Shuichi Soeda

Preferential and Comprehensive Reconstitution of Severely Damaged Sciatic Nerve Using Murine Skeletal Muscle-Derived Multipotent Stem Cells

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, the potential of skeletal muscle-derived multipotent stem cells (Sk-MSCs) was examined as a novel alternative cell source for peripheral nerve regeneration. Cultured/expanded Sk-MSCs were injected into severely crushed sciatic nerve corresponding to serious neurotmesis. After 4 weeks, engrafted Sk-MSCs preferentially differentiated into not only Schwann cells, but also perineurial/endoneurial cells, and formed myelin sheath and perineurium/endoneurium, encircling the regenerated axons. Increased vascular formation was also observed, leading to a favorable blood supply and waste product excretion. In addition, engrafted cells expressed key neurotrophic and nerve/vascular growth factor mRNAs; thus, endocrine/paracrine effects for the donor/recipient cells were also expected. Interestingly, skeletal myogenic capacity of expanded Sk-MSCs was clearly diminished in peripheral nerve niche. The same differentiation and tissue reconstitution capacity of Sk-MSCs was sufficiently exerted in the long nerve gap bridging the acellular conduit, which facilitated nerve regeneration/reconnection. These effects represent favorable functional recovery in Sk-MSC-treated mice, as demonstrated by good corduroy walking. We also demonstrated that these differentiation characteristics of the Sk-MSCs were comparable to native peripheral nerve-derived cells, whereas the therapeutic capacities were largely superior in Sk-MSCs. Therefore, Sk-MSCs can be a novel/suitable alternative cell source for healthy nerve autografts.

A Long-Gap Peripheral Nerve Injury Therapy Using Human Skeletal Muscle-Derived Stem Cells (Sk-SCs): An Achievement of Significant Morphological, Numerical and Functional Recovery

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.

3D reconstitution of nerve–blood vessel networks using skeletal muscle-derived multipotent stem cell sheet pellets

AIM To cover the large tissue deficits associated with significant loss of function following surgery, a 3D gel-patch-like nerve-vascular reconstitution system was developed using the skeletal muscle-derived multipotent stem cell (Sk-MSC) sheet pellet. MATERIALS & METHODS The Sk-MSC sheet pellet was prepared from GFP transgenic mice by the collagenase extraction and 7 days expansion cell culture, and transplanted into a severe muscle damage model with large disruptions to muscle fibers, blood vessels and peripheral nerves. RESULTS At 4 weeks after transplantation, engrafted cells contributed to nerve-vascular regeneration associated with cellular differentiation into Schwann cells, perineurial/endoneurial cells, vascular endothelial cells and pericytes. However, skeletal myogenic differentiation was scarcely observed. Paracrine effects regarding donor cells/tissues could also be expected, because of the active expression of neurogenic and vasculogenic factor mRNAs in the sheet pellet. CONCLUSION These results indicate that the vigorous skeletal myogenic potential of Sk-MSCs was clearly reduced in the sheet pellet preparation and this method may be a useful adjuvant for nerve-vascular regeneration in various tissue engineering applications.

Origin and hierarchy of basal lamina-forming and -non-forming myogenic cells in mouse skeletal muscle in relation to adhesive capacity and Pax7 expression in vitro

As a novel approach to distinguish skeletal myogenic cell populations, basal lamina (BL) formation of myogenic cells was examined in the mouse compensatory enlarged plantaris muscles in vivo and in fiber-bundle cultures in vitro. MyoD+ myogenic cells located inside the regenerative muscle fiber BL were laminin− but interstitial MyoD+ cells were laminin+. This was also confirmed by electron microscopy as structural BL formation. Similar trends were observed in the fiber-bundle cultures including satellite cells and interstitial myogenic cells and laminin+ myogenic cells predominantly showed non-adhesive (non-Ad) behavior with Pax7−, whereas laminin− cells were adhesive (Ad) with Pax7+. Moreover, non-Ad/laminin+ and Ad/laminin− myotubes were also observed and the former type showed spontaneous contractions, while the latter type did not. The origin and hierarchy of Ad/Pax7+/laminin− and non-Ad/Pax7−/laminin+ myogenic cells were also examined using skeletal muscle interstitium-derived CD34+/45− (Sk-34) and CD34−/45− (Sk-DN) multipotent stem cells, which were composed of non-committed myogenic cells with a few (<1%) Pax7+ cells in the Sk-DN cells at fresh isolation. Both cell types were separated by Ad/non-Ad capacity in repetitive culture. As expected, both Ad/Pax7+/laminin− and non-Ad/Pax7−/laminin+ myogenic cells consistently appeared in the Ad and non-Ad cell culture. However, Ad/Pax7+/laminin− cells were repeatedly detected in the non-Ad cell culture, while the opposite phenomenon did not occur. This indicates that the source of non-Ad/ Pax7−/laminin+ myogenic cells was present in the Sk-34 and Sk-DN stem cells and they were able to produce Ad/ Pax7+/ laminin− myogenic cells during myogenesis as primary myoblasts and situated hierarchically upstream of the latter cells.

Functional Nerve-Vascular Reconstitution of the Bladder-Wall; Applicationof Patch Transplantation of Skeletal Muscle-Derived Multipotent StemCell Sheet-Pellets

A three-dimensional gel-patch-like nerve-vascular reconstitution system using the Skeletal Muscle-Derived Multipotent Stem Cell (Sk-MSC) sheet-pellet was applied to the reconstitution of the severely damaged bladder wall as a non-skeletal muscle tissue, but has high demand for function. The Sk-MSC sheet-pellet was prepared by the mild detachment of expanded/confluent cells in culture with EDTA, then, collected in a tube and centrifuged. The sheetpellet was pasted on the open thin-walled region of the damaged bladder wall made by myotomy (remove one-third of serosal smooth muscle layer associate with large disruptions of nerve-blood vessel networks retaining the mucosal layer). At 4 wk after transplantation, significant prevention of the reduction in the passive wall-tension, and the positive wall-contraction via electrical stimulation was observed in the transplanted group. Supporting these functional results, immunohistochemical and immunoelectron microscopic analysis revealed that the engrafted cells actively contributed to the reconstitution of blood vessels and peripheral nerves with differentiation into pericytes, endothelial cells, and Schwann cells. However, skeletal and smooth muscle formation was not observed. Thus, this method is potentially useful for the reconstitution of nerve-vascular networks in the bladder-wall to be retaining function such as passive tension and contractile function.

Voluntary Exercise Positively Affects the Recovery of Long-Nerve Gap Injury Following Tube-Bridging with Human Skeletal Muscle-Derived Stem Cell Transplantation

The therapeutic effects of voluntary exercise on the recovery of long-gap nerve injury following the bridging of an acellular conduit filled with human skeletal muscle-derived stem cells (Sk-SCs) have been described. Human Sk-SCs were sorted as CD34+/45− (Sk-34) cells, then cultured/expanded under optimal conditions for 2 weeks. Surgery to generate a long-gap sciatic nerve injury was performed in athymic nude mice, after which the mice were divided into exercise (E) and non-exercise (NE) groups. The mice were housed in standard individual cages, and voluntary exercise wheels were introduced to the cages of the E group one week after surgery. After 8 weeks, the human Sk-34 cells were actively engrafted, and showed differentiation into Schwann cells and perineurial cells, in both groups. The recovery in the number of axons and myelin in the conduit and downstream tibial nerve branches, and the lower hindlimb muscle mass and their tension output, was consistently higher by 15–25% in the E group. Moreover, a significantly higher innervation ratio of muscle spindles, reduced pathological muscle fiber area, and acceleration of blood vessel formation in the conduit were each observed in the E group. These results showed that the combined therapy of tube-bridging, Sk-34 cell transplantation, and voluntary exercise is a potentially practical approach for recovery following long-gap nerve injury.

Purified Human Skeletal Muscle-derived Stem Cells Enhance the Repair and Regeneration in the Damaged Urethra

Background Postoperative damage of the urethral rhabdosphincter and nerve-vascular networks is a major complication of radical prostatectomy and generally causes incontinence and/or erectile dysfunction. The human skeletal muscle-derived stem cells, which have a synchronized reconstitution capacity of muscle-nerve-blood vessel units, were applied to this damage. Methods Cells were enzymatically extracted from the human skeletal muscle, sorted using flow cytometry as CD34+/45− (Sk-34) and CD29+/34−/45− (Sk-DN/29+) fractions, and separately cultured/expanded in appropriate conditions within 2 weeks. Urethral damage was induced by manually removing one third of the wall of the muscle layer in nude rats. A mixture of expanded Sk-34 and Sk-DN/29+ cells was applied on the damaged portion for the cell transplantation (CT) group. The same amount of media was used for the non-CT (NT) group. Urethral pressure profile was evaluated via electrical stimulation to assess functional recovery. Cell engraftments and differentiations were detected using immunohistochemistry and immunoelectron microscopy. Expression of angiogenic cytokines was also analyzed using reverse transcriptase-polymerase chain reaction and protein array. Results At 6 weeks after transplantation, the CT group showed a significantly higher functional recovery than the NT group (70.2% and 39.1%, respectively; P < 0.05). Histological analysis revealed that the transplanted human cells differentiated into skeletal muscle fibers, nerve-related Schwann cells, perineuriums, and vascular pericytes. Active paracrine angiogenic cytokines in the mixed cells were also detected with enhanced vascular formation in vivo. Conclusions The transplantation of Sk-34 and Sk-DN/29+ cells is potentially useful for the reconstitution of postoperative damage of the urethral rhabdosphincter and nerve-vascular networks.

Regeneration of Transected Recurrent Laryngeal Nerve Using Hybrid-Transplantation of Skeletal Muscle-Derived Stem Cells and Bioabsorbable Scaffold

Hybrid transplantation of skeletal muscle-derived multipotent stem cells (Sk-MSCs) and bioabsorbable polyglyconate (PGA) felt was studied as a novel regeneration therapy for the transected recurrent laryngeal nerve (RLN). Sk-MSCs were isolated from green fluorescence protein transgenic mice and then expanded and transplanted with PGA felt for the hybrid transplantation (HY group) into the RLN transected mouse model. Transplantation of culture medium (M group) and PGA + medium (PGA group) were examined as controls. After eight weeks, trans-oral video laryngoscopy demonstrated 80% recovery of spontaneous vocal-fold movement during breathing in the HY group, whereas the M and PGA groups showed wholly no recoveries. The Sk-MSCs showed active engraftment confined to the damaged RLN portion, representing favorable prevention of cell diffusion on PGA, with an enhanced expression of nerve growth factor mRNAs. Axonal re-connection in the HY group was confirmed by histological serial sections. Immunohistochemical analysis revealed the differentiation of Sk-MSCs into Schwann cells and perineurial/endoneurial cells and axonal growth supportive of perineurium/endoneurium. The number of axons recovered was over 86%. These results showed that the stem cell and cytokine delivery system using hybrid transplantation of Sk-MSCs/PGA-felt is a potentially practical and useful approach for the recovery of transected RLN.