Skeletal muscle resident fibroblastic cells are considered to be the origin of the intermuscular adiposity that develops in pathophysiological conditions such as myopathies, type-2 diabetes, and obesity (Contreras et al 2021). Isolated human skeletal muscle TE7+/collagen VI+/PDGFRα+ fibroblastic cells have been shown to have the potential to differentiate into adipocytes when exposed to media containing fatty acids and/or an adipocyte inducing medium (AIM; Agley et al 2013). However, it is not clear if similar behaviour is apparent in fibroblastic cells from other tissues. The aim of the present study was to compare marker expression and response to an adipogenic stimulus of human primary fibroblastic cells from different tissues (muscle, skin and fat) compared to pre-adipocytes.
Human skeletal muscle fibroblastic cells were isolated from muscle biopsies from the vastus lateralis of young healthy adult volunteers (n=6, aged 22.3±3.2 yrs). Human primary skin and lung fibroblasts, as well as human primary visceral pre-adipocytes were purchased and cultured as per manufacturer’s instructions (n=3 for each, PromoCell). For adipogenic differentiation, all cell types were grown for seven days in skeletal muscle growth medium before applying AIM+oleic treatment. This consisted of a 3-day incubation in a pre-adipocyte differentiation medium followed by a 15-day incubation in an adipocyte nutrition medium (PromoCell) supplemented with 600 µM oleic acid and 15 mg/ml BSA (AIM+oleic). Protein expression and cell morphological changes were analysed using immunocytochemistry. Expression of secreted adipokines, adipsin and adiponectin were analysed from conditioned media collected over 24 hours before and after treatment using a Luminex platform.
Expression of fibroblast marker proteins TE-7, PDGFRα, collagen VI, fibronectin, vimentin and TCF4 were indistinguishable between the four cell types under proliferation conditions. After AIM+oleic treatment three of six skeletal muscle fibroblastic and all pre-adipocyte cell populations showed protein expression of adipocyte marker perilipin (34.3±4.8% and 22.7±3.1% perilipin+ cells respectively, p<0.01), exhibited protein expression of other adipocyte protein markers acetyl-CoA carboxylase and fatty acid synthase, as well as having a classical adipocyte signate ring morphology. None of the skin or lung-derived fibroblasts showed perilipin expression or other adipocyte marker expression. Indeed, although all cell types increased secretion of adipsin (skin:17276±7225 pg/ml, lung:1211±6837 pg/ml, muscle:18504±359 pg/ml, pre-adipocyte: 12183±2592 pg/ml, n=3, p<0.05) and showed visible signs of increased lipid accumulation. Only the groups of perilipin+ skeletal muscle fibroblastic cells and pre-adipocyte populations reached statistical significance for total cellular lipid accumulation as measured by integrated density of oil red O staining (3.3×107±2.0×107 AU and 4.8×107±1.3×107 AU respectively, n=3, p<0.05). These cells also showed secretion of adiponectin (1722±318 pg/ml and 748±715 pg/ml for muscle fibroblasts and pre-adipocytes respectively, p<0.05). Non perilipin+ cell populations were characterised by accumulating multiple small lipid droplets whereas lipid-filled the cytoplasm of perilipin+ cell populations.
These data show that despite being indistinguishable on the basis of their fibroblast marker expression, skin and lung cells differ in their responses to an adipogenic stimulus compared with skeletal muscle origin fibroblastic cells. However, only muscle origin fibroblastic cells derived from some individuals exhibited the full potential for differentiation into adipocytes.