A clonal culture assay for human cord blood lymphohematopoietic progenitors

We describe a two-step clonal culture assay system for human lymphohematopoietic progenitors present in umbilical cord blood which are capable of differentiation along both myeloid and B-lymphoid lineages. Human cord blood CD34+ cells were plated in methylcellulose in the presence of stem cell factor (SCF), granulocyte-colony stimulating factor (G-CSF), interleukin (IL)-7, and the murine stroma cell line, MS-5. The growing primary colonies were individually examined for their potentials to differentiate along both myeloid and B-lymphoid lineages by reculturing aliquots of the primary colonies in methylcellulose culture containing IL-3, G-CSF and erythropoietin (Epo), and on a monolayer of MS-5 in the presence of SCF and G-CSF. Approximately 10-15% of the primary colonies generated various combinations of myeloid cells and CD19+ sIgM+ cells. Subsequent studies using micromanipulated single CD34+ cells unequivocally demonstrated the clonal origin of the lymphohematopoietic progenitors. This culture system should prove valuable for elucidation of the mechanisms regulating early stages of human lymphohematopoiesis.