PTCH1-null induced pluripotent stem cells exclusively differentiate into immature ectodermal cells with large areas of medulloblastoma-like tissue.

Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder with an increased incidence of tumors, such as basal cell carcinomas and medulloblastomas. The PTCH1 gene, responsible for NBCCS, suppresses the hedgehog signaling pathway, which is recognized as one of the important pathways in tumorigenesis and, thus, is a therapeutic target in cancer. In the present study, we generated PTCH1-/- induced pluripotent stem cells (iPSCs) from NBCCS patient-derived iPSCs (PTCH1+/-) by gene editing. The proliferation of PTCH1-/- iPSCs was accelerated due to the activation of the hedgehog signaling pathway. When PTCH1-/- iPSCs were subcutaneously injected into immunodeficient mice, the resulting teratomas almost exclusively contained immature ectodermal lineage cells expressing medulloblastoma markers, and the percentages of the area occupied by medulloblastoma-like tissue were larger in PTCH1-/- teratomas than in PTCH1+/- teratomas. In contrast, in PTCH1+/+ teratomas, medulloblastoma-like tissue positive for all of these medulloblastoma markers was not observed. The present results indicate the importance of PTCH1 in medulloblastoma formation and the suitability of these gene-edited iPSCs and PTCH1-/- teratomas as models for the formation of tumors, such as medulloblastomas and Hh-related tumors.

Gorlin syndrome-induced pluripotent stem cells form medulloblastoma with loss of heterozygosity in PTCH1.

Gorlin syndrome is a rare autosomal dominant hereditary disease with a high incidence of tumors such as basal cell carcinoma and medulloblastoma. Disease-specific induced pluripotent stem cells (iPSCs) and an animal model have been used to analyze disease pathogenesis. In this study, we generated iPSCs derived from fibroblasts of four patients with Gorlin syndrome (Gln-iPSCs) with heterozygous mutations of the PTCH1 gene. Gln-iPSCs from the four patients developed into medulloblastoma, a manifestation of Gorlin syndrome, in 100% (four out of four), of teratomas after implantation into immunodeficient mice, but none (0/584) of the other iPSC-teratomas did so. One of the medulloblastomas showed loss of heterozygosity in the PTCH1 gene while the benign teratoma, i.e. the non-medulloblastoma portion, did not, indicating a close clinical correlation between tumorigenesis in Gorlin syndrome patients and Gln-iPSCs.

Establishment of a Gorlin syndrome model from induced neural progenitor cells exhibiting constitutive GLI1 expression and high sensitivity to inhibition by smoothened (SMO).

The hedgehog signaling pathway is a vital factor for embryonic development and stem cell maintenance. Dysregulation of its function results in tumor initiation and progression. The aim of this research was to establish a disease model of hedgehog-related tumorigenesis with Gorlin syndrome-derived induced pluripotent stem cells (GS-iPSCs). Induced neural progenitor cells from GS-iPSCs (GS-NPCs) show constitutive high GLI1 expression and higher sensitivity to smoothened (SMO) inhibition compared with wild-type induced neural progenitor cells (WT-NPCs). The differentiation process from iPSCs to NPCs may have similarity in gene expression to Hedgehog signal-related carcinogenesis. Therefore, GS-NPCs may be useful for screening compounds to find effective drugs to control Hedgehog signaling activity.

Fetal Therapy Model of Myelomeningocele with Three-Dimensional Skin Using Amniotic Fluid Cell-Derived Induced Pluripotent Stem Cells.

Myelomeningocele (MMC) is a congenital disease without genetic abnormalities. Neurological symptoms are irreversibly impaired after birth, and no effective treatment has been reported to date. Only surgical repairs have been reported so far. In this study, we performed antenatal treatment of MMC with an artificial skin using induced pluripotent stem cells (iPSCs) generated from a patient with Down syndrome (AF-T21-iPSCs) and twin-twin transfusion syndrome (AF-TTTS-iPSCs) to a rat model. We manufactured three-dimensional skin with epidermis generated from keratinocytes derived from AF-T21-iPSCs and AF-TTTS-iPSCs and dermis of human fibroblasts and collagen type I. For generation of epidermis, we developed a protocol using Y-27632 and epidermal growth factor. The artificial skin was successfully covered over MMC defect sites during pregnancy, implying a possible antenatal surgical treatment with iPSC technology.

Somatic mosaicism containing double mutations in PTCH1 revealed by generation of induced pluripotent stem cells from nevoid basal cell carcinoma syndrome.

BACKGROUND: Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterised by developmental defects and tumorigenesis, such as medulloblastomas and basal cell carcinomas, caused by mutations of the patched-1 (PTCH1) gene. In this article, we seek to demonstrate a mosaicism containing double mutations in PTCH1 in an individual with NBCCS. METHODS AND RESULTS: A de novo germline mutation of PTCH1 (c.272delG) was detected in a 31-year-old woman with NBCCS. Gene analysis of two out of four induced pluripotent stem cell (iPSC) clones established from the patient unexpectedly revealed an additional mutation, c.274delT. Deep sequencing confirmed a low-prevalence somatic mutation (5.5%-15.6% depending on the tissue) identical to the one found in iPSC clones. CONCLUSIONS: This is the first case of mosaicism unequivocally demonstrated in NBCCS. Furthermore, the mosaicism is unique in that the patient carries one normal and two mutant alleles. Because these mutations are located in close proximity, reversion error is likely to be involved in this event rather than a spontaneous mutation. In addition, this study indicates that gene analysis of iPSC clones can contribute to the detection of mosaicism containing a minor population carrying a second mutation.

How Orthography Modulates Morphological Priming: Subliminal Kanji Activation in Japanese.

The current study investigates to what extent masked morphological priming is modulated by language-particular properties, specifically by its writing system. We present results from two masked priming experiments investigating the processing of complex Japanese words written in less common (moraic) scripts. In Experiment 1, participants performed lexical decisions on target verbs; these were preceded by primes which were either (i) a past-tense form of the same verb, (ii) a stem-related form with the epenthetic vowel -i, (iii) a semantically-related form, and (iv) a phonologically-related form. Significant priming effects were obtained for prime types (i), (ii), and (iii), but not for (iv). This pattern of results differs from previous findings on languages with alphabetic scripts, which found reliable masked priming effects for morphologically related prime/target pairs of type (i), but not for non-affixal and semantically-related primes of types (ii), and (iii). In Experiment 2, we measured priming effects for prime/target pairs which are neither morphologically, semantically, phonologically nor - as presented in their moraic scripts-orthographically related, but which-in their commonly written form-share the same kanji, which are logograms adopted from Chinese. The results showed a significant priming effect, with faster lexical-decision times for kanji-related prime/target pairs relative to unrelated ones. We conclude that affix-stripping is insufficient to account for masked morphological priming effects across languages, but that language-particular properties (in the case of Japanese, the writing system) affect the processing of (morphologically) complex words.

The effect of an auxiliary stimulation on motor function restoration by FES.

Functional Electrical Stimulation (FES) is a technology to generate neural activity in an artificial way to activate muscles. However, as reported by some researchers, the human responses to FES are likely to be affected by several factors, such as spasticity, muscle fatigue, nerve habituation and so forth. Consequently, the function restoration by FES is neither durable, nor stable. In order to realize long-term and stable FES assistance, this study investigated whether and why an Auxiliary Stimulation (AS) to the Gastrocnemius, with current frequency ranged from 2000 to 6000 Hz, could alleviate the symptom of spasticity and muscle fatigue caused by the stimulation to the Tibialis Anterior. We have developed a portable auxiliary stimulator, and performed experiments to verify its effectiveness. The results showed that our approach enabled comparatively stable and durable function restoration assistance. Moreover, for understanding underlying neuromuscular processes elicited by the AS and its qualitative nature, this study also measured the Hoffmann-reflex (H-reflex) in soleus muscle before and after the AS, to interpret the effect of the Auxiliary Stimulation.

A study on balance maintenance strategies during walking - a simulation study.

Walking assist systems should cope with both the external perturbation caused by slips, uneven terrain, slopes, and obstacles, and local function impairment caused by internal factors, like spastic paralysis. It is known that humans are able to cope with these difficulties by different strategies. One is that in the case when external perturbation occurs, especially when the occurrence cannot be predicted or perceived in advance, humans rely on reflexes, which cause unconscious, relatively fixed muscular response patterns to perturbations within a short period of time. Another is that in the case of local function impairment, humans generally develop compensated gait to overcome the falling-down risky factors. In this study, we investigated the both strategies by constructing a human walking model that consists of Central Pattern Generator (CPG) module. As the reflexive mechanism, we used muscle activity profiles acquired from human gait experiments, together with a CPG-phase-modulation, and we examined of the roles of these two reflexive mechanisms. Besides, we modeled a spastic hemiplegic gait by modulating some specific neuron's output, in order to study another strategy, compensated walking. The results indicated that the simulation model could display behavior resembling that of normal human walking, and on the occurrence of a slip-perturbation, together with the CPG-phase-modulation, the rapid muscular response could improve perturbation-resistance and maintain balance for the simulated walker. Moreover, the pes equinus caused by the spasticity in Gastrocnemius could be modelled and falling down risk resulted from the pes equinus could be reduced by the simulated compensated walking.

The roles of CPG phase modulation and reflexive muscular patterns in balance recovery reflexive responses to perturbation during walking - a simulation study.

Most walking assist systems reported are not ready for using in real-world environment, where there are frequent perturbations resulted from slips, uneven terrain, slopes and obstacles. Our ultimate goal is to realize artificial reflexes to real-world walking support systems for those paralyzed people. This goal needs both qualitative and quantitative understanding of human reflexive mechanism. Our approach includes 1) acquiring muscle activity profiles during normal walking and slip-perturbed walking by recording and processing Electromyographic (EMG) of several walking-related muscles, in a human gait experiment; 2) developing a central-pattern-generator (CPG) based neuro-musculo-skeletal simulation model; 3) using muscle activity profiles of reflexive muscle responses together with a CPG-phase-modulation mechanism, to construct a rapid responding pathway, and investigating the effects of the pathway on the simulated walker, as well as verifying several hypotheses on the underlying neuro-mechanism. Experiments were performed in a walking simulation model to investigate the roles of the two functional aspects. Results showed that, 1) CPG phase modulation alone could also improve the perturbation resistance on the occurrence of a comparatively small slip perturbation, by interacting with a pre-wired sensory-feedback mechanism; 2) on the occurrence of a big slip-perturbation, together with the CPG phase modulation, the rapid muscular responses could improve the perturbation-resistance and maintain balance for the simulated walker.

An artificial reflex improves the perturbation-resistance of a human walking simulator.

Most walking assist systems reported are not available for real-world environment, where frequent perturbations are caused by slips, uneven terrain, slopes and obstacles. On the other hand, it is evident that human beings cope with those perturbations, especially when the perturbations cannot be predicted or perceived in advance, with reflexes, which cause relatively fixed muscular responsive patterns to perturbations unconsciously within a short period of time ranging from several 10 to 200 ms. Our ultimate goal is to realize artificial reflexes in real-world walking support systems for those paralyzed people, whose afferent and efferent neural pathways are usually weakened, so that the reflexive system is also impaired to a certain degree. This goal needs both qualitative and quantitative understanding of human reflexive mechanism during walking. However, except for some hypotheses about the underlying neural mechanisms of the reflexes during walking, there is no widely accepted unified theory, nor are there clear experimental results that could be directly quoted in the disciplines of physiology and motor control. Our approach includes (1) acquiring muscle activity profiles during normal walking and slip-perturbed walking by recording and processing Electromyographic (EMG) signals of several walking-related muscles, in human gait experiments; (2) developing a central-pattern-generator (CPG) based neuro-musculo-skeletal simulation model; (3) comparing joint trajectories of the simulation model with those of a human subject during normal walking to verify the simulation model's conformity with human walking; (4) using muscle activity profiles of reflexive responses to slip-perturbation during walking to construct a rapid responding pathway. The results showed that, (1) The simulation model could show behavior resembling that of normal human walking; (2) in the case of occurrence of slip-perturbation, the rapid responding pathway could improve the perturbation-resistance and maintain the balance for the walking; (3) using the simulation model, several hypotheses on underlying neuro-mechanism were investigated. These reveal the possibility to realize the artificial reflex for the paralyzed people.