There is a crucial need for alternatives to native vein or artery for vascular surgery. The clinical efficacy of synthetic, allogeneic or xenogeneic vessels has been limited by thrombosis, rejection, chronic inflammation and poor mechanical properties. Using adult human fibroblasts extracted from skin biopsies harvested from individuals with advanced cardiovascular disease, we constructed tissue-engineered blood vessels (TEBVs) that serve as arterial bypass grafts in long-term animal models. These TEBVs have mechanical properties similar to human blood vessels, without relying upon synthetic or exogenous scaffolding. The TEBVs are antithrombogenic and mechanically stable for 8 months in vivo. Histological analysis showed complete tissue integration and formation of vasa vasorum. The endothelium was confluent and positive for von Willebrand factor. A smooth muscle-specific alpha-actin-positive cell population developed within the TEBV, suggesting regeneration of a vascular media. Electron microscopy showed an endothelial basement membrane, elastogenesis and a complex collagen network. These results indicate that a completely biological and clinically relevant TEBV can be assembled exclusively from an individual's own cells.
Multinucleated myofibers are the functional contractile units of skeletal muscle. In adult muscle, mononuclear satellite cells, located between the basal lamina and the plasmalemma of the myofiber, are the primary myogenic stem cells. This chapter describes protocols for isolation, culturing and immunostaining of myofibers from mouse skeletal muscle. Myofibers are isolated intact and retain their associated satellite cells. The first protocol discusses myofiber isolation from the flexor digitorum brevis (FDB) muscle. These short myofibers are cultured in dishes coated with PureCol collagen (formerly known as Vitrogen) using a serum replacement medium. Employing such culture conditions, satellite cells remain associated with the myofibers, undergoing proliferation and differentiation on the myofiber surface. The second protocol discusses the isolation of longer myofibers from the extensor digitorum longus (EDL) muscle. Different from the FDB preparation, where multiple myofibers are processed together, the longer EDL myofibers are typically processed and cultured individually in dishes coated with Matrigel using a growth factor rich medium. Under these conditions, satellite cells initially remain associated with the parent myofiber and later migrate away, giving rise to proliferating and differentiating progeny. Myofibers from other types of muscles, such as diaphragm, masseter, and extraocular muscles can also be isolated and analyzed using protocols described herein. Overall, cultures of isolated myofibers provide essential tools for studying the interplay between the parent myofiber and its associated satellite cells. The current chapter provides background, procedural, and reagent updates, and step-by-step images of FDB and EDL muscle isolations, not included in our 2005 publication in this series.
Background:The cause of leiomyosarcoma (LMS) is unknown. Results: Experimental modulation of versican levels in LMS cells resulted in altered cell proliferation, adhesion, migration, and tumor growth. Conclusion: Versican regulates the growth of LMS tumors in a mouse model. Significance: Collectively, these results suggest targeting versican in the treatment of LMS.
The thoracic diaphragm is a unique skeletal muscle composed of costal, crural, and central tendon domains. Although commonly described in medical textbooks, newer insights into the diaphragm cell composition are scarce. Here, using reporter mice, combined with gene expression analyses of whole tissues and primary cultures, we compared the diaphragm domains and their myogenic progenitors (i.e., Pax3/7 satellite cells). The outcomes of these analyses underscore the similarities between the myogenic aspects of the costal and crural domains. Expression levels of all myogenic genes examined (except Pax3) were strongly affected in mdx (dystrophin-null) mice and accompanied by an increase in fibrosis- and adiposity-related gene expression. Cell culture studies further indicated the presence of a non-myogenic Pax3-expressing population, potentially related to vascular mural cells. We additionally investigated the diaphragm vasculature. XLacZ4 and Sca1-GFP transgenes allowed a fine definition of the arterial and microvasculature network based on reporter expression in mural cells and capillary endothelium, respectively. We also provide insights into the organization of the diaphragm venous system, especially apparent in the central tendon and exhibiting arcades lined with fat-containing cells. The novel information in this “contemporary atlas” can be further explored in the context of diaphragm pathology and genetic disorders.
A promising method to fabricate tissue-engineered blood vessels is to have cells synthesize the supportive extracellular matrix scaffold of the tissue-engineered blood vessel; however, a shortcoming of this method has been limited elastogenesis. Previously, we found that arterial smooth muscle cells (ASMCs) produced significant quantities of elastin when transduced with splice variant 3 of the proteoglycan versican (V3). In this study, we assessed whether elastogenesis and the structural properties of entirely cell-derived engineered vascular constructs could be improved by the incorporation of V3-transduced rat ASMCs. After 18 weeks of culture, V3 constructs had more tropoelastin, more elastin crosslinks, higher burst strengths, greater elasticity, and thicker collagen fiber bundles compared with empty-vector controls. The expression of elastin and elastin-associated proteins was increased in V3 and control ASMC monolayer cultures when ascorbic acid, which promotes collagen synthesis and inhibits elastogenesis, was removed from the medium. Engineered vascular constructs with ascorbate withdrawn for 14 weeks, after an initial 4-week exposure to ascorbate, exhibited increased elastin, desmosine content, elasticity, and burst strength compared with constructs exposed continuously to ascorbate. Our results show that V3 coupled with limited exposure to ascorbate promotes elastogenesis and improves the structural and functional properties of engineered vascular constructs.
Multinucleated myofibers, the functional contractile units of adult skeletal muscle, harbor mononuclear Pax7 myogenic progenitors on their surface between the myofiber basal lamina and plasmalemma. These progenitors, known as satellite cells, are the primary myogenic stem cells in adult muscle. This chapter describes our laboratory protocols for isolating, culturing, and immunostaining intact myofibers from mouse skeletal muscle as a means for studying satellite cell dynamics. The first protocol discusses myofiber isolation from the flexor digitorum brevis (FDB) muscle. These short myofibers are plated in dishes coated with PureCol collagen (formerly known as Vitrogen) and maintained in a mitogen-poor medium (± supplemental growth factors). Employing such conditions, satellite cells remain at the surface of the parent myofiber while synchronously undergoing a limited number of proliferative cycles and rapidly differentiate. The second protocol discusses the isolation of longer myofibers from the extensor digitorum longus (EDL) muscle. These EDL myofibers are routinely plated individually as adherent myofibers in wells coated with Matrigel and maintained in a mitogen-rich medium, conditions in which satellite cells migrate away from the parent myofiber, proliferate extensively, and generate numerous differentiating progeny. Alternatively, these EDL myofibers can be plated as non-adherent myofibers in uncoated wells and maintained in a mitogen-poor medium (± supplemental growth factors), conditions that retain satellite cell progeny at the myofiber niche similar to the FDB myofiber cultures. However, the adherent myofiber format is our preferred choice for monitoring satellite cells in freshly isolated (Time 0) myofibers. We conclude this chapter by promoting the Nestin-GFP transgenic mouse as an efficient tool for direct analysis of satellite cells in isolated myofibers. While satellite cells have been often detected by their expression of the Pax7 protein or the Myf5 knockin reporter (approaches that are also detailed herein), the Nestin-GFP reporter distinctively permits quantification of satellite cells in live myofibers, which enables linking initial Time 0 numbers and subsequent performance upon culturing. We additionally point out to the implementation of the Nestin-GFP transgene for monitoring other selective cell lineages as illustrated by GFP expression in capillaries, endothelial tubes and neuronal cells. Myofibers from other types of muscles, such as diaphragm, masseter, and extraocular, can also be isolated and analyzed using protocols described herein. Collectively, this chapter provides essential tools for studying satellite cells in their native position and their interplay with the parent myofiber.
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