Satoko Yamawaki

Drug Screening for ALS Using Patient-Specific Induced Pluripotent Stem Cells

Anacardic acid attenuates mutant TDP-43–associated abnormalities in motor neurons derived from ALS patient–specific induced pluripotent stem cells. A Stepping Stone to ALS Drug Screening Amyotrophic lateral sclerosis (ALS) is an untreatable disorder in which the motor neurons degenerate, resulting in paralysis and death. Induced pluripotent stem cell (iPSC) technology makes it possible to analyze motor neurons from patients with ALS and to use them for screening new candidate drugs. In new work, Egawa et al. obtained motor neurons by inducing differentiation of iPSC lines derived from several patients with familial ALS. These patients carried disease-causing mutations in the gene encoding Tar DNA binding protein-43 (TDP-43). The ALS motor neurons in culture recapitulated cellular and molecular abnormalities associated with ALS. For example, the authors found that mutant TDP-43 in the ALS motor neurons perturbed RNA metabolism and that the motor neurons were more vulnerable to cellular stressors such as arsenite. The researchers then used the ALS motor neurons in a drug screening assay and identified a compound called anacardic acid, a histone acetyltransferase inhibitor, that could reverse some of the ALS phenotypes observed in the motor neurons. The new work provides an encouraging step toward using motor neurons generated from iPSCs derived from ALS patients to learn more about what triggers the death of motor neurons in this disease and to identify new candidate drugs that may be able to slow or reverse the devastating loss of motor neurons. Amyotrophic lateral sclerosis (ALS) is a late-onset, fatal disorder in which the motor neurons degenerate. The discovery of new drugs for treating ALS has been hampered by a lack of access to motor neurons from ALS patients and appropriate disease models. We generate motor neurons from induced pluripotent stem cells (iPSCs) from familial ALS patients, who carry mutations in Tar DNA binding protein-43 (TDP-43). ALS patient–specific iPSC–derived motor neurons formed cytosolic aggregates similar to those seen in postmortem tissue from ALS patients and exhibited shorter neurites as seen in a zebrafish model of ALS. The ALS motor neurons were characterized by increased mutant TDP-43 protein in a detergent-insoluble form bound to a spliceosomal factor SNRPB2. Expression array analyses detected small increases in the expression of genes involved in RNA metabolism and decreases in the expression of genes encoding cytoskeletal proteins. We examined four chemical compounds and found that a histone acetyltransferase inhibitor called anacardic acid rescued the abnormal ALS motor neuron phenotype. These findings suggest that motor neurons generated from ALS patient–derived iPSCs may provide a useful tool for elucidating ALS disease pathogenesis and for screening drug candidates.

Elastic fiber assembly is disrupted by excessive accumulation of chondroitin sulfate in the human dermal fibrotic disease, keloid

Keloid is a fibrotic disease characterized by abnormal accumulation of extracellular matrix in the dermis. The keloid matrix contains excess collagen and glycosaminoglycans (GAGs), but lacks elastic fiber. However, the roles of these matrix components in the pathogenesis of keloid are largely unknown. Here, we show that elastin and DANCE (also known as fibulin-5), a protein required for elastic fiber formation, are not deposited in the extracellular matrix of keloids, due to excess accumulation of chondoitin sulfate (CS), although the expression of elastin and DANCE is not affected. Amount of CS accumulated in the keloid legion was 6.9-fold higher than in normal skin. Fibrillin-1, a scaffold protein for elastic fiber assembly, was abnormally distributed in the keloid matrix. Addition of purified CS to keloid fibroblast culture resulted in abnormal deposition of fibrillin-1, concomitant with significantly decreased accumulation of elastin and DANCE in the extracellular matrix. We propose that CS plays a crucial role in the development of keloid lesions through inhibition of elastic fiber assembly.

Rapid magnetic resonance imaging for assessment of velopharyngeal muscle movement on phonation

The levator veli palatini muscle is the most important muscle for velopharyngeal (VP) closure in speech. I-3 Patients with speech abnormalities related to cleft palate are treated by reconstruction to achieve normal muscle realignment; however, in patients with abnormal speech related to cleft or noncleft palate, the structure and dynamic movement of the levator veli palatini muscle are unable to be observed by methods such as roentgenography, videofluoroscopy, and nasopharyngoscopy, which are used to evaluate the appearance of overall pharyngeal movement. Recently, rapid magnetic resonance imaging (MRI) has been developed, which permits the imaging of 5 slices within 14 seconds. Using it, we could obtain clear sequence images of the levator muscle sling itself during phonation. The purpose of this article is to present the possibility of assessment of the dynamics of VP closure associated with the levator muscle. MRI was performed as follows: The machine was a superconductive

Keloids can be forced into remission with surgical excision and radiation, followed by adjuvant therapy.

We have treated keloids using a combination of surgical excision and postoperative irradiation. The objective of this study was to evaluate the results of our treatment over 12 years. From 1995 until 2006, we treated keloids using the aforementioned treatment. If we identified a sign of recurrence during the follow-up period, we started an intralesional injection of triamcinolone acetonide immediately. We selected 91 keloids for which we had more than 2 years of follow-up data for this study and assessed the results according to our original scale (Kyoto scar scale) based on objective and subjective symptoms. In all, 51 keloids (56.0%) were cured completely by a combination of surgical excision and postoperative irradiation without additional treatment, and finally 81 keloids (89.0%) showed good results with additional treatment. Keloids are a controllable condition when treated with combination therapy, involving surgical excision with postoperative irradiation and early conservative treatment after the detection of recurrence.

A case of giant naevus followed up for 22 years after treatment with artificial dermis

We present an ultra-long followed-up case in which an artificial dermis was used for the treatment of a giant naevus. A 5-year-old boy had a giant naevus on his lower back and both buttocks. The light black pigmentation extended to the lower abdomen and both upper thighs. The lesions on the lower back and both buttocks were treated using the artificial dermis Pelnac in three operative series every 2 years. After removal of the lesion, Pelnac was placed onto the skin defect. Three weeks postoperatively, the silicone film was peeled off and a thin split-thickness skin graft (STSG) taken from the upper-middle back was placed on the regenerated dermis-like connective tissue. Thin STSGs were harvested from the same upper back area repeatedly. The lesions on both posteromedial upper thighs and the lower abdomen were treated in three operative series using tissue expanders. Finally, tissue expanders were inserted subcutaneously in both buttocks where Pelnac had been used 5 years or 7 years before. The lesions around the anus were reconstructed using the expanded skin and local skin flaps. Twenty-two years after the first operation, both grafted and donor sites keep good condition not only cosmetically but also functionally.

Chondroitinase injection improves keloid pathology by reorganizing the extracellular matrix with regenerated elastic fibers

Keloids are a proliferative fibrotic disease characterized by abnormal accumulation of extracellular matrix in the dermis. Keloid lesions lack skin plasticity due to deficiencies in elastic fiber formation in the extracellular matrix. The loss of elastic fiber is caused by excessive accumulation of chondroitin sulfate (CS), a sulfated glycosaminoglycan. However, there is no radical cure for keloids. Using a model system, we show herein that treatment of keloid tissues with chondroitinase ABC, an enzyme that specifically digests CS, improves clinical features of keloids. Keloid tissues obtained from patients were grafted on nude mice, and chondroitinase ABC was injected into the grafted keloid tissues. Chondroitinase ABC treatment significantly reduced the volume of keloid implants concomitant with recovery of elastic fiber formation. These results suggest that chondroitinase ABC injection is an effective therapy for keloid.

Open rhinoplasty using conchal cartilage during childhood to correct unilateral cleft-lip nasal deformities

BACKGROUND Although many articles have investigated the optimal method for the primary correction of nasal deformities during unilateral cleft lip repair, residual nasal deformities often remain. Such residual deformities are commonly corrected around the age of 5. We have started performing two-stage nasal repair operations for unilateral cleft lip patients. During the first stage, the nasal floor is elevated, and the alar base is brought into the correct position by muscle reconstruction during primary lip repair. During the second stage, the slanting nasal apex and drooping alar rim are corrected at pre-school age. This article describes the methods and results of second-stage open rhinoplasty. METHODS Open rhinoplasty is performed. The conchal cartilage is harvested and used as a strut to strengthen and extend the septum. The lower lateral cartilages are sutured to the grafted cartilage and fixed in the correct position. The operative results of 38 patients were evaluated photogrammetrically. The nasal height, nostril height and the columella angle on a basilar view of the nose were measured. RESULTS In most patients, the nose was refined and became less distorted. Poorly projecting nasal tips and drooping alar rims were corrected. The reformed configuration was relatively well maintained for many years. Photogrammetric analysis demonstrated increases in both the nasal height to nasal width ratio and the nostril height to nostril width ratio, and improvement of the columella angle. CONCLUSION Performing open rhinoplasty using conchal cartilage during childhood effectively improves unilateral cleft-lip nasal deformities.

Response to comment on "Drug screening for ALS using patient-specific induced pluripotent stem cells".

Our work and the study of Bilican et al. highlight the need for complementary assays to detect subtle phenotypic differences between control and mutant induced pluripotent stem cell lines.

Multipotent stem cells are effectively collected from adult human cheek skin.

Skin-derived precursor (SKP) cells are a valuable resource for tissue engineering and regenerative medicine, because they represent multipotent stem cells that differentiate into neural and mesodermal progenies. Previous studies suggest that the stem cell pool decreases with age. Here, we show that human multipotent SKP cells can be efficiently collected from adult cheek/chin skin, even in aged individuals of 70-78years. SKP cells were isolated from 38 skin samples by serum-free sphere culture and examined for the ability to differentiate into neural and mesodermal lineages. The number of spheres obtained from adult facial skin was significantly higher than that of trunk or extremity skin. SKP cells derived from cheek/chin skin exhibited a high ability to differentiate into neural and mesodermal cells relative to those derived from eyelid, trunk, or extremity skin. Furthermore, cheek/chin skin SKP cells were shown to express markers for undifferentiated stem cells, including a high expression level of the Sox9 gene. These results indicate that cheek/chin skin is useful for the recovery of multipotent stem cells for tissue engineering and regenerative therapy.

The combined use of hyoid bone flap and radial forearm free flap for reconstruction following partial laryngopharyngectomy.

In hypopharyngeal carcinoma, even partial laryngopharyngectomy, results in functional disorders involving swallowing and speaking. We reconstructed partial defects following partial laryngopharyngectomy using a combined flap of a hyoid bone flap and radial forearm free flap. Before ablative surgery, we prepared an ipsilateral hyoid bone as a bone flap with sternohyoidal muscle. Then a radial forearm free flap was prepared simultaneously with tumor surgery. Thereafter, we reconstructed the epiglottis and pyriform recess using the combined flap. We successfully reconstructed 7 patients suffering from squamous cell carcinoma of the hypopharynx. The tracheostoma in all patients could be closed. Six patients could swallow without dysphagia within 48 days. This is the first report of the successful combined use of a hyoid bone flap and radial forearm free flap for hypopharyngeal carcinoma.