Clinical isolation and functional characterization of cord blood CD133+hematopoietic progenitor cells

BACKGROUND: Human cord blood is a relevant source of CD133+ HPCs. Clinical-scale isolation of human umbilical cord blood (UCB) CD133+ HPCs using immunomagnetic microbeads and the CliniMACS clinical cell isolator is reported. CD133+ HPCs isolated after large-scale processing 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 x 10(6) 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 comparable 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 primitive HPCs with high hematopoietic activity and in-vitro mesenchymal potential.