Heterogeneity of in vitroecultured CD34D cells isolated from peripheral blood

Background aims. For transplantation, hematopoietic stem cells (HSC) are obtained from bone marrow, cord blood and mobilized adult peripheral blood. HSCs are present in the blood of healthy adults and can be recovered in leuko-reduction system chambers, with a potential yield of 1 to 4 � 10 6 CD34þ cells per unit. Some groups have investigated this valuable source of stem cells; however, investigations are still needed to support their use. Methods. CD34þ cells were purified from leuko-reduction system chambers and cultured with a defined custom medium without animal protein and supplemented with interleukin-3, interleukin-6, Fms-like tyrosine kinase 3, stem cell factor and thrombopoietin. Cells were cultured under 8% and 21% oxygen levels. With the use of multiparametric flow cytometry analysis, the phenotypes of emerging populations were compared between oxygen levels and resting CD34þ cells. Both conventional gating and clustering analysis were used to visualize the cellular outcome. Results. A maximum expansion of 20-fold was obtained without major differences in viability, number of cells or cellular heterogeneity between atmospheric and physiologic oxygen conditions. Worthy of note, phenotype analysis revealed that megakaryocyte and erythrocyte progenitors were favored, albeit more moderately when submitted to 8% O2. Conclusions. This study suggests that the bias of cultured blood CD34þ cells toward megakaryocyte and erythrocyte progenitor cells can be reduced by use of 8% pO2. It also shows how clustering software, such as SPADE, can help visualize the complexity of stem cell differentiation.