Leukotriene B₄(LTB4) is lipid mediator derived from membrane phospholipids during the process of inflammation, having many roles(ie; inducer of chemotaxis, the production of nitric oxide, transepithelial migration of neutrophil). The major activities of LTB4 include the recruitment and activation of leukocytes, suggesting that it may involve the process for transendothelial migration of nuclear cells in bone marrow environment. Reactive Oxygen Species (ROS) have a cell signaling roles that are involved in signal transduction cascades of numerous growth factor-, cytokine-, and hormone-mediated pathways, and regulate many biological systems. In this present study, we focused on the role of LTB4 and ROS on transmigration of bone marrow nuclear cells across endothelial or stromal cell monolayer.

MS-5, murine stromal cell line cells, or bEnd.3, murine microvascular cell line cells, were grown to confluence on microporous transwell membrane. Murine marrow cells were placed on top of the prepared transwell membrane. The transwells were then seated in wells containing media and LTB4 with or without pretreatment of N-acetylcysteine(NAC), an oxygen free radical scavenger, or diphenylene iodonium(DPI), an inhibitor of NADPH oxidase-like flavoproteins. Cells that migrated through the stromal or endothelial layer into the wells were assayed for transendothelial migration.

The numbers of migrated bone marrow nuclear cells through the bEnd.3 were increased by treatment of LTB4(control, 12.5±0.2%; 50nM, 22.7±0.9%; 100nM, 44.3±1.4%; 200 nM, 36.3±0.9%; p<0.05). The numbers of migrated bone marrow nuclear cells through the MS-5 were also increased by treatment of LTB4(control, 11.0±0.9%; 50nM, 25.7±0.9%; 100nM, 35.8±1.8%; 200nM, 32.1±0.9%; p<0.05). However, increasing effect of LTB4 to the transmigration of bone marrow nuclear cells through the MS-5 or bEnd.3 were inhibited by pretreatment of NAC or DPI.

Through our data, it is suggested that LTB4 could induce the transmigration of bone marrow nuclear cells and ROS might be involved on the transendothelial migration of bone marrow nuclear cells by LTB4. It would be very interesting to test the effects of LTB4 and ROS on stem cell mobilization and homing in the future.

Stable gene transfer to human pluripotent hematopoietic stem cells is an attractive strategy for the curative treatment of many genetic hematologic disorders. In clinical tral, the level of gene transfer to this cell population have generally been low.

In this study we have evaluated the efficiency of gene transfer to human umbilical cord blood(UCB) CD34+cells using vesicular stomatitis virus glycoprotein G(VSV-G) pseudotyped HIV-1 vector.

High titers of replication-defective VSV-G pseudotyped HIV-1 based vector encoding the enhanced yellow fluorescent protein were produced by transient transfection. Human CD34+cells purified from UCB were incubated with pseudotyped HIV supernatants for 24-48 hours. The transduction efficiency were measured by marker gene expression under the microscopy and flow cytometry.

Transduction rates into CD34+ were low at 0 and 24 hours, reflecting 4.7±2.4% at 24 hours, they were increased to 5.7±2.7% at 48 hours.

We demonstrate efficient transduction of purified human UCB CD34+ by HIV vectors pseudotyped with VSV-G. The results extend the lentiviral vector to clinical gene therapy using human pluripotent hematopoietic stem cells.