Retroviral gene transfer into hematopoietic stem cells derived from murine bone marrow and human umbilical cord blood

ABSTRACT

Both bone marrow [BM] and umbilical cord blood [UCB] CD,; cells represent an attractive source to approach gene therapy for treatment of many disorders that affect gastrointestinal tract [GIT], urinary bladder and liver which lead to different diseases and finally malignancies. Hematotoxicity is a major and frequently dose-limiting side effect of cancer chemotherapeutic drugs. Methylguanine-DNA-methyItransferase [MGMT] gene transfer into stem cells was considered as an efficient method for protection against hematotoxicity caused by chemotherapeutic drugs. In the present study a retroviral [RV] vector containing plasmids of both MGMT and eGFP [enhanced green fluorescent protein] was transfected, under the control of 1 alpha-promotor, into stem cells derived from murine BM and human UCB CD[34] cells. The retrovira1 gene transfer rate into both types of stem cells was detected by Flow cytometry at different concentrations of RV supernatant dilutions after 24 hours and 48 hours. The percentage positivity of transduction rate after 24 hours was 94.4% when RV supematant was added undiluted to hUCB CD[34] stem cells and at 110, 1100, 11000 dilutions, the positivity rate was 88.7%, 33.6%, 6.6% respectively. When the RV supernatant was added undiluted to mBM stein cells, the percentage positivity of transduction rate was 78.6%, and at 110, 1100, 11000 dilutions, the positivity rate was 72.8%, 31%, 6.3% respectively. After 48 hours, percentage positivity of undiluted RV supernatant was 96%, when transduced into hUCB CD[34] stem cells, and at dilutions 110, 1100, 11000, the percentage was 91.2%, 35%, 6.9% respectively. The positivity of gene transfer into mBM stem cells was 82.3% when RV supernatant was added undiluted, and at dilutions 110, 1100, 11000 the percentage positivity was 75.4%, 32.6%, 6.7% respectively. DNA detection by PCR has been performed to estimate both types of stem cells after gene transfer. Generally, these results conclude that gene therapy can be fulfilled by transducing a retroviral vector carrying special characters inside either bone marrow or umbilical cord blood stem cells. So it can be used for curing different diseases and malignancies