A Distinct Subpopulation of Bone Marrow Mesenchymal Stem Cells, Muse Cells, Directly Commit to the Replacement of Liver Components.

Genotyping graft livers by short tandem repeats after human living-donor liver transplantation (n = 20) revealed the presence of recipient or chimeric genotype cases in hepatocytes (6 of 17, 35.3%), sinusoidal cells (18 of 18, 100%), cholangiocytes (15 of 17, 88.2%) and cells in the periportal areas (7 of 8, 87.5%), suggesting extrahepatic cell involvement in liver regeneration. Regarding extrahepatic origin, bone marrow mesenchymal stem cells (BM-MSCs) have been suggested to contribute to liver regeneration but compose a heterogeneous population. We focused on a more specific subpopulation (1-2% of BM-MSCs), called multilineage-differentiating stress-enduring (Muse) cells, for their ability to differentiate into liver-lineage cells and repair tissue. We generated a physical partial hepatectomy model in immunodeficient mice and injected green fluorescent protein (GFP)-labeled human BM-MSC Muse cells intravenously (n = 20). Immunohistochemistry, fluorescence in situ hybridization and species-specific polymerase chain reaction revealed that they integrated into regenerating areas and expressed liver progenitor markers during the early phase and then differentiated spontaneously into major liver components, including hepatocytes (≈74.3% of GFP-positive integrated Muse cells), cholangiocytes (≈17.7%), sinusoidal endothelial cells (≈2.0%), and Kupffer cells (≈6.0%). In contrast, the remaining cells in the BM-MSCs were not detected in the liver for up to 4 weeks. These results suggest that Muse cells are the predominant population of BM-MSCs that are capable of replacing major liver components during liver regeneration.

Peripheral nerve regeneration through the space formed by a chitosan gel sponge.

The clinical treatment of traumatized peripheral nerves often requires grafting of autologous cutaneous nerves. However, there are drawbacks in sacrificing healthy nerves and tissue scarring. In this study, an artificial material, freeze-dried chitosan gel sponge, was examined as a scaffold for nerve regeneration in rats. An 8-mm gap was made by removing a segment of the sciatic nerve, and the distal and proximal stumps were sandwiched by chitosan gel sponge. Rats were killed at 4, 7, 14, and 28 days, and 2 and 4 months after the operation and histological and morphometric evaluations were performed. Regenerating axons were observed at 4 days after the operation. Regenerating nerves extended the distal stump at 14 days after surgery. By electron microscopy, numerous macrophages appeared to phagocyte chitosan, and made a dense cell layer on the chitosan. Regenerating axons did not touch the chitosan, and extended through the space surrounded by macrophage-stacked chitosan. Regenerating nerves were well-myelinated 2 months after surgery. Regenerating nerves were on average 2.45 and 2.75 microm in diameter at 2 and 4 months, respectively, after surgery. These results indicate that the chitosan gel sponge sandwich might be suitable as a graft for peripheral nerve regeneration.

Insights into autotransplantation: the unexpected discovery of specific induction systems in bone marrow stromal cells.

Many kinds of cells, including embryonic stem cells and tissue stem cells, have been considered candidates for transplantation therapy for neuro- and muscle-degenerative diseases. Bone marrow stromal cells (MSCs) also have great potential as therapeutic agents since they are easily isolated and can be expanded from patients without serious ethical or technical problems. Recently, new methods for the highly efficient and specific induction of functional neurons and skeletal muscle cells have been developed for MSCs. These induced cells were transplanted into animal models of stroke, Parkinson's disease and muscle degeneration, resulting in the successful integration of transplanted cells and improvement in the behavior of the transplanted animals. Here I describe the discovery of these induction systems and focus on the potential use of MSC-derived cells for 'auto-cell transplantation therapy' in neuro- and muscle-degenerative diseases.

Effect of GDNF gene transfer into axotomized retinal ganglion cells using in vivo electroporation with a contact lens-type electrode.

We developed an in vivo electroporation method to introduce foreign genes into retinal ganglion cells (RGCs). After the intravitreous injection of the plasmid gene (20 mug), five electric pulses (6 V/cm, 100 ms duration) were each delivered twice with 5 min interval to the rat eye using a contact lens-type electrode (cathodal) attached to the cornea and a needle electrode (anodal) inserted to the middle of the forehead. The efficiency of the genetic introduction into RGCs and tissue damage to the eyeball was evaluated using a green fluorescent protein (GFP) gene, TUNEL and histological observation. DiI retrograde labeling revealed that 24.4 +/- 4.7% of all RGCs were electrointroduced with the GFP gene. TUNEL and histological analysis showed a few tissue damages in the cornea, lens and retina. To confirm whether this method can actually rescue damaged RGCs, glial cell line-derived neurotrophic factor (GDNF) was electrointroduced into RGCs after optic nerve transection. After the electrointroduction, a significant increase in the number of surviving RGCs was observed 2 and 4 weeks after the optic nerve transection, and the decrease of caspase 3 and 9 was detected by RT-PCR. These results suggest that this method may be useful for the delivery of genes into RGCs with simplicity and minimal tissue damage.

Gene transfer into retinal ganglion cells by in vivo electroporation: a new approach.

We developed a new in vivo electroporation method to deliver genes into retinal ganglion cells (RGCs). Efficiency and degree of tissue damage were evaluated using green fluorescent protein (GFP) gene and TUNEL. Soon after the intravitreous injection of the GFP gene, electroporation (five electric pulses of 99 ms duration each and 12V/cm delivered twice 5 min apart) was carried out on the adult rat eyeball with the aid of tweezer-type disc electrodes attached to corneal (cathode) and scleral (anode) surfaces. GFP expression, exhibiting a maximum on day 7, was detectable for up to 21 days. DiI retrograde labeling of RGCs showed that 41.5% of the total ganglion cells in the electroinjected area were GFP-positive. Therefore, this new method may be a useful tool for the delivery of genes into RGCs.

Protection of retinal ganglion cells from ischemia-reperfusion injury by electrically applied Hsp27.

PURPOSE: To determine whether the Hsp27 protein can rescue retinal ganglion cells (RGCs) of rats from ischemia-reperfusion injury. METHODS: Retinal ischemia was induced in rats by clamping the ophthalmic artery within the dural sheath of the optic nerve. Immediately after removing the clamp and beginning the reperfusion, Hsp27 protein solution was injected into the vitreous, and electroporation was applied. To determine whether Hsp27 entered the RGCs, anti-Hsp27 immunohistochemistry was performed. The retinal damage was evaluated by counting the number of RGCs retrogradely labeled by 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine percholorate (diI) injected into the superior colliculus, and also by comparing the ratio of TUNEL-positive to all RGCs in the RGC layer. RESULTS: Electroporation successfully delivered Hsp27 protein into RGCs. In the Hsp27 electroinjected group, the number of RGCs 7 days after ischemia-reperfusion was significantly higher than in the control groups. The ratio of TUNEL-positive cells to all RGCs was lower in the group electroinjected with Hsp27 protein. CONCLUSIONS: Electroporation of Hsp27 protein into RGCs increased the resistance of the RGCs to the apoptosis induced by ischemia-reperfusion injury.

Effects of light and darkness on cell deaths in damaged retinal ganglion cells of the carp retina.

Effects of light and darkness on the apoptosis of retinal ganglion cells (RGCs) in young carp were measured by TUNEL method after transection of the optic nerve. Following the operation, the fish were kept under one of four regimens; constant darkness (DD), constant light (LL), 12 hr light and 12 hr dark (LD) and 3 hr of flickering light followed by 21 hr in the dark (FL). On day 3, the highest ratio of apoptotic RGCs was seen under conditions of DD, followed by LL, LD, and FL. On day 6, the percentages of apoptotic RGCs were lower under every experimental condition than what they had been earlier on day 3, but the same ranking order was maintained. Immunohistochemically it could be shown that phosphorylated ERKs were more intensively localized in FL rather than DD retinas. The results suggest that illumination regimens, and in particular cyclic diurnal light/dark changes, have an influence on the degree of apoptosis of damaged RGCs, and that inhibition of apoptosis is correlated with the higher expression of phosphorylated ERKs.

Optic nerve regeneration within artificial Schwann cell graft in the adult rat.

We investigate whether an artificial graft made by cultured Schwann cell, extracellular matrix (ECM) and trophic factors can provide the environment for the regeneration of retinal ganglion cell (RGC) axons in adult rats. Six kinds of artificial grafts were used: ECM (control); ECM and Schwann cells; ECM, Schwann cells and either nerve growth factor, brain-derived neurotrophic factor (BDNF) and neurotrophin-4 (NT-4); ECM, Schwann cells, BDNF and NT-4, combined with intravitreal injection of BDNF. The grafts were transplanted onto the transected optic nerve. RGC regeneration was evaluated by dil retrograde labeling, immunohistochemistry, and electron microscopy at 3 weeks post-operation. The degree of dil labeled RGC was approximately 2% for ECM alone, and 10% for ECM and Schwann cells (p < 0.01). The labeling increased to approximately 20% by administration of neurotrophins. The addition of intravitreous BDNF injection resulted in highest labeling percentage of 30%. Immunohistochemical study showed that axons were association with GAP-43 and cell adhesion molecules. Neurotrophin receptors (Trk-A and Trk-B) were detected in nerve fibers both in the retina and in the graft. Remyelination was seen by electron microscopic observation. These results demonstrate that the regeneration of RGC axons is induced with the use of cultured Schwann cells and ECM as promoting factors for regrowth. The degree of regeneration was significantly increased by neurotrophins in the grafts and in the vitreous.

Sciatic nerve regeneration in rats induced by transplantation of in vitro differentiated bone-marrow stromal cells.

Bone marrow stromal cells (MSCs) are multipotent stem cells that have the potential to differentiate into bone, cartilage, fat and muscle. We now demonstrate that MSCs can be induced to differentiate into cells with Schwann cell characteristics, capable of eliciting peripheral nervous system regeneration in adult rats. MSCs treated with beta-mercaptoethanol followed by retinoic acid and cultured in the presence of forskolin, basic-FGF, PDGF and heregulin, changed morphologically into cells resembling primary cultured Schwann cells and expressing p75, S-100, GFAP and O4. The MSCs were genetically engineered by transduction with retrovirus encoding green fluorescent protein (GFP), and then differentiated by treatment with factors described above. They were transplanted into the cut ends of sciatic nerves, which then responded with vigorous nerve fibre regeneration within 3 weeks of the operation. Myelination of regenerated fibers by GFP-expressing MSCs was recognized using confocal and immunoelectron microscopy. The results suggest that MSCs are able to differentiate into myelinating cells, capable of supporting nerve fibre re-growth, and they can therefore be applied to induce nerve regeneration.

The interaction and adhesive mechanisms between axon and Schwann cell during central and peripheral nerve regeneration.

It is well known that the injured mammalian PNS can successfully regenerate, while the CNS such as the optic nerve of adult mammals is incapable of regeneration. It is now generally accepted that the inability of CNS neurons to regenerate appears to be caused by the glial environment made up of astrocytes and oligodendrocytes. However, recent studies show that such CNS neurons have the intrinsic capacity to regenerate which is triggered by an experimental replacement of inhibitorial glial environment to peripheral nerve segment. Thus, the PNS environment is suitable not only for the regeneration of PNS itself, but also for the elicitation of CNS regeneration. Schwann cell is the major component of PNS, which plays a central role both in PNS and CNS regeneration by producing various kinds of functional substances. The contact of axons to Schwann cells based upon the structural and molecular linkages seems to be indispensable for stable and successful regeneration. In addition to cell adhesion molecules, Schwann cells utilize short focal tight junctions to provide morphological stabilization of the contact with the elongating axon, as well as small scale gap junctions to facilitate traffic of substances between them. Thus, nerve regeneration is not a simple phenomenon of axonal elongation on the part of the Schwann cell membrane, but is based on direct and dynamic communication between the axon and the neighboring Schwann cell, which may be partly associated with the mechanisms of neural regeneration.

Role of Schwann cells in retinal ganglion cell axon regeneration.

It is a well known fact that the injured PNS can successfully regenerate, on the other hand, the CNS such as retinal ganglion cell (RGC) axons of adult mammals is incapable of regeneration. After injury, RGC axons rapidly degenerate and most cell bodies go through the process of cell death, while glial cells at the site of injury undergo a series of responses which underlie the so-called glial scar formation. However, it has become apparent that RGCs do have an intrinsic capacity to regenerate which can be elicited by experimental replacement of the inhibitory glial environment with a permissive peripheral nerve milieu. Schwann cells are a major component of the PNS and play a role in regeneration, by producing various kinds of functional substances such as diffusible neurotrophic factors, extracellular matrix and cell adhesion molecules. RGC regeneration can be induced by cooperation of these substances. The contact of RGC axons to Schwann cells based upon the structural and molecular linkages seems to be indispensable for the stable and successful regeneration. In addition to cell adhesion molecules such as NCAM and L1, data from our laboratory show that Schwann cells utilize short focal tight junctions to provide morphological stabilization of the contact with the elongating axon, as well as a small scale of gap junctions to facilitate traffic of substances between them. Moreover, our results show that modifications of functional properties in neighboring glial cells of optic nerve are induced by transplantation of Schwann cells. Astrocytes usually considered to form a glial scar guide the regenerating axons in cooperation with Schwann cells. A decrease of the oligodendrocyte marker O4 and migration of ED-1 positive macrophages is observed within the optic nerve stump. Accordingly, RGC regeneration is not a simple phenomenon of axonal elongation on the Schwann cell membrane, but is based on direct and dynamic communication between the axon and the Schwann cell, and is also accompanied by changes and responses among the glial cell populations, which may be partly associated with the mechanisms of optic nerve regeneration.

Increase in nucleoli after x-radiation of fibroblasts of patients with Gorlin syndrome.

Gorlin syndrome (GS) is an autosomal dominant disorder in which patients are abnormally susceptible to ionizing radiation with radiotherapeutic doses. Radiogenic basal cell carcinomas may develop with a short latent period in patients. The mechanisms underlying the abnormal radiosusceptibility of cells in patients with GS has not been well characterized. In this study we report an increase in the number of nucleoli in fibroblast cells from 3 patients with GS after x-radiation. In GS fibroblasts, the increase in nucleolus number concomitant with the increase of ribonucleoprotein immunoreactive aggregates within the nucleus was observed after x-radiation, whereas significant change was not found in normal fibroblasts derived from healthy donors. This increase disappeared when cells were cultured with the RNA synthesis inhibitor actinomycin D after x-radiation but not when they were cultured with cycloheximide or aphydicolin, which are protein and DNA synthesis inhibitors, respectively. Ultraviolet exposure did not induce remarkable changes in the GS nucleoli. Thus the increase in nucleoli was induced after x-radiation of GS fibroblasts, and this increase seemed to be related to RNA synthesis metabolism.

Glial cells in degenerating and regenerating optic nerve of the adult rat.

The glial cell reaction both in degenerating and regenerating adult rat optic nerve was studied by immunohistochemistry and electron microscopy. Degeneration in the optic nerve was achieved by complete transection, and the retinal stump was then analyzed. The regeneration was observed by autotransplantation of a sciatic nerve segment to the transected retinal stump. In both cases, optic nerve axons were labeled anterogradely with rhodamine, followed by immunohistochemical staining. Glial fibrillary acidic protein-positive astrocytes covered the transected end of degenerating optic nerve, whereas in the regenerating optic nerve they enwrapped axonal bundles emerging from the optic nerve stump and migrated together into the transitional zone intervening between the retinal stump and graft. In electron microscopy, direct attachment of astrocyte and Schwann cell was found within the transitional zone, whereby these cells were holding axons between them. Decrease of 04 immunoreactivity, which labels oligodendrocytes, was apparent in the transected end of retinal stump during the regeneration. The ED1 -positivity, which labels microglia/macrophages, was found in cells accumulated in the transitional zone of degenerating optic nerve, whereas during regeneration, ED1-immunoreactive cells were also distributed in the retinal stump. These results suggest that astrocytes, usually considered to interfere with optic nerve regeneration, change their characteristics in the presence of peripheral nerve graft and guide the regenerating axons in cooperation with Schwann cells. The response of oligodendrocytes and microglia/macrophages may also be modulated by peripheral nerve.

Haemophilus influenzae has a GM1 ganglioside-like structure and elicits Guillain-Barré syndrome.

The authors report a patient with an axonal Guillain-Barré syndrome (acute motor axonal neuropathy) associated with anti-GM1 antibody after Haemophilus influenzae infection. The result of ELISA inhibition studies and cytochemical staining with cholera toxin suggest the presence of a GM1-like structure on the surface of H. influenzae isolated from the patient. A particular strain of H. influenzae may have a GM1-like structure and may elicit an axonal Guillain-Barré syndrome.

Immunohistochemical study of E-cadherin and ZO-1 in allergic nasal epithelium of the guinea pig.

Nasal epithelial damage during allergic inflammation was studied by observing the distribution of cell adhesion molecule E-cadherin and tight junction (zonula occludens) cell-cell contact associated protein ZO-1. The guinea pig model of nasal allergy, sensitized with intraperitoneally administered ovalbumin (OA) and subsequently challenged with OA intranasally, was used. In control epithelium, E-cadherin immunoreactivity was detected continuously along neighboring epithelial cell borders. ZO-1 spot-like immunoreactivity was detected in the apicolateral portion of epithelial cells corresponding to the tight junction (TJ) position, but no changes in immunoreactivity were found between control and challenged epithelia. In the challenged epithelium of sensitized animals, marked infiltration of eosinophils and structural changes, such as widening of the intercellular spaces and detachment of adjacent epithelial cells, were observed concurrently. In addition, spots negative for E-cadherin immunoreactivity were noted in the epithelium, associated with the extracellular deposition of eosinophil granule proteins. Immunoelectron microscopy revealed a decrease or disappearance of E-cadherin immunoreactivity, which took place not only in regions where intercellular spaces were wide and adjacent epithelial cells were detached, but also at the point of contact between infiltrating eosinophils and epithelial cells. Approximately 87% of eosinophils observed in the challenged epithelium were associated with such loss of E-cadherin immunoreactivity. These results suggest that the intimate epithelial cell contact mediated by E-cadherin is loosened as a consequence of eosinophil infiltration, which may trigger the initial step of subsequent epithelial destruction in allergic states.

Putative gap junctional communication between axon and regenerating Schwann cells during mammalian peripheral nerve regeneration.

Gap junctions are intercellular channels which mediate the traffic of ions and a variety of molecular messengers between contiguous cells. Here, we report on the possibility that atypical gap junctions develop between heterologous tissues, such as regenerating nerve axons and Schwann cells, during peripheral nerve regeneration in adult rats. After a complete transection and subsequent regeneration in the rat sciatic nerve distal segment, a small scale gap junction-like structure was observed between the regenerating axons and adjoining Schwann cells. Immunoelectron microscopy showed that one of the gap junctional proteins, connexin32, was located at a small region of contact between the axon and Schwann cells. Biocytin, a small molecular weight dye, was transported from regenerating axons into adjoining Schwann cells. The present findings suggest that regenerating axons communicate directly with adjacent Schwann cells through small gap junctions, which may play a role in the mechanism of regeneration following nerve transection.

Abnormal DNA synthesis activity induced by X-rays in nevoid basal cell carcinoma syndrome cells.

DNA synthesis activity was examined in fibroblasts and isolated nuclei derived from patients with nevoid basal cell carcinoma syndrome (NBCCS) upon exposure to X-ray and ultraviolet (UV). The DNA synthesis activity in NBCCS fibroblasts increased after X-ray irradiation, i.e., to twice that on mock-irradiation, while it decreased in healthy donor-derived fibroblasts. The DNA synthesis activity in isolated nuclei of X-ray irradiated NBCCS fibroblasts also increased, i.e., more than twice that on mock-irradiated. In the experiments using synchronized cells, DNA synthesis activity showed the most marked increase when the fibroblasts at S phase were irradiated with X-rays. In contrast, UV-irradiated NBCCS fibroblasts showed no such increase in DNA synthesis. These results revealed that DNA synthesis is abnormally induced in X-ray irradiated NBCCS cells and that this abnormality might be related with the tendency of tumorigenesis in NBCCS patients after exposure to X-ray.

A novel pathogenesis of megacolon in Ncx/Hox11L.1 deficient mice.

The Ncx/Hox11L.1 gene, a member of the Hox11 homeobox gene family, is mainly expressed in neural crest-derived tissues. To elucidate the role of Ncx/Hox11L.1, the gene has been inactivated in embryonic stem cells by homologous recombination. The homozygous mutant mice were viable. These mice developed megacolon with enteric ganglia by age 3-5 wk. Histochemical analysis of the ganglia revealed that the enteric neurons hyperinnervated in the narrow segment of megacolon. Some of these neuronal cells degenerated and neuronal cell death occurred in later stages. We propose that Ncx/Hox11L.1 is required for maintenance of proper functions of the enteric nervous system. These mutant mice can be used to elucidate a novel pathogenesis for human neuronal intestinal dysplasia.

The role of Schwann cells during retinal ganglion cell regeneration induced by peripheral nerve transplantation.

PURPOSE: To investigate the key role of Schwann cells in retinal ganglion cell regeneration elicited by peripheral nerve autotransplantation. METHODS: Three kinds of autografts, Schwann-cell graft (intact sciatic nerve, consisting of living Schwann cells and their basal laminae). Schwann-cell-eliminated graft (consisting mainly of Schwann cell basal laminae) and partial Schwann-cell graft (consisting of basal laminae and diffusible factors secreted by Schwann cells) were prepared and autotransplanted to the adult rat optic nerve. The membrane specialization between regenerating axons and Schwann cells was observed by electron microscopy. The expression of cell adhesion molecules was demonstrated by Western blot analysis and immunohistochemistry. RESULTS: Retinal ganglion cell axons were observed to regenerate into the Schwann-cell graft in contact with Schwann cells but not into the Schwann-cell-eliminated graft. The regeneration was not observed in the empty basal laminae of the partial Schwann-cell graft. Most of regenerating axons contacted astrocytes in the optic nerve segment, and Schwann cells in the graft. At the interface of regenerating axon and Schwann cell, in addition to immunoreactivity of N-CAM and LI, short focal tight junctions were observed. CONCLUSIONS: These results suggested that viable Schwann cells are good substrate for retinal ganglion cell regeneration, the intimate contact with viable Schwann cell surface plays an important role in retinal ganglion cell regeneration, tight junctions, and cell adhesion molecules (LI, N-CAM) are observed between the regenerating axon and Schwann cell.