Derivation and characterisation of the human embryonic stem cell lines, NOTT1 and NOTT2

  • Helen Priddle
  • Cinzia Allegrucci
  • Paul Burridge
  • Maria Munoz
  • Nigel M. Smith
  • Lyndsey Devlin
  • Cecilia Sjoblom
  • Sarah Chamberlain
  • Sue Watson
  • Lorraine E. Young
  • Chris Denning
Article

DOI: 10.1007/s11626-010-9290-6

Cite this article as:
Priddle, H., Allegrucci, C., Burridge, P. et al. In Vitro Cell.Dev.Biol.-Animal (2010) 46: 367. doi:10.1007/s11626-010-9290-6

Abstract

The ability to maintain human embryonic stem cells (hESCs) during long-term culture and yet induce differentiation to multiple lineages potentially provides a novel approach to address various biomedical problems. Here, we describe derivation of hESC lines, NOTT1 and NOTT2, from human blastocysts graded as 3BC and 3CB, respectively. Both lines were successfully maintained as colonies by mechanical passaging on mouse embryonic feeder cells or as monolayers by trypsin-passaging in feeder-free conditions on Matrigel. Undifferentiated cells retained expression of pluripotency markers (OCT4, NANOG, SSEA-4, TRA-1-60 and TRA-1-81), a stable karyotype during long-term culture and could be transfected efficiently with plasmid DNA and short interfering RNA. Differentiation via formation of embryoid bodies resulted in expression of genes associated with early germ layers and terminal lineage specification. The electrophysiology of spontaneously beating NOTT1-derived cardiomyocytes was recorded and these cells were shown to be pharmacologically responsive. Histological examination of teratomas formed by in vivo differentiation of both lines in severe immunocompromised mice showed complex structures including cartilage or smooth muscle (mesoderm), luminal epithelium (endoderm) and neuroectoderm (ectoderm). These observations show that NOTT1 and NOTT2 display the accepted characteristics of hESC pluripotency.

Keywords

Human embryonic stem cells NOTT1 NOTT2 Pluripotency Differentiation Cardiomyocytes Micro-electrode array Genetic modification 

Copyright information

© The Society for In Vitro Biology 2010

Authors and Affiliations

  • Helen Priddle
    • 1
  • Cinzia Allegrucci
    • 2
    • 3
  • Paul Burridge
    • 2
  • Maria Munoz
    • 2
  • Nigel M. Smith
    • 4
  • Lyndsey Devlin
    • 1
  • Cecilia Sjoblom
    • 1
  • Sarah Chamberlain
    • 1
  • Sue Watson
    • 5
  • Lorraine E. Young
    • 2
  • Chris Denning
    • 2
  1. 1.NURTUREQueen’s Medical CentreNottinghamUK
  2. 2.Wolfson Centre for Stem Cells, Tissue Engineering and Modelling (STEM), Centre for Biomolecular SciencesUniversity of NottinghamNottinghamNG7 2RDUK
  3. 3.School of Veterinary Medicine and Science, Sutton Bonington CampusUniversity of NottinghamLoughboroughUK
  4. 4.Department of Cytogenetics, Centre for Medical GeneticsNottingham City Hospital NHS TrustNottinghamUK
  5. 5.School of Clinical Sciences, Faculty of Medicine & Health SciencesQueen’s Medical CentreNottinghamUK

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