Abstract
The isolation and culture of neural progenitor cells (NPCs) from pluripotent stem cells has facilitated in vitro mechanistic studies of diseases related to the nervous system, as well as discovery of new medicine. In addition, NPCs are envisioned to play a crucial role in future cell replacement therapy. The pig has become recognized as an important large animal model and establishment of in vitro-derived porcine NPCs would allow for preclinical safety testing by transplantation in a porcine biomedical model. In this chapter, a detailed method for isolation and in vitro culture of porcine NPCs from porcine embryos or induced pluripotent stem cells is presented. The neural induction is performed in coculture and the isolation of rosette structures is carried out manually to ensure a homogenous population of NPCs. Using this method, multipotent NPCs can be obtained in approximately 1 month. The cells have the potential of long-term culture and the ability to differentiate into neural and glial cells.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Temple S (2001) The development of neural stem cells. Nature 414:112–117
Conti L, Pollard SM, Gorba T, Reitano E, Toselli M, Biella G, Sun Y, Sanzone S, Ying QL, Cattaneo E, Smith A (2005) Niche-independent symmetrical self-renewal of a mammalian tissue stem cell. PLoS Biol 3:283
Temple S (1989) Division and differentiation of isolated CNS blast cells in microculture. Nature 340:471–473
Okabe S, Forsberg-Nilsson K, Spiro AC, Segal M, McKay RD (1996) Development of neuronal precursor cells and functional postmitotic neurons from embryonic stem cells in vitro. Mech Dev 59:89–102
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
Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131:861–872
Reubinoff BE, Itsykson P, Turetsky T, Pera MF, Reinhartz E, Itzik A, Ben-Hur T (2001) Neural progenitors from human embryonic stem cells. Nat Biotechnol 19:1134–1140
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 27:275–280
Shi Y, Kirwan P, Smith J, MacLean G, Orkin SH, Livesey FJ (2012) A human stem cell model of early Alzheimer’s disease pathology in Down syndrome. Sci Transl Med 4:124ra29
Sánchez-Danés A, Richaud-Patin Y, Carballo-Carbajal I, Jiménez-Delgado S, Caig C, Mora S, Di Guglielmo C, Ezquerra M, Patel B, Giralt A, Canals JM, Memo M, Alberch J, López-Barneo J, Vila M, Cuervo AM, Tolosa E, Consiglio A, Raya A (2012) Disease-specific phenotypes in dopamine neurons from human iPS-based models of genetic and sporadic Parkinson’s disease. EMBO Mol Med 4:380–395
Dimos JT, Rodolfa KT, Niakan KK, Weisenthal LM, Mitsumoto H, Chung W, Croft GF, Saphier G, Leibel R, Goland R, Wichterle H, Henderson CE, Eggan K (2008) Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 321:1218–1221
Wolf XA, Rasmussen MA, Schauser K, Jensen AT, Schmidt M, Hyttel P (2011) OCT4 expression in outgrowth colonies derived from porcine inner cell masses and epiblasts. Reprod Domest Anim 46:385–392
Puy L, de Sousa C, Lopes SM, Haagsman HP, Roelen BA (2010) Differentiation of porcine inner cell mass cells into proliferating neural cells. Stem Cells Dev 19:61–70
Rasmussen MA, Hall VJ, Carter TF, Hyttel P (2011) Directed differentiation of porcine epiblast-derived neural progenitor cells into neurons and glia. Stem Cell Res 7:124–136
Yang JY, Mumaw JL, Liu Y, Stice SL, West FD (2013) SSEA4 positive pig induced pluripotent stem cells are primed for differentiation into neural cells. Cell Transplant 22(6):945–959
Perrier AL, Tabar V, Barberi T, Rubio ME, Bruses J, Topf N, Harrison NL, Studer L (2004) Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci U S A 101:12543–12548
Dyce PW, Zhu H, Craig J, Li J (2004) Stem cells with multilineage potential derived from porcine skin. Biochem Biophys Res Commun. Apr 9;316(3):651–8. PubMed PMID: 15033449
Zeng L, Rahrmann E, Hu Q, Lund T, Sandquist L, Felten M, O’Brien TD, Zhang J, Verfaillie C. Multipotent adult progenitor cells from swine bone marrow. Stem Cells. 2006 Nov;24(11):2355–66. Epub 2006 Aug 24. Erratum in: Stem Cells. 2007 Sep;25(9):2387. PubMed PMID: 16931778
Kuijk EW, du Puy L, van Tol HT, Haagsman HP, Colenbrander B, Roelen BA. Validation of reference genes for quantitative RT-PCR studies in porcine oocytesand preimplantation embryos. BMC Dev Biol. 2007 May 31;7:58. PubMed PMID: 17540017; PubMed Central PMCID: PMC1896162
Acknowledgement
The Danish National Advanced Technology Foundation (HTF) 047-2011-1, The Danish Council for Independent Research, Natural Sciences (FNU), 11-106627, and The Danish Council for Strategic Research (DSF)—The Program Commission on Individuals, Disease and Society.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, New York
About this protocol
Cite this protocol
Rasmussen, M.A., Hall, V.J., Hyttel, P. (2013). Isolation and Culture of Porcine Neural Progenitor Cells from Embryos and Pluripotent Stem Cells. In: Alberio, R. (eds) Epiblast Stem Cells. Methods in Molecular Biology, vol 1074. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-628-3_14
Download citation
DOI: https://doi.org/10.1007/978-1-62703-628-3_14
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-627-6
Online ISBN: 978-1-62703-628-3
eBook Packages: Springer Protocols