Telocytes within human skeletal muscle stem cell niche

Abstract

Human skeletal muscle tissue displays specific cellular architecture easily damaged during individual existence, requiring multiple resources for regeneration. Congruent with local prerequisites, heterogeneous muscle stem cells (MuSCs) are present in the muscle interstitium. In this study, we aimed to characterize the properties of human muscle interstitial cells that had the characteristic morphology of telocytes (TCs). Immunocytochemistry and immunofluorescence showed that cells with TC morphology stained positive for c-kit/CD117 and VEGF. C-kit positive TCs were separated with magnetic-activated cell sorting, cultured in vitro and expanded for study. These cells exhibited high proliferation capacity (60% expressed endoglin/CD105 and 80% expressed nuclear Ki67). They also exhibited pluripotent capacity limited to Oct4 nuclear staining. In addition, 90% of c-kit positive TCs expressed VEGF. C-kit negative cells in the MuSCs population exhibited fibroblast-like morphology, low trilineage differential potential and negative VEGF staining. These results suggested that c-kit/CD117 positive TCs represented a unique cell type within the MuSC niche.

Fig 1

Morphological aspect of skeletal muscle-derived primary cell culture obtained using explant method. TCs are identified based on their characteristics (arrows) within the heterogeneous cell population. Contrast-phase microscopy; magnification 200×.

Fig 2

The cytoskeleton protein, vimentin, was ubicuitary expressed in stromal cells. Telocytes were recognized by their distinct morphology of an oval body shape with telopodes. Arrows point to MuSC and telocytes, which confirm the heterogeneity of primary cell cultures obtained from skeletal muscle tissue; magnification 200×.

Fig 3

Immunohistochemical analysis of CD117 (c-kit) expressing TCs. Telocytes were isolated by magnetic separation with CD117-specific beads. (A) Single cells show long moniliform prolongations (telopodes); magnification 600×. (B) Overview of in vitro expanded CD117+ population of TCs; magnification 100×.

Fig 4

Immunofluorescence of CD117+ TCs, expanded during in vitro culture. A large fraction of cells (60%) showed proliferation. Proliferation was indicated by (A) the presence of Ki67 in the cell nucleus; and (B) endoglin/CD105 expression, at various intensities. (C) Pluripotency-associated transcription factor Oct4 stained positive in the considered cell population. (D) c-kit expression in TCs associated with an intermediate level of VEGF presence in the cytoplasm; magnification 400×.

Fig 5

Eighty percent of primary cell cultures readily differentiated into adipocytes (FABP4 positive staining), when they maintained contact with supporting interstitial TCs; magnification 600×.