Characterization of CM-Dil-labeled Muse cells in culture and in skin wounds in rats.

To investigate the characteristics of multilineage-differentiating stress-enduring (Muse) cells labeled with chloromethyl dialkylcarbocyanine (CM-Dil) in culture and in skin wounds of rats. Normal human dermal fibroblasts (NHDFs) were obtained from foreskins and were confirmed by immunocytochemistry with vimentin. Muse cells were derived from NHDFs using long-term trypsinization (LTT), were confirmed using immunocytochemistry with antibodies against stage specific embryonic antigen-3 (SSEA-3) and CD105 and were expanded in suspension cultures. The Muse cells were labeled with CM-Dil and were further evaluated with respect to their biological properties using CCK-8 assays and scratch tests. One hundred µl CM-Dil-labeled Muse cells at a concentration of 5 × 103/µl were injected subcutaneously at the edges of skin wounds in adult male SD rats. At weeks 1, 3 and 5 after the injection, the distribution of CM-Dil-labeled Muse cells in skin tissues was observed using immunofluorescence microscopy. Muse cells were double-positive for CD105 and SSEA-3. ALP staining of the M-clusters were positive and they displayed orange-red fluorescence after labelling with CM-Dil, which had no adverse effects on their viability, migration or differentiation capacity. One week after the subcutaneous injection of CM-Dil-labeled Muse cells, many cells with orange-red fluorescence were observed at the edges of the skin injuries; those fluorescent spots gradually decreased over time, and only a few Muse cells with fluorescence could be detected by week 5. CM-Dil can be used to label Muse cells without affecting their proliferation, migration or differentiation, and can be used for short-term tracking of Muse cells for the treatment of skin wounds in a rat model.

Characteristics of multilineage-differentiating stress-enduring cell clusters in different culture conditions.

OBJECTIVE: To observe the morphological characteristics of clusters of Muse cells from normal human dermal fibroblasts (NHDFs) under different culture conditions. METHODS: Muse cells were sorted by magnetic activated cell sorting (MACS) from NHDFs, and were evaluated by flow cytometry. Muse cells were cultured in suspension and in adherent conditions to obtain Muse cell clusters (M-clusters), which were further characterized by alkaline phosphatase (AP) staining, immunofluorescence (IF) staining and transmission electron microscopy (TEM). The M-clusters were further cultured on Lando artificial dermal regeneration matrix (LADRM) for analysis by scanning electron microscopy (SEM) and IF staining of frozen sections. RESULTS: The proportion of SSEA3 and CD105 double-positive cells obtained by MACS was 87.4%. The sorted cells rapidly formed M-clusters after suspension culture, and showed internal characteristics of stem cells under TEM. After adherent culture, M-clusters stained positively for AP, SSEA-3 and OCT-4. Each M-cluster on the surface of the LADRM displayed an outer membrane of amorphous materials under SEM. Frozen sections and fluorescence staining of LADRM loaded with M-clusters showed an uneven fluorescence intensity of SSEA-3 within the clusters. CONCLUSIONS: Muse cells sorted by MACS from NHDFs could generate M-clusters, which included cells of different stemness and are wrapped in membrane-like structures.