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<p>The development of a functional tissue-engineered human skeletal muscle model in
vitro would provide an excellent platform on which to study the process of myogenesis,
various musculoskeletal disease states, and drugs and therapies for muscle toxicity.
We developed a protocol to culture human skeletal muscle bundles in a fibrin hydrogel
under static conditions capable of exerting active contractions. Additionally, we
demonstrated the use of joint miR-133a and miR-696 inhibition for acceleration of
muscle differentiation, elevation of active contractile force amplitudes, and increasing
Type II myofiber formation in vitro. </p><p>The global hypothesis that motivated this
research was that joint inhibition of miR-133a and miR-696 in isolated primary human
skeletal myoblasts would lead to accelerated differentiation of tissue-engineered
muscle constructs with higher proportion of Type I myofibers and that are capable
of significantly increased active contractile forces when subjected to electrical
stimulus. The proposed research tested the following specific hypotheses: (1) that
HSkM would require different culture conditions than those optimal for C2C12 culture
(8% equine serum in differentiation medium on uncoated substrates), as measured by
miR expression, (2) that joint inhibition of miR-133a and miR-696 would result in
2D human skeletal muscle cultures with accelerated differentiation and increased Type
I muscle fibers compared to control and individual inhibition of each miR, as measured
by protein and gene expression, (3) that joint inhibition of miR-133a and miR-696
in this functional 3D human skeletal muscle model would result in active contraction
significantly higher than control and individual inhibition by each miR, as measured
by isometric force testing, and finally (4) that specific co-culture conditions could
support a lamellar co-culture model in 3D of human cord blood-derived endothelial
cells (hCB-ECs) and HSkM capable of active contraction, as measured by isometric force
testing and immunofluorescence. </p><p>Major results of the dissertation are as follows.
Culture conditions of 100 μg/mL growth factor reduced-Matrigel-coated substrates
and 2% equine serum in differentiation medium were identified to improve human skeletal
myoblast culture, compared to conditions optimal for C2C12 cell culture (uncoated
substrates and 8% equine serum media). Liposomal transfection of human skeletal myoblasts
with anti-miR-133a and anti-miR-696 led to increased protein presence of sarcomeric
alpha-actinin and PGC-1alpha when cells were cultured in 2D for 2 weeks. Presence
of mitochondria and distribution of fiber type did not change with miR transfection
in a 2D culture. Joint inhibition also resulted in increased PPARGC1A gene expression
after 2 weeks of 2D culture. For muscle bundles in 3D, results suggest there exists
a myoblast seeding density threshold for the production of functional muscle. 5 x
106 myoblasts/mL did not produce active contraction, while 10 x 106 myoblasts/mL and
above were successful. Of the seeding densities studied, 15 x 106 myoblasts/mL resulted
in constructs that exerted the highest twitch and tetanus forces. Engineering of human
skeletal muscle from transfected cells led to significant increases in force amplitude
in joint inhibition compared to negative control (transfection with scrambled miR
sequence). Joint inhibition in myoblasts seeded into 3D constructs led to decreased
presence of slow myosin heavy chain and increased fast myosin heavy chain. Finally,
co-culture of functional human skeletal muscle with human cord blood-derived endothelial
cells is possible in 3.3% FBS in DMEM culture conditions, with significant increases
in force amplitudes at 48 and 96 hours of co-culture.</p>
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