Establishment of serum-free pre-colony forming unit assays for differentiation of primitive hematopoietic progenitors: serum induces early macrophagedifferentiation and inhibits early erythroid differentiation of CD34(++)CD38(-) cells

In this report we show that serum has differentiation-inducing effects on p rimitive hematopoietic progenitor cells with the CD34(++)CD38(-) immunophen otype. Using the pre-colony forming unit (pre-CFU) assay as a model for ear ly myelopoiesis, we compared the effects of serum-containing and serum-free media and evaluated different cytokine cocktails [interleukin (IL)-1, IL-3 , IL-6, kit ligand with and without the Flt3/ Flk2 ligand (FL)]. In this as say, pre-CFUs are defined as cells unable to form colonies when plated dire ctly in semi-solid assays, but which can differentiate into CFUs when cultu red in liquid medium containing early-acting, cytokines. In one of the inve stigated serum-free media, the average myeloid expansion in liquid medium r eached up to more than 50% of that obtained in serum-containing medium. In addition, our experiments revealed differences in the clonogenic output bet ween cells cultured in serum-free medium and those cultured in serum-contai ning, medium, demonstrating that serum has a monocyte differentiation-induc ing effect on primitive hematopoietic progenitors. Also in serum-free mediu m, higher proportions of erythroid progenitors were generated. These differ entiation-inducing effects of serum further emphasize the need for serum-fr ee culture protocols for hematopoietic graft engineering. Addition of FL to the culture media ameliorated cellular expansion and resulted in a decreas e in the proportion of erythroid and granulocyte progenitors and an increas e in the proportion of monocyte progenitors. In conclusion, this study show s that good serum-free conditions are available for differentiation assays with primitive hematopoietic progenitors and demonstrates that serum and FL have biasing effects on the initial phase of hematopoietic differentiation , favoring the monocyte lineage.