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Optimization of isolation and further characterization of umbilical cord blood-derived very small embryonic/ epiblast-like stem cells (VSELs)


Mariusz Z. Ratajczak, MD, PhD, Professor and Director, Stem Cell Institute, James Graham Brown Cancer Center, University of Louisville, 500 S. Floyd Street, Louisville, KY 40202, USA. Tel: +502 852 1788; Fax: +502 852 3032; e-mail:


Because of their small size and density, umbilical cord blood (UCB)-derived very small embryonic/epiblast-like stem cells (VSELs) are usually lost at various steps of UCB preparation. Accordingly, we noticed that a significant number of these cells, which are smaller than erythrocytes, are lost during gradient centrifugation over Ficoll-Paque as well as during routine volume depletion of UCB units before freezing. To preserve these cells in final UCB preparations, we propose a relatively short and economical three-step isolation protocol that allows recovery of approximately 60% of the initial number of Lin/CD45/CD133+ UCB-VSELs present in freshly harvested UCB units. In this novel approach (i) UCB is lysed in a hypotonic ammonium chloride solution to deplete erythrocytes; (ii) CD133+ including VSELs cells are enriched by employing immunomagnetic beads; and subsequently (iii) Lin/CD45/CD133+ cells are sorted by fluorescence-activated cell sorting. The whole isolation procedure takes approximately 2–3 h per UCB unit and isolated cells are highly enriched for an Oct-4+ and SSEA-4+ population of small Lin/CD45/CD133+ cells.