Preclinical transplantation and safety of HS/PCs expanded from human umbilical cord blood

Figure 1 Serum-free co-culture system improves the proliferation of hematopoietic/progenitor stem cells. A: the in vitro expansion kinetics of hematopoietic/progenitor stem cells (HS/PCs) in three culture systems between day 0 and day 5; B: The in vitro expansion kinetics of HS/PCs in three culture systems between day 5 and day 10; C: The ratio of CD34⁺ cells in the mononuclear cells (MNCs) before expansion; D: The ratio of CD34⁺ cells in the MNCs after expansion in the serum-free co-culture system; E: The expansion of granulocyte-macrophage colony-forming cells (GM-CFCs) and erythroid colony-forming cells (E-CFCs) before and after expansion. ^aP < 0.05. TNCs: Total nuclear cells.

Figure 2 The determination of exogenous virus and stem cell factor residual in hematopoietic/progenitor stem cell product. A: The cellular morphology of human fibroblasts cultured in α-MEM (× 400); B: The cellular morphology of human fibroblasts cultured in α-MEM with the cell suspension of hematopoietic/progenitor stem cell (HS/PC) product (× 400); C: The standard curve of residual stem cell factor (SCF) in the HS/PC product. It was detected by ELISA. After a curve (dotted line) was constructed by plotting the mean absorbance against the concentration, a best fitting curve (solid line) through the points on the graph was drawn.

Figure 3 Engraftment of cells from hematopoietic/progenitor stem cell product in NOD/SCID mice. A: The recovery of WBC population in irradiated NOD/SCID mice transplanted with the expanded hematopoietic/progenitor stem cells (HS/PCs) and the combination of expanded HS/PCs and BM-mesenchymal stem cells (MSCs). Group A: WBC of mice transplanted with expanded HS/PCs, Group B: WBC of mice transplanted with the combination of expanded HS/PCs and BM-MSCs, Group C: WBC of mice in the control group. BL: Baseline of WBCs before irradiation; B: RT-PCR detection of human-specific Alu sequence fragment in NOD/SCID mice bone marrow. The GAPDH mRNA was used as a housekeeper gene to normalize the threshold cycle measurements for the Alu product. The result was showed as the concentration ratio of PCR product to that of GAPDH mRNA. ^aP < 0.05; C: The percentage of human CD45⁺ cells in the bone marrow of mice detected by the flow cytometry. White diamond indicates the percentage of human CD45+ cells in the bone marrow of mice transplanted with the combination of HS/PC product and BM-MSCs. Black diamond indicates the percentage of human CD45⁺ cells in the bone marrow of mice transplanted only with HS/PC product.