Protocol | Published:

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

Nature Protocols volume 3, pages 10461055 (2008) | Download Citation



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.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


  1. 1.

    et al. Production of stem cells with embryonic characteristics from human umbilical cord blood. Cell Prolif. 38, 245–255 (2005).

  2. 2.

    et al. Morphological and molecular characterization of novel population of CXCR4+ SSEA-4+ Oct-4+ very small embryonic-like cells purified from human cord blood: preliminary report. Leukemia 21, 297–303 (2007).

  3. 3.

    & Potential for access to embryonic-like cells from human umbilical cord blood. Cell Prolif. 41 (suppl. 1): 31–40 (2008).

  4. 4.

    & Hematopoietic transplantation by means of fetal (cord) blood. A new method. Va. Med. Mon. 99, 276–280 (1972).

  5. 5.

    , , , & Human cord blood-derived cells attain neuronal and glial features in vitro. J. Cell Sci. 115, 2131–2138 (2002).

  6. 6.

    et al. Neurogenic potential of human umbilical cord blood: neural-like stem cells depend on previous long-term culture conditions. J. Neurosci. Res. 83, 627–637 (2006).

  7. 7.

    et al. Trophic factor induction of human umbilical cord blood cells in vitro and in vivo. J. Neural. Eng. 4, 130–145 (2007).

  8. 8.

    et al. Long-term cultured human umbilical cord neural-like cells transplanted into the striatum of NOD SCID mice. Brain Res. Bull. 74, 155–163 (2007).

  9. 9.

    et al. A 37-year-old spinal cord-injured female patient, transplanted of multipotent stem cells from human UC blood, with improved sensory perception and mobility, both functionally and morphologically: a case study. Cytotherapy 7, 368–373 (2005).

  10. 10.

    et al. Intravenous versus intrastriatal cord blood administration in a rodent model of stroke. J. Neurosci. Res. 73, 296–307 (2003).

  11. 11.

    et al. Embryonic-like stem cells from umbilical cord blood and potential for neural modeling. Acta Neurobiol. Exp. (Wars) 66, 321–329 (2006).

  12. 12.

    et al. Umbilical cord blood-derived stem cells and brain repair. Ann. NY Acad. Sci. 1049, 67–83 (2005).

  13. 13.

    & Nanog and transcriptional networks in embryonic stem cell pluripotency. Cell Res. 17, 42–49 (2007).

  14. 14.

    et al. Early appearance of stem/progenitor cells with neural-like characteristics in human cord blood mononuclear fraction cultured in vitro. Exp. Hematol. 34, 914–925 (2006).

  15. 15.

    , , & Thrombopoietin, flt3-ligand and c-kit-ligand modulate HOX gene expression in expanding cord blood CD133 cells. Cell Prolif. 37, 295–306 (2004).

  16. 16.

    et al. In vitro culture conditions favoring selection of chromosomal abnormalities in human ES cells. J. Cell. Biochem. 99, 508–516 (2006).

  17. 17.

    , , , & Neural stem-like cell line derived from a nonhematopoietic population of human umbilical cord blood. Stem Cells Dev. 15, 391–406 (2006).

  18. 18.

    , , , & Cord blood revelations: the importance of being a first born girl, big, on time and to a young mother. Early Hum. Dev. 83, 733–741 (2007).

  19. 19.

    , , , & Function of ID1 protein in human cord blood-derived neural stem-like cells. J. Neurosci. Res. 84, 993–1002 (2006).

Download references


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.

Author information


  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, 1000 Ljubljana, Slovenia.

    • Marko Strbad


  1. Search for Colin McGuckin in:

  2. Search for Marcin Jurga in:

  3. Search for Hamad Ali in:

  4. Search for Marko Strbad in:

  5. Search for Nicolas Forraz in:

Corresponding author

Correspondence to Colin McGuckin.

About this article

Publication history



Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.