Abstract
Human embryonic stem cells represent a renewable source of pluripotent cells which can give rise to endodermal, mesodermal and ectodermal lineages. Not surprisingly, they have received enormous attention in the context of regenerative medicine. However, harnessing their tremendous potential requires a thorough understanding of their biology and the mechanisms that govern their pluripotency. Directed in vitro differentiation of human embryonic stem cells is typically based on recapitulating biological processes that take place during normal mammalian development. Unfortunately, differentiation of human embryonic stem cells to specific cell types is plagued by low yields and heterogeneous cell populations. This chapter will describe how the differentiation of human embryonic stem cells can be tracked using recombinant bacterial artificial chromosomes expressing fluorescent reporters driven by developmentally regulated promoters. As an example, we will describe how this technology can be applied within the neural lineage to track differentiation to neural stem cells and spinal motor neurons.
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References
Ables JL, Breunig JJ, Eisch AJ, Rakic P (2011) Not(ch) just development: Notch signalling in the adult brain. Nat Rev Neurosci 12:269–283
Arber S, Han B, Mendelsohn M, Smith M, Jessell TM, Sockanathan S (1999) Requirement for the homeobox gene Hb9 in the consolidation of motor neuron identity. Neuron 23:659–674
Chambers SM, Fasano CA, Papapetrou EP, Tomishima M, Sadelain M, Studer L (2009) Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol 27:275–80
Elkabetz Y, Panagiotakos G, Al Shamy G, Socci ND, Tabar V, Studer L (2008) Human ES cell-derived neural rosettes reveal a functionally distinct early neural stem cell stage. Genes Dev 22:152–165
Fu Y, Maye P (2011) Engineering BAC reporter gene constructs for mouse transgenesis. Methods Mol Biol 693:163–179
Goldman S (2005) Stem and progenitor cell-based therapy of the human central nervous system. Nat Biotechnol 23:862–871
Gong S, Yang XW, Li C, Heintz N (2002) Highly efficient modification of bacterial artificial chromosomes (BACs) using novel shuttle vectors containing the R6Kgamma origin of replication. Genome Res 12:1992–1998
Gong S, Zheng C, Doughty ML, Losos K, Didkovsky N, Schambra UB, Nowak NJ, Joyner A, Leblanc G, Hatten ME, Heintz N (2003) A gene expression atlas of the central nervous system based on bacterial artificial chromosomes. Nature 425:917–925
Gong S, Kus L, Heintz N (2010) Rapid bacterial artificial chromosome modification for large-scale mouse transgenesis. Nat Protoc 5:1678–1696
Hu BY, Zhang SC (2009) Differentiation of spinal motor neurons from pluripotent human stem cells. Nat Protoc 4:1295–1304
Kageyama R, Ohtsuka T, Kobayashi T (2008) Roles of Hes genes in neural development. Dev Growth Differ 50(Suppl 1):S97–S103
Lee EC, Yu D, Martinez de Velasco J, Tessarollo L, Swing DA, Court DL, Jenkins NA, Copeland NG (2001) A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA. Genomics 73:56–65
Lee H, Shamy GA, Elkabetz Y, Schofield CM, Harrsion NL, Panagiotakos G, Socci ND, Tabar V, Studer L (2007) Directed differentiation and transplantation of human embryonic stem cell-derived motoneurons. Stem Cells 25:1931–1939
Li X, Meng G, Krawetz R, Liu S, Rancourt DE (2008) The ROCK inhibitor Y-27632 enhances the survival rate of human embryonic stem cells following cryopreservation. Stem Cells Dev 17:1079–1085
Peljto M, Wichterle H (2011) Programming embryonic stem cells to neuronal subtypes. Curr Opin Neurobiol 21:43–51
Placantonakis DG, Tomishima MJ, Lafaille F, Desbordes SC, Jia F, Socci ND, Viale A, Lee H, Harrison N, Tabar V, Studer L (2009) BAC transgenesis in human embryonic stem cells as a novel tool to define the human neural lineage. Stem Cells 27:521–532
Roy NS, Nakano T, Keyoung HM, Windrem M, Rashbaum WK, Alonso ML, Kang J, Peng W, Carpenter MK, Lin J, Nedergaard M, Goldman SA (2004) Telomerase immortalization of neuronally restricted progenitor cells derived from the human fetal spinal cord. Nat Biotechnol 22:297–305
Sasai Y, De Robertis EM (1997) Ectodermal patterning in vertebrate embryos. Dev Biol 182:5–20
Shirasaki R, Pfaff SL (2002) Transcriptional codes and the control of neuronal identity. Annu Rev Neurosci 25:251–281
Shizuya H, Birren B, Kim UJ, Mancino V, Slepak T, Tachiiri Y, Simon M (1992) Cloning and stable maintenance of 300-kilobase-pair fragments of human DNA in Escherichia coli using an F-factor-based vector. Proc Natl Acad Sci U S A 89:8794–8797
Smith JR, Vallier L, Lupo G, Alexander M, Harris WA, Pedersen RA (2008) Inhibition of Activin/Nodal signaling promotes specification of human embryonic stem cells into neuroectoderm. Dev Biol 313:107–117
Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147
Tomishima MJ, Hadjantonakis AK, Gong S, Studer L (2007) Production of green fluorescent protein transgenic embryonic stem cells using the GENSAT bacterial artificial chromosome library. Stem Cells 25:39–45
Vazin T, Freed WJ (2010) Human embryonic stem cells: derivation, culture, and differentiation: a review. Restor Neurol Neurosci 28:589–603
Wang Z, Engler P, Longacre A, Storb U (2001) An efficient method for high-fidelity BAC/PAC retrofitting with a selectable marker for mammalian cell transfection. Genome Res 11:137–142
Wang Z, Longacre A, Engler P (2004) Retrofitting BACs with a selectable marker for transfection. Methods Mol Biol 256:69–76
Watanabe K, Ueno M, Kamiya D, Nishiyama A, Matsumura M, Wataya T, Takahashi JB, Nishikawa S, Nishikawa S, Muguruma K, Sasai Y (2007) A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol 25:681–686
Wichterle H, Lieberam I, Porter JA, Jessell TM (2002) Directed differentiation of embryonic stem cells into motor neurons. Cell 110:385–397
Yang XW, Gong S (2005) An overview on the generation of BAC transgenic mice for neuroscience research. Curr Protoc Neurosci. 5.20.1-5.20.11
Yang XW, Model P, Heintz N (1997) Homologous recombination based modification in Escherichia coli and germline transmission in transgenic mice of a bacterial artificial chromosome. Nat Biotechnol 15:859–865
Yu PB, Deng DY, Lai CS, Hong CC, Cuny GD, Bouxsein ML, Hong DW, McManus PM, Katagiri T, Sachidanandan C, Kamiya N, Fukuda T, Mishina Y, Peterson RT, Bloch KD (2008) BMP type I receptor inhibition reduces heterotopic ossification. Nat Med 14:1363–1369
Zhang SC, Wernig M, Duncan ID, Brüstle O, Thomson JA (2001) In vitro differentiation of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol 19:1129–1133
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We thank Aram Modrek for critically reading the manuscript.
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Tokcaer-Keskin, Z., Placantonakis, D.G. (2013). Genetic Identification of Human Embryonic Stem Cell-Derived Neural Cell Types Using Bacterial Artificial Chromosomes. In: Hayat, M. (eds) Stem Cells and Cancer Stem Cells, Volume 10. Stem Cells and Cancer Stem Cells, vol 10. Springer, Dordrecht. https://doi.org/10.1007/978-94-007-6262-6_11
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DOI: https://doi.org/10.1007/978-94-007-6262-6_11
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