These data indicate that the myocardin protein is a strong inducer of both smooth and cardiac muscle genes, but that additional factors are necessary to fully commit cells to either cardiac or smooth muscle cell fates.

Bone marrow-derived human mesenchymal stem cells (hMSCs) lack the Coxsackie-adenovirus (Ad) receptor and thus are poorly transduced by vectors based on human Ad serotype 5 (Ad5). We investigated whether this problem could be overcome by using tropism-modified Ad5 vectors carrying fiber shaft domains and knobs of different human species B Ads (Ad5FBs). To allow quantitative analyses, these vectors coded for the enhanced green fluorescent protein (eGFP). Transgene expression analysis showed superior transduction of hMSCs by all Ad5FBs tested as compared with conventional Ad5 vectors. This was evident both by the frequency of eGFP-positive cells and by the eGFP level per cell. Highly efficient transduction of hMSCs, with limited variability between cells from different donors, was achieved with vectors displaying fiber domains of Ad serotypes 50, 35, and 16. These findings could not be reconciled with the very low levels of CD46, a recently identified receptor for species B Ads, on hMSCs, suggesting that AdFBs probably use receptors other than CD46 to enter these cells. We further observed that high eGFP levels were maintained in replication-restricted hMSCs for more than 30 days. In dividing hMSCs, foreign DNA delivered by Ad5FBs was expressed in a large fraction of the cells for approximately 3 weeks without compromising their replication capacity. Importantly, the transduced hMSCs retained their capacity to differentiate into adipocytes and osteoblasts when exposed to the appropriate stimuli.

Duchenne muscular dystrophy (DMD) is the most prevalent inheritable muscle disease. It is caused by mutations in the approximately 2.5-megabase dystrophin (Dys) encoding gene. Therapeutic attempts at DMD have relied on injection of allogeneic Dys-positive myoblasts. The immune rejection of these cells and their limited availability have prompted the search for alternative therapies and sources of myogenic cells. Stem cell-based gene therapy aims to restore tissue function by the transplantation of gene-corrected autologous cells. It depends on (i) the capacity of stem cells to participate in tissue regeneration and (ii) the efficient genetic correction of defective autologous stem cells. We explored the potential of bone marrow-derived human mesenchymal stem cells (hMSCs) genetically modified with the full-length Dys-coding sequence to engage in myogenesis. By tagging hMSCs with enhanced green fluorescent protein (EGFP) or the membrane dye PKH26, we demonstrated that they could participate in myotube formation when cultured together with differentiating human myoblasts. Experiments performed with EGFP-marked hMSCs and DsRed-labeled DMD myoblasts revealed that the EGFP-positive DMD myotubes were also DsRed-positive indicating that hMSCs participate in human myogenesis through cellular fusion. Finally, we showed that hMSCs transduced with a tropism-modified high-capacity hybrid viral vector encoding full-length Dys could complement the genetic defect of DMD myotubes.

Duchenne muscular dystrophy (DMD) is caused by mutations in the X chromosome-linked DMD gene, which encodes the sarcolemma-stabilizing protein-dystrophin. Initial attempts at DMD therapy deployed muscle progenitor cells from healthy donors. The utilization of these cells is, however, hampered by their immunogenicity, while those from DMD patients are scarce and display limited ex vivo replication. Nonmuscle cells with myogenic capacity may offer valuable alternatives especially if, to allow autologous transplantation, they are amenable to genetic intervention. As a paradigm for therapeutic gene transfer by heterotypic cell fusion we are investigating whether human mesenchymal stem cells (hMSCs) can serve as donors of recombinant DMD genes for recipient human muscle cells. Here, we show that forced MyoD expression in hMSCs greatly increases their tendency to participate in human myotube formation turning them into improved DNA delivery vehicles. Efficient loading of hMSCs with recombinant DMD was achieved through a new tropism-modified high-capacity adenoviral (hcAd) vector directing striated muscle-specific synthesis of full-length dystrophin. This study introduces the principle of genetic complementation of gene-defective cells via directed cell fusion and provides an initial framework to test whether transient MyoD synthesis in autologous, gene-corrected hMSCs increases their potential for treating DMD and, possibly, other muscular dystrophies.

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