galactosidase. Biotechniques, 21, 520-524.
[22] Xiong, J.W., Battaglino, R., Leahy, A. and Stuhlmann, H. (1998) Large-scale screening for developmental genes in embryonic stem cells and embryoid bodies using retroviral entrapment vectors. Developmental Dynamics, 212, 181-197. doi:10.1002/(SICI)1097-0177(199806)212:2<181::AID-AJA4>3.0.CO;2-D
[23] Laposa, R. (2011) Stem cells for drug screening. Journal of Cardiovascular Pharmacology, 58, 240-245. doi:10.1097/FJC.0b013e31821823f5
[24] Barbaric, I., Jones, M., Harley, D.J., Gokhale, P.J. and Andrews, P.W. (2011) High-content screening for chemical modulators of embryonal carcinoma cell differentiation and survival. Journal of Biomolecular Screening, 16, 603-617. doi:10.1177/1087057111406547
[25] Schmitt, R.M., Bruyns, E. and Snodgrass, H.R. (1991) Hematopoietic development of embryonic stem cells in vitro: Cytokine and receptor gene expression. Genes & Development, 5, 728-740. doi:10.1101/gad.5.5.728
[26] Risau, W., Sariola, H., Zerwes, H.G., Sasse, J., Ekblom, P., Kemler, R. and Doetschman, T. (1988) Vasculogenesis and angiogenesis in embryonic-stem-cell-derived embryoid bodies. Development, 102, 471-478.
[27] Wang, R., Clark, R. and Bautch, V.L. (1992) Embryonic stem cell-derived cystic embryoid bodies form vascular channels: An in vitro model of blood vessel development. Development, 114, 303-316.
[28] O’Shea, K.S. (1999) Embryonic stem cell models of development. Anatomical Record, 257, 32-41. doi:10.1002/(SICI)1097-0185(19990215)257:1<32::AID-AR6>3.0.CO;2-2
[29] Kane, N.M., Xiao, Q., Baker, A.H., Luo, Z., Xu, Q. and Emanueli, C. (2011) Pluripotent stem cell differentiation into vascular cells: A novel technology with promises for vascular re(generation). Pharmacology & Therapeutics, 129, 29-49. doi:10.1016/j.pharmthera.2010.10.004
[30] Vallier, L., Mancip, J., Markossian, S., Lukaszewicz, A., Dehay, C., Metzger, D., Chambon, P., Samarut, J. and Savatier, P. (2001) An efficient system for conditional gene expression in embryonic stem cells and in their in vitro and in vivo differentiated derivatives. Proceedings of the National Academy of Sciences of the United States of America, 98, 2467-2472. doi:10.1073/pnas.041617198
[31] Doss, M.X., Koehler, C.I., Gissel, C., Hescheler, J. and Sachinidis, A. (2004) Embryonic stem cells: A promising tool for cell replacement therapy. Journal of Cellular and Molecular Medicine, 8, 465-473. doi:10.1111/j.1582-4934.2004.tb00471.x
[32] Odorico, J.S., Kaufman, D.S. and Thomson, J.A. (2001) Multilineage differentiation from human embryonic stem cell lines. Stem Cells, 19, 193-204. doi:10.1634/stemcells.19-3-193
[33] Sauer, H., Gunther, J., Hescheler, J., and Wartenberg, M. (2000) Thalidomide inhibits angiogenesis in embryoid bodies by the generation of hydroxyl radicals. American Journal of Pathology, 156, 151-158. doi:10.1016/S0002-9440(10)64714-1

comments powered by Disqus

Copyright © 2020 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.