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

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Abstract
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.