Gentaro Kumagai

Therapeutic potential of appropriately evaluated safe-induced pluripotent stem cells for spinal cord injury

Various types of induced pluripotent stem (iPS) cells have been established by different methods, and each type exhibits different biological properties. Before iPS cell-based clinical applications can be initiated, detailed evaluations of the cells, including their differentiation potentials and tumorigenic activities in different contexts, should be investigated to establish their safety and effectiveness for cell transplantation therapies. Here we show the directed neural differentiation of murine iPS cells and examine their therapeutic potential in a mouse spinal cord injury (SCI) model. “Safe” iPS-derived neurospheres, which had been pre-evaluated as nontumorigenic by their transplantation into nonobese diabetic/severe combined immunodeficiency (NOD/SCID) mouse brain, produced electrophysiologically functional neurons, astrocytes, and oligodendrocytes in vitro. Furthermore, when the safe iPS-derived neurospheres were transplanted into the spinal cord 9 d after contusive injury, they differentiated into all three neural lineages without forming teratomas or other tumors. They also participated in remyelination and induced the axonal regrowth of host 5HT+ serotonergic fibers, promoting locomotor function recovery. However, the transplantation of iPS-derived neurospheres pre-evaluated as “unsafe” showed robust teratoma formation and sudden locomotor functional loss after functional recovery in the SCI model. These findings suggest that pre-evaluated safe iPS clone-derived neural stem/progenitor cells may be a promising cell source for transplantation therapy for SCI.

Roles of ES Cell-Derived Gliogenic Neural Stem/ Progenitor Cells in Functional Recovery after Spinal Cord Injury

Transplantation of neural stem/progenitor cells (NS/PCs) following the sub-acute phase of spinal cord injury (SCI) has been shown to promote functional recovery in rodent models. However, the types of cells most effective for treating SCI have not been clarified. Taking advantage of our recently established neurosphere-based culture system of ES cell-derived NS/PCs, in which primary neurospheres (PNS) and passaged secondary neurospheres (SNS) exhibit neurogenic and gliogenic potentials, respectively, here we examined the distinct effects of transplanting neurogenic and gliogenic NS/PCs on the functional recovery of a mouse model of SCI. ES cell-derived PNS and SNS transplanted 9 days after contusive injury at the Th10 level exhibited neurogenic and gliogenic differentiation tendencies, respectively, similar to those seen in vitro. Interestingly, transplantation of the gliogenic SNS, but not the neurogenic PNS, promoted axonal growth, remyelination, and angiogenesis, and resulted in significant locomotor functional recovery after SCI. These findings suggest that gliogenic NS/PCs are effective for promoting the recovery from SCI, and provide essential insight into the mechanisms through which cellular transplantation leads to functional improvement after SCI.

Comparative Study of Methods for Administering Neural Stem/Progenitor Cells to Treat Spinal Cord Injury in Mice:

To investigate potential cures for spinal cord injury (SCI), several researchers have transplanted neural stem/progenitor cells (NS/PCs) into the injured spinal cord by different procedures, including intralesional (IL), intrathecal (IT), and intravenous (IV) injection. However, there are no reports quantifying or comparing the number of cells successfully transplanted to the lesion site by each procedure in vivo. The purpose of the present study was to determine the optimal method of cell transplantation to the SCI site in terms of grafted cell survival and safety. For this purpose, we developed mouse NS/PCs that expressed a novel Venus-luciferase fusion protein that enabled us to detect a minimum of 1,000 grafted cells in vivo by bioluminescence imaging (BLI). After inducing contusive SCI at the T10 level in mice, NS/PCs were transplanted into the injured animals three different ways: by IL, IT, or IV injection. Six weeks after the transplantation, BLI analysis showed that in the IL group, the luminescence intensity of the grafted cells had decreased to about 10% of its initial level, and appeared at the site of injury. In the IT group, the luminescence of the grafted cells, which was distributed throughout the entire subarachnoid space immediately after transplantation, was detected at the injured site 1 week later, and by 6 weeks had gradually decreased to about 0.3% of its initial level. In the IV group, no grafted cells were detected at the site of injury, but all of these mice showed luminescence in the bilateral chest, suggesting pulmonary embolism. In addition, one third of these mice died immediately after the IV injection. In terms of grafted cell survival and safety, we conclude that the IL application of NS/PCs is the most effective and feasible method for transplanting NS/PCs into the SCI site.

Cervical range of motion and alignment after laminoplasty preserving or reattaching the semispinalis cervicis inserted into axis.

Study Design A radiographic study in 111 patients using radiographs was conducted. Objective To clarify whether the modified laminoplasty with C3 laminectomy preserving the semispinalis cervicis (SSC) inserted into C2 could maintain the postopertive range of motion (ROM) and sagittal alignment compared with conventional C3-C7 laminoplasty reattaching the muscle to C2. Summary of Background Data Intraoperative injury of the SSC is relevant to the significant loss of ROM and the malalignment after laminoplasty. To expose the C3 lamina, however, the SSC inserted into C2 could not be preserved in conventional C3-C7 laminoplasty. Methods The ROM and sagittal alignment of 70 patients (group A) (52 men, 18 women, mean age 59 y, mean follow-up period 1 y and 7 mo) with C4-C7 laminoplasty with C3 laminectomy were compared with those of 41 patients (group B) (28 men, 13 women, mean age 59 y, mean follow-up period 2 y and 6 mo) with C3-C7 laminoplasty using radiographs of the cervical spine. Results Regarding C2-C7 ROM, the postoperative ROM was larger (P=0.003) and the decrease rate of ROM was smaller (P=0.0006), and decreased ROM in extension was smaller (P<0.0001) in group A. Regarding O-C2 ROM, the increased ROM was smaller (P=0.043) and increased ROM in extension was smaller (P=0.001) in group A. Regarding O-C7 ROM, the postoperative ROM was larger (P=0.029) in group A. Regarding the cervical alignment, the increased lordotic angle at O-C2 was smaller (P=0.046) in group A. Conclusions This modified laminoplasty preserving the SSC inserted into C2 is an effective procedure for maintaining postoperative ROM, especially in extension, and sagittal alignment of the upper cervical spine well.

Simple foot tapping test as a quantitative objective assessment of cervical myelopathy.

Study Design. A clinical and cohort study. Objective. The first purpose of this study was to investigate the standard value of a simple foot tapping test (FTT) in a large healthy population. The second purpose was to elucidate the validity of FTT as a quantitative assessment of lower extremity motor function for cervical compressive myelopathy. Summary of Background Data. Several clinical performance tests have been reported as objective assessments for the severity of cervical myelopathy. The FTT is the simplest and easiest method for a quantitative analysis of lower limb motor dysfunction in the upper motor neuron diseases. However, there were few studies about the FTT in cervical myelopathy. Methods. We recruited 252 patients who were diagnosed with cervical myelopathy and 792 healthy volunteers who participated in a health promotion project. Among the patients, 126 who underwent surgery were evaluated both before and 1 year after surgery. We performed the FTT and grip and release test and evaluated the modified Japanese Orthopaedic Association (JOA) score for cervical myelopathy. Results. The mean value of FTT was 23.8 ± 7.2 in myelopathic patients, which was significantly lower than 31.7 ± 6.4 in healthy controls and decreased with age. The value of FTT significantly correlated with the lower extremity motor function of modified JOA score and the value of grip and release test. Among the patients who underwent surgery, the average value of FTT was 22.4 ± 7.0 preoperatively and improved to 28.4 ± 8.1 at 1 year postoperatively. Postoperative gain of FTT significantly correlated with the gain of JOA score. Conclusion. The FTT results correlated with those of other tests for cervical myelopathy, and the FTT scores were improved by surgery. The FTT is an easy and useful quantitative assessment method for lower extremity motor function in patients with cervical myelopathy, especially those who cannot walk.

Mesenchymal stem cell isolation and characterization from human spinal ligaments.

Mesenchymal stem cells (MSCs) have a fibroblast-like morphology, multilineage potential, long-term viability and capacity for self-renewal. While several articles describe isolating MSCs from various human tissues, there are no reports of isolating MSCs from human spinal ligaments, and their localization in situ. If MSCs are found in human spinal ligaments, they could be used to investigate hypertrophy or ossification of spinal ligaments. To isolate and characterize MSCs from human spinal ligaments, spinal ligaments were harvested aseptically from eight patients during surgery for lumbar spinal canal stenosis and ossification of the posterior longitudinal ligament. After collagenase digestion, nucleated cells were seeded at an appropriate density to avoid colony-to-colony contact. Cells were cultured in osteogenic, adipogenic or chondrogenic media to evaluate their multilineage differentiation potential. Immunophenotypic analysis of cell surface markers was performed by flow cytometry. Spinal ligaments were processed for immunostaining using MSC-related antibodies. Cells from human spinal ligaments could be extensively expanded with limited senescence. They were able to differentiate into osteogenic, adipogenic or chondrogenic cells. Flow cytometry revealed that their phenotypic characteristics met the minimum criteria of MSCs. Immunohistochemistry revealed the localization of CD90-positive cells in the collagenous matrix of the ligament, and in adjacent small blood vessels. We isolated and expanded MSCs from human spinal ligaments and demonstrated localization of MSCs in spinal ligaments. These cells may play an indispensable role in elucidating the pathogenesis of numerous spinal diseases.

Postoperative changes at the lower end of cervical laminoplasty : For preservation of the C7 spinous process in laminoplasty

Introduction C3-C7 laminoplasty has been the standard treatment for cervical myelopathy, although several recent reports described C3-C6 laminoplasty for preserving the muscles inserting in C7 and reducing postoperative axial symptoms. However, postoperative changes at C6/C7 of the lower end of C3-C6 laminoplasty, especially regarding a possibility of postoperative spinal canal narrowing have not been measured. The purpose of this study was to clarify postoperative changes at the lower end of laminoplasty. Methods Pre and postoperative spinal dura diameter at the lower end of conventional C3-C7 laminoplasty using MRI, and the related factors for spinal dura diameter and the causes of postoperative dura narrowing were investigated. Results At the last follow-up after C3-C7 laminoplasty, dura diameter at C7/T1 was significantly wider after operation than before operation, and postoperative narrowing of dura diameter, which was found in 20% of patients, was a maximum amount of one millimeter. No pre and postoperative factor significantly correlated with dura diameter at C7/T1. The causes of postoperative narrowing at the lower end of laminoplasty were disc protrusion and/or posterior scar, or segmental angulation of the spinal cord. Conclusion In conclusion, the presence of preoperative subarachnoid space over one millimeter at C6/C7 may be able to be one of the radiological indications for C3-C6 laminoplasty.

The relationship between the anatomy of the nuchal ligament and postoperative axial pain after cervical laminoplasty: cadaver and clinical study.

Study Design. A cadaver and clinical study investigated the attachment of the nuchal ligament to the cervical spinous process. Objective. To investigate the anatomical details of the attachment of the nuchal ligament to the spinous process and the relationship between the morphology of the nuchal ligament and postoperative axial pain after laminoplasty. Summary of Background Data. The relationship between the length of the C6 spinous process and the morphology of the nuchal ligament and occurrence of postoperative axial pain has not been elucidated. Methods. The morphology of the nuchal ligament was investigated in 35 cadavers and 60 patients on preoperative computed tomography and magnetic resonance imaging. The lengths of the C6 and C7 spinous processes were measured, and the C6:C7 ratio (C6 spinous process length/C7 spinous process length) was calculated. The relationship between the morphology of the attachment of nuchal ligament to the C6 spinous process and the C6:C7 ratio were investigated. In addition, the effects of the anatomy of the nuchal ligament around the C6 spinous process and different procedures of surgical invasion to C6 or C7 on postoperative axial pain were investigated for 113 patients who underwent laminoplasty. Results. The nuchal ligament was attached to not only the C7 spinous process, but also the C6 spinous process when the C6:C7 ratio was more than 0.8. When the nuchal ligament was attached to the C6 spinous process and to C7, postoperative axial pain after C3–C7 laminoplasty occurred more often compared with C3–C6 laminoplasty for patients without the nuchal ligament attached to the C6 spinous process. Conclusion. This study shows that there is an association between the individual anatomical differences of the nuchal ligament and the occurrence of postoperative axial pain after laminoplasty. Careful attention should be paid to the morphology of the attachment of the nuchal ligament to the C6 spinous process to reduce postoperative axial pain.

Perioperative cardiopulmonary complications after cervical spine surgery in the prone position: the relationship between age and preoperative testing.

BackgroundCardiac arrest during spine surgery in the prone position is difficult to manage as poor access makes cardiopulmonary resuscitation and defibrillation difficult. Advanced age is the maximal risk factor for cardiac arrest. Therefore, we wanted to determine the relationship between age and cardiac risk factors/pre-operating tests for cervical spine surgery in the prone position.MethodsThe inclusion criteria for this study specified 88 patients scheduled should undergo cervical spine surgery in the prone position. The patients were divided into two groups: Paients in group A (50 patients) were aged 69 and under, Group B (38 patients) 70 and above. All patients responded to a medical interview about eight cardiac risk factors including past history, chest symptoms, diabetes mellitus, hypertension, hyperlipidemia, obesity, smoking, and family history. All patients underwent physical examination and 24-h Holter ECG and echocardiography performed by two cardiologists before surgery. We analyzed relationships between cardiac risk factors and ECG/echocardiography and investigated intra- and postoperative cardiovascular complications.ResultsAlthough there were no significant differences in the number of cardiac risk factors between the two groups, the frequency of hypertension was significantly greater in Group B than in Group A. The frequency of abnormal ECG and echocardiography findings especially was significantly greater in Group B than in Group A. In ECG and echocardiography, three patients in Group B who had no cardiac risk factors before surgery showed abnormal findings, and one of the three patients had the amalgamation of arrhythmia after the operation. Also, in Group B, cardiovascular complications occurred in one case during operation.ConclusionThese results suggested that patients aged 70 and above should undergo ECG and echocardiography examination before cervical spine surgery in the prone position whether they have cardiac risk factors or not . A prospective, randomized multi-center study with a larger patient sample is warranted to ultimately demonstrate how patients should be tested before spine surgery in the prone position.

High Osteogenic Potential of Adipose- and Muscle-derived Mesenchymal Stem Cells in Spinal-ossification Model Mice

Study Design. Basic experiments in a mouse model of ossification of the posterior longitudinal ligament (OPLL). Objective. To assess the osteogenic potential of mesenchymal stem cells (MSCs) obtained from muscle and adipose tissue in Tiptoe-walking (ttw) mice, in which cervical OPLL compresses the spinal cord and causes motor and sensory dysfunction. Summary of Background Data. In humans, MSCs have been implicated in the pathogenesis of cervical OPLL. Cervical OPLL in ttw mice causes chronic compression of the spinal cord. Few studies have compared the MSC osteogenic potential with behavioral changes in an OPLL animal model. Methods. We compared the osteogenic potential and behavioral characteristics of MSCs from ttw mice (4 to 20 weeks old) with those from control wild-type mice (without hyperostosis). Ligament ossification was monitored by micro-computed tomography and pathology; tissues were double stained with fluorescent antibodies against markers for MSCs (CD45 and CD105), at 8 weeks. The Basso Mouse Scale was used to assess motor function, and heat and mechanical tests to assess sensory function. The osteogenic potential of adipose and muscle MSCs was assessed by Alizarin Red S absorbance, staining for osteogenic mineralization, and real-time quantitative polymerase chain reaction for osteogenesis-related genes. Results. Spinal-ligament ossification began in ttw mice at 8 weeks of age, and the ossified area increased with age. Immunofluorescence staining identified MSCs in the ossification area. The ttw mice became hyposensitive at 8 weeks of age, and Basso Mouse Scale scores showed motor-function deficits starting at 12 weeks of age. Alizarin Red S staining for mineralization showed a higher osteogenic potential in the adipose- and muscle-derived MSCs from ttw mice than from wild-type mice at 4, 8, and 20 weeks of age. Real-time quantitative polymerase chain reaction showed that ttw MSCs strongly expressed osteogenesis-related genes. Conclusion. MSCs derived from muscle and adipose tissue in ttw mice had a high osteogenic potential. Level of Evidence: N/A