Dynamics of acute local inflammatory response after autologous transplantation of muscle-derived cells into the skeletal muscle

Abstract

The vast majority of myoblasts transplanted into the skeletal muscle die within the first week after injection. Inflammatory response to the intramuscular cell transfer was studied in allogeneic but not in autologous model. The aim of this study was to evaluate immune reaction to autotransplantation of myogenic cells and to assess its dynamics within the first week after injection. Muscle-derived cells or medium alone was injected into the intact skeletal muscles in autologous model. Tissue samples were collected 1, 3, and 7 days after the procedure. Our analysis revealed the peak increase of the gene expression of all evaluated cytokines (Il-1α, Il-1 β, Il-6, Tgf-β, and Tnf-α) at day 1. The mRNA level of analyzed cytokines normalized in subsequent time points. The increase of Il- β gene expression was further confirmed at the protein level. Analysis of the tissue sections revealed rapid infiltration of injected cell clusters with neutrophils and macrophages. The inflammatory infiltration was almost completely resolved at day 7. The survived cells were able to participate in the muscle regeneration process. Presented results demonstrate that autotransplanted muscle-derived cells induce classical early immune reaction in the site of injection which may contribute to cellular graft elimination.

Figure 1

Identification of isolated muscle-derived cells. (a) The appearance of muscle-derived colony 6 days after isolation; (b and c) fluorescence microscopy. Desmin protein stained with Alexa 594 conjugated Ab (red) in undifferentiated MDCs (b) and in MDCs induced to myogenic differentiation for 3 days (c); (d) the presence of lipid accumulating cells within MDC population, red lipid droplets indicated with white arrowhead (Oil Red O staining), and cell nuclei stained with DAPI (blue). A multinucleated myotube can be observed in the same field of view (arrow). Scale bars in (a) 200 μm, (b and c) 50 μm, and (d) 20 μm.

Figure 2

The analysis of cytokines gene expression in the muscle tissue determined by real-time PCR. The graphs represent relative gene expression of Il-1α, Il-1 β , Il-6, Tgf- β ,and Tnf-α in animals' muscles from different groups: untreated control (CTRL), after vehicle injection (VEH), and after MDCs transplantation (MDC) 1, 3, and 7 days after surgery. Results were analyzed in pairs (untreated control versus VEH and VEH versus MDC) using nonparametric U Mann-Whitney test. * indicates statistically significant difference between MDC and VEH groups and ^#indicates statistically significant difference between VEH and CTRL groups in certain time points.

Figure 3

The concentration of cytokines: IL-1α (a), IL-1β (b) in the muscle tissue: untreated control (CTRL), after vehicle injection (VEH), and after MDCs transplantation (MDC) 1, 3, and 7 days after surgery. Protein concentrations of cytokines in tissue were determined using ELISA tests. Results were analyzed in pairs (untreated control versus VEH and VEH versus MDC) using nonparametric U Mann-Whitney test. * indicate statistically significant difference between MDC and VEH groups and ^#indicate statistically significant difference between VEH and CTRL groups in certain time points.

Figure 4

Immunohistochemical staining in different time points. Macrophages (CD68 antigen stained with Alexa 488 (green)) are infiltrating the cluster of transplanted MDC (PKH26 (red)). Cell nuclei stained with DAPI (blue). Scale bars: 50 μm.

Figure 5

Immunohistochemical staining in different time points. Leukocytes (CD43 antigen stained with Alexa 488 (green)) are infiltrating the cluster of transplanted MDC (PKH26 (red)). Cell nuclei stained with DAPI (blue) in different time points. Scale bars: 50 μm.

Figure 6

The skeletal muscle stained with hematoxylin and eosin. Images present the area of injection at days 1, 3, and 7 after procedure in subsequent rows. (a and b) Cross sections from transplanted group in two different magnifications. Distinct clusters of injected cells are visible. Dashed lines drawn on images in column A indicate approximate area of clusters located between muscle fibers. At day 1, neutrophils can be recognized within the cluster (arrows). At day 3, identification of neutrophils is not obvious; large mononuclear cells dominate in the cluster. At day 7, inflammatory infiltration is not clearly visible anymore; (c and d) cross sections from group injected with vehicle only in two different magnifications. Some inflammatory cells could be recognized in samples collected at day 1 time point.

Figure 7

In vivo imaging of a representative animal in subsequent time points. 2.5 × 10⁵ DiD-labeled MDCs were injected into the gastrocnemius muscle (limb on the left). Contralateral limb was injected with the equal volume of vehicle and constituted internal control. Optical imaging was carried out at 10 min, days 1, 3, and 7 after the injection. Read-out scale was unified between the subsequent images, as presented on the right panel. Values in blue rectangles indicate total radiant efficiency of DiD-derived signal measured in subsequent time points in equal regions of interest (ROI).

Figure 8

Immunohistochemical staining of samples collected 7 days after autologous MDCs injection. (a and b) PKH26 positive cells (red) can be recognized between and within muscle fibers (arrow), desmin stained with Alexa 488 (green); (c and d) PKH26 positive cells (red) can be recognized in the central position of muscle fiber (arrowhead) indicating the ability to contribute muscle regeneration. Few macrophages (CD68 stained with Alexa 488 (green)) are still present in the site of transplantation. Scale bars: 50 μm.