Abstract
Losses in vital functions of the somatic motor and sensory nervous system are induced by severe long-gap peripheral nerve transection injury. In such cases, autologous nerve grafts are the gold standard treatment, despite the unavoidable sacrifice of other healthy functions, whereas the prognosis is not always favorable. Here, we use human skeletal muscle-derived stem cells (Sk-SCs) to reconstitute the function after long nerve-gap injury. Muscles samples were obtained from the amputated legs from 9 patients following unforeseen accidents. The Sk-SCs were isolated using conditioned collagenase solution, and sorted as CD34+/45- (Sk-34) and CD34-/45-/29+ (Sk-DN/29+) cells. Cells were separately cultured/expanded under optimal conditions for 2 weeks, then injected into the athymic nude mice sciatic nerve long-gap model (7-mm) bridging an acellular conduit. After 8–12 weeks, active cell engraftment was observed only in the Sk-34 cell transplanted group, showing preferential differentiation into Schwann cells and perineurial/endoneurial cells, as well as formation of the myelin sheath and perineurium/endoneurium surrounding regenerated axons, resulted in 87% of numerical recovery. Differentiation into vascular cell lineage (pericyte and endothelial cells) were also observed. A significant tetanic tension recovery (over 90%) of downstream muscles following electrical stimulation of the sciatic nerve (at upper portion of the gap) was also achieved. In contrast, Sk-DN/29+ cells were completely eliminated during the first 4 weeks, but relatively higher numerical (83% vs. 41% in axon) and functional (80% vs. 60% in tetanus) recovery than control were observed. Noteworthy, significant increase in the formation of vascular networks in the conduit during the early stage (first 2 weeks) of recovery was observed in both groups with the expression of key factors (mRNA and protein levels), suggesting the paracrine effects to angiogenesis. These results suggested that the human Sk-SCs may be a practical source for autologous stem cell therapy following severe peripheral nerve injury.
Highlights
Severe nerve transection with a long gap is an irreparable injury to the living body, leading to permanent loss of related motor and sensory functions
We recently reported the potential therapeutic use of mouse skeletal muscle-derived multipotent stem cells (Sk-MSCs) in long-gap nerve treatment with bridging by an acellular conduit [8] based on their capacity for synchronized reconstitution of the muscle-nerve-blood vessel unit [9]
We studied the optimal methods for the therapeutic isolation and fractionation of human skeletal muscle-derived cells (Sk-Cs), and established appropriate cell expansion culture methods [16]
Summary
Severe nerve transection with a long gap is an irreparable injury to the living body, leading to permanent loss of related motor and sensory functions. In such cases, autologous nerve grafts have been used as the gold standard treatment [1], with the expectation of proliferation and activation of associated Schwann cells and production of neurotrophic factors and cytokines [2]. It appears clear that use of these conduits alone does not facilitate nerve regeneration across long gaps [3]. Even the combined use of the iPSderived neurospheres with bioabsorbable conduit transplantation showed only about 5% axonal recovery in long-gap nerve injury after 12 weeks [7]
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