Isolation, characterization and generation of functional macrophages from human cord blood-derived cd34+ hematopoietic cells

BACKGROUND: Umbilical cord blood (UCB) has become an alternative source of hematopoietic progenitor cells for transplantation. The aim of this study was to test the effectiveness of some modifications of human hematopoietic stem cells isolation protocols with the intention of improving the output and viability of CD34+ cells and progenitor subpopulations progeny that can be obtained from a sample of human umbilical cord blood and keeping the ability of longterm self-renewal and the capacity to give rise to one or more types of differentiated progeny. METHODS: Human cord blood samples were transformed to buffy coat prior to the isolation of HSCs which was performed by two steps involving CD34 pre-enrichment using human cord blood CD34 positive selection kit and an immunomagnetic cell separation, targeting CD34 surface antigen. CD34+ cells were immunophenotyped by four-color fluorescence, using a large panel of monoclonal antibodies (CD34/PE, CD45/FITC, CD38/APC, CD33/Per-Cy, HLA-DR/PE, CD117/APC, CD123/Per-Cy, CD105-FITC, CD56/PE, CD14/ Per-Cy, CD19/Per-Cy and CD3/APC) recognizing different lineage or activation antigens. The isolated CD34+ cells were also studied for proliferation and differentiation in liquid cultures in the presence of Flt3L, SCF, IL-3, IL-6 and MCSF. Using differing combinations of growth factors the effect on cell proliferation and differentiation was determined. The macrophage-like phenotype was confirmed by analyses of surface markers, histo-morphology and phagocytosis. RESULTS: Our results showed that the percentage of CD34+ cells in whole human cord blood samples was 0.02% of total cells. After isolation by two-step, combining CD34 preenrichment and immunomagnetic isolation, the frequency of CD34+ stem cells represented 0.65% among total MNCs and 83.53% among total isolated cells. This isolation leaded to a purity of over 95% and viability of 98.60%. CONCLUSIONS: We conclude that our modified technique enabled us to obtain an important proportion of primitive hematopoietic progenitors, as suggested by the absence of HLA-DR and CD38, as well as the presence of CD117, CD33, CD123, and CD105 on their surface. These cells are recognized as having long-term reconstitution capacity within the human CD34+ cell population.

Author(s): Souad Al-Okla

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