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Background Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by heterotopic bone formation in the posterior longitudinal ligament of the spine. Although the patients with OPLL are more common in the 60s and 70s, we know that there are markedly young patients (e.g., early 40s). However, to the best of our knowledge, there is few reports characterize young patients with cervical OPLL in terms of the imaging features, subjective symptoms, and ADL problems. Methods This is the multicenter cross-sectional study. Two hundred and thirty-seven Japanese symptomatic patients with cervical OPLL confirmed by standard X-rays collected from 16 institutions belonging to the Japanese Multicenter Research Organization for Ossification of the Spinal Ligament formed by the Japanese Ministry of Health, Labor and Welfare were recruited. Whole spine CT data as well as demographic data such as age, gender, patients-based evaluations, and the 36-item Short Form Health Survey (SF-36) were evaluated. Results Young group (≦ 45 years old) consisted of 23 patients (8 females and 15 males), accounting for 9.7% of the total. Their characteristics were high body mass index (BMI), significant involvement of trauma in the onset and deterioration of symptoms, and the predominance of thoracic OPLL. The patient-based evaluations did not show a significant difference between the young and non-young groups, or between the genders in the young group except for bodily pain (BP) of SF-36. Female patients in young group had significantly lower BP score of SF-36 than that of male in young group. Conclusions Characteristics of young patients with cervical OPLL were high BMI, significant involvement of trauma in the onset and deterioration of symptoms, lower BP score of SF-36 in female, and the predominance of thoracic OPLL.

BACKGROUND Spinal extradural arachnoid cysts are thought to be pouches that communicate with the intraspinal subarachnoid space through a dural defect. The treatment for these cysts is resection of the cyst wall followed by obliteration of the communicating defect, which is often elusive. OBSERVATIONS The authors report the case of a 22-year-old man with an extradural arachnoid cyst with claudication and progressive motor weakness. Regular magnetic resonance imaging (MRI) and computed tomography did not reveal the location of the defect in the cyst. However, three-dimensional (3D) phase-contrast MRI clearly indicated the location of the defect and the flow of cerebrospinal fluid into the cyst. These findings allowed the authors to perform the least invasive surgery; the patient recovered motor function and could walk more smoothly. LESSONS 3D phase-contrast MRI can reveal a subtle dural defect in patients with spinal extradural arachnoid cysts.

Background Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by heterotopic bone formation in the posterior longitudinal ligament of the spine. We know that the size and distribution of the ossified lesions in patients with OPLL are different in each case. However, the characteristics of the patients with radiologically severe cervical OPLL remain unknown. Methods The participants of our study were symptomatic patients with cervical OPLL who were diagnosed by standard radiographs of the cervical spine. Whole-spine CT data and demographic data such as age and sex were obtained from 20 institutions belonging to the Japanese Multicenter Research Organization for Ossification of the Spinal Ligament. According to the number of the levels involved by OPLL, we stratified the patients into two subgroups: severe group (S-group) and non-severe group (NS-group) to delineate the characteristics of radiologically severe patients with cervical OPLL. We also evaluated the most compressed level and the degree of occupying ratio of cervical spinal canal by OPLL at the most compressed level. Results A total of 234 patients with a mean age of 65 years were recruited. The S-group consisted of 48 patients (21%, 12 females and 36 males) and the NS-group consisted of 92 patients (79%, 22 females and 70 males). The mean age of males in the S-group (68 years old) was significantly higher than that of males in the NS-group (64 years old); however there was no significant difference in the mean age in females between the S-group (69 years old) and the NS-group (66 years old). No significant difference of body mass index, ossification of the nuchal ligament-positivity and presence of diabetes mellitus were found between the S- and the NS-group. Conclusions It is likely that the manner of extension of cervical OPLL is different between male and female patients.

Background: Although diffuse idiopathic skeletal hyperostosis (DISH) is known to coexist with the ossification of spinal ligaments (OSLs), details of the radiographic relationship remain unclear. Methods: We prospectively collected data of 239 patients with symptomatic cervical ossification of the posterior longitudinal ligament (OPLL) and analyzed the DISH severity on whole-spine computed tomography images, using the following grades: grade 0, no DISH, grade 1, DISH at T3–T10, grade 2, DISH at both T3–T10 and C6–T2 and/or T11–L2, and grade 3, DISH beyond C5 and/or L3. Ossification indices were calculated as the sum of vertebral and intervertebral levels with OSL for each patient. Results: DISH was found in 107 patients (44.8%), 65 (60.7%) of whom had grade 2 DISH. We found significant associations of DISH grade with the indices for cervical OPLL (r = 0.45, p &lt, 0.0001), thoracic ossification of the ligamentum flavum (OLF, r = 0.41, p &lt, 0.0001) and thoracic ossification of the supra/interspinous ligaments (OSIL, r = 0.53, p &lt, 0.0001). DISH grade was also correlated with the index for each OSL in the whole spine (OPLL: r = 0.29, p &lt, 0.0001, OLF: r = 0.40, p &lt, OSIL: r = 0.50, p &lt, 0.0001). Conclusion: The DISH grade correlated with the indices of OSL at each high-prevalence level as well as the whole spine.

Background: This study investigated how diffuse idiopathic skeletal hyperostosis (DISH) influences clinical characteristics in patients with cervical ossification of the posterior longitudinal ligament (OPLL). Although DISH is considered unlikely to promote neurologic dysfunction, this relationship remains unclear. Methods: Patient data were prospectively collected from 16 Japanese institutions. In total, 239 patients with cervical OPLL were enrolled who had whole-spine computed tomography images available. The primary outcomes were visual analog scale pain scores and the results of other self-reported clinical questionnaires. Correlations were sought between clinical symptoms and DISH using the following grading system: 1, DISH at T3-T10, 2, DISH at both T3–10 and C6–T2 and/or T11–L2, and 3, DISH beyond the C5 and/or L3 levels. Results: DISH was absent in 132 cases, grade 1 in 23, grade 2 in 65, and grade 3 in 19. There were no significant correlations between DISH grade and clinical scores. However, there was a significant difference in the prevalence of neck pain (but not in back pain or low back pain) among the three grades. Interestingly, DISH localized in the thoracic spine (grade 1) may create overload at the cervical spine and lead to neck pain in patients with cervical OPLL. Conclusion: This study is the first prospective multicenter cross-sectional comparison of subjective outcomes in patients with cervical OPLL according to the presence or absence of DISH. The severity of DISH was partially associated with the prevalence of neck pain.

Background: The purpose of this study was to clarify the clinical features of ossification of the posterior longitudinal ligament (OPLL) and extreme ossification at multiple sites. Methods: This prospective study involved patients with a diagnosis of cervical OPLL at 16 institutions in Japan. Patient-reported outcome measures, including responses on the Japanese Orthopaedic Association (JOA) Cervical Myelopathy Evaluation Questionnaire (JOA-CMEQ), JOA Back Pain Evaluation Questionnaire (JOA-BPEQ), and visual analog scale pain score, were collected to investigate clinical status. In each patient, the sum of the levels at which OPLL was located (OP index) was evaluated on whole-spine computed tomography, along with ossification of other spinal ligaments including the anterior longitudinal ligament (OALL), ligament flavum (OLF), supra- and intraspinous ligaments (SSL), and diffuse idiopathic skeletal hyperostosis (DISH). The distribution of OP index values in the study population was investigated, and the clinical and radiologic characteristics of patients in the top 10% were assessed. Results: In total, 236 patients (163 male, 73 female; mean age 63.5 years) were enrolled. Twenty-five patients with OP index ≥ 17 were categorized into a high OP index group and the remainder into a moderate/low OP index group. There were significantly more women in the high OP index group. Patients in the high OP index group also had significantly poorer scores for lower extremity function and quality of life on the JOA-CMEQ and in each domain but not for body pain on the JOA-BPEQ compared with those in the moderate/low OP index group. Patients in the high OP index group had more OALL in the cervical spine and more OLF and SSL in the thoracic spine. The prevalence of DISH was also significantly higher in the high OP index group. In the high OP index group, interestingly, OPLL was likely to be present adjacent to DISH in the cervicothoracic and thoracolumbar spine, especially in men, and often coexisted with DISH in the thoracic spine in women. Conclusion: This prospective cohort registry study is the first to demonstrate the clinical and radiologic features of patients with OPLL and a high OP index. In this study, patients with a high OP index had poorer physical function in the lumbar spine and lower extremities and were also predisposed to extreme ossification of spinal ligaments other than the OPLL.

Progress in regenerative medicine is realizing the possibility of neural regeneration and functional recovery in spinal cord injury (SCI). Recently, rehabilitation has attracted much attention with respect to the synergistic promotion of functional recovery in combination with neural stem/progenitor cell (NS/PC) transplantation, even in the chronic refractory phase of SCI. Nevertheless, sensory disturbance is one of the most prominent sequelae, even though the effects of combination or single therapies have been investigated mostly in the context of motor recovery. To determine how combination therapy with treadmill training (TMT) and NS/PC transplantation affects the manifestation of thermal allodynia and tactile hyperalgesia in chronic phase SCI, four groups of SCI mice were used to assess pain-related behavior and histological changes: combined transplantation and TMT therapy, transplantation only, TMT only, and control groups. Thermal allodynia and coarse touch-pressure hyperalgesia exhibited significant recovery in the combined therapy group in comparison with controls, whereas there were no significant differences with fine touch-pressure hyperalgesia and motor function. Further investigation revealed fewer fibers remaining in the posterior funiculus, which contained the tracts associated with the two modalities showing less recovery; that is, touch-pressure hyperalgesia and motor function. A significant correlation was only observed between these two modalities. Although no remarkable histological recovery was found within the lesion epicenter, changes indicating amelioration of pain were observed in the lumbar enlargement of the combination therapy group. Our results suggest that amelioration of thermal allodynia and tactile hyperalgesia can be brought about by the additive effect of NS/PC transplantation and TMT. The degree of recovery seems dependent on the distribution of damage.

This was a retrospective multicenter study.To clarify the progression of diffuse idiopathic skeletal hyperostosis (DISH) using whole-spine computed tomography in patients with cervical ossification of the posterior longitudinal ligament (OPLL).DISH and cervical OPLL frequently coexist, and can cause ankylosing spinal fractures due to biomechanical changes and fragility of the affected vertebrae. The epidemiology and pathophysiology of DISH occurring with cervical OPLL are unclear.We used whole-spine computed tomography to determine the prevalence of DISH in 234 patients with a diagnosis of cervical OPLL based on plain cervical radiographs. We established a novel system for grading the progression of DISH based on a cluster analysis of the DISH distribution along the spine. We calculated the correlation coefficient between this grading system and patient age.The prevalence of DISH in patients with cervical OPLL was 48.7%. Patients with DISH were significantly older than those who did not have DISH (67.3 vs. 63.4 y; P=0.005). Cluster analysis classified the DISH distribution into 6 regions, based on the levels affected: C2-C5, C3-T1, C6-T5, T3-10, T8-L2, and T12-S1. DISH was observed most frequently at T3-T10. We defined a system for grading DISH progression based on the number of regions involved, from grade 0 to 6. DISH was distributed at T3-T10 in60% of the grade 1 patients, whereas most patients with DISH at the cervical or lumbar spine were grade 4 or 5. There was a weak but significant correlation between the DISH grade and patient age.DISH was present in nearly half of the patients with cervical OPLL. DISH was more common in older patients. DISH developed at the thoracic level and progressed into the cervical and/or lumbar spine with age.Level III.

Study design Retrospective comparative study. Objectives The present study investigated radiographical changes in global spinal sagittal alignment (GSSA) and clinical outcomes following tumor resection using spinous process-splitting laminectomy (SPSL) approach without fixation in patients with conus medullaris (CM) or cauda equina (CE) tumor. Methods Forty-one patients with CM or CE tumor (19 males, 22 females, mean age at surgery of 52.9 ± 13.0 years) were included in this study. The variations of outcome variables were analyzed in various GSSA profiles using radiographic outcomes. The clinical outcomes were assessed using Japan Orthopaedic Association (JOA) score and JOA back pain evaluation questionnaire (JOABPEQ). Results In all cases, the various GSSA parameters (sagittal vertical axis, C2–7 lordosis, T1 slope, thoracic kyphosis, T10–L2 kyphosis, lumbar lordosis [LL; upper, middle, and lower], sacral slope, pelvic incidence, and pelvic tilt) did not significantly change in the 2-years postoperative period. Moreover, age at surgery, the number of resected laminae, preoperative T12–L2 kyphosis, or LL did not affect the postoperative changes in T12–L2 kyphosis or LL, and had no statistically significant correlation among them. The scores of each postoperative JOA domain and the Visual Analogue Scale included in the JOABPEQ were significantly improved. There was no statistical significant group difference in each sagittal profile or clinical outcomes between CM and CE groups postoperatively. Conclusions Tumor resection using SPSL approach did not affected the various GSSA parameters examined and resulted in satisfactory clinical outcomes, indicating that SPSL approach is a suitable surgical technique for patients with CM or CE tumor.

Diabetes is the main cause of gastroparesis accompanying decreased neuronal nitric oxide synthase (nNOS) in myenteric ganglia of the stomach. Decreased nNOS expression in the stomach also results from defects in apolipoprotein E (ApoE), which is secreted by astrocytes and has neuroprotective effects on the central nervous system. However, the roles of ApoE and enteric glial cells on gastric motility are uncertain. In this study, ApoE and enteric glial cell alterations in gastroparesis were investigated. Type 2 diabetic (db/db) mice and ApoE-knockout mice were analyzed. Gastric emptying was measured using the 13C acetic acid breath test. Expression levels of the pan-neuronal marker, protein gene product 9.5 (PGP 9.5), and glial marker, glial fibrillary acidic protein (GFAP) were examined by immunohistochemistry. Neural stem cells (NSCs) were injected into the gastric antral wall of ApoE-knockout mice. Delayed gastric emptying was observed in 27% of db/db mice with significant decreases in serum ApoE levels and GFAP expression in the gastric antrum. Gastric emptying was also delayed in ApoE-knockout mice, with a significant decrease in GFAP expression, but no change in PGP 9.5 expression. Transplantation of NSCs improved gastric emptying in ApoE-knockout mice through supplementation of GFAP-positive cells. Our results suggest that decreased enteric glial cells in ApoE-knockout mice are crucial for development of delayed gastric emptying, and NSC transplantation is effective in restoring myenteric ganglia and gastric motility.

The complication rate for palliative surgery in spinal metastasis is relatively high, and major complications can impair the patient's activities of daily living. However, surgical indications are determined based primarily on the prognosis of the cancer, with the risk of complications not truly considered. We aimed to identify the risk predictors for perioperative complications in palliative surgery for spinal metastasis.A multicentered, retrospective review of 195 consecutive patients with spinal metastasis who underwent palliative surgeries with posterior procedures from 2001 to 2016 was performed. We evaluated the type and incidence of perioperative complications within 14 days after surgery. Patients were categorized into either the complication group (C) or no-complication group (NC). Univariate and multivariate analyses were used to identify potential predictors for perioperative complications.Thirty patients (15%) experienced one or more complications within 14 days of surgery. The most frequent complications were surgical site infection (4%) and motor weakness (3%). A history of diabetes mellitus (C; 37%, NC; 9%: p 0.01) and surgical time over 300 min (C; 27%, NC; 12%: p 0.05) were significantly associated with complications according to univariate analysis. Increased blood loss and non-ambulatory status were determined to be potential risk factors. Of these factors, multivariate logistic regression revealed that a history of diabetes mellitus (OR: 6.6, p 0.001) and blood loss over 1 L (OR: 2.7, p 0.05) were the independent risk factors for perioperative complications. There was no difference in glycated hemoglobin A1c between the diabetes patients with and without perioperative complications.Diabetes mellitus should be used for the risk stratification of surgical candidates regardless of the treatment status, and strict prevention of bleeding is needed in palliative surgeries with posterior procedures to mitigate the risk of perioperative complications.

Ossification of the posterior longitudinal ligament (OPLL) is a progressive disease. The bridging of ossified lesions to the vertebral body gradually increases, thereby decreasing the mobility of the cervical spine; thus, cervical spine function may decrease over time. However, cervical spine function in patients with cervical OPLL has not been evaluated in large prospective studies. Therefore, we conducted a prospective multicenter study to clarify whether ossification spread can influence cervical spine function and quality of life (QOL) in patients with cervical OPLL. In total, 238 patients (162 men, 76 women; mean age, 63.9 years) were enrolled from 16 institutions. Each patient underwent whole spine computed tomography and was evaluated cervical spine function and QOL using the Japanese Orthopaedic Association Cervical Myelopathy Evaluation Questionnaire (JOACMEQ). In the multivariate regression analysis, a higher neck VAS score and a larger number of bridge formations of OPLL in the whole spine were significant predictors of adverse outcomes related to cervical spine function. This is the first prospective multicenter study to reveal the impact of ossification spread on cervical spine function. These findings are important to understand the natural course of OPLL and can serve as controls when evaluating postoperative cervical spine function.

This study aimed to clarify whether ossification predisposition influences clinical symptoms including pain, restriction of activities of daily living, and quality of life in patients with cervical ossification of the posterior longitudinal ligament (OPLL). Cervical ossification predisposition potentially causes neurologic dysfunction, but the relationship between clinical symptoms and radiologic severity of OPLL has not yet been investigated. Data were prospectively collected from 16 institutions across Japan. We enrolled 239 patients with cervical OPLL. The primary outcomes were patient-reported outcomes, including visual analog scale (VAS) pain scores and other questionnaires. Whole-spine computed tomography images were obtained, and correlations were investigated between clinical symptoms and radiologic findings, including the distribution of OPLL, the sum of the levels where OPLL was present (OP-index), and the canal narrowing ratio (CNR) grade. The cervical OP-index was Grade 1 in 113 patients, Grade 2 in 90, and Grade 3 in 36. No significant correlations were found between radiologic outcomes and VAS pain scores. The cervical OP-index was associated with lower extremity function, social dysfunction, and locomotive function. The CNR grade was not correlated with clinical symptoms, but Grade 4 was associated with lower extremity dysfunction. Thickness and extension of ossified lesions may be associated with lower extremity dysfunction in cervical OPLL.

Background Previous studies have shown that patients with cervical ossification of the posterior longitudinal ligament (OPLL) often have co-existing ossification of the nuchal ligament (ONL). However, no studies have focused on ONL and its relevance to the severity of OPLL or ossification of other spinal ligaments, such as anterior longitudinal ligament (OALL), ligamentum flavum (OLF), and supraspinous/interspinous ligament (OSIL). Methods In this multicenter study, we investigated ossification of the spinal ligaments in the whole spine computed tomography (CT) images of 233 cervical OPLL patients. The severity of ossification was evaluated using ossification index for each spinal ligament, calculated as the sum of the level of ossification. We compared the severity of ossification in each spinal ligament between patients with ONL and those without ONL. Furthermore, we investigated how the number of segments, where ONL exists, affects the severity of ossification in each spinal ligament. Results One hundred thirty patients (55.8%) had co-existing ONL in the cervical OPLL patients included in this study. The ONL (+) group included more male and aged patients. The cervical ossification indexes of OPLL and OALL were higher in ONL (+) patients than in ONL (−) patients. The thoracolumbar ossification indexes of OALL and OSIL were also higher in ONL (+) patients. Logistic regression analysis revealed that age, gender and cervical OA-index were independent factors correlating to the existence of ONL. In the cervical spine, both the ossification indexes of OALL and OPLL increased as the levels of ONL increased. Similarly, in the thoracolumbar spine, both the ossification indexes of OALL and OSIL were increased as the levels of cervical ONL increased. In the multiple regression analysis, cervical OA-index and thoracolumbar OSI-index showed significant correlation with the number of ONL levels. Conclusions Co-existence of ONL in cervical OPLL patients was associated with the severity of spinal hyperostosis especially in cervical OPLL, OALL, thoracolumbar OALL and OSIL.

Previous studies have demonstrated that transplantation of neural stem/progenitor cells (NS/PCs) into the lesioned spinal cord can promote functional recovery following incomplete spinal cord injury (SCI) in animal models. However, this strategy is insufficient following complete SCI because of the gap at the lesion epicenter. To obtain functional recovery in a mouse model of complete SCI, this study uses a novel collagen-based microfiber as a scaffold for engrafted NS/PCs. We hypothesized that the NS/PC-microfiber combination would facilitate lesion closure as well as transplant survival in the transected spinal cord. NS/PCs were seeded inside the novel microfibers, where they maintained their capacity to differentiate and proliferate. After transplantation, the stumps of the transected spinal cord were successfully bridged by the NS/PC-laden microfibers. Moreover, the transplanted cells migrated into the host spinal cord and differentiated into three neural lineages (astrocytes, neurons, and oligodendrocytes). However, the NS/PC-laden scaffold could not achieve a neural connection between the rostral end of the injury and the intact caudal area of the spinal cord, nor could it achieve recovery of motor function. To obtain optimal functional recovery, a microfiber design with a modified composition may be useful. Furthermore, combinatorial therapy with rehabilitation and/or medications should also be considered for practical success of biomaterial/cell transplantation-based approaches to regenerative medicine.

Background Posterior epidural migration of lumbar disc fragments (PEMLDF) is extremely rare. It is often confused with other posterior lesions and is usually diagnosed intraoperatively. We here describe the use of preoperative discography in the diagnosis of PEMLDF. Case presentation A 78-year-old man presented with acute low back pain, gait disturbance, and paresthesia in both legs. Magnetic resonance imaging showed a mass located posteriorly and laterally to the left aspect of the dural sac at the L3 level. The initial diagnosis indicated PEMLDF, malignancy, spontaneous hematoma, or epidural abscess. L3/4 discography clearly showed leakage of the contrast medium into the posterior dural space, indicating PEMLDF. The lesion was identified intraoperatively as a herniated-disc fragment, consistent with the preoperative discography. Conclusion PEMDLF is difficult to diagnose preoperatively. Discography is useful for the definitive diagnosis of PEMDLF prior to surgery.

Degenerative spondylolisthesis of the cervical spine is relative rare. We present a relative rare case of severe degenerative cervical spondylolisthesis with posterior corrective fusion. A 70-year-old female complained of neck pain and gait disturbance with cervical spondylotic myelopathy. Plain radiographs showed 8.3 mm anterolisthesis of C3/ C4, which gradually progressed during four years. The slip was not reduced by flexion and extension position. After releasing C3/C4 facet joint and yellow ligament, posterior reduction with instrumentation were performed and resulted in successful cervical correction and fusion.

Previous studies have demonstrated that transplantation of neural stem/progenitor cells (NS/PCs) into the lesioned spinal cord can promote functional recovery following incomplete spinal cord injury (SCI) in animal models. However, this strategy is insufficient following complete SCI because of the gap at the lesion epicenter. To obtain functional recovery in a mouse model of complete SCI, this study uses a novel collagen-based microfiber as a scaffold for engrafted NS/PCs. We hypothesized that the NS/PC-microfiber combination would facilitate lesion closure as well as transplant survival in the transected spinal cord. NS/PCs were seeded inside the novel microfibers, where they maintained their capacity to differentiate and proliferate. After transplantation, the stumps of the transected spinal cord were successfully bridged by the NS/PC-laden microfibers. Moreover, the transplanted cells migrated into the host spinal cord and differentiated into three neural lineages (astrocytes, neurons, and oligodendrocytes). However, the NS/PC-laden scaffold could not achieve a neural connection between the rostral end of the injury and the intact caudal area of the spinal cord, nor could it achieve recovery of motor function. To obtain optimal functional recovery, a microfiber design with a modified composition may be useful. Furthermore, combinatorial therapy with rehabilitation and/or medications should also be considered for practical success of biomaterial/cell transplantation-based approaches to regenerative medicine.

Our previous work reported functional recovery after transplantation of mouse and human induced pluripotent stem cell-derived neural stem/progenitor cells (hiPSC-NS/PCs) into rodent models of spinal cord injury (SCI). Although hiPSC-NS/PCs proved useful for the treatment of SCI, the tumorigenicity of the transplanted cells must be resolved before they can be used in clinical applications. The current study sought to determine the feasibility of ablation of the tumors formed after hiPSC-NS/PC transplantation through immunoregulation. Tumorigenic hiPSC-NS/PCs were transplanted into the intact spinal cords of immunocompetent BALB/cA mice with or without immunosuppressant treatment. In vivo bioluminescence imaging was used to evaluate the chronological survival and growth of the transplanted cells. The graft survival rate was 0% in the group without immunosuppressants versus 100% in the group with immunosuppressants. Most of the mice that received immunosuppressants exhibited hind-limb paralysis owing to tumor growth at 3 months after iPSC-NS/PC transplantation. Histological analysis showed that the tumors shared certain characteristics with low-grade gliomas rather than with teratomas. After confirming the progression of the tumors in immunosuppressed mice, the immunosuppressant agents were discontinued, resulting in the complete rejection of iPSC-NS/PC-derived masses within 42 days after drug cessation. In accordance with the tumor rejection, hind-limb motor function was recovered in all of the mice. Moreover, infiltration of microglia and lymphocytes was observed during the course of tumor rejection, along with apoptosis of iPSC-NS/PC-generated cells. Thus, immune rejection can be used as a fail-safe system against potential tumorigenicity after transplantation of iPSC-NS/PCs to treat SCI.

Transplantation of neural stem/progenitor cells (NS/PCs) promotes functional recovery after spinal cord injury (SCI); however, few studies have examined the optimal site of NS/PC transplantation in the spinal cord. The purpose of this study was to determine the optimal transplantation site of NS/PCs for the treatment of SCI. Wild-type mice were generated with contusive SCI at the T10 level, and NS/PCs were derived from fetal transgenic mice. These NS/PCs ubiquitously expressed ffLuc-cp156 protein (Venus and luciferase fusion protein) and so could be detected by in vivo bioluminescence imaging 9 days postinjury. NS/PCs (low: 250,000 cells per mouse; high: 1 million cells per mouse) were grafted into the spinal cord at the lesion epicenter (E) or at rostral and caudal (RC) sites. Phosphate-buffered saline was injected into E as a control. Motor functional recovery was better in each of the transplantation groups (E-Low, E-High, RC-Low, and RC-High) than in the control group. The photon counts of the grafted NS/PCs were similar in each of the four transplantation groups, suggesting that the survival of NS/PCs was fairly uniform when more than a certain threshold number of cells were transplanted. Quantitative RT-PCR analyses demonstrated that brain-derived neurotropic factor expression was higher in the RC segment than in the E segment, and this may underlie why NS/PCs more readily differentiated into neurons than into astrocytes in the RC group. The location of the transplantation site did not affect the area of spared fibers, angiogenesis, or the expression of any other mediators. These findings indicated that the microenvironments of the E and RC sites are able to support NS/PCs transplanted during the subacute phase of SCI similarly. Optimally, a certain threshold number of NS/PCs should be grafted into the E segment to avoid damaging sites adjacent to the lesion during the injection procedure.

Previous studies have demonstrated that neural stem/progenitor cells (NS/PCs) promote functional recovery in rodent animal models of spinal cord injury (SCI). Because distinct differences exist in the neuroanatomy and immunological responses between rodents and primates, it is critical to determine the effectiveness and safety of allografted embryonic stem cell (ESC)-derived NS/PCs (ESC-NS/PCs) in a nonhuman primate SCI model. In the present study, common marmoset ESC-NS/PCs were grafted into the lesion epicenter 14 days after contusive SCI in adult marmosets (transplantation group). In the control group, phosphate-buffered saline was injected instead of cells. In the presence of a low-dose of tacrolimus, several grafted cells survived without tumorigenicity and differentiated into neurons, astrocytes, or oligodendrocytes. Significant differences were found in the transverse areas of luxol fast blue-positive myelin sheaths, neurofilament-positive axons, corticospinal tract fibers, and platelet endothelial cell adhesion molecule-1-positive vessels at the lesion epicenter between the transplantation and control groups. Immunoelectron microscopic examination demonstrated that the grafted ESC-NS/PC-derived oligodendrocytes contributed to the remyelination of demyelinated axons. In addition, some grafted neurons formed synaptic connections with host cells, and some transplanted neurons were myelinated by host cells. Eventually, motor functional recovery significantly improved in the transplantation group compared with the control group. In addition, a mixed lymphocyte reaction assay indicated that ESC-NS/PCs modulated the allogeneic immune rejection. Taken together, our results indicate that allogeneic transplantation of ESC-NS/PCs from a nonhuman primate promoted functional recovery after SCI without tumorigenicity. Significance This study demonstrates that allogeneic embryonic stem cell (ESC)-derived neural stem/progenitor cells (NS/PCs) promoted functional recovery after transplantation into the injured spinal cord in nonhuman primates. ESC-NS/PCs were chosen because ESC-NS/PCs are one of the controls for induced pluripotent stem cell-derived NS/PCs and because ESC derivatives are possible candidates for clinical use. This translational research using an allograft model of a nonhuman primate is critical for clinical application of grafting NS/PCs derived from various allogeneic pluripotent stem cells, especially induced pluripotent stem cells, into injured spinal cord at the subacute phase.

Recent studies have demonstrated that transplantation of induced pluripotent stem cell-derived neurospheres can promote functional recovery after spinal cord injury in rodents, as well as in nonhuman primates. However, the potential tumorigenicity of the transplanted cells remains a matter of apprehension prior to clinical applications. As a first step to overcome this concern, this study established a glioblastoma multiforme xenograft model mouse. The feasibility of controlling immune suppression to ablate the grafted cells was then investigated. The human glioblastoma multiforme cell line U251 MG was transplanted into the intact spinal cords of immunodeficient NOD/SCID mice or into those of immunocompetent C57BL/6J H-2kb mice treated with or without immunosuppressants [FK506 plus anticluster of differentiation (CD) 4 antibody (Ab), or FK506 alone]. In vivo bioluminescent imaging was used to evaluate the chronological survival of the transplanted cells. The graft survival rate was 100% ( n = 9/9) in NOD/SCID mice, 0% ( n = 6/6) in C57BL/6J mice without immunosuppressant treatment, and 100% ( n = 37/37) in C57BL6/J mice with immunosuppressant treatment. After confirming the growth of the grafted cells in the C57/BL6J mice treated with immunosuppressants, immune suppression was discontinued. The grafted cells were subsequently rejected within 3 days in C57BL/6J mice treated with FK506 alone, as opposed to 26 days in C57BL/6J mice treated with FK506 plus anti-CD4 Ab. Histological evaluation confirmed the ablation of the grafted cells. Although this work describes a xenograft setting, the results suggest that this immunomodulatory strategy could provide a safety lock against tumor formation stemming from transplanted cells.

Spinal cord injury (SCI) is a devastating condition with no established treatment. To better understand the pathology and develop a treatment modality for SCI, an understanding of the physiological changes following SCI at the molecular level is essential. However, studies on SCI have primarily used rodent models, and few studies have examined SCI in non-human primates. In this study, we analyzed the temporal changes in gene expression patterns following SCI in common marmosets (Callithrix jacchus) using microarray analysis and mRNA deep sequencing. This analysis revealed that, although the sequence of events is comparable between primates and rodents, the inflammatory response following SCI is significantly prolonged and the onset of glial scar formation is temporally delayed in primates compared with rodents. These observations indicate that the optimal time window to treat SCI significantly differs among different species. This study provides the first extensive analysis of gene expression following SCI in non-human primates and will serve as a valuable resource in understanding the pathology of SCI.

Background The transplantation of neural stem/progenitor cells (NS/PCs) at the sub-acute phase of spinal cord injury, but not at the chronic phase, can promote functional recovery. However, the reasons for this difference and whether it involves the survival and/or fate of grafted cells under these two conditions remain unclear. To address this question, NS/PC transplantation was performed after contusive spinal cord injury in adult mice at the sub-acute and chronic phases. Results Quantitative analyses using bio-imaging, which can noninvasively detect surviving grafted cells in living animals, revealed no significant difference in the survival rate of grafted cells between the sub-acute and chronic transplantation groups. Additionally, immunohistology revealed no significant difference in the differentiation phenotypes of grafted cells between the two groups. Microarray analysis revealed no significant differences in the expression of genes encoding inflammatory cytokines or growth factors, which affect the survival and/or fate of grafted cells, in the injured spinal cord between the sub-acute and chronic phases. By contrast, the distribution of chronically grafted NS/PCs was restricted compared to NS/PCs grafted at the sub-acute phase because a more prominent glial scar located around the lesion epicenter enclosed the grafted cells. Furthermore, microarray and histological analysis revealed that the infiltration of macrophages, especially M2 macrophages, which have anti-inflammatory role, was significantly higher at the sub-acute phase than the chronic phase. Ultimately, NS/PCs that were transplanted in the sub-acute phase, but not the chronic phase, promoted functional recovery compared with the vehicle control group. Conclusions The extent of glial scar formation and the characteristics of inflammation is the most remarkable difference in the injured spinal cord microenvironment between the sub-acute and chronic phases. To achieve functional recovery by NS/PC transplantation in cases at the chronic phase, modification of the microenvironment of the injured spinal cord focusing on glial scar formation and inflammatory phenotype should be considered.

Murine and human iPSC-NS/PCs (induced pluripotent stem cell-derived neural stem/progenitor cells) promote functional recovery following transplantation into the injured spinal cord in rodents. However, for clinical applicability, it is critical to obtain proof of the concept regarding the efficacy of grafted human iPSC-NS/PCs (hiPSC-NS/PCs) for the repair of spinal cord injury (SCI) in a non-human primate model. This study used a pre-evaluated “safe” hiPSC-NS/PC clone and an adult common marmoset (Callithrix jacchus) model of contusive SCI. SCI was induced at the fifth cervical level (C5), followed by transplantation of hiPSC-NS/PCs at 9 days after injury. Behavioral analyses were performed from the time of the initial injury until 12 weeks after SCI. Grafted hiPSC-NS/PCs survived and differentiated into all three neural lineages. Furthermore, transplantation of hiPSC-NS/PCs enhanced axonal sparing/regrowth and angiogenesis, and prevented the demyelination after SCI compared with that in vehicle control animals. Notably, no tumor formation occurred for at least 12 weeks after transplantation. Quantitative RT-PCR showed that mRNA expression levels of human neurotrophic factors were significantly higher in cultured hiPSC-NS/PCs than in human dermal fibroblasts (hDFs). Finally, behavioral tests showed that hiPSC-NS/PCs promoted functional recovery after SCI in the common marmoset. Taken together, these results indicate that pre-evaluated safe hiPSC-NS/PCs are a potential source of cells for the treatment of SCI in the clinic.

Background. Although the pathogenesis of delayed gastric emptying, namely gastroparesis, is still unclear, diabetes mellitus is major etiology of gastroparesis. Decreased neuronal nitric oxide synthase (nNOS), which induces relaxation of gastric smooth muscle through the production of nitric oxide, is supposed to be one of the mechanisms of gastroparesis (Gastroenterology 113:1535, 1997). However, the effective treatment for gastroparesis has not been developed. While dyslipidemia is a risk factor of diabetic neuropathy, the defect of apolipoprotein E (apoE), a lipid transporter secreted from glial cell, decreased the expression of nNOS in the stomach (Dig Dis Sci. 57:1504, 2012). Furthermore, transplantion of neural stem cell into gastric antral wall was reported to improve delayed gastric emptying in nNOS knockout mouse (Gastroenterology 129:1817, 2005). The aim of the present study is to explore the role of apoE on gastric emptying and evaluate the effect of neural stem cell transplantation against the model of gastroparesis. Methods. The model of type2 diabetes, db/db mice, and apoE knockout mice, which is the model of arteriosclerosis and hyperlipidemia, were used. For measurement of gastric emptying, 0.48 mg of 13C-acetic acid with 0.15 ml liquid meal (Lacol®) was orally administered. The gastric half emptying time (t1/2) measured by 13C breath test was used for evaluation. The expression levels of nNOS, pan-neuronal marker, PGP 9.5, glial fibrillary acidic protein (GFAP), and glia derived neurotrophic factor (GDNF) were examined by immunohistochemistry andWestern blotting. Neural stem cells were isolated from ffLuc-transgenic mouse, which fluorescent protein, venus, and luciferase gene was fused (BBRC 419; 188, 2012). After tertiary neurosphere was formed, it was injected into gastric antral wall by glass needle. Results. Delayed gastric emptying was observed in 27% of db/db mice, while the levels of blood glucose and body weight were not significantly different. The expression of nNOS in gastric antrum and serum level of apoE were significantly decreased in db/db mice which presented delayed gastric emptying. In apoE knockout mice, the gastric emptying was also delayed compared with control. Although, the expression of PGP 9.5 was not changed, the expression of nNOS was significantly decreased in apoE knockout mice. Furthermore, the expression levels of GFAP and GDNF were significantly decreased in apoE knockout mice. Transplantation of neural stem cell significantly improved the delayed gastric emptying in apoE knockout mice. Conclusion. Neural stem cell transplantation restored the delayed gastric emptying in apoE knockout mice, suggesting a promising application of regenerative therapy against gastroparesis.

Stem cells represent a potential cellular resource in the development of regenerative medicine approaches to the treatment of pathologies in which specific cells are degenerated or damaged by genetic abnormality, disease, or injury. Securing sufficient supplies of cells suited to the demands of cell transplantation, however, remains challenging, and the establishment of safe and efficient cell banking procedures is an important goal. Cryopreservation allows the storage of stem cells for prolonged time periods while maintaining them in adequate condition for use in clinical settings. Conventional cryopreservation systems include slow-freezing and vitrification both have advantages and disadvantages in terms of cell viability and/or scalability. In the present study, we developed an advanced slow-freezing technique using a programmed freezer with a magnetic field called Cells Alive System (CAS) and examined its effectiveness on human induced pluripotent stem cell-derived neural stem/progenitor cells (hiPSC-NS/PCs). This system significantly increased cell viability after thawing and had less impact on cellular proliferation and differentiation. We further found that frozen-thawed hiPSC-NS/PCs were comparable with non-frozen ones at the transcriptome level. Given these findings, we suggest that the CAS is useful for hiPSC-NS/PCs banking for clinical uses involving neural disorders and may open new avenues for future regenerative medicine.

Background Supra/interspinous ligaments connect adjacent spinous processes and act as a stabilizer of the spine. As with other spinal ligaments, it can become ossified. However, few report have discussed ossification supra/interspinous ligaments (OSIL), so its epidemiology remains unknown. We therefore aimed to investigate the prevalence and distribution of OSIL in symptomatic patients with cervical ossification of the posterior longitudinal ligament (OPLL). Methods The participants of our study were symptomatic patients with cervical OPLL who were diagnosed by standard radiographs of the cervical spine. The whole spine CT data as well as clinical parameters such as age and sex were obtained from 20 institutions belong to the Japanese Multicenter Research Organization for Ossification of the Spinal Ligament (JOSL). The prevalence and distribution of OSIL and the association between OSIL and clinical parameters were reviewed. The sum of the levels involved by OPLL (OP-index) and OSIL (OSI-index) as well as the prevalence of ossification of the nuchal ligament (ONL) were also investigated. Results A total of 234 patients with a mean age of 65 years was recruited. The CT-based evidence of OSIL was noted in 68 (54 males and 14 females) patients (29%). The distribution of OSIL showed a significant thoracic preponderance. In OSIL-positive patients, single-level involvement was noted in 19 cases (28%), whereas 49 cases (72%) presented multi-level involvement. We found a significant positive correlation between the OP-index grade and OSI-index. ONL was noted at a significantly higher rate in OSIL-positive patients compared to negative patients. Conclusions The prevalence of OSIL in symptomatic patients with cervical OPLL was 29%. The distribution of OSIL showed a significant thoracic preponderance.