In Vitro Model of Human Skeletal Muscle Tissue for the Study of Resident Macrophages and Stem Cells

Abstract

Findings from studies of muscle regeneration can significantly contribute to the treatment of age-related loss of skeletal muscle mass, which may predispose older adults to severe morbidities. We established a human experimental model using excised skeletal muscle tissues from reconstructive surgeries in eight older adults. Muscle samples from each participant were preserved immediately or maintained in agarose medium for the following 5, 9, or 11 days. Immunofluorescence analyses of the structural proteins, actin and desmin, confirmed the integrity of muscle fibers over 11 days of maintenance. Similarly, the numbers of CD80-positive M1 and CD163-positive M2 macrophages were stable over 11 days in vitro. However, the numbers of PAX7-positive satellite cells and MYOD-positive myoblasts changed in opposite ways, suggesting that satellite cells partially differentiated in vitro. Further experiments revealed that stimulation with unsaturated fatty acid C18[2]c (linoleic acid) increased resident M1 macrophages and satellite cells specifically. Thus, the use of human skeletal muscle tissue in vitro provides a direct experimental approach to study the regulation of muscle tissue regeneration by macrophages and stem cells and their responses to therapeutic compounds.

Unsaturated Long-Chain Fatty Acids Activate Resident Macrophages and Stem Cells in a Human Skeletal Muscle Tissue Model

“…To analyze temporal changes in the resident cell counts, we maintained two sections of donor samples ( n = 20) in vitro for 9 and 11 days, as described previously [ 16 ]. All samples were subjected to HE, DAPI, and immunofluorescence staining, to obtain the numbers of cells, as described above ( Section 3.1 ).…”

“…After being dehydrated and embedded in paraffin, tissue blocks were cut into 5 μm sections using a SLIDE4003E microtome (pfm Medical, Cologne, Germany). The sections were then fixed onto adhesive microscope slides and deparaffinized, before being stained via an automated slide-staining station (Gemini, manufactured by Thermo Fisher Scientific, Waltham, MA, USA), as previously described [ 16 ]. After being stained in hematoxylin for 5–10 min, the slides were rinsed in warm water for 10 min, stained in 0.3% eosin for 5 min, rinsed again with tap water, and successively dehydrated in 70%, 96%, and 100% ethanol.…”

“…Previously, we established an in vitro model of human skeletal muscle tissue using surgical specimens [ 16 ]. Here, we used this model to compare the effects of different FA species on muscle-tissue-resident macrophages and stem cells in 20 donors of different ages.…”

The Role of Supporting Cell Populations in Satellite Cell Mediated Muscle Repair

“…Another abundant leukocyte found within the skeletal muscle microenvironment is macrophages. Tissue-resident macrophages regulate skeletal muscle homeostasis and selfrenew without the recruitment of blood-derived monocytes [66,[68][69][70][71]. These immune cells can be triggered by muscle damage, and after the initial injury response by neutrophils, macrophages become the primary cell type in the regenerating muscle tissue.…”