Tissue-engineered human bioartificial muscles expressing a foreign recombinant protein for gene therapy.
Murine skeletal muscle cells transduced with foreign genes and tissue engineered in vitro into bioartificial muscles (BAMs) are capable of long-term delivery of soluble growth factors when implanted into syngeneic mice (Vandenburgh et al., 1996b). With the goal of developing a therapeutic cell-based protein delivery system for humans, similar genetic tissue-engineering techniques were designed for human skeletal muscle stem cells. Stem cell myoblasts were isolated, cloned, and expanded in vitro from biopsied healthy adult (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 significantly different between the two patient groups. Percent myoblasts per biopsy (73 +/- 6%), number of myoblast doublings prior to senescence in vitro (37 +/- 2), and myoblast doubling time (27 +/- 1 hr) were also not significantly different between the two patient groups. Fusion kinetics of the myoblasts were similar for the two groups after 20-22 doublings (74 +/- 2% myoblast fusion) when the biopsy samples had been expanded to 1 to 2 billion muscle cells, a number acceptable for human gene therapy use. The myoblasts from the two groups could be equally transduced ex vivo with replication-deficient retroviral expression vectors to secrete 0.5 to 2 microg of a foreign protein (recombinant human growth hormone, rhGH)/10(6) cells/day, and tissue engineered into human BAMs containing parallel arrays of differentiated, 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 implantable living protein secretory devices for therapeutic use.
Powell
C
C
Department of Molecular Pharmacology, Physiology, and Biotechnology, Brown University, Providence, RI 02912, USA.
Shansky
J
J
Del Tatto
M
M
Forman
D E
DE
Hennessey
J
J
Sullivan
K
K
Zielinski
B A
BA
Vandenburgh
H H
HH
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001187:Artificial Organs; D002454:Cell Differentiation; D002455:Cell Division; D005260:Female; D015316:Genetic Therapy; D013006:Growth Hormone / Q000235:genetics* / Q000627:therapeutic use; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D008875:Middle Aged; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism*; D011863:Radioimmunoassay; D011994:Recombinant Proteins / Q000235:genetics / Q000627:therapeutic use; D014161:Transduction, Genetic
D011994:Recombinant Proteins; D013006:Growth Hormone