Cindy S. Cheng

Physiology and metabolism of tissue-engineered skeletal muscle

Skeletal muscle is a major target for tissue engineering, given its relative size in the body, fraction of cardiac output that passes through muscle beds, as well as its key role in energy metabolism and diabetes, and the need for therapies for muscle diseases such as muscular dystrophy and sarcopenia. To date, most studies with tissue-engineered skeletal muscle have utilized murine and rat cell sources. On the other hand, successful engineering of functional human muscle would enable different applications including improved methods for preclinical testing of drugs and therapies. Some of the requirements for engineering functional skeletal muscle include expression of adult forms of muscle proteins, comparable contractile forces to those produced by native muscle, and physiological force–length and force–frequency relations. This review discusses the various strategies and challenges associated with these requirements, specific applications with cultured human myoblasts, and future directions.

Design considerations for an integrated microphysiological muscle tissue for drug and tissue toxicity testing.

Microphysiological systems provide a tool to simulate normal and pathological function of organs for prolonged periods. These systems must incorporate the key functions of the individual organs and enable interactions among the corresponding microphysiological units. The relative size of different microphysiological organs and their flow rates are scaled in proportion to in vivo values. We have developed a microphysiological three-dimensional engineered human skeletal muscle system connected to a circulatory system that consists of a tissue-engineered blood vessel as part of a high-pressure arterial system. The engineered human skeletal muscle tissue reproduces key mechanical behaviors of skeletal muscle in vivo. Pulsatile flow is produced using a novel computer-controlled magnetically activated ferrogel. The system is versatile and the muscle unit can be integrated with other organ systems. Periodic monitoring of biomechanical function provides a non-invasive assessment of the health of the tissue and a way to measure the response to drugs and toxins.

Conditions that promote primary human skeletal myoblast culture and muscle differentiation in vitro

Conditions under which skeletal myoblasts are cultured in vitro are critical to growth and differentiation of these cells into mature skeletal myofibers. We examined several culture conditions that promoted human skeletal myoblast (HSkM) culture and examined the effect of microRNAs and mechanical stimulation on differentiation. Culture conditions for HSkM are different from those that enable rapid C2C12 myoblast differentiation. Culture on a growth factor-reduced Matrigel (GFR-MG)-coated surface in 2% equine serum-supplemented differentiation medium to promote HSkM differentiation under static conditions was compared with culture conditions used for C2C12 cell differentiation. Such conditions led to a >20-fold increase in myogenic miR-1, miR-133a, and miR-206 expression, a >2-fold increase in myogenic transcription factor Mef-2C expression, and an increase in sarcomeric α-actinin protein. Imposing ±10% cyclic stretch at 0.5 Hz for 1 h followed by 5 h of rest over 2 wk produced a >20% increase in miR-1, miR-133a, and miR-206 expression in 8% equine serum and a >35% decrease in 2% equine serum relative to static conditions. HSkM differentiation was accelerated in vitro by inhibition of proliferation-promoting miR-133a: immunofluorescence for sarcomeric α-actinin exhibited accelerated development of striations compared with the corresponding negative control, and Western blotting showed 30% more α-actinin at day 6 postdifferentiation. This study showed that 100 μg/ml GFR-MG coating and 2% equine serum-supplemented differentiation medium enhanced HSkM differentiation and myogenic miR expression and that addition of antisense miR-133a alone can accelerate primary human skeletal muscle differentiation in vitro.

Cell Density and Joint microRNA-133a and microRNA-696 Inhibition Enhance Differentiation and Contractile Function of Engineered Human Skeletal Muscle Tissues.

To utilize three-dimensional (3D) engineered human skeletal muscle tissue for translational studies and in vitro studies of drug toxicity, there is a need to promote differentiation and functional behavior. In this study, we identified conditions to promote contraction of engineered human skeletal muscle bundles and examined the effects of transient inhibition of microRNAs (miRs) on myogenic differentiation and function of two-dimensional (2D) and 3D cultures of human myotubes. In 2D cultures, simultaneously inhibiting both miR-133a, which promotes myoblast proliferation, and miR-696, which represses oxidative metabolism, resulted in an increase in sarcomeric α-actinin protein and the metabolic coactivator PGC-1α protein compared to transfection with a scrambled miR sequence (negative control). Although PGC-1α was elevated following joint inhibition of miRs 133a and 696, there was no difference in myosin heavy chain (MHC) protein isoforms. 3D engineered human skeletal muscle myobundles seeded with 5 × 10(6) human skeletal myoblasts (HSkM)/mL and cultured for 2 weeks after onset of differentiation consistently did not contract when stimulated electrically, whereas those seeded with myoblasts at 10 × 10(6) HSkM/mL or higher did contract. When HSkM were transfected with both anti-miRs and seeded into fibrin hydrogels and cultured for 2 weeks under static conditions, twitch and tetanic specific forces after electrical stimulation were greater than for myobundles prepared with HSkM transfected with scrambled sequences. Immunofluorescence and Western blots of 3D myobundles indicate that anti-miR-133a or anti-miR-696 treatment led to modest increases in slow MHC, but no consistent increase in fast MHC. Similar to results in 2D, only myobundles prepared with myoblasts treated with anti-miR-133a and anti-miR-696 produced an increase in PGC-1α mRNA. PGC-1α targets were differentially affected by the treatment. HIF-2α mRNA showed an expression pattern similar to that of PGC-1α mRNA, but COXII mRNA levels were not affected by the anti-miRs. Overall, joint inhibition of miR-133a and miR-696 accelerated differentiation, elevated the metabolic coactivator PGC-1α, and increased the contractile force in 3D engineered human skeletal muscle bundles.

Private Transnational Governance of Economic Development: International Development Aid

This paper examines the role of private actors in international development aid, focusing on four new actors or actors who have in recent years taken on new roles: (1) transnational aid NGOs as a channel of delivery for public (governmental) development aid; (2) transnational aid NGOs as development agenda-setters; (3) foundations and corporations as sources of development aid; (4) transnational aid NGOs as private providers of privately funded aid. For each of them, we discuss the sources of their power and influence and examine how ideas about development and aid have shaped the rise of these new players, identifying throughout promising and important areas for future research. In the final section, we consider peer-to-peer development aid and other innovative attempts to solve pervasive accountability problems in development aid. The paper was written as a chapter for the forthcoming Handbook on Global Economic Governance (Routledge), for which is has been accepted for publication in 2013. The posted version is the pre-copyedit manuscript.

Endothelial Progenitor Cells for Vascular Repair

Endothelial progenitor cells (EPCs), present in the blood and bone marrow, represent a potential source of endothelial cells for repair of injured blood vessels, neovascularization, and tissue engineering. EPCs are present at low levels in peripheral blood, although their numbers increase in response to cytokines, VEGF, and statins. There are at least two types of EPCs characterized following in vitro culture: colony-forming unit ECs (CFU-ECs) and endothelial colony-forming cells (ECFCs). CFU-ECs appear early in culture, have limited ability to proliferate, and share markers for endothelial cells and monocytes. In contrast, ECFCs appear later in culture, grow rapidly, and to large numbers express only endothelial cell markers. This chapter examines the properties of these EPCs, in vitro and in vivo studies using these two cell types, and the potential of these EPCs for therapeutic applications.