Recently, we experienced the certain case of the paracrine effects in the experimental therapies of the severely damaged peripheral nerve with a log-gap, using mouse and human skeletal muscle-derived stem cells (Sk-SCs). Because of this background, the paracrine effects of these cells before and after transplantation are discussed in this commentary, with the comparison with the case of bone-marrow-derived stromal cells (BMSCs).

Loss of vital functions in the somatic motor and sensory nervous systems can be induced by severe peripheral nerve transection with a long gap following trauma. In such cases, autologous nerve grafts have been used as the gold standard, with the expectation of activation and proliferation of graft-concomitant Schwann cells associated with their paracrine effects. However, there are a limited number of suitable sites available for harvesting of nerve autografts due to the unavoidable sacrifice of other healthy functions. To overcome this problem, we examined the potential of Sk-SCsas a novel alternative cell source for peripheral nervere generation therapy [1,2].The reason is that the Sk-SCs including CD34⁺/45^- (Sk-34) [3] and CD34^-/45^- (Sk-DN) [4] cells, which showed differentiation potential into mesodermal cells (skeletal muscle cells, vascular smooth muscle cells, pericytes and endothelial cells) and ectodermal cells (Schwann cells and perineurial cells) in vivo, and typically exerted synchronized reconstitution of the muscular, vascular and peripheral nervous system in the severely damaged skeletal muscle[5-7]. The potential was not changed, when both Sk-34 and Sk-DN cells were used by the mixed cells, as the Sk-SCs [8-10]. Because of this background, we made sciatic nerve long-gap transection model of mice (7-mm) and rats (12-mm), and bridged using anacellular conduit made from separated esophageal submucous membrane from mice after 3-days of 70% ethanol treatment. Then, the cells were injected in the bridging-conduit [2]. As we expected in the mouse-to-mouse experiment, the transplanted Sk-SCs differentiated into all the peripheral nerve support cells(such as Schwann, endoneurial/perineurial cells), and contributed to the recovery of the number of axon for almost 90% and close to 60% of myelin (Figure 1A and 1B). Interestingly, myogenic cells were also observed in this nerve niche, but the skeletal myogenic capacity of expanded Sk-SCs had been diminished gradually, and completely disappeared at 4-weeks after the transplantation [2]. Similarly, BMSCs transplantation was also performed, however, the results showed almost no effects (wholly comparable to the level of medium control) for the recoveries of the number of axons and myelin (Figure 1A and B). In addition, transplanted BMSCs disappeared during first one week. Therefore, no cell engraftment with no effect is reasonable in this case.

The observed differences in both groups are considered due to the prolonged survival/engraftment/paracrine of Sk-34 cells and their differentiation into all the nerve support cells. However, it is also certain that the complete elimination of the human Sk-DN cells occurred during 4-weeks after transplantation, but significantly higher morphological and functional recoveries than the control was achieved. Therefore, this is reasonable to consider that this result may be due to the paracrine effects of human Sk-DN cells during 4-weeks after transplantation. Putative paracrine capacities of human Sk-34 and Sk-DN cells, which was supposed by expressions of mRNAs were shown in Figure 3. Both cells consistently expressed 8 analyzed nerves and 5 analyzed vascular growth factors just before the transplantation, and a comparable paracrine effect can be expected. In fact, these expressions of mRNAs have wholly kept in the Sk-34 cells even at 4-weeks after transplantation; this was confirmed by the analysis for the engrafted Sk-34 cells, which was re-isolated enzymatically, and sorted using human-specific antibody (data not shown). In the comparison of the above mouse and human cell studies, both the mouse BMSCs and the human Sk-DN cells were eliminated in the peripheral nerve niche, but the former showed no effects for nerve regeneration, and the latter showed a significant contribution for the recovery. What makes a difference between them; that is the term of elimination, thus how long stay in the damaged tissue is considered to be the limiting factor of the paracrine effects following stem cell transplantation, because the expressions of nerve-vascular growth factor mRNAs were similar in both the mouse BMSCs [2] and present human Sk-DN cells (Figure 3) at just before the transplantation. In these view points, it is likely that one week of the cell retaining-period is insufficient, but 4-weeks retaining, with continuous expressions of growth factors after the onset of the damage, may have a beneficial effect on the nerve regeneration. However, we still not perform a more quantitative analysis (such as real-time RT-PCR) for the expressions of nerve-vascular growth factors in Sk-34 and Sk-DN cells. Therefore, further experiments, to clarify which factors may play a more prominent paracrine role during regeneration process, should be necessary.

From the other point of view, it was also suggested that the paracrine substances of skeletal-myogenic cells exert facilitative effects for the peripheral nerve regeneration process. This concept further suggests that the skeletal muscle fiber and peripheral nerve regeneration process may share the large number of essential factors, and they co-working together. This notion also supports the previous results of Sk-SCs transplantation that asynchronized reconstitution of muscle-nerve-blood vessels were induced in the severe muscle injury with a sizable defect [5,6].

Finally, the cytokine supply associate with the stem cell transplantation was proposed in this commentary. However, administration therapy of recombinant cytokines, which will be selected appropriately in the near future, should be also considered. In particular, the nerve injury categorized in the Seddons’s axonotmesis and/or the Sunderland’s fourth degree, because the continuity of the epineurium (the most outer layer) is maintained, but involves loss of axons, endoneurial tubes, perineurial fasciculi and vascular networks, thus, associated with a bad prognosis. In addition, a development of the nerve bridging materials, which enable a sustained-release of cytokines, such as the biodegradable tubes, is also much-needed.

CONCLUSION

Through these studies, it is supposed that the first 2-3 weeks from the onset of nerve damage may be a critical period to supply nerve-vascular growth factors to obtain the better nerve regeneration. This notion is found to be useful for future cytokine therapy for the severe peripheral nerve injury.