Abstract
Genetic modification of human adipose tissue–derived multilineage progenitor cells (hADMPCs) is highly valuable for their exploitation in therapeutic applications. Here, we have developed a novel single tet-off lentiviral vector platform. This vector combines (1) a modified tetracycline (tet)-response element composite promoter, (2) a multi-cistronic strategy to express an improved version of the tet-controlled transactivator and the blasticidin resistance gene under the control of a ubiquitous promoter, and (3) acceptor sites for easy recombination cloning of the gene of interest. In the present study, we used the cytomegalovirus (CMV) or the elongation factor 1 α (EF-1α) promoter as the ubiquitous promoter, and EGFP was introduced as the gene of interest. hADMPCs transduced with a lentiviral vector carrying either the CMV promoter or the EF-1α promoter were effectively selected by blasticidin without affecting their stem cell properties, and EGFP expression was strictly regulated by doxycycline (Dox) treatment in these cells. However, the single tet-off lentiviral vector carrying the EF-1α promoter provided more homogenous expression of EGFP in hADMPCs. Intriguingly, differentiated cells from these Dox-responsive cell lines constitutively expressed EGFP only in the absence of Dox. This single tet-off lentiviral vector thus provides an important tool for applied research on hADMPCs.
Highlights
Human adipose tissue–derived mesenchymal stem cells (MSCs), referred to as human adipose tissue–derived multilineage progenitor cells, are multipotent stem cells that can differentiate into various types of cells, including hepatocytes [1], cardiomyoblasts [2], pancreatic cells [3], and neuronal cells [4,5,6]
We examined 2 ubiquitous promoters commonly used in mammalian systems: the CMV promoter and the human polypeptide chain elongation factor 1 a (EF-1a) promoter, to determine which promoter is more efficient in human adipose tissue–derived multilineage progenitor cells (hADMPCs)
Duan et al reported that the angiogenic effect of MSCs could be enhanced by adenovirus-mediated HGF overexpression in the treatment of cardiac ischemia injury [14]
Summary
Human adipose tissue–derived mesenchymal stem cells (MSCs), referred to as human adipose tissue–derived multilineage progenitor cells (hADMPCs), are multipotent stem cells that can differentiate into various types of cells, including hepatocytes [1], cardiomyoblasts [2], pancreatic cells [3], and neuronal cells [4,5,6]. MSCs, including hADMPCs, have the ability to migrate to injured areas and secrete a wide variety of cytokines and growth factors necessary for tissue regeneration [7,8,9,10,11]. Because of their hypoimmunogenicity and immune modulatory effects, hADMPCs are good candidates for gene delivery vehicles for therapeutic purposes [12]. It has been reported that the CMV promoter is silenced in rat bone marrow–derived MSCs [18,19], suggesting that consideration of promoter used in the lentiviral vector is one of the most critical issues
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