Effect of tpo on expansion and differentiation of cd34+ derived dendritic cells

CD34⁺ cells immuno-magnetically separated from 5 bone marrows and 4 G-CSF mobilised apheresis (PBSC) were grown for 14 days in serum-free medium (X VIVO - 15) in the presence of a cocktail of cytokines including SCF, Flt-3 ligand, TNFa, TGFβ, GM-CSF to generate highly purified dendritic cells (DC). The addition of TPO induced a slight increase in DC expansion, but a more immature phenotype. The effect of TPO on the cultures could be explained both by an increased cell survival and a decreased apoptosis rate. DC grown with TPO showed a lower expression of maturation markers such as CD83 and a higher capacity of dextrane-FITC uptake, which is typical of more immature DC. Furthermore, DC grown with TPO showed a lower immunostimulatory capacity in the mixed leukocyte reaction (MLR). The DC obtained with the two different cytokine combinations were then FACS sorted on the basis of their light scatter properties. Two populations of "large" and "small' cells respectively were separated: the "large" cells showed a higher expression of dendritic markers such as CD1a, CD83 and costimulatory molecules like CD80 and CD86, and a higher antigen presenting capacity in MLR. The "small" cells were early myeloid precursors (CD1a^-/CD14^-/CD15^-/CD33⁺/CD13 ⁼) incapable of further DC differentiation and antigen presentation in MLR assay. Our data, taken together, seem to indicate that the addition of TPO to our cytokine cocktail induced the generation of a larger number of DC with an immature phenotype. These culture conditions and the FACS sorting allow the generation of a highly pure DC population which could further facilitate DC-based immunotherapy protocols.