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. 2013 Sep;2(9):690-702.
doi: 10.5966/sctm.2013-0035. Epub 2013 Aug 9.

Enhancing in Vivo Survival of Adipose-Derived Stromal Cells Through Bcl-2 Overexpression Using a Minicircle Vector

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Free PMC article

Enhancing in Vivo Survival of Adipose-Derived Stromal Cells Through Bcl-2 Overexpression Using a Minicircle Vector

Jeong Hyun et al. Stem Cells Transl Med. .
Free PMC article

Abstract

Tissue regeneration using progenitor cell-based therapy has the potential to aid in the healing of a diverse range of pathologies, ranging from short-gut syndrome to spinal cord lesions. However, there are numerous hurdles to be overcome prior to the widespread application of these cells in the clinical setting. One of the primary barriers to effective stem cell therapy is the hostile environment that progenitor cells encounter in the clinical injury wound setting. In order to promote cellular survival, stem cell differentiation, and participation in tissue regeneration, relevant cells and delivery scaffolds must be paired with strategies to prevent cell death to ensure that these cells can survive to form de novo tissue. The Bcl-2 protein is a prosurvival member of a family of proteins that regulate the mitochondrial pathway of apoptosis. Using several strategies to overexpress the Bcl-2 protein, we demonstrated a decrease in the mediators of apoptosis in vitro and in vivo. This was shown through the use of two different clinical tissue repair models. Cells overexpressing Bcl-2 not only survived within the wound environment at a statistically significantly higher rate than control cells, but also increased tissue regeneration. Finally, we used a nonintegrating minicircle technology to achieve this in a potentially clinically applicable strategy for stem cell therapy.

Keywords: Adult stem cells; Apoptosis; Cellular therapy; Gene delivery systems in vivo or in vitro.

Figures

Figure 1.
Figure 1.
Bcl-2 overexpression affects hASC proliferation and apoptosis. (A): Viability assay. hASCs with Ad-Bcl-2, Sh-Bcl-2, and control Ad-GFP were treated with staurosporine (1 μM), resulting in significantly increased survival for hASCs with Bcl-2 overexpression (*, p < .05). (B): Fluorescence-activated cell sorting plot with live cells staining high with red fluorescence (PE-Texas Red: y-axis) and apoptotic cells staining high with green fluorescence (FITC: x-axis) showing significantly increased survival of hASCs with Ad-Bcl-2 over Sh-Bcl-2 and control Ad-Luc. (C): Bcl-2 slightly decreased hASC proliferation, as shown through Bcl-2 overexpression and Sh-Bcl-2 at 24 hours. (D): Apoptosis array analyzing the relative expression of 35 apoptotic genes with cell lysates from hASCs with Ad-Bcl-2, Ad-GFP, and Sh-Bcl-2 treated with staurosporine (blots on left). (E): Analysis/photodensity plots of six key proteins: Bcl-2, Procaspase-3, Caspase 3, cytochrome c, Smac/DIABLO, and XIAP (*, p < .05). Abbreviations: Ad, adenovirus; Cont., control; FITC, fluorescein isothiocyanate; GFP, green fluorescence protein; hASC, human adipose-derived stromal cell; PE, R-phycoerythrin; Sh, short hairpin RNA.
Figure 2.
Figure 2.
Expression of key apoptosis mediators at the RNA and protein levels. (A): Quantitative real-time polymerase chain reaction analysis of gene transcripts for XIAP, BAX, Smac/DIABLO, APAF, Caspase 3, and Caspase 7 (*, p < .05) with Bcl-2, Sh-Bcl-2, and GFP-control hASCs after apoptotic assay versus untreated control gene transcripts. (B): Enzyme-linked immunosorbent assay showing relative protein concentration in cell lysates for Bax, APAF, cytochrome c, and Smac/DIABLO (*, p < .05) after apoptotic assay for the Bcl-2, sh-Bcl-2, GFP-control, and untreated control groups. (C): Bioluminescent assay for active Caspase 3/7 with representative images on the right (*, p < .05). Abbreviations: Ad, adenovirus; GFP, green fluorescence protein; hASC, human adipose-derived stromal cell; Sh, short hairpin RNA.
Figure 3.
Figure 3.
Bcl-2 overexpression increases calvarial skeletal regeneration. (A): Representative images of bioluminescent imaging with hASCs transduced with luciferase vector and control (top row), sh-Bcl-2 (middle row), and Bcl-2 (bottom row). (B): Quantification of luminescence for 10 days. Values are expressed as mean ± SE. hASCs with Bcl-2 (blue) had significantly increased luminescence versus control (beige) and sh-Bcl-2 (red). (C): Coronal T2 weighted magnetic resonance images show a hypointense (dark) signal of iron-labeled cells (red arrows) in the calvarial defect area as opposed to a hyperintense (bright) signal of unlabeled hASCs (top), control labeled hASCs (middle), and labeled hASCs with Bcl-2 (bottom). (D): MRI quantification corresponding to (C) showing Bcl-2-labeled cells (blue) as having higher density (*, p < .05). (E): MicroCT quantification of calvarial regeneration with hASC control (left), sh-Bcl-2 (middle), and hASC-Bcl-2 (right) for time points of 1, 2, and 4 weeks, with higher bony regeneration at week 4 for hASC-Bcl-2. (F): MicroCT quantification of calvarial regeneration, with significantly (*, p < .05) higher regeneration at week 4 for hASC-Bcl-2. Abbreviations: Ad, adenovirus; ASC, adipose-derived stromal cell; Cont, control; HA-PLGA, hydroxyapatite-coated poly(lactic-co-glycolic acid); hASC, human adipose-derived stromal cell; MicroCT, micro-computed tomography; MRI, magnetic resonance imaging; Sh, short hairpin RNA.
Figure 4.
Figure 4.
Bcl-2 overexpression decreases apoptosis mediators in vivo. (A): Apoptosis in situ terminal deoxynucleotidyl transferase dUTP nick-end labeling assay (left) showing DNA-based fragmentation (*, p < .05). Apoptosis was decreased in hASCs with Ad-Bcl-2 with corresponding quantification (right) in the area of the calvarial defect. Scale bars = 50 μm. (B): Immunohistochemistry of human nuclear antigen (left) and quantification of staining (right). A higher percentage of human cells was present in the calvarial defect area in hASCs with Ad-Bcl-2 (*, p < .05) in the area of the calvarial defect. Scale bars = 50 μm. (C–E): Immunofluorescent staining of the calvarial defect by confocal microscopy (magnification, ×63) of cytochrome c (C), Smac/DIABLO (D), and Caspase 3 (E). Top rows: No primary control. Second rows: hASCs with control-Luc. Third rows: hASCs with sh-Bcl-2. Bottom rows: hASCs with Ad-Bcl-2. Left columns: Combined DAPI and immunofluorescent Alexa Fluor 488 (green). Middle columns: DAPI (blue). Right columns: Alexa Fluor 488 (green). Scale bars = 20 μm. Abbreviations: Ad, adenovirus; DAPI, 4′,6-diamidino-2-phenylindole; hASC, human adipose-derived stromal cell; Sh, short hairpin RNA.
Figure 5.
Figure 5.
Bcl-2 overexpression accelerates wound healing curve. (A): Representative images of bioluminescent imaging with PBS control (top row) hASCs transduced with luciferase vector (second row), sh-Bcl-2 (third row), and Bcl-2 (bottom row). (B): Quantification of luminescence for 10 days. Values are expressed as mean ± SE, with wounds with PBS control (gray) and hASCs with Bcl2 (blue) having significantly increased luminescence (*, p < .05) versus control (beige) and sh-Bcl-2 (red). (C): Representative images of wound closure. hASCs with Bcl-2 overexpression showed increased closure of wound healing (bottom row). (D): Percentage of open wound evaluated every 2 days postwounding. hASC-Ad-Bcl-2 produced significantly (*, p < .05) accelerated wound healing compared with hASCs with Luc control and hASCs with sh-Bcl-2 or PBS control. Values are expressed as mean ± SE. Abbreviations: Ad, adenovirus; hASC, human adipose-derived stromal cell; PBS, phosphate-buffered saline; Sh, short hairpin RNA.
Figure 6.
Figure 6.
Increased calvarial regeneration with hASCs with Bcl-2 minicircle vector. (A): Quantitative real-time polymerase chain reaction analysis of gene transcripts for BCL-2 to evaluate minicircle transfection with increased (*, p < .05) BCL-2 gene transcript with minicircle vector compared with control. (B): ELISA showing relative protein concentration in cell lysates for hASCs with minicircle-Bcl-2 and minicircle control with increased Bcl-2 protein expression for the Bcl-2-minicircle vector (*, p < .05). (C): Immunohistochemistry of Bcl-2 protein showing increased in vivo staining of Bcl-2 in hASCs with Bcl-2 minicircle. Scale bars = 100 μm. (D): MicroCT quantification of calvarial regeneration with hASCs with minicircle control (top row) and hASCs with minicircle-Bcl-2 (bottom row) showing higher bony regeneration at week 4 for hASC minicircle-Bcl-2. (E): MicroCT quantification of calvarial regeneration with significantly (*, p < .05) higher regeneration at week 4 for hASC-Bcl-2 (right columns). Abbreviations: Cont, control; ELISA, enzyme-linked immunosorbent assay; hASC, human adipose-derived stromal cell; MC, minicircle; MicroCT, micro-computed tomography.

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