Experimental Studies On Ex Vivo Expansion Of Umbilical Cord Blood Hematopoietic Stem Cells

Umbilical cord blood (UCB) is one of the hematopoietic stem cell resources with advantages of easy access and obtainment. The transplantation related morbility and mortality, expecially graft-versus-host disease (GvHD) is lower in the setting of transplantation. However, the number of hematopoietic stem cell in a single cord blood donation is limited, and the manipulation of separation, cryopreservation and thawing of cord blood will result in some losses of HSC/HPC, which limit the clinical application of transplantation of cord blood in adult. Therefore, in order to enhance the number of hematopoietic stem cell in cord blood, ex vivo expansion of umbilical cord blood stem cells could be used.The losses of cord blood CD34+ cells during separation and purification was discussed, the differentiation of cord blood CD34+ cells cultured with different series-peculiar cytokines and ex vivo expansive fold of CD34+ cells and NC were studied.The recovery rate of MNC from UCB was 54.51 ± 14.29% by 3% gelatin and Ficoll-Hypaque(FH) separation, and the enrichment of CD34+cells was 36.22 folds by MACS isolation system after MNC incubation for 120 min to remove the adherent cell layer.Cord blood CD34+ cells could differentiate to committed hematopoietic progenitors with the induction of series-peculiar cytokines. The expression of CD7 and CD 10 declined from 8.84%, 1.38% to 0.96%, 0.72% respectively during culluring. However, the expression of CD 13, CD41 and Gly-A increased from 6.28%, 5.35% and 4.57% to 35.86%, 25.66% and 34.68% respectively at 7 days.Different combination group of cytokines had different expansive ability on cord blood CD34+ cell. The best combination group for NC expansion is FL+TPO+SCF+G-CSF+IL-6+GM-CSF+EPO+IL-3, with 328.96?.59 folds at 14 days, and the best combination group for CD34+ cells expansion isFL+TPO+SCF+G-CSF+IL-6+GM-CSF, with 25.23 ± 8.46folds at 7 days.The studies showed that the separation with 3% gelatin+FH could enhance harvest of cord blood MNC. MACS separation system was able to promote purification rate of CD34+ cells after the adherent cells were removed. The best combination group of cytokines for NC expansion was FL+TPO+SCF+G-CSF+IL-6+GM-CSF+EPO+IL-3, and the best combination group for CD34+ cells expansion was FL+TPO+SCF+G-CSF+IL-6+GM-CSF.