Abstract
Here, we present a highly efficient and reproducible method for the establishment of mouse embryonic stem cells (mESCs) from embryonic day 3.5 (E3.5) whole blastocysts. This protocol involves the use of small molecules SB431542 and PD0325901, which inhibit transforming growth factor-β (TGF-β) and extracellular signal-regulated kinases (ERK1/2), respectively. This protocol is universal in the derivation of mESC lines from NMRI, C57BL/6, BALB/c, DBA/2, and FVB/N strains, which have previously been considered refractory or non-permissive for ESC establishment. The efficiency of mESC lines generation is 100%, regardless of genetic background.
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References
Martin GR (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. Proc Natl Acad Sci USA 78:7634–7638
Evans MJ, Kaufman MH (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature 292:154–156
Yu J, Thomson JA (2008) Pluripotent stem cell lines. Genes Dev 22:1987–1997
Nichols J, Smith A (2009) Naive and primed pluripotent states. Cell Stem Cell 4:487–492
Hanna J, Markoulaki S, Mitalipova M, Cheng AW, Cassady JP, Staerk J, Carey BW, Lengner CJ, Foreman R, Love J, Gao Q, Kim J, Jaenisch R (2009) Metastable pluripotent states in NOD-mouse-derived ESCs. Cell Stem Cell 4: 513–524
Buecker C, Geijsen N (2010) Different flavors of pluripotency, molecular mechanisms, and practical implications. Cell Stem Cell 7:559–564
Hassani SN, Totonchi M, Farrokhi A, Taei A, Larijani MR, Gourabi H, Baharvand H (2012) Simultaneous suppression of TGF-beta and ERK signaling contributes to the highly efficient and reproducible generation of mouse embryonic stem cells from previously considered refractory and non-permissive strains. Stem Cell Rev 8(2):472–481
Ying QL, Wray J, Nichols J, Batlle-Morera L, Doble B, Woodgett J, Cohen P, Smith A (2008) The ground state of embryonic stem cell self-renewal. Nature 453:519–523
Acevedo N, Wang X, Dunn RL, Smith GD (2007) Glycogen synthase kinase-3 regulation of chromatin segregation and cytokinesis in mouse preimplantation embryos. Mol Reprod Dev 74:178–188
Tighe A, Ray-Sinha A, Staples OD, Taylor SS (2007) GSK-3 inhibitors induce chromosome instability. BMC Cell Biol 8:34
Michalska E (2007) Isolation and propagation of mouse embryonic fibroblasts and preparation of mouse embryonic feeder layer cells. Curr Protoc Stem Cell Biol Chapter 1: Unit1C 3
Meissner A, Eminli S, Jaenisch R (2009) Derivation and manipulation of murine embryonic stem cells. Methods Mol Biol 482:3–19
Wong ES, Ban KH, Mutalif R, Jenkins NA, Copeland NG, Stewart CL (2010) A simple procedure for the efficient derivation of mouse ES cells. Methods Enzymol 476:265–283
Hogan B (1994) Manipulating the mouse embryo: a laboratory manual. Cold Spring Harbor Laboratory Press, Plainview, NY, USA
Batlle-Morera L, Smith A, Nichols J (2008) Parameters influencing derivation of embryonic stem cells from murine embryos. Genesis 46(12):758–767
Bryja V, Bonilla S, Arenas E (2006) Derivation of mouse embryonic stem cells. Nat Protoc 1:2082–2087
Baharvand H, Matthaei KI (2004) Culture condition difference for establishment of new embryonic stem cell lines from the C57BL/6 and BALB/c mouse strains. In Vitro Cell Dev Biol Anim 40:76–81
Umehara H, Kimura T, Ohtsuka S, Nakamura T, Kitajima K, Ikawa M, Okabe M, Niwa H, Nakano T (2007) Efficient derivation of embryonic stem cells by inhibition of glycogen synthase kinase-3. Stem Cells 25:2705–2711
Schoonjans L, Kreemers V, Danloy S, Moreadith RW, Laroche Y, Collen D (2003) Improved generation of germline-competent embryonic stem cell lines from inbred mouse strains. Stem Cells 21:90–97
Cinelli P, Casanova EA, Uhlig S, Lochmatter P, Matsuda T, Yokota T, Rulicke T, Ledermann B, Burki K (2008) Expression profiling in transgenic FVB/N embryonic stem cells over expressing STAT3. BMC Dev Biol 8:57
Acknowledgments
This study was funded by grants provided from Royan Institute, the Iranian Council of Stem Cell Technology, and the Iran National Science Foundation (INSF).
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Baharvand, H., Hassani, SN. (2013). A New Chemical Approach to the Efficient Generation of Mouse Embryonic Stem Cells. In: Lakshmipathy, U., Vemuri, M. (eds) Pluripotent Stem Cells. Methods in Molecular Biology, vol 997. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-348-0_2
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DOI: https://doi.org/10.1007/978-1-62703-348-0_2
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