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Directed Fusion of Mesenchymal Stem Cells with Cardiomyocytes via VSV-G Facilitates Stem Cell Programming.

Kouris NA, Schaefer JA, Hatta M, Freeman BT, Kamp TJ, Kawaoka Y, Ogle BM - Stem Cells Int (2012)

Bottom Line: That stem cells can be programmed, or somatic cells reprogrammed, in this fashion suggests that stem cell fusion holds promise as a therapeutic approach for the repair of damaged tissues, especially tissues not readily capable of functional regeneration, such as the myocardium.In an attempt to increase the frequency of stem cell fusion and, in so doing, increase the potential for cardiac tissue repair, we expressed the fusogen of the vesicular stomatitis virus (VSV-G) in human MSCs.In vivo, vMSCs delivered to damaged mouse myocardium via a collagen patch were able to home to the myocardium and fuse to cells within the infarct and peri-infarct region of the myocardium.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, University of Wisconsin at Madison, Madison, WI 53706, USA.

ABSTRACT
Mesenchymal stem cells (MSCs) spontaneously fuse with somatic cells in vivo, albeit rarely, and the fusion products are capable of tissue-specific function (mature trait) or proliferation (immature trait), depending on the microenvironment. That stem cells can be programmed, or somatic cells reprogrammed, in this fashion suggests that stem cell fusion holds promise as a therapeutic approach for the repair of damaged tissues, especially tissues not readily capable of functional regeneration, such as the myocardium. In an attempt to increase the frequency of stem cell fusion and, in so doing, increase the potential for cardiac tissue repair, we expressed the fusogen of the vesicular stomatitis virus (VSV-G) in human MSCs. We found VSV-G expressing MSCs (vMSCs) fused with cardiomyocytes (CMs) and these fusion products adopted a CM-like phenotype and morphology in vitro. In vivo, vMSCs delivered to damaged mouse myocardium via a collagen patch were able to home to the myocardium and fuse to cells within the infarct and peri-infarct region of the myocardium. This study provides a basis for the investigation of the biological impact of fusion of stem cells with CMs in vivo and illustrates how viral fusion proteins might better enable such studies.

No MeSH data available.


Related in: MedlinePlus

Expression of VSV-G in MSCs. VSV-G expression was analyzed via immunofluorescence using an anti-VSV-G-FITC antibody. (a) Representative image analysis of untransfected MSCs and vMSCs; VSV-G (green); DAPI (blue). Scale bar = 25 μm. (b) Transfection efficiency was defined as the number of VSV-G-positive cells divided by the total number of nuclei and is reported as the mean ± standard deviation. A low level of nonspecific binding was associated with the anti-VSV-G antibody and is reflected in the percentage of positive cells reported in the untransfected population of MSCs (2.3% ± 3.4%). (c) Dissociation reagent impacts VSV-G expression. Trypsin treatment reduces detection of VSV-G expressing cells to that of untransfected MSCs. Accutase treatment retains a significantly greater number of cells expressing VSV-G than treatment with trypsin; **P < 0.005. (d) Dissociation reagent impacts the number of VSV-G proteins per cell. The number of VSV-G proteins expressed per cell is significantly reduced with trypsin treatment as compared with Accutase treatment, which was quantified utilizing Quantum Simply Cellular standards; **P < 0.005.
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fig1: Expression of VSV-G in MSCs. VSV-G expression was analyzed via immunofluorescence using an anti-VSV-G-FITC antibody. (a) Representative image analysis of untransfected MSCs and vMSCs; VSV-G (green); DAPI (blue). Scale bar = 25 μm. (b) Transfection efficiency was defined as the number of VSV-G-positive cells divided by the total number of nuclei and is reported as the mean ± standard deviation. A low level of nonspecific binding was associated with the anti-VSV-G antibody and is reflected in the percentage of positive cells reported in the untransfected population of MSCs (2.3% ± 3.4%). (c) Dissociation reagent impacts VSV-G expression. Trypsin treatment reduces detection of VSV-G expressing cells to that of untransfected MSCs. Accutase treatment retains a significantly greater number of cells expressing VSV-G than treatment with trypsin; **P < 0.005. (d) Dissociation reagent impacts the number of VSV-G proteins per cell. The number of VSV-G proteins expressed per cell is significantly reduced with trypsin treatment as compared with Accutase treatment, which was quantified utilizing Quantum Simply Cellular standards; **P < 0.005.

Mentions: MSCs were induced to express VSV-G via transfection by electroporation. Low transfection efficiency would limit the ability of VSV-G to promote fusion and so VSV-G expression on MSCs was determined following electroporation. Twenty-four hours after transfection control MSCs and MSCs transfected with VSV-G (vMSCs) were probed with an anti-VSV-G antibody conjugated to fluorescein isothiocyanate (FITC) and visualized with fluorescence microscopy to determine the percentage of cells expressing VSV-G. The average transfection efficiency was 32% ± 5% (n = at least 6 optical fields per sample per trial for 3 trials, Figure 1(a)). Since vMSCs will be harvested for in vivo studies, we also assessed VSV-G expression via flow cytometry after removal from culture plates with trypsin. We found expression of VSV-G plummeted to 5% ± 2% (n = 1 replicate per sample per trial for 3 trials, Figure 1(b)). This is perhaps not surprising as others have reported decreased stability of VSV-G with trypsin treatment [50, 51]. Trypsin is a serine protease that cleaves carboxyl groups on the cell surface to remove cells from a culture surface. VSV-G is a cell surface protein that would be exposed to the dissociation reagent [52]. The disruption to VSV-G by trypsin was corroborated by evaluating the number of VSV-G proteins per cell. The administration of trypsin significantly reduced the number of VSV-G proteins on the cell surface of vMSCs (Figure 1(d)). Thus we replaced trypsin with Accutase, a mixture of proteases and collagenases that has been shown to improve cell viability compared to trypsin [53]. With Accutase treatment, the average number of cells expressing VSV-G after cell harvest was 21% ± 7%, a significant improvement over treatment with trypsin and at a level high enough to discern whether expression of VSV-G can impact MSC-CM fusion (n = 1 replicate per sample per trial for 3 trials).


Directed Fusion of Mesenchymal Stem Cells with Cardiomyocytes via VSV-G Facilitates Stem Cell Programming.

Kouris NA, Schaefer JA, Hatta M, Freeman BT, Kamp TJ, Kawaoka Y, Ogle BM - Stem Cells Int (2012)

Expression of VSV-G in MSCs. VSV-G expression was analyzed via immunofluorescence using an anti-VSV-G-FITC antibody. (a) Representative image analysis of untransfected MSCs and vMSCs; VSV-G (green); DAPI (blue). Scale bar = 25 μm. (b) Transfection efficiency was defined as the number of VSV-G-positive cells divided by the total number of nuclei and is reported as the mean ± standard deviation. A low level of nonspecific binding was associated with the anti-VSV-G antibody and is reflected in the percentage of positive cells reported in the untransfected population of MSCs (2.3% ± 3.4%). (c) Dissociation reagent impacts VSV-G expression. Trypsin treatment reduces detection of VSV-G expressing cells to that of untransfected MSCs. Accutase treatment retains a significantly greater number of cells expressing VSV-G than treatment with trypsin; **P < 0.005. (d) Dissociation reagent impacts the number of VSV-G proteins per cell. The number of VSV-G proteins expressed per cell is significantly reduced with trypsin treatment as compared with Accutase treatment, which was quantified utilizing Quantum Simply Cellular standards; **P < 0.005.
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Related In: Results  -  Collection

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fig1: Expression of VSV-G in MSCs. VSV-G expression was analyzed via immunofluorescence using an anti-VSV-G-FITC antibody. (a) Representative image analysis of untransfected MSCs and vMSCs; VSV-G (green); DAPI (blue). Scale bar = 25 μm. (b) Transfection efficiency was defined as the number of VSV-G-positive cells divided by the total number of nuclei and is reported as the mean ± standard deviation. A low level of nonspecific binding was associated with the anti-VSV-G antibody and is reflected in the percentage of positive cells reported in the untransfected population of MSCs (2.3% ± 3.4%). (c) Dissociation reagent impacts VSV-G expression. Trypsin treatment reduces detection of VSV-G expressing cells to that of untransfected MSCs. Accutase treatment retains a significantly greater number of cells expressing VSV-G than treatment with trypsin; **P < 0.005. (d) Dissociation reagent impacts the number of VSV-G proteins per cell. The number of VSV-G proteins expressed per cell is significantly reduced with trypsin treatment as compared with Accutase treatment, which was quantified utilizing Quantum Simply Cellular standards; **P < 0.005.
Mentions: MSCs were induced to express VSV-G via transfection by electroporation. Low transfection efficiency would limit the ability of VSV-G to promote fusion and so VSV-G expression on MSCs was determined following electroporation. Twenty-four hours after transfection control MSCs and MSCs transfected with VSV-G (vMSCs) were probed with an anti-VSV-G antibody conjugated to fluorescein isothiocyanate (FITC) and visualized with fluorescence microscopy to determine the percentage of cells expressing VSV-G. The average transfection efficiency was 32% ± 5% (n = at least 6 optical fields per sample per trial for 3 trials, Figure 1(a)). Since vMSCs will be harvested for in vivo studies, we also assessed VSV-G expression via flow cytometry after removal from culture plates with trypsin. We found expression of VSV-G plummeted to 5% ± 2% (n = 1 replicate per sample per trial for 3 trials, Figure 1(b)). This is perhaps not surprising as others have reported decreased stability of VSV-G with trypsin treatment [50, 51]. Trypsin is a serine protease that cleaves carboxyl groups on the cell surface to remove cells from a culture surface. VSV-G is a cell surface protein that would be exposed to the dissociation reagent [52]. The disruption to VSV-G by trypsin was corroborated by evaluating the number of VSV-G proteins per cell. The administration of trypsin significantly reduced the number of VSV-G proteins on the cell surface of vMSCs (Figure 1(d)). Thus we replaced trypsin with Accutase, a mixture of proteases and collagenases that has been shown to improve cell viability compared to trypsin [53]. With Accutase treatment, the average number of cells expressing VSV-G after cell harvest was 21% ± 7%, a significant improvement over treatment with trypsin and at a level high enough to discern whether expression of VSV-G can impact MSC-CM fusion (n = 1 replicate per sample per trial for 3 trials).

Bottom Line: That stem cells can be programmed, or somatic cells reprogrammed, in this fashion suggests that stem cell fusion holds promise as a therapeutic approach for the repair of damaged tissues, especially tissues not readily capable of functional regeneration, such as the myocardium.In an attempt to increase the frequency of stem cell fusion and, in so doing, increase the potential for cardiac tissue repair, we expressed the fusogen of the vesicular stomatitis virus (VSV-G) in human MSCs.In vivo, vMSCs delivered to damaged mouse myocardium via a collagen patch were able to home to the myocardium and fuse to cells within the infarct and peri-infarct region of the myocardium.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, University of Wisconsin at Madison, Madison, WI 53706, USA.

ABSTRACT
Mesenchymal stem cells (MSCs) spontaneously fuse with somatic cells in vivo, albeit rarely, and the fusion products are capable of tissue-specific function (mature trait) or proliferation (immature trait), depending on the microenvironment. That stem cells can be programmed, or somatic cells reprogrammed, in this fashion suggests that stem cell fusion holds promise as a therapeutic approach for the repair of damaged tissues, especially tissues not readily capable of functional regeneration, such as the myocardium. In an attempt to increase the frequency of stem cell fusion and, in so doing, increase the potential for cardiac tissue repair, we expressed the fusogen of the vesicular stomatitis virus (VSV-G) in human MSCs. We found VSV-G expressing MSCs (vMSCs) fused with cardiomyocytes (CMs) and these fusion products adopted a CM-like phenotype and morphology in vitro. In vivo, vMSCs delivered to damaged mouse myocardium via a collagen patch were able to home to the myocardium and fuse to cells within the infarct and peri-infarct region of the myocardium. This study provides a basis for the investigation of the biological impact of fusion of stem cells with CMs in vivo and illustrates how viral fusion proteins might better enable such studies.

No MeSH data available.


Related in: MedlinePlus