Therapeutic efficacy and safety of TRAIL-producing human adipose tissue-derived mesenchymal stem cells against experimental brainstem glioma

Abstract

Mesenchymal stem cells (MSCs) have an extensive migratory capacity for gliomas, which is comparable to that of neural stem cells. Among the various types of MSCs, human adipose tissue-derived MSCs (hAT-MSC) emerge as one of the most attractive vehicles for gene therapy because of their high throughput, lack of ethical concerns, and availability and ease of isolation. We evaluated the therapeutic potential and safety of genetically engineered hAT-MSCs encoding the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) against brainstem gliomas. Human AT-MSCs were isolated from human fat tissue, characterized, and transfected with TRAIL using nucleofector. The therapeutic potential of TRAIL-producing hAT-MSCs (hAT-MSC.TRAIL) was confirmed using in vitro and in vivo studies. The final fate of injected hAT-MSCs was traced in long-survival animals. The characterization of hAT-MSCs revealed the expression of MSC-specific cell-type markers and their differentiation potential into mesenchymal lineage. Short-term outcomes included a 56.3% reduction of tumor volume (P < .001) with increased apoptosis (3.03-fold, P < .05) in animals treated with hAT-MSC.TRAIL compared with the control groups. Long-term outcomes included a significant survival benefit in the hAT-MSC.TRAIL-treated group (26 days of median survival in the control group vs 84 days in the hAT-MSC.TRAIL-treated group, P < .0001), without any evidence of mesenchymal differentiation in vivo. Our study demonstrated the therapeutic efficacy and safety of nonvirally engineered hAT-MSCs against brainstem gliomas and showed the possibility of stem-cell-based targeted gene therapy for clinical application.

Fig. 1.

Characterization and differentiation of hAT-MSCs analyzed by flow cytometry and microscopy. The MSC-specific markers CD73, CD90, and CD105 were expressed in hAT-MSCs, whereas the hematopoietic stem-cell markers CD14, CD34, and CD45 were not (A). hAT-MSCs showed a fibroblast-like morphology and had the ability for adipogenic, osteogenic, and chondrogenic differentiation in differentiation induction medium (B). Differentiated cells were stained with oil red O, alizarin red S, and alcian blue.

Fig. 2.

Confirmation of TRAIL expression in hAT-MSC after nucleofection. Expression of the TRAIL transcript was confirmed by RT–PCR. GAPDH controls confirmed equal protein loading. The TRAIL transcript was expressed in hAT-MSC.TRAIL cells but not in hAT-MSC cells (A). Expression of TRAIL protein (green) was detected 24 hours after nucleofection using fluorescence microscopy (B). FACS analysis of nucleofected hMSCs revealed that 76.5% of cells expressed GFP Day 3 and 90.4% Day 7 (C).

Fig. 3.

In vitro quantitative analysis of TRAIL expression in hAT-MSC.TRAIL cells and their therapeutic efficacy on F98 cells. TRAIL protein secreted into hAT-MSC.TRAIL medium was quantified using ELISA. hAT-MSC.TRAIL supernatant contained increased levels of TRAIL, in a cell number-dependent fashion. Approximately 5 × 10³ hAT-MSC.TRAIL cells secreted 1 ηg/mL of TRAIL protein (A). The therapeutic efficacy of hAT-MSC-TRAIL cells was analyzed in coculture experiments. F98 cells cultured in the presence of hAT-MSC-TRAIL cells exhibited significant growth inhibition; however, there was no significant growth inhibition in coculture with hAT-MSC cells (B). Columns represent cell viability as a percentage of the control viability; bars, SE (P < .05, Kruskal–Wallis test with post hoc analysis).

Fig. 4.

Short-term therapeutic efficacy of hAT-MSC.TRAIL in vivo. (A) Representative histological images (H&E staining; magnification, ×1) (A). A 56.3% decrease in tumor volume was observed in hAT-MSC.TRAIL–treated animals compared with PBS-treated animals (P < .001; Kruskal–Wallis test) (B). Representative immunofluorescence images. Primary antibodies included anti-Ki67 nuclear antigen (for the detection of proliferating cells; red color) and anticleaved caspase-3 (for the detection of apoptosis; red color) (C). Sections were counterstained with DAPI (blue). Bars indicate 50 μm. Immunofluorescence analysis revealed that the proliferative indices were not different among the groups. In contrast, the number of apoptotic cells was increased 3.01-fold in the hAT-MSC.TRAIL-treated group compared with the control groups (P < .05; Kruskal–Wallis test) (D).

Fig. 5.

Long-term therapeutic efficacy and safety of hAT-MSC.TRAIL in vivo. Kaplan–Meier plots revealed a significant survival gain in hAT-MSC.TRAIL-treated animals (median survival, 84 days) compared with animals treated with PBS (median survival, 26 days) or hAT-MSC (median survival, 29 days; P < .0001), within 100 days of survival endpoint (A). Two rats treated with hAT-MSC.TRAIL survived for 100 days after tumor cell injection. hAT-MSC.TRAIL cells (green color) injected into these long-term survival rats exhibited neuronal differentiation (red color, white arrow) but lacked mesenchymal differentiation (red color). Sections were counterstained with DAPI (blue) (B).