Shinsuke Shibata

Dysfunction of fibroblasts of extrarenal origin underlies renal fibrosis and renal anemia in mice

In chronic kidney disease, fibroblast dysfunction causes renal fibrosis and renal anemia. Renal fibrosis is mediated by the accumulation of myofibroblasts, whereas renal anemia is mediated by the reduced production of fibroblast-derived erythropoietin, a hormone that stimulates erythropoiesis. Despite their importance in chronic kidney disease, the origin and regulatory mechanism of fibroblasts remain unclear. Here, we have demonstrated that the majority of erythropoietin-producing fibroblasts in the healthy kidney originate from myelin protein zero-Cre (P0-Cre) lineage-labeled extrarenal cells, which enter the embryonic kidney at E13.5. In the diseased kidney, P0-Cre lineage-labeled fibroblasts, but not fibroblasts derived from injured tubular epithelial cells through epithelial-mesenchymal transition, transdifferentiated into myofibroblasts and predominantly contributed to fibrosis, with concomitant loss of erythropoietin production. We further demonstrated that attenuated erythropoietin production in transdifferentiated myofibroblasts was restored by the administration of neuroprotective agents, such as dexamethasone and neurotrophins. Moreover, the in vivo administration of tamoxifen, a selective estrogen receptor modulator, restored attenuated erythropoietin production as well as fibrosis in a mouse model of kidney fibrosis. These findings reveal the pathophysiological roles of P0-Cre lineage-labeled fibroblasts in the kidney and clarify the link between renal fibrosis and renal anemia.

Sema3A regulates bone-mass accrual through sensory innervations

Semaphorin 3A (Sema3A) is a diffusible axonal chemorepellent that has an important role in axon guidance. Previous studies have demonstrated that Sema3a−/− mice have multiple developmental defects due to abnormal neuronal innervations. Here we show in mice that Sema3A is abundantly expressed in bone, and cell-based assays showed that Sema3A affected osteoblast differentiation in a cell-autonomous fashion. Accordingly, Sema3a−/− mice had a low bone mass due to decreased bone formation. However, osteoblast-specific Sema3A-deficient mice (Sema3acol1−/− and Sema3aosx−/− mice) had normal bone mass, even though the expression of Sema3A in bone was substantially decreased. In contrast, mice lacking Sema3A in neurons (Sema3asynapsin−/− and Sema3anestin−/− mice) had low bone mass, similar to Sema3a−/− mice, indicating that neuron-derived Sema3A is responsible for the observed bone abnormalities independent of the local effect of Sema3A in bone. Indeed, the number of sensory innervations of trabecular bone was significantly decreased in Sema3asynapsin−/− mice, whereas sympathetic innervations of trabecular bone were unchanged. Moreover, ablating sensory nerves decreased bone mass in wild-type mice, whereas it did not reduce the low bone mass in Sema3anestin−/− mice further, supporting the essential role of the sensory nervous system in normal bone homeostasis. Finally, neuronal abnormalities in Sema3a−/− mice, such as olfactory development, were identified in Sema3asynasin−/− mice, demonstrating that neuron-derived Sema3A contributes to the abnormal neural development seen in Sema3a−/− mice, and indicating that Sema3A produced in neurons regulates neural development in an autocrine manner. This study demonstrates that Sema3A regulates bone remodelling indirectly by modulating sensory nerve development, but not directly by acting on osteoblasts.

Significance of Remyelination by Neural Stem/Progenitor Cells Transplanted into the Injured Spinal Cord†‡§

Previous reports of functional recovery from spinal cord injury (SCI) in rodents and monkeys after the delayed transplantation of neural stem/progenitor cells (NS/PCs) have raised hopes that stem cell therapy could be used to treat SCI in humans. More research is needed, however, to understand the mechanism of functional recovery. Oligodendrocytes derived from grafted NS/PCs remyelinate spared axons in the injured spinal cord. Here, we studied the extent of this remyelinations contribution to functional recovery following contusive SCI in mice. To isolate the effect of remyelination from other possible regenerative benefits of the grafted cells, NS/PCs obtained from myelin‐deficient shiverer mutant mice (shi‐NS/PCs) were used in this work alongside wild‐type NS/PCs (wt‐NS/PCs). shi‐NS/PCs behaved like wt‐NS/PCs in vitro and in vivo, with the exception of their myelinating potential. shi‐NS/PC‐derived oligodendrocytes did not express myelin basic protein in vitro and formed much thinner myelin sheaths in vivo compared with wt‐NS/PC‐derived oligodendrocytes. The transplantation of shi‐NS/PCs promoted some locomotor and electrophysiological functional recovery but significantly less than that afforded by wt‐NS/PCs. These findings establish the biological importance of remyelination by graft‐derived cells for functional recovery after the transplantation of NS/PCs into the injured spinal cord. STEM CELLS 2011;29:1983–1994.

The Neural Stem/Progenitor Cell Marker Nestin Is Expressed in Proliferative Endothelial Cells, but Not in Mature Vasculature:

Nestin is an intermediate filament protein that is known as a neural stem/progenitor cell marker. It is expressed in undifferentiated central nervous system (CNS) cells during development, but also in normal adult CNS and in CNS tumor cells. Additionally, nestin is expressed in endothelial cells (ECs) of CNS tumor tissues and of adult tissues that replenish by angiogenesis. However, the regulation of nestin expression in vascular endothelium has not been analyzed in detail. This study showed that nestin expression was observed in proliferating endothelial progenitor cells (EPCs), but not in mature ECs. In adherent cultured cells derived from bone marrow cells, EPCs that highly expressed nestin also expressed the endothelial marker CD31 and the proliferation marker Ki67. ECs cultured without growth factors showed attenuated nestin immunoreactivity as they matured. Trans-genic mice that carried the enhanced green fluorescent protein under the control of the CNS-specific second intronic enhancer of the nestin gene showed no reporter gene expression in EPCs. This indicated that the mechanisms of nestin gene expression were different in EPCs and CNS cells. Immunohistochemistry showed nestin expression in neovascular cells from two distinct murine models. Our results demonstrate that nestin can be used as a marker protein for neovascularization.

The dual origin of the peripheral olfactory system: placode and neural crest

BackgroundThe olfactory epithelium (OE) has a unique capacity for continuous neurogenesis, extending axons to the olfactory bulb with the assistance of olfactory ensheathing cells (OECs). The OE and OECs have been believed to develop solely from the olfactory placode, while the neural crest (NC) cells have been believed to contribute only the underlying structural elements of the olfactory system. In order to further elucidate the role of NC cells in olfactory development, we examined the olfactory system in the transgenic mice Wnt1-Cre/Floxed-EGFP and P0-Cre/Floxed-EGFP, in which migrating NC cells and its descendents permanently express GFP, and conducted transposon-mediated cell lineage tracing studies in chick embryos.ResultsExamination of these transgenic mice revealed GFP-positive cells in the OE, demonstrating that NC-derived cells give rise to OE cells with morphologic and antigenic properties identical to placode-derived cells. OECs were also positive for GFP, confirming their NC origin. Cell lineage tracing studies performed in chick embryos confirmed the migration of NC cells into the OE. Furthermore, spheres cultured from the dissociated cells of the olfactory mucosa demonstrated self-renewal and trilineage differentiation capacities (neurons, glial cells, and myofibroblasts), demonstrating the presence of NC progenitors in the olfactory mucosa.ConclusionOur data demonstrates that the NC plays a larger role in the development of the olfactory system than previously believed, and suggests that NC-derived cells may in part be responsible for the remarkable capacity of the OE for neurogenesis and regeneration.

Sox10- Venus mice: a new tool for real-time labeling of neural crest lineage cells and oligodendrocytes

BackgroundWhile several mouse strains have recently been developed for tracing neural crest or oligodendrocyte lineages, each strain has inherent limitations. The connection between human SOX10 mutations and neural crest cell pathogenesis led us to focus on the Sox10 gene, which is critical for neural crest development. We generated Sox10- Venus BAC transgenic mice to monitor Sox10 expression in both normal development and in pathological processes.ResultsTissue fluorescence distinguished neural crest progeny cells and oligodendrocytes in the Sox10- Venus mouse embryo. Immunohistochemical analysis confirmed that Venus expression was restricted to cells expressing endogenous Sox10. Time-lapse imaging of various tissues in Sox10- Venus mice demonstrated that Venus expression could be visualized at the single-cell level in vivo due to the intense, focused Venus fluorescence. In the adult Sox10- Venus mouse, several types of mature and immature oligodendrocytes along with Schwann cells were clearly labeled with Venus, both before and after spinal cord injury.ConclusionsIn the newly-developed Sox10- Venus transgenic mouse, Venus fluorescence faithfully mirrors endogenous Sox10 expression and allows for in vivo imaging of live cells at the single-cell level. This Sox10- Venus mouse will thus be a useful tool for studying neural crest cells or oligodendrocytes, both in development and in pathological processes.

Beneficial compaction of spinal cord lesion by migrating astrocytes through glycogen synthase kinase-3 inhibition

The migratory response of astrocytes is essential for restricting inflammation and preserving tissue function after spinal cord injury (SCI), but the mechanisms involved are poorly understood. Here, we observed stimulation of in vitro astrocyte migration by the new potent glycogen synthase kinase‐3 (GSK‐3) inhibitor Ro3303544 and investigated the effect of Ro3303544 administration for 5 days following SCI in mice. This treatment resulted in accelerated migration of reactive astrocytes to sequester inflammatory cells that spared myelinated fibres and significantly promoted functional recovery. Moreover, the decreased extent of chondroitin sulphate proteoglycans and collagen IV demonstrated that scarring was reduced in Ro3303544‐treated mice. A variety of in vitro and in vivo experiments further suggested that GSK‐3 inhibition stimulated astrocyte migration by decreasing adhesive activity via reduced surface expression of β1‐integrin. Our results reveal a novel benefit of GSK‐3 inhibition for SCI and suggest that the stimulation of astrocyte migration is a feasible therapeutic strategy for traumatic injury in the central nervous system.

Induction of neural crest cells from mouse embryonic stem cells in a serum-free monolayer culture

The neural crest (NC) is a group of cells located in the neural folds at the boundary between the neural and epidermal ectoderm. NC cells differentiate into a vast range of cells,including neural cells, smooth muscle cells, bone and cartilage cells of the maxillofacial region, and odontoblasts. The molecular mechanisms underlying NC induction during early development remain poorly understood. We previously established a defined serum-free culture condition for mouse embryonic stem (mES) cells without feeders. Here, using this defined condition, we have developed a protocol to promote mES cell differentiation into NC cells in an adherent monolayer culture. We found that adding bone morphogenetic protein (BMP)-4 together with fibroblast growth factor (FGF)-2 shifts mES cell differentiation into the NC lineage. Furthermore, we have established a cell line designated as P0-6 that is derived from the blastocysts of P0-Cre/Floxed-EGFP mice expressing EGFP in an NC-lineage-specific manner. P0-6 cells cultured using this protocol expressed EGFP. This protocol could be used to help clarify the mechanisms by which cells differentiate into the NC lineage and to assist the development of applications for clinical therapy.

Schwann cell plasticity after spinal cord injury shown by neural crest lineage tracing

After spinal cord injury (SCI), various cell types are recruited to the lesion site, including Schwann cells, which originate in the neural crest and normally myelinate axons in the peripheral nervous system. Here, we investigated the differentiation states, migration patterns, and roles of neural crest derivatives following SCI, using two transgenic mouse lines carrying neural crest‐specific reporters, P0‐Cre/Floxed‐EGFP and Wnt1‐Cre/Floxed‐EGFP. In these mice, EGFP is expressed only in the neural crest cell lineage. Immunohistochemical analysis revealed that most of the EGFP+ cells that infiltrated the lesion site after SCI were Schwann cells. Seven days after SCI, the P0‐positive, mature Schwann cells residing at the nerve roots had dedifferentiated into P0−/p75+ immature Schwann cells, which proliferated and began migrating into the lesion site. The dedifferentiation of the Schwann cells was corroborated by their expression of phosphorylated c‐Jun, which promotes dedifferentiation and inhibits the expression of myelin‐associated genes in the peripheral nerves. Thereafter, the number of EGFP+/p75+ immature Schwann cells decreased and that of EGFP+/P0+ mature cells increased gradually, indicating that the cells redifferentiated into mature Schwann cells within the lesion site. This study draws on the advantages offered by transgenic mouse lines bearing a genetic cell‐lineage marker and extends previous work by describing the origins and behavior of the neural crest‐derived cells that contribute to endogenous repair after SCI. This process, involving Schwann cell plasticity, is a novel repair mechanism for the lesioned mammalian spinal cord.

The Semaphorin 3A Inhibitor SM-345431 Accelerates Peripheral Nerve Regeneration and Sensitivity in a Murine Corneal Transplantation Model

Background Peripheral nerve damage of the cornea is a complication following surgery or infection which may lead to decreased visual function. We examined the efficacy of the semaphorin 3A inhibitor, SM-345431, in promoting regeneration of peripheral nerves in a mouse corneal transplantation model. Methodology/Principal Findings P0-Cre/Floxed-EGFP mice which express EGFP in peripheral nerves cells were used as recipients of corneal transplantation with syngeneic wild-type mouse cornea donors. SM-345431 was administered subconjunctivally every 2 days while control mice received vehicle only. Mice were followed for 3 weeks and the length of regenerating nerves was measured by EGFP fluorescence and immunohistochemistry against βIII tubulin. Cornea sensitivity was also measured by the Cochet-Bonnet esthesiometer. CD31 staining was used to determine corneal neovascularization as a possible side effect of SM-345431. Regeneration of βIII tubulin positive peripheral nerves was significantly higher in SM-345431 treated mice compared to control. Furthermore, corneal sensitivity significantly improved in the SM-345431 group by 3 weeks after transplantation. Neovascularization was limited to the peripheral cornea with no difference between SM-345431 group and control. Conclusions/Significance Subconjunctival injections of SM-345431 promoted a robust network of regenerating nerves as well as functional recovery of corneal sensation in a mouse keratoplasty model, suggesting a novel therapeutic strategy for treating neurotrophic corneal disease.