ABSTRACT
BACKGROUND: In vitro generated cells from cord blood (CB) CD34+ cells increase cell dose and may reduce the severity and the duration of pancytopenia after transplantation. But safe engraftment for adolescents and adults is still not predictable and standardized. We attempted to establish a clinically application of in vitro generated cells by investigating the use of cytokines for the culture of CB CD34+ cells. METHODS: CD34+ cells, purified from four separate human CB units by magnetic bead selection, were cultured in IMDM with several cytokines. Differentiation of the in vitro generated cells has been confirmed by flowcytometry with specific erythroid, myeloid and megakaryocytic lineage surface markers. And apoptosis of these cells also was analysed with annexin V staining and morphologic analysis under the electron microscopic examination was done, simultaneously. RESULTS: Purified CB CD34+ cells were expanded with differentiation from CD38- to CD38+ expression with time. CB CD34+ cells could be terminally differentiated into erythroid (GPA+) and myeloid (CD33+/CD15+) lineage cells without apoptosis (annexin-V - ) in the presence of EPO and G-CSF, respectively. Megakaryocytic differentiation was partially arrested in early stage due to apoptosis in the presence of TPO. Morphological examination using electron microscope revealed all stages of erythroid and myeloid lineage cells without apoptosis, and apoptotic megakaryocytic lineage cells of early stage. But we could observe the small number of fully maturated platelets. CONCLUSION: CB CD34+ cells were terminally differentiated to erythroid, myeloid, and megakaryocytic lineage cells with or without apoptosis by several cytokines.