The Study Of HOXB4 Expression For The Cord Blood CD34～+ Cells Expanded In Vitro

ObjectiveTo investigate the change of self-renewal potential of the cord blood CD34~+ cells expanded in vitro via the detection of HOXB4 expression level. And to explored the regulatory function of the BM-MSCs on the self-renewal potential maintenance of the cord blood CD34~+ cells expanded in vitro. MethodsCord blood CD34~+ cells were cultured in vitro in the serum-free medium containing thrombopoietin (TPO ), stem cell factor (SCF ), flt3/flk2 ligand (FL) and granulocyte-colony stimulating factor (G-CSF) with or without BM-MSCs. The output of total cells (TC) and CD34~+ cells were evaluated after 14 days' expansion in vitro. The HOXB4 expression level of expanded cells was assayed by real time PCR on the 7th day and 14th day. ResultsTC and CD34~+ cells increased as many as 470.4 and 38.3 folds after co-cultured with BM-MSCs and 4 cytokines for 14 days. While HOXB4 expression level decreased gradually to 45.8% and 18.1% of its pre-cultured level when they were expanded on the 7th day and 14th day.By contrast, when cultured merely in the presence of 4 cytokines, the TC and CD34~+ cells increased 74.4 and 10.9 folds, respectively. However, the HOXB4 expression level of cells cultured for 7 or 14 days was only 17.8% or 2.95% of its former level. There is significant difference between two groups (P<0.05 ) concerning cells proliferation and HOXB4 expression. ConclusionThe HOXB4 expression level of cord blood CD34~+ cells was down-regulated when they were expanded in vitro. It implied that expanded cells gradually lost their self-renewal potential as the culture time extended. In BM-MSCs and 4 cytokines co-culture system, the increase folds of TC and CD34~+ cells, together with the HOXB4 expression level were higher than the culture system without BM-MSCs. It indicated BM-MSCs might be helpful to the expansion of cord blood CD34~+ cells and slow down the loss of their self-renewal potential.