iPSC-derived mesenchymal stem cells attenuate cerebral ischemia-reperfusion injury by inhibiting inflammatory signaling and oxidative stress.

Induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) hold great promise as a cell source for transplantation into injured tissues to alleviate inflammation. However, the therapeutic efficacy of iMSC transplantation for ischemic stroke remains unknown. In this study, we evaluated the therapeutic effects of iMSC transplantation on brain injury after ischemia-reperfusion using a rat transient middle cerebral artery occlusion model and compared its therapeutic efficacy with that of bone marrow mesenchymal stem cells (BMMSCs). We showed that iMSCs and BMMSCs reduced infarct volumes after reperfusion and significantly improved motor function on days 3, 7, 14, 28, and 56 and cognitive function on days 28 and 56 after reperfusion compared with the vehicle group. Furthermore, immunological analyses revealed that transplantation of iMSCs and BMMSCs inhibited microglial activation and expression of proinflammatory cytokines and suppressed oxidative stress and neuronal cell death in the cerebral cortex at the ischemic border zone. No difference in therapeutic effect was observed between the iMSC and BMMSC groups. Taken together, our results demonstrate that iMSC therapy can be a practical alternative as a cell source for attenuation of brain injury and improvement of neurological function because of the unlimited supply of uniform therapeutic cells.

Smaller cross-sectional areas of the hamstring tendon measured from preoperative ultrasonography are likely to need additional gracilis harvesting for double-bundle anterior cruciate ligament reconstructions.

BACKGROUND/PURPOSE: Hamstring tendon autografts are commonly used for double-bundle anterior cruciate ligament reconstruction (DB-ACLR). If the volume of the semitendinosus (ST) tendon is insufficient, the gracilis (G) tendon is also harvested. Additional harvesting of the G autograft can affect patients' short-term postoperative outcome, such as muscle recovery; thus, preoperative information about whether an additional G autograft is needed would be useful. The purpose of this study was to investigate whether preoperative measurement of the ST tendon using ultrasonography could inform the intraoperative decision to harvest the G tendon. METHODS: We enrolled 20 patients (13 men and seven women) who underwent DB-ACLR between October 2017 and March 2019. The mean patient age was 28.5 years. The ipsilateral ST tendon was measured using ultrasonography before surgery. Measurements included the diameter and breadth of the short-axis image. The cross-sectional area (CSA) was calculated from these measurements. During surgery, when two grafts with diameters of ≥ 5.0 mm could not be made, the G tendon was also harvested. Patients were categorized into two groups: the ST group where only the ST tendon was harvested, and the semitendinosus gracilis tendon (STG) group where the ST and G tendons were both harvested. The CSA value was compared between the two groups, and the cutoff value was calculated. RESULTS: In the ST group (n = 8), the mean diameter and breadth of the semitendinosus tendon were 4.21 and 2.34 mm, respectively. In the STG group (n = 12), the mean diameter and breadth of the ST tendon were 3.39 and 1.78 mm, respectively. The CSAs calculated for the ST group and the STG group were 7.74 mm2 and 4.79 mm2, respectively. A cutoff value of 7.0 mm2 was found to correspond to a specificity and sensitivity to harvest the G tendon of 87.5% and 75.0%, respectively. CONCLUSIONS: The preoperative CSA of the ST tendon determined using ultrasonography can, therefore, be informative for deciding whether to harvest the G tendon for DB-ACLR. The results of this study provide valuable information for graft selection in anterior cruciate ligament reconstruction. LEVEL OF EVIDENCE: IV (Retrospective case series design).

Improved Intravitreal AAV-Mediated Inner Retinal Gene Transduction after Surgical Internal Limiting Membrane Peeling in Cynomolgus Monkeys.

The retina is an ideal target for gene therapy because of its easy accessibility and limited immunological response. We previously reported that intravitreally injected adeno-associated virus (AAV) vector transduced the inner retina with high efficiency in a rodent model. In large animals, however, the efficiency of retinal transduction was low, because the vitreous and internal limiting membrane (ILM) acted as barriers to transduction. To overcome these barriers in cynomolgus monkeys, we performed vitrectomy (VIT) and ILM peeling before AAV vector injection. Following intravitreal injection of 50 µL triple-mutated self-complementary AAV serotype 2 vector encoding EGFP, transduction efficiency was analyzed. Little expression of GFP was detected in the control and VIT groups, but in the VIT+ILM group, strong GFP expression was detected within the peeled ILM area. To detect potential adverse effects, we monitored the retinas using color fundus photography, optical coherence tomography, and electroretinography. No serious side effects associated with the pretreatment were observed. These results indicate that surgical ILM peeling before AAV vector administration would be safe and useful for efficient transduction of the nonhuman primate retina and provide therapeutic benefits for the treatment of retinal diseases.

Enzyme replacement in the CSF to treat metachromatic leukodystrophy in mouse model using single intracerebroventricular injection of self-complementary AAV1 vector.

Metachromatic leukodystrophy (MLD) is a lysosomal storage disease caused by a functional deficiency in human arylsulfatase A (hASA). We recently reported that ependymal cells and the choroid plexus are selectively transduced by intracerebroventricular (ICV) injection of adeno-associated virus serotype 1 (AAV1) vector and serve as a biological reservoir for the secretion of lysosomal enzymes into the cerebrospinal fluid (CSF). In the present study, we examined the feasibility of this AAV-mediated gene therapy to treat MLD model mice. Preliminary experiments showed that the hASA level in the CSF after ICV injection of self-complementary (sc) AAV1 was much higher than in mice injected with single-stranded AAV1 or scAAV9. However, when 18-week-old MLD mice were treated with ICV injection of scAAV1, the concentration of hASA in the CSF gradually decreased and was not detectable at 12 weeks after injection, probably due to the development of anti-hASA antibodies. As a result, the sulfatide levels in brain tissues of treated MLD mice were only slightly reduced compared with those of untreated MLD mice. These results suggest that this approach is potentially promising for treating MLD, but that controlling the immune response appears to be crucial for long-term expression of therapeutic proteins in the CSF.

Targeted gene transfer into ependymal cells through intraventricular injection of AAV1 vector and long-term enzyme replacement via the CSF.

Enzyme replacement via the cerebrospinal fluid (CSF) has been shown to ameliorate neurological symptoms in model animals with neuropathic metabolic disorders. Gene therapy via the CSF offers a means to achieve a long-term sustainable supply of therapeutic proteins within the central nervous system (CNS) by setting up a continuous source of transgenic products. In the present study, a serotype 1 adeno-associated virus (AAV1) vector was injected into a lateral cerebral ventricle in adult mice to transduce the gene encoding human lysosomal enzyme arylsulfatase A (hASA) into the cells of the CNS. Widespread transduction and stable expression of hASA in the choroid plexus and ependymal cells was observed throughout the ventricles for more than 1 year after vector injection. Although humoral immunity to hASA developed after 6 weeks, which diminished the hASA levels detected in CSF from AAV1-injected mice, hASA levels in CSF were maintained for at least 12 weeks when the mice were tolerized to hASA prior of vector injection. Our results suggest that the cells lining the ventricles could potentially serve as a biological reservoir for long-term continuous secretion of lysosomal enzymes into the CSF following intracerebroventricular injection of an AAV1 vector.

Serotype-independent method of recombinant adeno-associated virus (AAV) vector production and purification.

A variety of gene transfer strategies have been developed to treat inherited, degenerative, and acquired diseases. Among the different vector systems developed so far, recombinant adeno-associated viral (AAV) vectors have shown notable benefits, including prolonged gene expression, transduction of both dividing and nondividing cells, and a lack of pathogenicity caused by wild-type infections. Thanks to these features, the use of AAV vectors as a gene transfer tool has increased dramatically during the past several years, and several recent clinical trials have used AAV vectors. However, AAV vectors are more complicated to produce than are other viral vectors. With steady advances toward clinical application, much effort has been made to isolate novel AAV serotypes and to develop methods for their efficient, scalable, and versatile production and purification. Here we review state of the art methods for AAV vector production and purification, which we have refined in our laboratory.

Engrafted VHL peptide-delivered bone marrow stromal cells promote spinal cord repair in rats.

Stem cell-based therapy using bone marrow stromal cells (MSCs) has been expected to be a promising therapy for neuronal regeneration. To repair the injured spinal cord, neuronal differentiation of MSCs before transplantation has a more satisfactory effect. Recently, neuronal differentiation of neural progenitor/stem cells by an intracellular delivery of a pVHL-derived synthetic peptide (VHL peptide) has been shown. Here, we show that VHL peptide-delivered MSCs differentiated into neuron-like cells, and that engrafted VHL peptide-delivered MSCs more recovered the behaviors of the rats than that of nondelivered MSCs. Our result suggests that the use of VHL peptide-delivered MSCs would be a promising therapeutic strategy for repairing the injured spinal cord.

Transplantation of Von Hippel-Lindau peptide delivered neural stem cells promotes recovery in the injured rat spinal cord.

For transplantation of neural stem cells (NSCs) to repair the injured spinal cord, neuronal differentiation of NSCs before transplantation has more satisfactory effect because differentiation grafted NSCs are restricted to the glial lineage. Therefore, we focused on the Von Hippel-Lindau protein (VHL), which has the potential to induce neuronal differentiation of NSCs. Here, we show the transplantation of protein transduction domain-linked VHL peptide-delivered NSCs promotes the repair of the injured spinal cord. Transplantation of protein transduction domain -linked VHL peptide-delivered NSCs more recovered the behaviors of the rats than that of nondelivered NSCs, and engrafted NSCs differentiated to neuronal marker positive cells. Thus, our finding of the neuronal differentiation through VHL-peptide transfer has the great potential to cure the spinal cord injury.

Low-intensity pulsed ultrasound activates the phosphatidylinositol 3 kinase/Akt pathway and stimulates the growth of chondrocytes in three-dimensional cultures: a basic science study.

INTRODUCTION: The effect of low-intensity pulsed ultrasound (LIPUS) on cell growth was examined in three-dimensional-cultured chondrocytes with a collagen sponge. To elucidate the mechanisms underlying the mechanical activation of chondrocytes, intracellular signaling pathways through the Ras/mitogen-activated protein kinase (MAPK) and the integrin/phosphatidylinositol 3 kinase (PI3K)/Akt pathways as well as proteins involved in proliferation of chondrocytes were examined in LIPUS-treated chondrocytes. METHODS: Articular cartilage tissue was obtained from the metatarso-phalangeal joints of freshly sacrificed pigs. Isolated chondrocytes mixed with collagen gel and culture medium composites were added to type-I collagen honeycomb sponges. Experimental cells were cultured with daily 20-minute exposures to LIPUS. The chondrocytes proliferated and a collagenous matrix was formed on the surface of the sponge. Cell counting, histological examinations, immunohistochemical analyses and western blotting analysis were performed. RESULTS: The rate of chondrocyte proliferation was slightly but significantly higher in the LIPUS group in comparison with the control group during the 2-week culture period. Western blot analysis showed intense staining of type-IX collagen, cyclin B1 and cyclin D1, phosphorylated focal adhesion kinase, and phosphorylated Akt in the LIPUS group in comparison with the control group. No differences were detected, however, in the MAPK, phosphorylated MAPK and type-II collagen levels. CONCLUSION: LIPUS promoted the proliferation of cultured chondrocytes and the production of type-IX collagen in a three-dimensional culture using a collagen sponge. In addition, the anabolic LIPUS signal transduction to the nucleus via the integrin/phosphatidylinositol 3-OH kinase/Akt pathway rather than the integrin/MAPK pathway was generally associated with cell proliferation.

Interaction between the Spatiotemporal Learning Rule (STLR) and Hebb type (HEBB) in single pyramidal cells in the hippocampal CA1 Area.

The spatiotemporal learning rule (STLR), proposed as a non-Hebb type by Tsukada et al. (Neural Networks 9 (1996) 1357 and Tsukada and Pan (Biol. cyberm 92 (2005) 139), 2005), consists of two distinctive factors; "cooperative plasticity without a cell spike," and "its temporal summation". On the other hand, Hebb (The organization of behavior. John Wiley, New York, 1949) proposed the idea (HEBB) that synaptic modification is strengthened only if the pre- and post-cell are activated simultaneously. We have shown, experimentally, that both STLR and HEBB coexist in single pyramidal cells of the hippocampal CA1 area. The functional differences between STLR and HEBB in dendrite (local)-soma (global) interactions in single pyramidal cells of CA1 and the possibility of pattern separation, pattern completion and reinforcement learning were discussed.

Establishment of ten strains of genetically engineered Salmonella typhimurium TA1538 each co-expressing a form of human cytochrome P450 with NADPH-cytochrome P450 reductase sensitive to various promutagens.

We newly developed 10 Salmonela typhimurium TA1538 strains each co-expressing a form of human cytochrome P450s (P450 or CYP) together with NADPH-cytochrome P450 reductase (CPR) for highly sensitive detection of mutagenic activation of mycotoxins, polycyclic aromatic hydrocarbons, heterocyclic amines, and aromatic amines at low substrate concentrations. Each form of P450 (CYP1A1, CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 or CYP3A5) expressed in the TA1538 cells efficiently catalyzed the oxidation of a representative substrate. Aflatoxin B1 was mutagenically activated effectively by CYP1A1, CYP1A2, and CYP3A4 and weakly by CYP2A6 and CYP2C8 expressed in S. typhimurium TA1538. CYP1A1 and CYP1A2 were responsible for the mutagenic activation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 2-acetylaminofluorene. Benzo[a]pyrene was also activated efficiently by CYP1A1 and weakly by CYP1A2, CYP2C9, CYP2C19, and CYP3A4 expressed in TA1538. These results suggest that the newly developed S. typhimurium TA1538 strains are applicable for detecting the activation of promutagens of which mutagenic activation is not or weakly detectable with N-nitrosamine-sensitive YG7108 strains expressing human P450s.

Specific expression of inducible nitric oxide synthase in the synovium of the diseased temporomandibular joint.

OBJECTIVE: The expression of inducible nitric oxide synthase (iNOS) in temporomandibular joint (TMJ) specimens obtained arthroscopically from diseased TMJs was investigated by using immunohistochemistry and compared with clinical, arthroscopic, and histologic findings. STUDY DESIGN: Synovial biopsies were obtained arthroscopically from 18 TMJs in 15 patients with symptomatic internal derangement (ID) or osteoarthritis (OA). We also obtained arthroscopic biopsies from 8 control TMJs (3 with habitual luxation of the mandible, one with ID with clicking, and 4 with mandibular condyle fractures). The expression of iNOS was examined by immunohistochemistry and was compared with clinical, arthroscopic, and histologic findings. RESULTS: Definite or intense iNOS immunoreactivity was observed in both the synovial lining cells and the endothelial cells of TMJs with symptomatic ID or OA. Weaker immunoreactivity was present in synovial fibroblasts. In contrast, in TMJs without synovitis (eg, those with habitual luxation of the mandible) the expression of iNOS was weak or marginal. The expression of iNOS correlated significantly with arthroscopic evidence of synovitis (r = 0.406, P <.05) but not with cartilaginous degeneration (P >.05). The expression of iNOS also correlated with the histologic grade of the synovial lining cell layers (r = 0.530, P <.05). However, in patients with ID or OA of the TMJ, there was no statistically significant correlation between the intensity of iNOS immunoreactivity and clinical, arthroscopic, or histologic findings (P >.05). CONCLUSION: These data clearly suggest that nitric oxide is locally produced in the synovial lining of the TMJ in ID and OA.

Projection neurons originating from thermo- and hygrosensory glomeruli in the antennal lobe of the cockroach.

Most insects are equipped with specialized thermo- and hygroreceptors to locate a permissible range of ambient temperature and distant water sources, respectively. In the cockroach, Periplaneta americana, cold, moist, and dry receptor cells in the antennae send axons to particular sets of two or three glomeruli in the dorsocentral part of the antennal lobe (primary olfactory center), designated DC1-3 glomeruli. However, it is not known how thermo- and hygrosensory signals from these glomeruli are represented in higher-order centers, the protocerebrum, in any insect species. With the use of intracellular recording and staining techniques, we identified a new class of interneurons with dendrites almost exclusively in the DC1, DC2, or DC3 glomeruli and axons projecting to the protocerebrum in the cockroach. Remarkably, terminals of all these projection neurons (PNs) covered almost identical areas in the lateral protocerebrum (LP), although their termination areas outside the LP differed from neuron to neuron. The termination areas within the LP were distinct from, but close to, those of uniglomerular and macroglomerular PNs that transmitted signals concerning general odors and female sex pheromones, respectively. PNs originating from DC1, DC2, and DC3 glomeruli exhibited excitatory responses to cold, moist, and dry stimuli, respectively, probably due to excitatory synaptic input from cold, moist, and dry receptor cells, respectively, whereas their responses were often modulated by olfactory stimuli. These findings suggested that dorsocentral PNs participate in neural pathways that lead to behavioral responses to temperature or humidity changes.

Role of human cytochrome P450 (CYP) in the metabolic activation of nitrosamine derivatives: application of genetically engineered Salmonella expressing human CYP.

The role of human cytochrome P450 (CYP) in the metabolic activation of tobacco-related N-nitrosamines was examined by Salmonella mutation test using a series of genetically engineered Salmonella typhimurium YG7108 strains each co-expressing a form of CYP (CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5) together with human NADPH-cytochrome P450 reductase. Seven tobacco-related N-nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, N-nitrosodiethylamine, N-nitrosopyrrolidine, N-nitrosopiperidine, N-nitrosonornicotine, N-nitrosoanabasine, and N-nitrosoanatabine were used. The CYP2A6 was found to be responsible for the mutagenic activation of essentially all tobacco-related N-nitrosamines examined. On the basis of the evidence, genetic polymorphism of the CYP2A6 gene appeared to be one of the factors determining cancer susceptibility caused by smoking. Previously, we found the whole deletion of the CYP2A6 gene (CYP2A6*4C) as a type of genetic polymorphism in Japanese. We hypothesized that individuals possessing the gene homozygous for CYP2A6*4C were incapable of activating tobacco-related N-nitrosamines and showed lower susceptibility to lung cancer induced by tobacco smoke. Thus, the relationship between the CYP2A6*4C and the susceptibility to the lung cancer was evaluated. The frequency of the CYP2A6*4C was significantly lower in the lung cancer patients than healthy volunteers, suggesting that the subjects carrying the CYP2A6*4C alleles are resistant to carcinogenesis caused by N-nitrosamines because of the poor metabolic activation capacity. Taking these results into account, CYP2A6 is an enzyme enhancing lung cancer risk.