Cocultivation of umbilical cord blood CD34^＋ cells with retro-transduced hMSCs leads to effective amplification of long-term culture-initiating cells

AIM： To establish a novel coculture system for ex vivo expansion of umbilical cord blood（UCB） hematopoietic progenitors using thrombopoietin （TPO）/FIt-3 ligand （FL）-transduced human marrow-derived mesenchymal stem cells （tfhMSCs） as feeder. METHODS： UCB CD34^＋ cells were isolated and cultured using four culture systems in serum-containing or serumfree medium. Suitable aliquots of cultured cells were used to monitor cell production, clonogenic activity, and long-term culture-initiating culture （LTC-IC） output. Finally, the severe-combined immunodeficient （SCID） mouse-repopulating cell （SRC） assay was performed to confirm ability of the cultured cells to reconstitute longterm hematopoiesis. RESULTS： There were no significant differences in the number of total nudeated cells among different culture systems in serum-containing medium during 21-d culture. However, on d 14, the outputs of CD34^＋ cells, CFU-C and CFU-GEMM in tfhMSCs coculture system were significantly enhanced. LTC-IC assay demonstrated that the tfhMSCs coculture system had the most powerful activity. The severe-combined immunodeficient （SCID） mouse repopulating cell （SRC） assay confirmed extensive ability of the expanded cells to reconstitute long-term hematopoiesis. Furthermore, PCR analysis demonstrated the presence of human hematopoietic cells in the bone marrow and peripheral blood cells of NOD/SCID mice. CONCLUSION： The TPO/FL-transduced hMSCs, in combination with additive cytokines, can effectively expand hematopoietic progenitors from UCB in vitro and the tfhMSCs coculture system may be a suitable system for ex vivo manipulation of primitive progenitor cells under contact culture conditions.