hTERT-Immortalized Bone Mesenchymal Stromal Cells Expressing Rat Galanin via a Single Tetracycline-Inducible Lentivirus System

Abstract

The use of human telomerase reverse transcriptase-immortalized bone marrow mesenchymal stromal cells (hTERT-BMSCs) as vehicles to deliver antinociceptive galanin (GAL) molecules into pain-processing centers represents a novel cell therapy strategy for pain management. Here, an hTERT-BMSCs/Tet-on/GAL cell line was constructed using a single Tet-on-inducible lentivirus system, and subsequent experiments demonstrated that the secretion of rat GAL from hTERT-BMSCs/Tet-on/GAL was switched on and off under the control of an inducer in a dose-dependent manner. The construction of this cell line is the first promising step in the regulation of GAL secretion from hTERT-immortalized BMSCs, and the potential application of this system may provide a stem cell-based research platform for pain.

Figure 1

Plasmid profiles of the pLV.ExSi/Puro-EF1α-hTERT (a) and pLV.ExSi/ PGK-Puro (b) vectors with and without the hTERT gene, respectively, used for the immortalization of primary rat BMSCs (Cyagen Biosciences).

Figure 2

Plasmid profile of the single tetracycline-inducible lentiviral backbone vector system pLV.TetIIP-EGFP-/Ubi-TetR (Genechem, GV347). The system comprises an EGFP reporter gene and a tetracycline (Tet) response element under the control of separate promoters, the TetIIP and Ubi promoters. This construct drives the expression of rat galanin from transduced cells via Dox induction after cloning of the GAL gene into the multiple cloning sites (MCS).

Figure 3

Telomerase activity of HeLa cells, BMSCs, PGK-BMSCs, and hTERT-BMSCs (TRAP assay) after disposed with or without heat. There was little telomerase activity in all heat-treated negative control cells; however, in unheated cells, despite extensive doublings, the immortalized cells (hTERT-BMSCs) displayed significantly higher telomerase activity than their primary counterparts and PGK-BMSCs, except the positive control cells (HeLa) and the telomerase activity of the immortalized population remained stable (assessed at P30). Significance level is P < 0.05, indicated by ∗.

Figure 4

EdU proliferation assay of the effect of hTERT on the growth of BMSCs. The red fluorescent cells are in the S phase of mitosis, and the blue fluorescent cells represent all cells. (a) and (b) present images of P30 hTERT-BMSCs and P14 BMSCs, respectively. (c) Ratio of EdU-positive cells. Significance level is P < 0.05, indicated by ∗. Scale bar: 100 μm.

Figure 5

Identification of surface phenotype and neurogenesis of hTERT-BMSCs. (a–e) Expression of surface molecules detected using flow cytometry analysis. (f) Typical morphology was observed under an inverted phase contrast microscope. (g–i) The expression of neural specific markers on hTERT-immortalized BMSCs (note: nuclei were stained with Hoechst 33342 blue). Scale bar: 100 μm.

Figure 6

Regulatable EGFP expression from hTERT-BMSCs/Tet-on/GAL cells in the presence and absence of Dox in vitro (×400). hTERT-BMSCs transfected with LV.TetIIP-GAL-EGFP-/Ubi-TetR showed strong green fluorescence after treatment with 1 μg/mL Dox for 48 h (a), whereas very faint fluorescence was observed without Dox induction (b). Scale bar: 200 μm.

Figure 7

GAL secretion from cells after Dox induction in vitro. (a) The dose response of galanin production in cultured hTERT-BMSCs/Tet-on/GAL revealed a correlation between the Dox dose and the galanin secretion level. Significance level is ^∗∗P > 0.05, indicated by ∗∗. (b) Time course of galanin secretion from hTERT-BMSCs/Tet-on/GAL under the control of Dox. The arrows indicate the addition (+Dox) or removal (−Dox) of Dox from the culture medium.