Retrovirus-mediated transduction of a cytosine deaminase gene preserves the stemness of mesenchymal stem cells

Abstract

Human mesenchymal stem cells (MSCs) have emerged as attractive cellular vehicles to deliver therapeutic genes for ex-vivo therapy of diverse diseases; this is, in part, because they have the capability to migrate into tumor or lesion sites. Previously, we showed that MSCs could be utilized to deliver a bacterial cytosine deaminase (CD) suicide gene to brain tumors. Here we assessed whether transduction with a retroviral vector encoding CD gene altered the stem cell property of MSCs. MSCs were transduced at passage 1 and cultivated up to passage 11. We found that proliferation and differentiation potentials, chromosomal stability and surface antigenicity of MSCs were not altered by retroviral transduction. The results indicate that retroviral vectors can be safely utilized for delivery of suicide genes to MSCs for ex-vivo therapy. We also found that a single retroviral transduction was sufficient for sustainable expression up to passage 10. The persistent expression of the transduced gene indicates that transduced MSCs provide a tractable and manageable approach for potential use in allogeneic transplantation.

Dihydropyrimidine Dehydrogenase Is a Prognostic Marker for Mesenchymal Stem Cell-Mediated Cytosine Deaminase Gene and 5-Fluorocytosine Prodrug Therapy for the Treatment of Recurrent Gliomas

“…Since we introduced reporters in stem cells for visualization (Figure S2 ), we could validate that stem cell characteristics of MSC/CDs were unchanged (Figure 3 C). Preservation of stem cell characteristics in the CD-transduced MSCs was described in an earlier report 17 . Stem Cell characteristics were evaluated by monitoring the expression of CD29 (Integrin protein), CD44 (hyaluronate receptor), CD105 (endoglin protein), CD34 (hematopoietic progenitor cell marker), and CD45 (leukocyte common antigen) markers.…”

“…Since we anticipated that suicide gene therapy using CD-expressing MSCs with prodrug 5-FC may be a better option for the gliomas resistant to conventional therapy, we introduced MSC/CDs 16 , 17 in two glioblastoma cell lines and cells derived from a GBM patient (U87MG, U373, GBM28, GBM37). Our strategy was to use the tumor targeting property of MSC/CDs as well as the bystander effect generated by MSC/CDs by converting the non-toxic prodrug 5-FC to toxic 5-FU (Figure 3 A).…”

“…This indicates that the risk of using genetically-manipulated cells in clinical applications can be avoided by using cytosine deaminase, as the therapeutic cells could otherwise be depleted by the suicide effect. For safety issues, our previous study demonstrated that retroviral-infected MSCs maintain their original properties as well as genetic stability 17 . In addition, we also monitored tumorigenesis of retroviral-infected MSC cells (MSC/CD).…”

“…Aboody et al has shown preclinical results from combination therapy with neural stem cells-mediated CD gene and 5-FC, which led to the approval of a human trial for the treatment of recurrent high grade gliomas 14 , 15 . Instead of neuronal stem cells (NSCs), Suh-Kim et al have suggested that mesenchymal stem cells (MSCs) could be used as a vehicle to deliver CD gene to brain tumors 16 , 17 . Compared to NSCs, MSCs can be easily isolated from bone marrow and adipose tissues of patients, are highly proliferative, and therefore can be expanded in vitro suggesting the possibility of MSCs' production at an industrial scale as a cell therapeutic agent.…”

“…Compared to NSCs, MSCs can be easily isolated from bone marrow and adipose tissues of patients, are highly proliferative, and therefore can be expanded in vitro suggesting the possibility of MSCs' production at an industrial scale as a cell therapeutic agent. Furthermore, the lack of human leukocyte antigen, antigen D related (HLA-DR) on MSCs reduces their immunogenic property contributing to a reduced immune rejection response after allogeneic injection of therapeutic MSCs 17 , 18 .…”