Kosuke Saito

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.

Transoral en bloc resection of superficial laryngeal and pharyngeal cancers

The objective of this study was to evaluate the efficacy and safety of minimally invasive transoral en bloc resection of superficial pharyngeal and laryngeal cancers.

Therapeutic isolation and expansion of human skeletal muscle-derived stem cells for the use of muscle-nerve-blood vessel reconstitution

Skeletal muscle makes up 40–50% of body mass, and is thus considered to be a good adult stem cell source for autologous therapy. Although, several stem/progenitor cells have been fractionated from mouse skeletal muscle showing a high potential for therapeutic use, it is unclear whether this is the case in human. Differentiation and therapeutic potential of human skeletal muscle-derived cells (Sk-Cs) was examined. Samples (5–10 g) were obtained from the abdominal and leg muscles of 36 patients (age, 17–79 years) undergoing prostate cancer treatment or leg amputation surgery. All patients gave informed consent. Sk-Cs were isolated using conditioned collagenase solution, and were then sorted as CD34−/CD45−/CD29+ (Sk-DN/29+) and CD34+/CD45− (Sk-34) cells, in a similar manner as for the previous mouse Sk-Cs. Both cell fractions were appropriately expanded using conditioned culture medium for about 2 weeks. Differentiation potentials were then examined during cell culture and in vivo transplantation into the severely damaged muscles of athymic nude mice and rats. Interestingly, these two cell fractions could be divided into highly myogenic (Sk-DN/29+) and multipotent stem cell (Sk-34) fractions, in contrast to mouse Sk-Cs, which showed comparable capacities in both cells. At 6 weeks after the separate transplantation of both cell fractions, the former showed an active contribution to muscle fiber regeneration, but the latter showed vigorous engraftment to the interstitium associated with differentiation into Schwann cells, perineurial/endoneurial cells, and vascular endothelial cells and pericytes, which corresponded to previous observations with mouse SK-Cs. Importantly, mixed cultures of both cells resulted the reduction of tissue reconstitution capacities in vivo, whereas co-transplantation after separate expansion showed favorable results. Therefore, human Sk-Cs are potentially applicable to therapeutic autografts and show multiple differentiation potential in vivo.

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.

Reconstruction of Multiple Facial Nerve Branches Using Skeletal Muscle-Derived Multipotent Stem Cell Sheet-Pellet Transplantation.

Head and neck cancer is often diagnosed at advanced stages, and surgical resection with wide margins is generally indicated, despite this treatment being associated with poor postoperative quality of life (QOL). We have previously reported on the therapeutic effects of skeletal muscle-derived multipotent stem cells (Sk-MSCs), which exert reconstitution capacity for muscle-nerve-blood vessel units. Recently, we further developed a 3D patch-transplantation system using Sk-MSC sheet-pellets. The aim of this study is the application of the 3D Sk-MSC transplantation system to the reconstitution of facial complex nerve-vascular networks after severe damage. Mouse experiments were performed for histological analysis and rats were used for functional examinations. The Sk-MSC sheet-pellets were prepared from GFP-Tg mice and SD rats, and were transplanted into the facial resection model (ST). Culture medium was transplanted as a control (NT). In the mouse experiment, facial-nerve-palsy (FNP) scoring was performed weekly during the recovery period, and immunohistochemistry was used for the evaluation of histological recovery after 8 weeks. In rats, contractility of facial muscles was measured via electrical stimulation of facial nerves root, as the marker of total functional recovery at 8 weeks after transplantation. The ST-group showed significantly higher FNP (about three fold) scores when compared to the NT-group after 2–8 weeks. Similarly, significant functional recovery of whisker movement muscles was confirmed in the ST-group at 8 weeks after transplantation. In addition, engrafted GFP+ cells formed complex branches of nerve-vascular networks, with differentiation into Schwann cells and perineurial/endoneurial cells, as well as vascular endothelial and smooth muscle cells. Thus, Sk-MSC sheet-pellet transplantation is potentially useful for functional reconstitution therapy of large defects in facial nerve-vascular networks.

The potential risk of vessel infiltration and cervical lymph node metastasis in hypopharyngeal superficial squamous cell carcinoma: a retrospective observational study

Abstract Conclusion: The depth of hypopharyngeal superficial cancer may predict vessel infiltration and potential risk of cervical lymph node metastasis. Objectives: To elucidate the histopathological predictors of vessel infiltration and the risk of regional lymph node metastasis in hypopharyngeal superficial cancer. Methods: This study included 31 lesions from 30 patients who had undergone transoral en bloc resection in the hospital. Patients with intraepithelial neoplasia or muscular invasion were excluded. Patient characteristics, nodal status, state of vessel infiltration, state of perineural invasion, histopathological parameters, and post-operative cervical lymph node recurrence were retrospectively examined. The histopathological parameters measured were tumor diameter and the following three parameters: tumor thickness, depth from the mucosal surface, and depth from the basement membrane. Correlations between histopathological parameters and state of vessel infiltration were statistically analyzed. Results: Of the 31 lesions examined, four had vessel infiltration. Three of the four lesions with vessel infiltration had regional lymph node metastasis as well as subsequent lymph node metastasis. Lesions with vessel infiltration were significantly deeper than those without. In contrast, there was no significant difference in lesion diameters. In addition, there was no correlation between the depth and the diameter of the lesion.

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.

Evaluating the significance of level IIb neck dissection for hypopharyngeal cancer.

This study evaluated cervical lymph node metastases at level IIb in cases of hypopharyngeal cancer and analyzed the possibility of preservation of level IIb during neck dissection.

The predictive role of infiltrative growth pattern in early pharyngeal cancers.

Abstract Conclusion: The infiltrative growth pattern may predict tumor depth and lymph node metastasis. INF-a seems to fall into a low-risk category, and no additional treatment may be required immediately. Objectives: Tumor depth is a predictor of lymph node metastasis in early pharyngeal cancers. An infiltrative growth pattern is also a prognostic factor in other cancers. This study aimed to elucidate the predictive role of infiltrative growth pattern in early pharyngeal cancers. Methods: Thirty-eight lesions from 37 patients who had undergone trans-oral resection of pharyngeal cancers were included. According to the Japanese Classification of Esophageal Cancer, infiltrative growth pattern was classified into three groups; INF-a, INF-b, and INF-c. The correlation between infiltrative growth pattern and tumor depth, cervical lymph node metastasis was analyzed. Results: Of the 38 lesions, 25 were INF-a, nine were INF-b, and four were INF-c lesions. Lymph node metastasis was observed in three INF-b and one INF-c lesions. In contrast, no INF-a had lymph node metastasis. All INF-a lesions showed shallow invasion of the sub-epithelium; INF-b and INF-c lesions had significantly greater depth than INF-a.