Isolation and ex vivo expansion of human umbilical cord blood-derived CD34+ stem cells and their cotransplantation with or without mesenchymal stem cells.

Umbilical cord blood (UCB) contains a high number of primitive progenitor cells, allowing UCB to be used as a source of hematopoietic progenitors for clinical transplantation. However the rate of UCB CD34(+) stem cells graft is low. Mesenchymal stem cells (MSCs) have been implicated in playing an important role in hematopoietic stem cell engraftment. In this study we examined the effect of human MSC on engraftment of human UCB-derived CD34(+) cells in irradiated Balb/c mice. Human UCB CD34(+) cells were obtained from full-term normal deliveries by using an immunomagnetic separation technique and MSC were isolated by standard methodology from human bone marrow. Isolated CD34(+) cells were cultured in Stemline Hematopoietic stem cell expansion medium supplemented with 100 ng/ml stem cell factor (SCF), and 100 ng/ml thrombopoietin (TPO) in 24-well plates and incubated at 37 degrees C in a fully humidified atmosphere with 5% CO(2), and maintained over 3 weeks and half the medium was exchanged twice a week. Irradiated (7 Gy) Balb/c mice were transplanted intravenously with 0.1 x 10(6) to 10 x 10(6) human UCB CD34(+) cells in the presence or absence of 0.5 x 10(6) and 1 x 10(6) human bone marrow-derived MSC. After 11 days, in each group, the spleen was dissected and colony assay performed. Hematoxilin and eosin staining of the spleen colony was performed, and UCB CD34(+) cells labeled with super paramagnetic iron oxide (SPIO). After establishing the presence of colonies in spleen, Prussian blue staining was performed. Flow cytometry assay showed that up to 90% purity of CD34(+) cells and 96% for MSC. After 3 weeks the cell numbers showed a 1000-fold increase in CD34(+). Cotransplantation of low doses of UCB CD34(+) cells (0.2 x 10(6) and 0.3 x 10(6)) and MSC (0.5 x 10(6) and 1 x 10(6)) resulted in a significant increase in colony forming unit spleen, in comparison with engraftment of UCB CD34(+) stem cells without MSC after 11 days (p<0.01). In conclusion the results showed that two cytokines (SCF, TPO) were sufficient for expansion of UCB CD34(+) cells and cotransplantation of MSC with UCB CD34(+) cells, promoting engraftment of UCB CD34(+) cells.