Removal of CD34- cells to increase self-renewal symmetric division and expansion ex vivo of cord blood CD34+ cells through reducing the TGF-β1

Haematopoietic stem cells (HSCs) exist in CD34(+) cells and lose CD34 marker expression when they differentiate into CD34(-) cells during ex vivo expansion. In this work, periodical removal of CD34(-) cells with full medium replacement and full medium replacement were performed to investigate the influence of CD34(+) cells on the expansion of CD34(+) cells. After 10 days of culture, the percentages of CD34(+) cells with symmetric self-renewing division cultured according to these two strategies were significantly higher than those in the control group. As a result, by using these two strategies, the concentrations of total TGF-beta 1 and activated TGF-beta 1 were investigated and were significantly reduced compared with those in the control group. The expansion of CD34(+) cells was significantly greater than that of control cells. Importantly, the expansion of CD34(+)CD38(-)CD45RA(-)CD49D(+)CD90(+) cells was also significantly increased. Moreover, both of the two strategies better maintained the colony-forming ability and secondary expansion ability of cells. The qRT-PCR results further indicated that key genes involved in the self-renewal and function of HSCs were upregulated (pu.1, hoxa9, hoxb4, hes1 and bmi1). Therefore, we speculated that CD34(+) cells inhibited the expansion of CD34(+) cells by TGF-beta 1.