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Culture of embryonic-like stem cells from human umbilical cord blood and onward differentiation to neural cells in vitro

Nature Protocols volume 3, pages 1046–1055 (2008)Cite this article

Abstract

This 3-week protocol produces embryonic-like stem cells from human umbilical cord blood (CBEs) for neural differentiation using a three-step system (cell isolation/expansion/differentiation). The CBE isolation produces a highly purified fraction (CD45−, CD33−, CD7−, CD235a−) of small pluripotent stem cells (2–3 μm in diameter) coexpressing embryonic stem cell markers including Oct4 and Sox2. Initial CBE expansion is performed in high density (5–10 millions per ml) in the presence of extracellular matrix proteins and epidermal growth factor. Subsequent neural differentiation of CBEs requires sequential introduction of morphogenes, retinoic acid, brain-derived neurotrophic factor and cyclic AMP. Described methods emphasize defined media and reagents at all stages of the experiment comparable to protocols described for culturing human embryonic stem cells and cells from other somatic stem cell sources. Neural progenitor and cells generated from CBEs may be used for in vitro drug testing and cell-based assays and potentially for clinical transplantation.

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Figure 1: Separation of cord blood with Ficoll before (left tube) and after (right tube) centrifugation.
Figure 2: Contamination of platelets in culture.
Figure 3: CBEs contaminated with magnetic beads.
Figure 4: Embryonic-like stem cells freshly isolated from human umbilical cord blood.
Figure 5: Harvesting and expansion of CBEs in serum-free media.
Figure 6: Differentiation and maturation of CBEs in serum-free media.
Figure 7: Intracellular localization of markers of pluripotency.
Figure 8: Example of later stage neural phenotype from culture.

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Acknowledgements

Our work and the development of these protocols have been supported by: (i) the National Health Service Obstetrics and Gynaecology Directorate, Newcastle Hospitals Trust; (ii) Fondation Jerome LeJeune, Paris; (iii) Novus Sanguis adult stem cells charity, Paris; (iv) The Kuwaiti Government sponsorship of H.A.; and (v) The Blood Transfusion Service of Llubjana, Slovenia sponsorship of M.S.

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Authors and Affiliations

  1. Newcastle Centre for Cord Blood, North East England Stem Cell Institute and Institute of Human Genetics, Centre for Life, Central Parkway, Newcastle upon Tyne, NE1 3BZ, UK

    Colin McGuckin, Marcin Jurga, Hamad Ali, Marko Strbad & Nicolas Forraz

  2. Department of Immunohaematology, Blood Transfusion Centre of Slovenia, Slajmerjeva 6, Ljubljana, 1000, Slovenia

    Marko Strbad

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  1. Colin McGuckin
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  2. Marcin Jurga
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Correspondence to Colin McGuckin.

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McGuckin, C., Jurga, M., Ali, H. et al. Culture of embryonic-like stem cells from human umbilical cord blood and onward differentiation to neural cells in vitro. Nat Protoc 3, 1046–1055 (2008). https://doi.org/10.1038/nprot.2008.69

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