Fibroblasts influence muscle progenitor differentiation and alignment in contact independent and dependent manners in organized co-culture devices

Abstract

Myoblasts are precursor muscle cells that lie nascent to mature skeletal muscle. Once muscle is damaged, these cells migrate, fuse, and regenerate the muscle tissue. It is known that skeletal muscle can partially regenerate in vivo after muscle tissue damage. However, this regeneration does not always occur, especially in more severe injuries. Cellular therapy using tissue-engineering approaches has been shown to improve organ repair and function. To exploit potential benefits of using cell therapy as an avenue for skeletal muscle repair, it is important to understand the cellular dynamics underlying skeletal myocyte formation and growth. Cardiac fibroblasts have been shown to have a major influence on cardiomyocyte function, repair, and overall spatial distribution. However, little is known regarding fibroblasts’ role on skeletal myocyte function. In this study, we utilized a reconfigurable co-culture device to understand the contact and paracrine effects of fibroblasts on skeletal myocyte alignment and differentiation using murine myoblast and fibroblast cell lines. We demonstrate that myotube alignment is increased by direct contact with fibroblasts, while myotube differentiation is reduced both in the gap and contact configurations with fibroblasts after 6 days of co-culture. Furthermore, neutralizing antibodies to FGF-2 can block these effects of fibroblasts on myotube differentiation and alignment. Finally, bi-directional signaling is critical to the observed myoblast-fibroblast interactions, since conditioned media could not reproduce the same effects observed in the gap configuration. These findings could have direct implications on cell therapies for repairing skeletal muscle, which have only utilized skeletal myoblasts or stem cell populations alone.

Engineering an Injectable Muscle-Specific Microenvironment for Improved Cell Delivery Using a Nanofibrous Extracellular Matrix Hydrogel

“…11 We hypothesized that a nanofibrous ECM hydrogel derived from decellularized skeletal muscle would create a supportive microenvironment for cell delivery to skeletal muscle. To further create a supportive niche, we also examined co-delivery of skeletal muscle fibroblasts, stromal support cells, which have been previously shown to improve myoblast viability and maturation in vitro through both soluble and cell contact signaling events, 12 but have yet to be tested in vivo . Here we show that both the complex nanofibrous ECM hydrogel scaffold and delivery with a stromal support cell improves cell viability and differentiation in vitro , which translated to improved cell viability, engraftment, and ischemic limb perfusion in vivo .…”

Macro-to-Micro Interface for the Control of Cellular Organization

“…This device has proven to be a powerful biological tool and has played a critical role in recent discoveries in our laboratory related to cell-cell signaling in cultured liver tissue [7, 8] and muscle [16]. More broadly, this class of devices should prove to be generally applicable to many kinds of studies involving cell-cell interaction, for example stem cells and supportive feeder layers, or tumors and their surrounding stroma.…”

“…In cases when one comb happens to pop out of plane during assembly (<10% of cases), the misalignment can be readily detected by microscope inspection and quickly fixed by disassembly and reassembly. The positioning accuracy of the device has proven adequate for the study of many types of cell-cell interactions [7,8,16]. Contact-dependent signaling is achieved between cell populations on adjoining fingers in the contact configuration, and cells are unable to migrate across the 80 μm separation in the gap configuration.…”

Patterning of sharp cellular interfaces with a reconfigurable elastic substrate

“…Our previously reported comb device 4 consists of microfabricated silicon parts that are independently seeded with different cell types and then locked together to form patterned cocultures. This device has been successfully employed to dissect cell-cell interactions in the liver, 3, 4 modulate coculture interactions to drive the differentiation of stem cells, 2, 12 and interrogate tumor-stromal signaling. 5 However, while reconfigurable micromechanical substrates can pattern sharp cellular interfaces, the devices are costly, require a significant amount of training to operate, and are optically opaque, complicating their use with the inverted microscopes commonly employed by biologists.…”