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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

VSV-G facilitates vMSC-CM fusion. Four hours after pH-induced fusion, samples were harvested and analyzed using fluorescence deconvolution microscopy and flow cytometry for events displaying both CellTracker fluorescent probes. (a) Representative vMSC-CM fusion event (white arrow). CMs were labeled with CellTracker green and vMSCs were labeled with CellTracker red. Scale bar = 50 μm. (b) Effect of VSV-G on vMSC-CM fusion at pH = 5.5. Fusion events were increased 3.5-fold with VSV-G at low pH. (c) Effect of pH on VSV-G-mediated MSC-CM fusion. vMSC-CM fusion was inhibited by pH = 6.5 and 7.5 (inactive form of VSV-G) *P < 0.05.
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fig2: VSV-G facilitates vMSC-CM fusion. Four hours after pH-induced fusion, samples were harvested and analyzed using fluorescence deconvolution microscopy and flow cytometry for events displaying both CellTracker fluorescent probes. (a) Representative vMSC-CM fusion event (white arrow). CMs were labeled with CellTracker green and vMSCs were labeled with CellTracker red. Scale bar = 50 μm. (b) Effect of VSV-G on vMSC-CM fusion at pH = 5.5. Fusion events were increased 3.5-fold with VSV-G at low pH. (c) Effect of pH on VSV-G-mediated MSC-CM fusion. vMSC-CM fusion was inhibited by pH = 6.5 and 7.5 (inactive form of VSV-G) *P < 0.05.

Mentions: To determine whether MSCs expressing VSV-G are better equipped to fuse with CMs than unmanipulated counterparts, vMSCs or MSCs were cocultured with CMs. To distinguish cell types in cocultures, MSCs and vMSCs were stained with Red CellTracker, while CMs were stained with Green CellTracker fluorescent probes prior to being combined. Since VSV-G undergoes a conformational change from its inactive form to its active form at pH < 6.2 [54, 55], cocultures were briefly incubated (2 min) with fusion medium of pH = 5.5. Image analysis of vMSC-CM cocultures treated with fusion medium of pH = 5.5 revealed cells with colocalization of green and red fluorescence, indicating fusion events, while MSC-CM cocultures under the same pH condition exhibited limited colocalization (Figure 2(a)). To accurately assess the amount of cell fusion, cocultures were harvested 24 hours after seeding and analyzed via flow cytometry (double positive cells correspond to fusion events). vMSCs treated with acidic medium (pH 5.5) had significantly higher rates of fusion with CMs (4.7% ± 1.1%) than MSCs treated in the same way (i.e., spontaneous fusion, 1.4% ± 0.2%) (P < 0.05) (Figure 2(b)). Further, the percentage of fusion products identified in vMSC-CM cocultures exposed to fusion medium of pH = 6.5 or 7.5 (maintaining VSV-G in the inactive form) did not differ from MSC-CM cultures (n = 3 replicates per sample per trial for 3 trials, Figure 2(c)).


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)

VSV-G facilitates vMSC-CM fusion. Four hours after pH-induced fusion, samples were harvested and analyzed using fluorescence deconvolution microscopy and flow cytometry for events displaying both CellTracker fluorescent probes. (a) Representative vMSC-CM fusion event (white arrow). CMs were labeled with CellTracker green and vMSCs were labeled with CellTracker red. Scale bar = 50 μm. (b) Effect of VSV-G on vMSC-CM fusion at pH = 5.5. Fusion events were increased 3.5-fold with VSV-G at low pH. (c) Effect of pH on VSV-G-mediated MSC-CM fusion. vMSC-CM fusion was inhibited by pH = 6.5 and 7.5 (inactive form of VSV-G) *P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3369562&req=5

fig2: VSV-G facilitates vMSC-CM fusion. Four hours after pH-induced fusion, samples were harvested and analyzed using fluorescence deconvolution microscopy and flow cytometry for events displaying both CellTracker fluorescent probes. (a) Representative vMSC-CM fusion event (white arrow). CMs were labeled with CellTracker green and vMSCs were labeled with CellTracker red. Scale bar = 50 μm. (b) Effect of VSV-G on vMSC-CM fusion at pH = 5.5. Fusion events were increased 3.5-fold with VSV-G at low pH. (c) Effect of pH on VSV-G-mediated MSC-CM fusion. vMSC-CM fusion was inhibited by pH = 6.5 and 7.5 (inactive form of VSV-G) *P < 0.05.
Mentions: To determine whether MSCs expressing VSV-G are better equipped to fuse with CMs than unmanipulated counterparts, vMSCs or MSCs were cocultured with CMs. To distinguish cell types in cocultures, MSCs and vMSCs were stained with Red CellTracker, while CMs were stained with Green CellTracker fluorescent probes prior to being combined. Since VSV-G undergoes a conformational change from its inactive form to its active form at pH < 6.2 [54, 55], cocultures were briefly incubated (2 min) with fusion medium of pH = 5.5. Image analysis of vMSC-CM cocultures treated with fusion medium of pH = 5.5 revealed cells with colocalization of green and red fluorescence, indicating fusion events, while MSC-CM cocultures under the same pH condition exhibited limited colocalization (Figure 2(a)). To accurately assess the amount of cell fusion, cocultures were harvested 24 hours after seeding and analyzed via flow cytometry (double positive cells correspond to fusion events). vMSCs treated with acidic medium (pH 5.5) had significantly higher rates of fusion with CMs (4.7% ± 1.1%) than MSCs treated in the same way (i.e., spontaneous fusion, 1.4% ± 0.2%) (P < 0.05) (Figure 2(b)). Further, the percentage of fusion products identified in vMSC-CM cocultures exposed to fusion medium of pH = 6.5 or 7.5 (maintaining VSV-G in the inactive form) did not differ from MSC-CM cultures (n = 3 replicates per sample per trial for 3 trials, Figure 2(c)).

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