Toshihiro Nagai

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.

Vascular endothelial growth factor increases fenestral permeability in hepatic sinusoidal endothelial cells

Abstract: Vascular endothelial growth factor (VEGF) is an important regulator of vasculogenesis and vascular permeability. Hepatic sinusoidal endothelial cells (SECs) possess sieve‐like pores that form an anastomosing labyrinth structure by the deeply invaginated plasma membrane. Caveolin is the principal structural protein in caveolae. In this study, we examined the role of VEGF on the fenestration and permeability of SECs and the relation with caveolin‐1. SECs isolated from rat livers by collagenase infusion method were cultured for 24 h with (10 or 100 ng/ml) or without VEGF. The cells were then examined by transmission and scanning electron microscopy (EM). The expression of caveolin was investigated by confocal immunofluorescence, immunogold EM, and Western blot. Endocytosis and intracellular traffic was studied using horseradish peroxidase (HRP) reaction as a marker of fluid phase transport in SECs.

Rho modulates hepatic sinusoidal endothelial fenestrae via regulation of the actin cytoskeleton in rat endothelial cells

The presence of actin-like microfilaments in the vicinity of sinusoidal endothelial fenestrae (SEF) indicates that the cytoskeleton of sinusoidal endothelial cells (SEC) plays an important role in the modulation of SEF. Rho has emerged as an important regulator of the actin cytoskeleton, and consequently cell morphology. The present study aimed to examine how a Rho stimulator; lysophosphatidic acid (LPA), and a Rho inhibitor; bacterial toxin C3 transferase (C3-transferase), affect the morphology of SEF. Monolayers of SEC culture were established by infusing a rat liver with collagenase for 30 min and then culturing in RMPI medium for 24 h. The cells were separated into three groups; control, LPA-treated (15 μM), and C3-transferase-treated (15 μg/ml) groups. SEF morphology was observed by scanning electron microscopy. Formation of F-actin stress fibers was observed by confocal microscopy. Rho A and phosphorylated myosin light-chain kinase were analyzed by Western blotting. Active Rho was measured by Rens modification. Treatment of SECs with LPA contracted the SEF, concomitant with increases in F-actin stress fiber and actin microfilament, and high expression of phosphorylated myosin light-chain kinase. Following treatment with C3-transferase, SEF dilated and fused, concomitant with a loss of F-actin and microfilament, and low expression of phosphorylated myosin light chain. Rho A expression does not change by both treatments. In conclusion, these results indicate that Rho modulates fenestral changes in SEC via regulation of the actin cytoskeleton.

Caveolin‐1 and Rac regulate endothelial capillary‐like tubular formation and fenestral contraction in sinusoidal endothelial cells

Background/Aims: Rho guanidine triphosphatases (GTPases) are major regulators of cell migration. We investigated the cytoskeleton and Rho GTPases during cell migration and morphogenesis processes in isolated rat liver sinusoidal endothelial cells (LSECs) cultured on Matrigel while stimulated by the vascular endothelial growth factor (VEGF).

IGF2 Preserves Osteosarcoma Cell Survival by Creating an Autophagic State of Dormancy That Protects Cells against Chemotherapeutic Stress

Osteosarcoma is a malignant bone tumor in children and adolescents characterized by intrinsic therapeutic resistance. The IGF2 is expressed at elevated levels in osteosarcoma after treatment with chemotherapy, prompting an examination of its functional contributions to resistance. We found that continuous exposure to IGF2 or insulin in the absence of serum created a dormant growth state in osteosarcoma cells that conferred resistance to various chemotherapeutic drugs in vitro. Mechanistic investigations revealed that this dormant state correlated with downregulation of downstream signaling by the IGF1 receptor, heightened cell survival, enhanced autophagy, and the presence of extracellular glutamine. Notably, inhibiting autophagy or depleting glutamine was sufficient to increase chemotherapeutic sensitivity in osteosarcoma xenografts in mice. Clinically, we confirmed that IGF expression levels were elevated in human osteosarcoma specimens from patients who received chemotherapy. Together, our results suggest that activation of IGF or insulin signaling preserves the survival of osteosarcoma cells under chemotherapeutic stress, providing a drug-resistant population that may engender minimal residual disease. Attenuating this survival mechanism may help overcome therapeutic resistance in osteosarcoma.

Application of q-Space Diffusion MRI for the Visualization of White Matter.

White matter abnormalities in the CNS have been reported recently in various neurological and psychiatric disorders. Quantitation of non-Gaussianity for water diffusion by q-space diffusional MRI (QSI) renders biological diffusion barriers such as myelin sheaths; however, the time-consuming nature of this method hinders its clinical application. In the current study, we aimed to refine QSI protocols to enable their clinical application and to visualize myelin signals in a clinical setting. For this purpose, animal studies were first performed to optimize the acquisition protocol of a non-Gaussian QSI metric. The heat map of standardized kurtosis values derived from optimal QSI (myelin map) was then created. Histological validation of the myelin map was performed in myelin-deficient mice and in a nonhuman primate by monitoring its variation during demyelination and remyelination after chemical spinal cord injury. The results demonstrated that it was sensitive enough to depict dysmyelination, demyelination, and remyelination in animal models. Finally, its utility in clinical practice was assessed by a pilot clinical study in a selected group of patients with multiple sclerosis (MS). The human myelin map could be obtained within 10 min with a 3 T MR scanner. Use of the myelin map was practical for visualizing white matter and it sensitively detected reappearance of myelin signals after demyelination, possibly reflecting remyelination in MS patients. Our results together suggest that the myelin map, a kurtosis-related heat map obtainable with time-saving QSI, may be a novel and clinically useful means of visualizing myelin in the human CNS. SIGNIFICANCE STATEMENT Myelin abnormalities in the CNS have been gaining increasing attention in various neurological and psychiatric diseases. However, appropriate methods with which to monitor CNS myelin in daily clinical practice have been lacking. In the current study, we introduced a novel MRI modality that produces the “myelin map.” The myelin map accurately depicted myelin status in mice and nonhuman primates and in a pilot clinical study of multiple sclerosis patients, suggesting that it is useful in detecting possibly remyelinated lesions. A myelin map of the human brain could be obtained in <10 min using a 3 T scanner and it therefore promises to be a powerful tool for researchers and clinicians examining myelin-related diseases.

Accumulation of Secretory Vesicles in the Lacrimal Gland Epithelia Is Related to Non-Sjögren's Type Dry Eye in Visual Display Terminal Users

Previous observations in a rat model of a non-Sjögrens syndrome (non-SS) type of dry eye seen in users of visual display terminals (VDT) indicated that secretory vesicle (SV) accumulation in the lacrimal gland epithelia contributes to the condition. Here, to examine this possibility in humans, we compared the lacrimal gland histology and percent SV area in the cytoplasm of acinar epithelial cells using light microscopy and transmission electron microscopy, in patients with VDT work-related non-SS dry-eye (VDT group), SS-induced dry-eye, and autopsied normal controls. In addition, the VAMP8 (vesicle-associated membrane protein 8, an exocrine-pathway molecule) and Rab3D (mature vesicle marker) were histochemically examined in lacrimal gland tissue sections. The lacrimal gland acini were larger in the VDT group than in the SS group, and the percent SV area was significantly higher in the VDT group than in the normal controls (P = 0.021) or SS group (P = 0.004). Immunostaining revealed abnormal distributions of VAMP8 in the VDT and SS groups. Rab3D was more strongly expressed in the cytoplasm of acinar epithelial cells in the VDT group than in that of normal controls. The duration of VDT use was significantly longer in the VDT group than in the other groups. These findings suggest that excessive SV accumulation in the acinar epithelia may contribute to the reduced tear secretion in VDT users.

Connectomics: comprehensive approaches for whole-brain mapping

The aim of connectomics analysis is to understand whole-brain neural connections. This is accomplished using new biotechnologies. Here, we provide an overview of the recent progress in connectomics analysis. The entire neural network of an organism was revealed for the first time in the nematode. Caenorhabditis elegans (C. elegans) have an advantage of their limited number of neurons and their transparency, allowing the neural network to be visualized using light and electron microscopes (EMs). It is practically impossible to adopt the same approach for mammals because of the large number of neural cells and the opacity of the central nervous system. A variety of new technologies are being developed to perform computer-assisted high-throughput image acquisition and analysis to obtain whole-brain maps for higher species, including mammals. Diffusion tensor magnetic resonance imaging and tractography and three-dimensional imaging with the EM are examples of novel approaches to connectomics. These new technologies will soon be applied not only to Drosophila, C. elegans and rodent research, but also to comprehensive connectomics analysis in a wide range of species including humans and primates. In the near future, results from connectomics analysis will reveal the neural circuitry of the whole brain and enhance our understanding of the human mind and neuropsychiatric diseases.

Mucosal microvilli in dry eye patients with chronic GVHD

The ocular surface is a frequent target tissue of mucosal chronic GVHD (cGVHD). We investigated the histopathological features of the conjunctival microvilli in patients with cGVHD. Conjunctival tissue specimens from patients with cGVHD or Sjögrens syndrome (SS) or from healthy individuals were examined by light microscopy and EM, impression cytology, and immunohistochemistry. The cGVHD conjunctivae showed significantly more metaplasia and fewer goblet cells than the SS and normal conjunctivae. Abundant CD8+ T cells infiltrated the basal epithelia in the cGVHD conjunctiva. The microvilli per standard epithelial unit and the secretory vesicles were counted by analyzing electron micrographs. The mean number of mucosal microvilli was significantly lower in the cGVHD than that in the SS or normal specimens, and the microvilli were significantly shorter, with a smaller height–width ratio. The mean number of secretory vesicles was also significantly lower, and the membrane-spanning mucin thinner, in the cGVHD compared with the SS and normal specimens. Thus, the conjunctival mucosal microvilli of cGVHD patients were significantly different in number and morphology from those of SS and normal subjects. These may be important factors affecting the stability of the tear-film layer and its contribution to cGVHD-related dry eye.

H1foo Has a Pivotal Role in Qualifying Induced Pluripotent Stem Cells

Summary Embryonic stem cells (ESCs) are a hallmark of ideal pluripotent stem cells. Epigenetic reprogramming of induced pluripotent stem cells (iPSCs) has not been fully accomplished. iPSC generation is similar to somatic cell nuclear transfer (SCNT) in oocytes, and this procedure can be used to generate ESCs (SCNT-ESCs), which suggests the contribution of oocyte-specific constituents. Here, we show that the mammalian oocyte-specific linker histone H1foo has beneficial effects on iPSC generation. Induction of H1foo with Oct4, Sox2, and Klf4 significantly enhanced the efficiency of iPSC generation. H1foo promoted in vitro differentiation characteristics with low heterogeneity in iPSCs. H1foo enhanced the generation of germline-competent chimeric mice from iPSCs in a manner similar to that for ESCs. These findings indicate that H1foo contributes to the generation of higher-quality iPSCs.