Naosuke Kamei

BDNF, NT-3, and NGF released from transplanted neural progenitor cells promote corticospinal axon growth in organotypic cocultures.

Study Design. Experimental study of spinal cord injury using an organotypic slice culture. Objective. To clarify the mechanism of corticospinal axon regeneration following transplantation of neural progenitor cells (NPCs) in the injured spinal cord. Summary of Background Data. Several mechanisms underlying central nervous system regeneration after transplantation of NPCs have been proposed; however, the precise mechanism has not been clarified. Previously, we demonstrated that transplanted NPCs secreted humoral factors that in turn promoted corticospinal axon growth using the unique organotypic coculture system involving brain cortex and spinal cord from neonatal rats. Methods. Cultured NPCs were immunostained with antibodies against neurotrophic factors including brain-derived neurotrophic factor (BDNF), neurotrophin (NT)-3, nerve growth factor (NGF), and ciliary neurotrophic factor (CNTF) both before and after differentiation. To evaluate corticospinal axon growth quantitatively, we used the organotypic coculture system. The dissected brain cortex and spinal cord obtained from neonatal rats were aligned next to each other and cultured on a membrane. NPCs were transplanted onto the cocultures. Furthermore, neutralizing antibodies against BDNF, NT-3, NGF, or CNTF were added to the cocultures. Axon growth from the brain cortex into the spinal cord was assessed quantitatively using anterograde axon tracing with DiI. Results. The cultured NPCs were positively immunostained by antibodies against BDNF, NT3, NGF, and CTNF both before and after differentiation. Transplantation of NPCs promoted axon growth from the brain cortex into the spinal cord. The axon growth promoted by NPCs was significantly suppressed by the addition of neutralizing antibodies against BDNF, NT-3, and NGF but not CNTF. Conclusion. The neurotrophic factors, BDNF, NT-3, and NGF, secreted by transplanted NPCs, were involved in the promotion of corticospinal axon growth after transplantation of NPCs.

Mesenchymal‐stem‐cell‐derived exosomes accelerate skeletal muscle regeneration

Mesenchymal stem cell (MSC) transplantation is used for treatment of many diseases. The paracrine role of MSCs in tissue regeneration is attracting particular attention. We investigate the role of MSC exosomes in skeletal muscle regeneration. MSC exosomes promote myogenesis and angiogenesis in vitro, and muscle regeneration in an in vivo model of muscle injury. Although MSC exosomes had low concentrations of muscle‐repair‐related cytokines, a number of repair‐related miRNAs were identified. This study suggests that the MSC‐derived exosomes promote muscle regeneration by enhancing myogenesis and angiogenesis, which is at least in part mediated by miRNAs such as miR‐494.

CD34+ Cells Represent Highly Functional Endothelial Progenitor Cells in Murine Bone Marrow

Background Endothelial progenitor cells (EPCs) were shown to have angiogenic potential contributing to neovascularization. However, a clear definition of mouse EPCs by cell surface markers still remains elusive. We hypothesized that CD34 could be used for identification and isolation of functional EPCs from mouse bone marrow. Methodology/Principal Findings CD34+ cells, c-Kit+/Sca-1+/Lin− (KSL) cells, c-Kit+/Lin− (KL) cells and Sca-1+/Lin− (SL) cells were isolated from mouse bone marrow mononuclear cells (BMMNCs) using fluorescent activated cell sorting. EPC colony forming capacity and differentiation capacity into endothelial lineage were examined in the cells. Although CD34+ cells showed the lowest EPC colony forming activity, CD34+ cells exhibited under endothelial culture conditions a more adherent phenotype compared with the others, demonstrating the highest mRNA expression levels of endothelial markers vWF, VE-cadherin, and Flk-1. Furthermore, a dramatic increase in immediate recruitment of cells to the myocardium following myocardial infarction and systemic cell injection was observed for CD34+ cells comparing with others, which could be explained by the highest mRNA expression levels of key homing-related molecules Integrin β2 and CXCR4 in CD34+ cells. Cell retention and incorporation into the vasculature of the ischemic myocardium was also markedly increased in the CD34+ cell-injected group, giving a possible explanation for significant reduction in fibrosis area, significant increase in neovascularization and the best cardiac functional recovery in this group in comparison with the others. Conclusion These findings suggest that mouse CD34+ cells may represent a functional EPC population in bone marrow, which could benefit the investigation of therapeutic EPC biology.

Responses of microRNAs 124a and 223 following spinal cord injury in mice.

Study design:We investigated microRNA (miRNA) expression after spinal cord injury (SCI) in mice.Objectives:The recent discovery of miRNAs suggests a novel regulatory control over gene expression during plant and animal development. MiRNAs are short noncoding RNAs that suppress the translation of target genes by binding to their mRNAs, and play a central role in gene regulation in health and disease. The purpose of this study was to examine miRNA expression after SCI.Setting:Department of Orthopaedic Surgery, Graduate School of Biomedical Sciences, Hiroshima University.Methods:We examined the expression of miRNA (miR)-223 and miR-124a in a mouse model at 6 h, 12 h, 1 day, 3 days and 7 days after SCI using quantitative PCR. The miRNA expression was confirmed by in situ hybridization.Results:Quantitative PCR revealed two peaks of miR-223 expression at 6 and 12 h and 3 days after SCI. MiR-124a expression decreased significantly from 1 day to 7 days after SCI. In situ hybridization demonstrated the presence of miR-223 around the injured site. However, miR-124a, which was present in the normal spinal cord, was not observed at the injured site.Conclusion:Our results indicate a time-dependent expression pattern of miR-223 and miR-124a in a mouse model of SCI. In this study, the time course of miRNA-223 expression may be related to inflammatory responses after SCI, and the time course of decreased miR-124a expression may reflect cell death.

Postoperative segmental C5 palsy after cervical laminoplasty may occur without intraoperative nerve injury: a prospective study with transcranial electric motor-evoked potentials.

Study Design. Intraoperative neurophysiologic monitoring with transcranial electric motor-evoked potentials was performed on patients who underwent cervical laminoplasty at a university hospital in a prospective study. Objective. To evaluate the usefulness of intraoperative spinal cord monitoring with transcranial electric motor-evoked potentials for prediction of the occurrence of segmental motor paralysis after cervical laminoplasty. Summary of Background Data. Segmental motor paralysis occasionally occurs among patients who undergo expansive laminoplasty for cervical myelopathy, and it has been attributed to nerve root lesions caused by either a traumatic surgical technique or a tethering effect after decompression. Methods. Sixty-two consecutive patients (47 men and 15 women; mean age 64 years [range 32–89]) who were scheduled to undergo cervical laminoplasty under intraoperative spinal cord monitoring with transcranial electric motor-evoked potentials were included in this study. Transcranial electrical stimulations were delivered through pin-type electrodes, and the evoked potentials were recorded over the deltoid, biceps, and triceps muscles in the bilateral upper extremities and thoracic spinal cord. Results. Intraoperative evoked potentials were successfully recorded in all muscles in 57 patients (92%), and incomplete evoked potentials were recorded in the remaining 5 patients. No critical decrease in the amplitude of the evoked potentials was observed in any of the 62 patients. All patients showed sufficient postoperative recovery from their clinical symptoms; however, postoperative transient C5 palsy occurred in 3 patients. Conclusions. No abnormalities were observed on transcranial electric motor-evoked potential monitoring, even in those patients who developed postoperative transient C5 palsy. These results suggest that the development of postoperative C5 palsy after cervical laminoplasty is not associated with intraoperative injury of the nerve root or the spinal cord, although the precise mechanism of its development is still unclear. Surgeons should be aware that C5 palsy is a possible complication of cervical laminoplasty, even in the absence of intraoperative nerve injury.

Acceleration of skeletal muscle regeneration in a rat skeletal muscle injury model by local injection of human peripheral blood-derived CD133-positive cells.

Muscle injuries in sport activities can pose challenging problems in traumatology and sports medicine. The best treatment for muscle injury has not been clearly established except for the conservative treatment that is routinely performed. We investigated the potential of human adult CD133+ cells to contribute to skeletal muscle regeneration in an athymic rat model. We tested whether CD133+ cells locally transplanted to the skeletal muscle lacerated models could (a) induce vasculogenesis/angiogenesis, (b) differentiate into endothelial and myogenic lineages, and (c) finally promote histological and functional skeletal myogenesis. Granulocyte colony stimulating factor‐mobilized peripheral blood (PB) CD133+ cells, PB mononuclear cells, or phosphate‐buffered saline was locally injected after creating a muscle laceration in the tibialis anterior muscle in athymic rats. After treatment, histological and functional skeletal myogenesis was observed significantly in the CD133+ group. The injected CD133+ cells differentiated into endothelial and myogenic lineages. Using real‐time polymerase chain reaction analysis, we found that the gene expressions related to microenvironment conduction for host angiogenesis, fibrosis, and myogenesis were ideally up/downregulated. Our results show that CD133+ cells have the potential to enhance the histological and functional recovery from skeletal muscle injury rather via indirect contribution to environment conduction for muscular regeneration. It would be relatively easy to purify this cell fraction from PB, which could be a feasible and attractive autologous candidate for skeletal muscle injuries in a clinical setting. These advantages could accelerate the progression of cell‐based therapies for skeletal muscle injuries from laboratory to clinical implementation. STEM CELLS 2009;27:949–960

Concurrent Vasculogenesis and Neurogenesis From Adult Neural Stem Cells

Rationale: Recent reports have demonstrated that signals from vascular endothelial cells are necessary for organogenesis that may precede vasculogenesis. However, the origin of these neovascular cells in regenerating tissue has not been clarified. Objective: Here we tested the hypothesis that adult neural stem cells (NSCs) can differentiate into vascular lineage, as well as neural lineage, in the process of collaborative organogenesis. Methods and Results: NSCs, clonally isolated from mouse brain, were shown to develop endothelial and smooth muscle phenotypes in vitro. To elucidate whether NSCs can simultaneously differentiate into vascular and neural cells in vivo, genetically labeled NSCs were administered to mice with unilateral sciatic nerve crush injury or operatively induced brain and myocardial ischemia. Two weeks later, necropsy examination disclosed recruitment of the labeled NSCs to sites of injury differentiating into vascular cells (endothelial cells and vascular smooth muscle cells) and Schwann cells in regenerating nerve. Similarly, NSC-derived vascular cells/astrocytes and endothelial cells were identified in ischemic brain tissue and capillaries in myocardium 2 weeks following transplantation, respectively. Conclusions: These findings, concurrent vasculogenesis and neurogenesis from a common stem cell, suggest that certain somatic stem cells are capable of differentiating into not only somatic cells of identity but also into vascular cells for tissue regeneration.

MicroRNA-223 expression in neutrophils in the early phase of secondary damage after spinal cord injury

MicroRNA (miR)s are short non-coding RNAs that suppress the translation of target genes, and play an important role in gene regulation. Despite this prominence, there are few reports that refer to the expression of miRs after spinal cord injury (SCI). Previously, we reported on miR-223 expression after SCI in mice. The purpose of this study is to reveal the distribution of miR-223 and identify the cells that express miR-223 in the injured spinal cord. Quantitative polymerase chain reaction analysis revealed high expression of miR-223 at 12h after SCI. Double staining of in situ hybridization and immunohistochemistry showed that the signals of miR-223 merged with Gr-1 positive neutrophils. Our data indicate that miR-223 might regulate neutrophils in the early phase after SCI.

Lnk Deletion Reinforces the Function of Bone Marrow Progenitors in Promoting Neovascularization and Astrogliosis Following Spinal Cord Injury

Lnk is an intracellular adaptor protein reported as a negative regulator of proliferation in c‐Kit positive, Sca‐1 positive, lineage marker‐negative (KSL) bone marrow cells. The KSL fraction in mouse bone marrow is believed to represent a population of hematopoietic and endothelial progenitor cells (EPCs). We report here that, in vitro, Lnk−/− KSL cells form more EPC colonies than Lnk+/+ KSL cells and show higher expression levels of endothelial marker genes, including CD105, CD144, Tie‐1, and Tie2, than their wild‐type counterparts. In vivo, the administration of Lnk+/+ KSL cells to a mouse spinal cord injury model promoted angiogenesis, astrogliosis, axon growth, and functional recovery following injury, with Lnk−/− KSL being significantly more effective in inducing and promoting these regenerative events. At day 3 following injury, large vessels could be observed in spinal cords treated with KSL cells, and reactive astrocytes were found to have migrated along these large vessels. We could further show that the enhancement of astrogliosis appears to be caused in conjunction with the acceleration of angiogenesis. These findings suggest that Lnk deletion reinforces the commitment of KSL cells to EPCs, promoting subsequent repair of injured spinal cord through the acceleration of angiogenesis and astrogliosis. STEM CELLS 2010;28:365–375

Mesenchymal Stem Cell-Derived Exosomes Promote Fracture Healing in a Mouse Model

Paracrine signaling by bone‐marrow‐derived mesenchymal stem cells (MSCs) plays a major role in tissue repair. Although the production of regulatory cytokines by MSC transplantation is a critical modulator of tissue regeneration, we focused on exosomes, which are extracellular vesicles that contain proteins and nucleic acids, as a novel additional modulator of cell‐to‐cell communication and tissue regeneration. To address this, we used radiologic imaging, histological examination, and immunohistochemical analysis to evaluate the role of exosomes isolated from MSC‐conditioned medium (CM) in the healing process in a femur fracture model of CD9−/− mice, a strain that is known to produce reduced levels of exosomes. We found that the bone union rate in CD9−/− mice was significantly lower than wild‐type mice because of the retardation of callus formation. The retardation of fracture healing in CD9−/− mice was rescued by the injection of exosomes, but this was not the case after the injection of exosomes‐free conditioned medium (CM‐Exo). The levels of the bone repair‐related cytokines, monocyte chemotactic protein‐1 (MCP‐1), MCP‐3, and stromal cell‐derived factor‐1 in exosomes were low compared with levels in CM and CM‐Exo, suggesting that bone repair may be in part mediated by other exosome components, such as microRNAs. These results suggest that exosomes in CM facilitate the acceleration of fracture healing, and we conclude that exosomes are a novel factor of MSC paracrine signaling with an important role in the tissue repair process.