Simultaneous expansion and harvest of hematopoietic stem cells and mesenchymal stem cells derived from umbilical cord blood
Fan XB., Song KD., Liu TQ., Yin YQ., Hao YJ., Ma XH., Cui ZF.
Simultaneous expansion and harvest of hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) derived from umbilical cord blood (UCB) were carried out in a bioreactor. The coculture of UCB-HSCs and UCB-MSCs were investigated respectively in spinner flasks and rotating wall vessel bioreactor (RWVB) together with glass coated styrene copolymer (GCSC) microcarriers. The medium is IMDM without serum but supplemented with the combination of cytokines including SCF 15 ng·mL-1, FL 5 ng·mL-1, TPO 6 ng·mL-1, IL-3 15 ng·mL-1, G-CSF 1 ng·mL-1 and GM-CSF 5 ng·mL-1. The stromal cells derived from normal allogeneic adipose tissue were encapsulated in alginate chitosan (AC) beads and used as feeding cells. Immunophenotype analysis, methylcellulose colony assay and multi-lineage differentiation of UCB-MSCs were applied to evaluate the quality of the harvested UCB-HSCs and -MSCs. After 12 days culture, the expansions of total cell number, CFU-Cs, CD34+CD45+CD105- (HSCs) cells and CD34-CD45-CD105+ (MSCs) cells in RWVB are (3.7±0.3) fold, (5.1±1.2) fold, (5.2±0.4) fold and (13.9±1.2) fold, respectively, which is better than those cocultured in spinner flasks. Since UCB-MSCs are only adhered on the microcarriers, the UCB-HSCs and UCB-MSCs could be easily separated by gravity sedimentation after coculture. At the same time, the fibroblast-like cells appeared on the surface of GCSC microcarriers can be induced and differentiated into osteoblasts, chondrocytes and adipocytes, and can express MSCs-related surface markers of CD13, CD44, CD73 and CD105 and not hematpopietic cells-related surface markers of CD34, CD45 and HLA-DR, which is similar to MSCs derived from bone marrow. The results show that the simultaneous expansion and harvest of UCB-HSCs and UCB-MSCs can be realized in a feasible bioreactor culture system such as RWVB with GCSC microcarriers.