Limits...
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

Phenotypic and morphologic characteristics of vMSC-CM fusion products. After pH-induced fusion, fusion products display two distinct morphologies: CM-like (round) and MSC-like (spread). Fusion products were probed for markers indicative of MSC (CD105) and CM (MF20) phenotype. (a) MF20+/CD105+ cells are significantly increased with vMSCs at day 5 relative to untransfected MSCs, while the culture environment (MSC medium and CM medium) had no effect on the percent of dual positive events in vMSC-CM cocultures at day 5. *P < 0.05. (b) Morphology of MF20+/CD105+ cells was typically CM-like and culture environment did not alter this tendency. *P < 0.05. (c) Representative morphologies of MF20+/CD105+ cells. White arrows indicate MF20+/CD105+ cells. Scale bar = 25 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3369562&req=5

fig3: Phenotypic and morphologic characteristics of vMSC-CM fusion products. After pH-induced fusion, fusion products display two distinct morphologies: CM-like (round) and MSC-like (spread). Fusion products were probed for markers indicative of MSC (CD105) and CM (MF20) phenotype. (a) MF20+/CD105+ cells are significantly increased with vMSCs at day 5 relative to untransfected MSCs, while the culture environment (MSC medium and CM medium) had no effect on the percent of dual positive events in vMSC-CM cocultures at day 5. *P < 0.05. (b) Morphology of MF20+/CD105+ cells was typically CM-like and culture environment did not alter this tendency. *P < 0.05. (c) Representative morphologies of MF20+/CD105+ cells. White arrows indicate MF20+/CD105+ cells. Scale bar = 25 μm.

Mentions: Many studies have demonstrated that stem cell programming is influenced by the microenvironment [56–58]. To determine whether the phenotypic fate of vMSC-CM fusion products could be regulated by the microenvironment, following treatment with fusion media, we cultured vMSC-CM fusion products under either MSC-specific or CM-specific culture conditions and examined the incidence of fusion and morphology of MSC-CM fusion products. At days 5 and 7 following the induction of cell fusion, cocultures were probed with CM and MSC specific antibodies (anti-MF20 and anti-CD105, respectively, n = 1 replicate per sample per trial for 3 trials). At day 5, vMSC-CM cocultures contained a relatively high number of cells that expressed both MF20 and CD105 and the percentage of MF20+/CD105+ cells relative to the total cell number was significantly greater than that of MSC-CM cocultures for both culture conditions (P < 0.005) (Figure 3(a)). Of note, the percentage of MF20+/CD105+ cells was much higher than the percentage of double positive cells detected using CellTracker dyes and flow cytometry (Figure 2(b)). This could reflect the loss of VSV-G sustained by cell harvest, the different analytical approach (i.e., flow cytometry versus image analysis) and/or the behavior of fusion products between day 1 and day 5 (i.e., proliferation). By day 7, the percentage of MF20+/CD105+ cells decreased to levels not statistically different from controls for both culture conditions. At the same time, the number of cells expressing MF20 alone increased substantially for both culture conditions. The change in percentage of MF20+/CD105+ cells from day 5 to day 7 could reflect death of fusion products, or programming of the MSC fusion partner to a cardiomyocyte phenotype or both. If death of fusion products occurred, one would expect unfused CMs and MSCs to proliferate to fill the voids of the culture space. Interestingly, only the CM population increased from day 5 to day 7 and at rates significantly higher than that of control cultures, suggesting at least a portion of fusion products were maintained, and ultimately adopted a cardiomyocyte-like phenotype. This result was observed independent of the culture conditions. Of note, this experimental approach does not exclude the possibility that metaplasia rather than fusion occurred, that is, MSCs differentiate into CMs as a consequence of soluble factors in the coculture medium and maintain (at least transiently) expression of each cell type. However, MF20+/CD105+ cells were rare in MSC-CM cocultures, suggesting metaplasia alone cannot account for coexpression of MF20+/CD105+ or subsequent loss of MF20+/CD105+ cells. In addition, MF20+/CD105+ cells exhibited two distinct morphologies; some were long and spread, displaying MSC-like morphology (MSC medium = 16.59% ± 6.32%; CM medium = 14.03% ± 1.59%) while the majority (P < 0.05) were round and cobblestone-like, indicative of CM-like morphology (MSC medium = 80.49% ± 10.45%; CM medium = 85.97% ± 1.60%) (Figures 3(b) and 3(c), Supplementary Figure 1B). These results further support the possibility that CM nuclear material and cytoplasmic elements direct programming of MSC-CM fusion products independent of culture conditions.


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)

Phenotypic and morphologic characteristics of vMSC-CM fusion products. After pH-induced fusion, fusion products display two distinct morphologies: CM-like (round) and MSC-like (spread). Fusion products were probed for markers indicative of MSC (CD105) and CM (MF20) phenotype. (a) MF20+/CD105+ cells are significantly increased with vMSCs at day 5 relative to untransfected MSCs, while the culture environment (MSC medium and CM medium) had no effect on the percent of dual positive events in vMSC-CM cocultures at day 5. *P < 0.05. (b) Morphology of MF20+/CD105+ cells was typically CM-like and culture environment did not alter this tendency. *P < 0.05. (c) Representative morphologies of MF20+/CD105+ cells. White arrows indicate MF20+/CD105+ cells. Scale bar = 25 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3369562&req=5

fig3: Phenotypic and morphologic characteristics of vMSC-CM fusion products. After pH-induced fusion, fusion products display two distinct morphologies: CM-like (round) and MSC-like (spread). Fusion products were probed for markers indicative of MSC (CD105) and CM (MF20) phenotype. (a) MF20+/CD105+ cells are significantly increased with vMSCs at day 5 relative to untransfected MSCs, while the culture environment (MSC medium and CM medium) had no effect on the percent of dual positive events in vMSC-CM cocultures at day 5. *P < 0.05. (b) Morphology of MF20+/CD105+ cells was typically CM-like and culture environment did not alter this tendency. *P < 0.05. (c) Representative morphologies of MF20+/CD105+ cells. White arrows indicate MF20+/CD105+ cells. Scale bar = 25 μm.
Mentions: Many studies have demonstrated that stem cell programming is influenced by the microenvironment [56–58]. To determine whether the phenotypic fate of vMSC-CM fusion products could be regulated by the microenvironment, following treatment with fusion media, we cultured vMSC-CM fusion products under either MSC-specific or CM-specific culture conditions and examined the incidence of fusion and morphology of MSC-CM fusion products. At days 5 and 7 following the induction of cell fusion, cocultures were probed with CM and MSC specific antibodies (anti-MF20 and anti-CD105, respectively, n = 1 replicate per sample per trial for 3 trials). At day 5, vMSC-CM cocultures contained a relatively high number of cells that expressed both MF20 and CD105 and the percentage of MF20+/CD105+ cells relative to the total cell number was significantly greater than that of MSC-CM cocultures for both culture conditions (P < 0.005) (Figure 3(a)). Of note, the percentage of MF20+/CD105+ cells was much higher than the percentage of double positive cells detected using CellTracker dyes and flow cytometry (Figure 2(b)). This could reflect the loss of VSV-G sustained by cell harvest, the different analytical approach (i.e., flow cytometry versus image analysis) and/or the behavior of fusion products between day 1 and day 5 (i.e., proliferation). By day 7, the percentage of MF20+/CD105+ cells decreased to levels not statistically different from controls for both culture conditions. At the same time, the number of cells expressing MF20 alone increased substantially for both culture conditions. The change in percentage of MF20+/CD105+ cells from day 5 to day 7 could reflect death of fusion products, or programming of the MSC fusion partner to a cardiomyocyte phenotype or both. If death of fusion products occurred, one would expect unfused CMs and MSCs to proliferate to fill the voids of the culture space. Interestingly, only the CM population increased from day 5 to day 7 and at rates significantly higher than that of control cultures, suggesting at least a portion of fusion products were maintained, and ultimately adopted a cardiomyocyte-like phenotype. This result was observed independent of the culture conditions. Of note, this experimental approach does not exclude the possibility that metaplasia rather than fusion occurred, that is, MSCs differentiate into CMs as a consequence of soluble factors in the coculture medium and maintain (at least transiently) expression of each cell type. However, MF20+/CD105+ cells were rare in MSC-CM cocultures, suggesting metaplasia alone cannot account for coexpression of MF20+/CD105+ or subsequent loss of MF20+/CD105+ cells. In addition, MF20+/CD105+ cells exhibited two distinct morphologies; some were long and spread, displaying MSC-like morphology (MSC medium = 16.59% ± 6.32%; CM medium = 14.03% ± 1.59%) while the majority (P < 0.05) were round and cobblestone-like, indicative of CM-like morphology (MSC medium = 80.49% ± 10.45%; CM medium = 85.97% ± 1.60%) (Figures 3(b) and 3(c), Supplementary Figure 1B). These results further support the possibility that CM nuclear material and cytoplasmic elements direct programming of MSC-CM fusion products independent of culture conditions.

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