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Characterization of a Lineage-Negative Stem-Progenitor Cell Population Optimized for Ex Vivo Expansion and Enriched for LTC-IC

^a King-George Laboratory, St. George’s Hospital Medical School and Kingston University, London, UK;
^b School of Life Sciences, Kingston University, Kingston upon Thames, UK;
^c Department of Haematology, St. George’s Hospital Medical School, London, UK

Key Words. Negative selection • Stem cells • LTC-IC • CD34 • Ex vivo expansion

Colin P. McGuckin, Ph.D., Reader, School of Life Sciences, Faculty of Science, Kingston University, Penrhyn Road, Kingston upon Thames, Surrey KT1 2EE, United Kingdom. Telephone: 44-797-126-6764; Fax: 44-208-725-0245; e-mail: c.mcguckin{at}kingston.ac.uk

Current hematopoietic stem cell transplantation protocols rely heavily upon CD34⁺ cells to estimate hematopoietic stem and progenitor cell (HSPC) yield. We and others previously reported CD133⁺ cells to represent a more primitive cell population than their CD34⁺ counterparts. However, both CD34⁺ and CD133⁺ cells still encompass cells at various stages of maturation, possibly impairing long-term marrow engraftment. Recent studies demonstrated that cells lacking CD34 and hematopoietic lineage markers have the potential of reconstituting long-term in vivo hematopoiesis. We report here an optimized, rapid negative-isolation method that depletes umbilical cord blood (UCB) mononucleated cells (MNC) from cells expressing hematopoietic markers (CD45, glycophorin-A, CD38, CD7, CD33, CD56, CD16, CD3, and CD2) and isolates a discrete lineage-negative (Lin^-) cell population (0.10% ± 0.02% MNC, n = 12). This primitive Lin^- cell population encompassed CD34^+/- and CD133^+/- HSPC and was also enriched for surface markers involved in HSPC migration, adhesion, and homing to the bone marrow (CD164, CD162, and CXCR4). Moreover, our depletion method resulted in Lin^- cells being highly enriched for long-term culture-initiating cells when compared with both CD133⁺ cells and MNC. Furthermore, over 8 weeks in liquid culture stimulated by a cytokine cocktail optimized for HSPC expansion, TPOFLK (thrombopoietin 10 ng/ml, Flt3 ligand 50 ng/ml, c-Kit ligand 20 ng/ml) Lin^- cells underwent slow proliferation but maintained/expanded more primitive HSPC than CD133⁺ cells. Therefore, our Lin^- stem cell offers a promising alternative to current HSPC selection methods. Additionally, this work provides an optimized and well-characterized cell population for expansion of UCB for a wider therapeutic potential, including adult stem cell transplantation.

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