[Effects of HOXB4-transfecting directly and HOXB4-transfected HUCMSC on in vitro expansion of human umbilical cord blood CD34+ cells].

ABSTRACT

This study was purposed to investigate the difference of nucleated cell (NC) count, CD34(+) cell ratio and expansion multiple, cell cycle and colony formation capability in in vitro expanded human umbilical cord blood CD34(+) cells from HOXB4-transfecting directly and HOXB4-transfected human umbilical cord mesenchymal stem cells (HUCMSC) by means of prepared feeder layers of HUCMSC. The HUCMSC were divided into 2 groupsfirst group, in which HOXB4 gene was transfected into HUCMSC by using lentiviral vecfor, and feeder layers were set up; and second group in which feeder layers for HUCMSC of non-transfected HOXB4 gene were set up. The CD34(+) cells were separated from HUCB by magmatic activated cell sorting(MACS). After culture in medium with cytokines for 2 days, CD34(+) cells were divided into 5 groups, including control group and experimental group. The control groups included CD34(+) cells as group A (blank control group) and GFP-CD34(+) cells as group B (negative control group) and experimental groups included HOXB4-CD34(+) cells as group C, HUCMSC+CD34(+) cells as group D, HOXB4-HUCMSC+ CD34(+) cells as group E and cells in all groups were cultured in vitro. The number of nucleated cells were counted at day 6, 10, 14 of culture and CD34 immunophenotypes, cell cycle and colony forming capability were measured at day 10 of culture in different conditions. The results indicated that HOXB4 gene could be transfected into HUCMSC by lentiviral vector and feeder layers were set up successfully. After culture for 14 days, the nucleated cells in 5 groups could be amplified effectively, and the expansion levels in 5 groups were in order HOXB4-HUCMSC+CD34(+) cell group> HOXB4-CD34(+) cell group>HUCMSC+CD34(+) cell group> control groups (P < 0.05). At day 10 of in vitro expansion the CD34(+) cell percentage decreased significantly in all groups, while the number of CD34(+) cell increased in experiment groups, which were in order HOXB4-CD34(+) cells group> HOXB4-HUCMSC+CD34(+) cell group>HUCMSC+CD34(+) cell group>control groups (P < 0.05). The cell cycle detection showed that the percentage of cells in S+G2/M phase in experiment groups were higher than that in control groups (P < 0.05), and percentage of cells in HOXB4-HUCMSC+CD34(+) cells group was higher (41.57%) than that in HOXB4-CD34(+) cells group(37.87%) and HUCMSC+CD34(+) cell group (28.65%) (P < 0.05). There was no statistical difference in the CFU number between HOXB4-HUCMSC+CD34(+) cell group and HOXB4-CD34(+) cell group, which were both higher than that in HUCMSC+CD34(+) cell group and control groups (P < 0.05).It is concluded that the CD34(+) cells cultured on HOXB4-HUCMSC feeder layers can be amplified significantly and kept the characteristics of stem cells, The feeder lager of HOXB4-HUCMSC is relative safe for amplification of CD34(+) cells in vitro, it possesses the potential useful value.