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. 2011;6(10):e26381.
doi: 10.1371/journal.pone.0026381. Epub 2011 Oct 24.

Targeted Cell Fusion Facilitates Stable Heterokaryon Generation in Vitro and in Vivo

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Free PMC article

Targeted Cell Fusion Facilitates Stable Heterokaryon Generation in Vitro and in Vivo

Michael A Long et al. PLoS One. .
Free PMC article

Abstract

Induced cell fusion has enabled several important discoveries, including the phenomenon of nuclear reprogramming and may yet be applied as a novel therapy for degenerative diseases. However, existing fusogens lack the efficiency required to enable investigation of the epigenetic modifications underlying nuclear reprogramming and the specificity required for clinical application. Here we present a chimeric measles hemagglutinin, Hα7, which specifically and efficiently mediates the fusion of diverse cell types with skeletal muscle both in vitro and in vivo. When compared directly to polyethylene glycol, Hα7 consistently generated a substantial increase in heterokaryon yield and exhibited insignificant levels of toxicity. Moreover, this increased fusion efficiency enabled detection of chromatin modifications associated with nuclear reprogramming following Hα7-mediated fusion of human fibroblasts and mouse myotubes. Finally, Hα7 was also capable of increasing the contribution of transplanted fibroblasts to skeletal muscle repair in vivo, suggesting that this strategy could be used for therapeutic gene delivery.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Design, construction and characterization of Hα7.
(A–D) Evaluation of the anti-alpha7 integrin scFv by flow cytometry. The scFv (A, solid line) retains the ability of the parental monoclonal antibody (C, solid line) to stain C2C12 myoblasts, whereas neither antibody stains NIH/3T3 fibroblasts (B and D, solid line). In all plots, the staining level of cells incubated with secondary antibody alone is shown in dotted lines. (E) Schematic representation of Hα7, approximating the locations each blade (β1–β6) in the β-propeller fold as well as the location of mutations that abrogate CD46 binding (Y481A) and CD150 binding (R533A). The anti-alpha7 integrin scFv is displayed as a carboxy-terminal extension of the type II transmembrane glycoprotein. Standard one-letter abbreviations are used to denote amino acid residues. N: Amino-terminal cytoplasmic tail. TM: Transmembrane domain. (F) Hα7 mediates fusion of transfected 293T cells and differentiated C2C12 myotubes with an efficiency that is proportional to the amount of transfected plasmid and is dependent on the presence of the anti-alpha7 integrin scFv and the measles F protein. Data are shown as mean ± s.d. of three independent fusion experiments. (G) Morphology of myotubes following fusion. Scale bar, 100 µm
Figure 2
Figure 2. Verification of heterokaryon status following Hα7-mediated fusion.
(A) Two populations of 293T cells labeled with either GFP or mCherry and co-transfected with Hα7 and F do not fuse with one another. (B–D) Following co-culture of transfected human 293T cells with differentiated mouse C2C12 myotubes, elongated GFP-positive cells (B) express sarcomeric myosin heavy-chain (C) and contain multiple nuclei (D, merged). (E) Differential DAPI staining demonstrates the presence of both human and mouse nuclei within heterokaryons. Mouse nuclei contain dense chromocenters (arrow) while human nuclei stain diffusely and exhibit dark nucleoli (arrowhead). (F) Fluorescent in situ hybridization of human α-satellite DNA (red) and mouse γ-satellite DNA (green) further confirms the presence of both human and mouse nuclei within heterokaryons. Scale bars, 25 µm (A–D), 20 µm (E), 10 µm (F)
Figure 3
Figure 3. Comparison of Hα7 and PEG-mediated fusion efficiencies.
(A–C) The total number of myotubes (white bars) as well as the number of GFP-positive myotubes (green bars) was determined by visual inspection of randomly selected, low power (5x) fields at 1 day (A), 2 days (B) and 3 days (C) post-fusion. The number of GFP-positive myotubes observed following Hα7-mediated fusion was significantly higher on all days than the number observed following PEG-mediated fusion (green asterisks). The total number of myotubes surviving PEG treatment was significantly lower than the total number observed in co-cultures lacking any fusogen (black asterisks). ***: p<0.001 (unpaired t-test). Data are shown as mean ± s.d. of three independent fusion experiments.
Figure 4
Figure 4. Nuclear reprogramming following Hα7 or PEG-mediated fusion.
(A) Endpoint RT-PCR reactions demonstrating specificity of the primers used to amplify the human MyoD and myogenin transcripts. (B–C) Quantitative RT-PCR analysis reveals robust transcription of human MyoD following Hα7-mediated fusion (B) of MRC-5 cells and differentiating C2C12 myotubes, whereas the level of MyoD transcript observed following PEG-mediated fusion (C) is weak by comparison. (D) Likewise, the level of human myogenin transcript detected following Hα7-mediated fusion (black bars) was greater than the level detected following PEG-mediated fusion (white bars) at each timepoint. All quantitative RT-PCR values were normalized to β-actin transcript levels and subsequently to the mean expression level of Hα7-containing cultures on Day 1. (E) Endpoint PCR reactions demonstrating specificity of the primers used to amplify a segment of the human MyoD promoter (lanes 1 and 2), normal rabbit IgG chromatin immunoprecipitation (CHIP) control performed 8 days post-fusion (lane 3) and an αH3K9/K14 CHIP control performed on MRC-5 cells prior to fusion (lane 4). (F) Chromatin immunoprecipitation reveals early and stable induction of histone H3K9/K14 acetylation at the human MyoD promoter following Hα7-mediated fusion of MRC-5 cells and differentiating C2C12 myotubes. *: p<0.05 (unpaired t-test). All data are shown as mean ± s.d. of three independent fusion experiments.
Figure 5
Figure 5. Hα7-mediated fusion in vivo.
(A,B) Mouse embryonic fibroblasts infected with lentiviruses encoding Hα7-IRES-GFP and F-IRES-YFP (A) or Hα7-IRES-GFP alone (B) were purified by flow cytometry and transplanted intramuscularly into wild-type recipients. (C) Cells expressing Hα7 and F (MEFHα7/F) efficiently fused with host myofibers and this process was greatly augmented by co-injection of notexin. Conversely, cells expressing Hα7 alone (MEFHα7) did not fuse efficiently with host myofibers regardless of the presence or absence of notexin. Data are shown as mean ± s.d. **: p<0.01 (unpaired t-test). (D–F) Representative images demonstrating GFP-positive fibers (green) surrounded by a laminin sheath (red) in an undamaged recipient of MEFHα7/F cells (D), a notexin-damaged recipient of MEFHα7/F cells (E) and a notexin-damaged recipient of MEFHα7 cells (F). Scale bar, 100 µm.

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