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BACKGROUND:  Human cord blood is a relevant source of CD133+ HPCs. Clinical-scale isolation of human umbi-lical cord blood (UCB) CD133+ HPCs using immunomag-netic microbeads and the CliniMACS clinical cell isolator is reported. CD133+ HPCs isolated after large-scale pro-cessing were functionally characterized.

STUDY DESIGN AND METHODS:  Closed disposable sets were used to process nine different samples of RBC-reduced UCB nucleated cells. In-vitro hematopoietic assays and human xenografts in NOD/SCID mice were performed to assess the functional properties of isolated CD133+ cells. Different mixtures of human cytokines were tested for the ability to expand nascent CD133+ HPCs. Furthermore, freshly isolated CD133+ cells were conditioned in culture medium specifically tested to support in-vitro myogenesis or osteogenesis.

RESULTS:  Isolation procedures yielded the recovery of an average of 2.53 × 106 CD133+ HPCs with a mean recovery of 96 percent (referred to as RBC-reduced samples) and a final sample purity of 82 percent. Purified CD133+ cells had high cloning efficiency, had relevant long-term activity, and were capable of repopulating irradiated NOD/SCID mice. In 10-day stroma-free cultures, a 2-fold and 8.3-fold expansion of colony-forming cells (CFCs) and extended long-term culture-initiating cells, respectively, was obtained. Freshly isolated CD133+ cells differen-tiated into large nucleated cells expressing either myosin D or osteopontin (as revealed by RT-PCR and immuno-cytochemistry), with a protein/mRNA expression compar-able to or even higher than that observed in UCB CD133– nucleated cells in identical culture conditions.

CONCLUSION:  Collectively, clinical-scale isolation of UCB CD133+ cells provides a relevant amount of primi-tive HPCs with high hematopoietic activity and in-vitro mesenchymal potential.