Tissue-engineered human bioartificial muscles expressing a foreign recombinant protein for gene therapy

Murine skeletal muscle cells transduced with foreign genes and tissue engin eered in vitro into bioartificial muscles (BAMs) are capable of long-term d elivery of soluble growth factors when implanted into syngeneic mice (Vande nburgh et al,, 1996b), With the goal of developing a therapeutic cell-based protein delivery system for humans, similar genetic tissue-engineering tec hniques were designed for human skeletal muscle stem cells. Stem cell myobl asts were isolated, cloned, and expanded in vitro from biopsied healthy adu lt (mean age, 42 +/- 2 years), and elderly congestive heart failure patient (mean age, 76 +/- 1 years) skeletal muscle. Total cell yield varied widely between biopsies (50 to 672 per 100 mg of tissue, N = 10), but was not sig nificantly different between the two patient groups, Percent myoblasts per biopsy (73 +/- 6%), number of myoblast doublings prior to senescence in vit ro (37 +/- 2), and myoblast doubling time (27 +/- 1 hr) were also not signi ficantly different between the two patient groups, Fusion kinetics of the m yoblasts were similar for the two groups after 20-22 doublings (74 +/- 2% m yoblast fusion) when the biopsy samples had been expanded to 1 to 2 billion muscle cells, a number acceptable for human gene therapy use. The myoblast s from the two groups could be equally transduced ex vivo with replication- deficient retroviral expression vectors to secrete 0.5 to 2 mu g of a forei gn protein (recombinant human growth hormone, rhGH)/10(6) cells/day, and ti ssue engineered into human BAMs containing parallel arrays of differentiate d, postmitotic myofibers, This work suggests that autologous human skeletal myoblasts from a potential patient population can be isolated, genetically modified to secrete foreign proteins, and tissue engineered into implantab le living protein secretory devices for therapeutic use.