Transplantation of adipose tissue-derived stromal cells increases mass and functional capacity of damaged skeletal muscle.

The regenerating skeletal muscle environment is capable of inducing uncommitted progenitors to terminally differentiate. The aim of this work was to determine whether adipose tissue-derived stromal cells were able to participate in muscle regeneration and to characterize the effect on muscle mass and functional capacities after transplantation of these cells. Adipose tissue stromal cells labeled with Adv cyto LacZ from 3-day-old primary cultures (SVF1) were autotransplanted into damaged tibialis anterior muscles. Fifteen days later, beta-galactosidase staining of regenerated fibers was detected, showing participation of these cells in muscle regeneration. Two months after SVF1 cell transfer, muscles were heavier, showed a significantly larger fiber section area, and developed a significantly higher maximal force compared with damaged control muscles. These results are similar to those previously obtained after satellite cell transplantation. However, SVF1 transfer also generated a small amount of adipose tissue localized along the needle course. To minimize these adipose contaminants, we transferred cells from 7-day-old secondary cultures of the SVF1, containing only a small proportion of already engaged preadipocytes (SVF2). Under these conditions, no adipose tissue was observed in regenerated muscle but there was also no effect on muscle performances compared with damaged control muscles. This result provides further evidence for the existence of progenitor cells in the stromal fraction of freshly isolated adipose tissue cells, which, under our conditions, keep some of their pluripotent properties in primary cultures.

Changes in mass and performance in rabbit muscles after muscle damage with or without transplantation of primary satellite cells.

Changes in morphology, metabolism, myosin heavy chain gene expression, and functional performances in damaged rabbit muscles with or without transplantation of primary satellite cells were investigated. For this purpose, we damaged bilaterally the fast muscle tibialis anterior (TA) with either 1.5 or 2.6 ml cardiotoxin 10(-5) M injections. Primary cultures of satellite cells were autotransplanted unilaterally 5 days after muscle degeneration. Two months postoperation, the masses of damaged TAs, with or without transplantation, were significantly larger than those of the controls. Furthermore, damaged transplanted muscles weighed significantly more than damaged muscles only. The increase in muscle mass was essentially due to increased fiber size. These results were independent of the quantity of cardiotoxin injected into the muscles. Maximal forces were similar in control and 2.6 ml damaged TAs with or without satellite cell transfer. In contrast, 1.5 ml damaged TAs showed a significant decrease in maximal forces that reached the level of controls after transplantation of satellite cells. Fatigue resistance was similar in control and 1.5 ml damaged TAs independently of satellite cell transfer. Fatigue index was significantly higher in 2.6 ml damaged muscles with or without cell transplantation. These changes could be explained in part by muscle metabolism, which shifted towards oxidative activities, and by gene expression of myosin heavy chain isoforms, which presented an increase in type IIa and a decrease in type I and IIb in all damaged muscles with or without cell transfer. Under our experimental conditions, these results show that muscle damage rather than satellite cell transplantation changes muscle metabolism, myosin heavy chain isoform gene expression, and, to a lesser extent, muscle contractile properties. In contrast, muscle weight and fiber size are increased both by muscle damage and by satellite cell transfer.

Inhibition of myoblast differentiation by Sfrp1 and Sfrp2.

Secreted Frizzled-related proteins (Sfrps) are extracellular regulators of Wnt signalling and play important roles in developmental and oncogenic processes. They are known to be upregulated in regenerating muscle and in myoblast cultures but their function is unknown. Here, we show that the addition of recombinant Sfrp1 or Sfrp2 to C2C12 cell line cultures or to primary cultures of satellite cells results in the inhibition of myotube formation with no significant effect on the cell cycle or apoptosis. Even though at confluence, treated and untreated cultures are identical in appearance, analyses have shown that, for maximum effect, the cells have to be treated while they are proliferating. Furthermore, removal of Sfrp from the culture medium during differentiation restores normal myotube formation. We conclude that Sfrp1 and Sfrp2 act to prevent myoblasts from entering the terminal differentiation process.

Transplantation of adult myoblasts or adipose tissue precursor cells by high-density injection failed to improve reinnervated skeletal muscles.

We previously showed that transfer of adult myoblasts (MB) into cardiotoxin-damaged muscle improved the properties of reinnervated tibialis anterior muscle of rabbits. However, this cell therapy protocol cannot be applied to humans because of the hazardous effects of the myotoxin. To circumvent this approach, we used the recently developed high-density injection technique to autotransplant cultured cells 1 mm from each other into the tibialis anterior muscle without previous cardiotoxin-induced damage. Two months after transection and immediate suture of the common peroneal nerve, we transferred by this technique two types of precursor cells, MB or cells isolated from the adipose tissue stromal vascular fraction. In contrast to our previous results, muscles studied at 4 months showed no benefits in terms of function or morphology, whatever the transferred cells. These results, together with the results of earlier studies, emphasize the importance of delivery methods and the muscle environment in supporting cell integration into host tissues.

Short- or long-term effects of adult myoblast transfer on properties of reinnervated skeletal muscles.

Skeletal muscle demonstrates a force deficit after repair of injured peripheral nerves. Data from the literature indicate that myoblast transfer enhances recovery of muscle function. Thus, we tested the hypothesis that transfer of adult myoblasts improves the properties of reinnervated rabbit tibialis anterior (TA) muscles in both the short term (4 months) and long term (14 months). Two months after transection and immediate suture of the common peroneal nerve, TA muscles were made to degenerate by cardiotoxin injection and then transplanted with adult myoblasts cultured for 13 days. Under these conditions, muscles studied at 4 months were heavier, contained larger fibers, and developed a significantly higher maximal force than muscles that had only been denervated-reinnervated. In the long term, although muscles made to degenerate were heavier and developed a significantly higher maximal force than denervated-reinnervated muscles, myoblast transfer failed to improve these parameters. However, the overall characteristics of long-term operated muscles tended clearly to approach those of the controls. Taken together, these results may have significant implications in certain orthopedic contexts, particularly after immediate or delayed muscle reinnervation.

Transplantation of primary satellite cells improves properties of reinnervated skeletal muscles.

Skeletal muscle demonstrates a force deficit after repair of injured peripheral nerves. We tested the hypothesis that transplantation of satellite cells into reinnervated rabbit tibialis anterior (TA) muscles improves their properties. Adult rabbits underwent transection and immediate suture of the common peroneal nerve. In order to provide an environment favorable for cell transplantation, TA were then made to degenerate by cardiotoxin injection, either immediately or after a 2-month delay, which is sufficient for muscle reinnervation. In both cases, the injured TA were transplanted with cultured satellite cells 5 days after induction of muscle degeneration. When cells were transferred immediately after nerve repair, drastic morphological and functional muscle alterations were observed. However, when the muscles were allowed to become reinnervated before cell transplantation, muscles were heavier and developed a significantly higher maximal force compared to denervated-reinnervated muscles. Thus, application of the cell therapy protocol improved properties of denervated muscles only when they were allowed to become innervated. These results, which represent the application of cell therapy to improve force recovery of reinnervated muscles, will be of significant interest in certain clinical contexts, particularly after immediate or delayed muscle reinnervation.

Transplantation of Adipose Tissue-Derived Stromal Cells Increases Mass and Functional Capacity of Damaged Skeletal Muscle.

The regenerating skeletal muscle environment is capable of inducing uncommitted progenitors to terminally differentiate. The aim of this work was to determine whether adipose tissue-derived stromal cells were able to participate in muscle regeneration and to characterize the effect on muscle mass and functional capacities after transplantation of these cells. Adipose tissue stromal cells labeled with Adv cyto LacZ from 3-day-old primary cultures (SVF1) were autotransplanted into damaged tibialis anterior muscles. Fifteen days later, ß-galactosidase staining of regenerated fibers was detected, showing participation of these cells in muscle regeneration. Two months after SVF1 cell transfer, muscles were heavier, showed a significantly larger fiber section area, and developed a significantly higher maximal force compared with damaged control muscles. These results are similar to those previously obtained after satellite cell transplantation. However, SVF1 transfer also generated a small amount of adipose tissue localized along the needle course. To minimize these adipose contaminants, we transferred cells from 7-day-old secondary cultures of the SVF1, containing only a small proportion of already engaged preadipocytes (SVF2). Under these conditions, no adipose tissue was observed in regenerated muscle but there was also no effect on muscle performances compared with damaged control muscles. This result provides further evidence for the existence of progenitor cells in the stromal fraction of freshly isolated adipose tissue cells, which, under our conditions, keep some of their pluripotent properties in primary cultures.

Changes in Mass and Performance in Rabbit Muscles after Muscle Damage with or without Transplantation of Primary Satellite Cells.

Changes in morphology, metabolism, myosin heavy chain gene expression, and functional performances in damaged rabbit muscles with or without transplantation of primary satellite cells were investigated. For this purpose, we damaged bilaterally the fast muscle tibialis anterior (TA) with either 1.5 or 2.6 ml cardiotoxin 10-5 M injections. Primary cultures of satellite cells were autotransplanted unilaterally 5 days after muscle degeneration. Two months postoperation, the masses of damaged TAs, with or without transplantation, were significantly larger than those of the controls. Furthermore, damaged transplanted muscles weighed significantly more than damaged muscles only. The increase in muscle mass was essentially due to increased fiber size. These results were independent of the quantity of cardiotoxin injected into the muscles. Maximal forces were similar in control and 2.6 ml damaged TAs with or without satellite cell transfer. In contrast, 1.5 ml damaged TAs showed a significant decrease in maximal forces that reached the level of controls after transplantation of satellite cells. Fatigue resistance was similar in control and 1.5 ml damaged TAs independently of satellite cell transfer. Fatigue index was significantly higher in 2.6 ml damaged muscles with or without cell transplantation. These changes could be explained in part by muscle metabolism, which shifted towards oxidative activities, and by gene expression of myosin heavy chain isoforms, which presented an increase in type IIa and a decrease in type I and IIb in all damaged muscles with or without cell transfer. Under our experimental conditions, these results show that muscle damage rather than satellite cell transplantation changes muscle metabolism, myosin heavy chain isoform gene expression, and, to a lesser extent, muscle contractile properties. In contrast, muscle weight and fiber size are increased both by muscle damage and by satellite cell transfer.