Abstract
The trophoblast is the first lineage to undergo differentiation during mammalian development. In the preimplantation blastocyst embryo, two cell types are present including the inner cell mass (ICM) and the trophectoderm (TE). ICM cells exhibit pluripotent potential, or the capacity to give rise to all cells represented in the adult organism, while TE cells are multipotent and are therefore only capable of differentiating into trophoblast lineages represented in the placenta. The TE is essential for implantation of the embryo into the uterine tissue, formation of trophoblast lineages represented in the placenta, and exchange of nutrients and waste between the embryo and the mother. Trophoblast stem (TS) cells, which can be derived from the TE of preimplantation embryos in the presence of external signals such as FGF4, can self-renew indefinitely, and because they are capable of differentiating into epithelial lineages of the trophoblast, TS cells are a useful in vitro model to study the biology of the trophoblast including epigenetic regulation of gene expression. In this chapter we describe protocols for derivation of TS cells from mouse blastocysts, culture conditions that promote self-renewal and differentiation, and methods to transduce TS cells with lentiviral particles encoding shRNAs. These protocols are sufficient for efficient derivation of TS cells and robust RNAi knockdown of target genes in TS cells.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Cross JC, Baczyk D, Dobric N, Hemberger M, Hughes M, Simmons DG, Yamamoto H, Kingdom JC (2003) Genes, development and evolution of the placenta. Placenta 24(2–3): 123–130
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 U S A 78(12): 7634–7638
Tanaka S, Kunath T, Hadjantonakis AK, Nagy A, Rossant J (1998) Promotion of trophoblast stem cell proliferation by FGF4. Science 282(5396):2072–2075
Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D, Chambers I, Scholer H, Smith A (1998) Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell 95(3): 379–391
Avilion AA, Nicolis SK, Pevny LH, Perez L, Vivian N, Lovell-Badge R (2003) Multipotent cell lineages in early mouse development depend on SOX2 function. Genes Dev 17(1):126–140
Mitsui K, Tokuzawa Y, Itoh H, Segawa K, Murakami M, Takahashi K, Maruyama M, Maeda M, Yamanaka S (2003) The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells. Cell 113(5):631–642
Chambers I, Colby D, Robertson M, Nichols J, Lee S, Tweedie S, Smith A (2003) Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 113(5):643–655
Strumpf D, Mao CA, Yamanaka Y, Ralston A, Chawengsaksophak K, Beck F, Rossant J (2005) Cdx2 is required for correct cell fate specification and differentiation of trophectoderm in the mouse blastocyst. Development 132(9):2093–2102. doi:10.1242/dev.01801, dev.01801 [pii]
Russ AP, Wattler S, Colledge WH, Aparicio SA, Carlton MB, Pearce JJ, Barton SC, Surani MA, Ryan K, Nehls MC, Wilson V, Evans MJ (2000) Eomesodermin is required for mouse trophoblast development and mesoderm formation. Nature 404(6773):95–99
Luo J, Sladek R, Bader JA, Matthyssen A, Rossant J, Giguere V (1997) Placental abnormalities in mouse embryos lacking the orphan nuclear receptor ERR-beta. Nature 388(6644):778–782. doi:10.1038/42022
Nishioka N, Yamamoto S, Kiyonari H, Sato H, Sawada A, Ota M, Nakao K, Sasaki H (2008) Tead4 is required for specification of trophectoderm in pre-implantation mouse embryos. Mech Dev 125(3–4):270–283
Kidder BL, Palmer S (2010) Examination of transcriptional networks reveals an important role for TCFAP2C, SMARCA4, and EOMES in trophoblast stem cell maintenance. Genome Res 20(4):458–472. doi:10.1101/gr.101469. 109, gr.101469.109 [pii]
Kidder BL, Palmer S (2012) HDAC1 regulates pluripotency and lineage specific transcriptional networks in embryonic and trophoblast stem cells. Nucleic Acids Res 40(7):2925–2939. doi:10.1093/nar/gkr1151, gkr1151 [pii]
Kidder BL, Oseth L, Miller S, Hirsch B, Verfaillie C, Coucouvanis E (2008) Embryonic stem cells contribute to mouse chimeras in the absence of detectable cell fusion. Cloning Stem Cells 10(2):231–248
Yan J, Tanaka S, Oda M, Makino T, Ohgane J, Shiota K (2001) Retinoic acid promotes differentiation of trophoblast stem cells to a giant cell fate. Dev Biol 235(2):422–432. doi:10.1006/dbio.2001.0300S0012-1606(01)90300-8 [pii]
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2014 Springer Science+Business Media New York
About this protocol
Cite this protocol
Kidder, B.L. (2014). Derivation and Manipulation of Trophoblast Stem Cells from Mouse Blastocysts. In: Kidder, B. (eds) Stem Cell Transcriptional Networks. Methods in Molecular Biology, vol 1150. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-0512-6_13
Download citation
DOI: https://doi.org/10.1007/978-1-4939-0512-6_13
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-0511-9
Online ISBN: 978-1-4939-0512-6
eBook Packages: Springer Protocols