Koshi Otsuki

Bioengineered trachea using autologous chondrocytes for regeneration of tracheal cartilage in a rabbit model.

In this study, a bioengineered trachea composed of autologous chondrocytes was developed, and its effect on cartilaginous regeneration was evaluated by implantation into tracheal defects in rabbits.

Potential for respiratory epithelium regeneration from induced pluripotent stem cells.

Objectives: In cases of laryngeal inflammatory lesions and tracheal invasion of a malignant tumor, autologous tissue implantation techniques using skin or cartilage are often applied. However, these techniques are both invasive and unstable. The purpose of this study was to evaluate the potential use of induced pluripotent stem (iPS) cells in the regeneration of respiratory epithelium. Methods: We seeded iPS cells on low-attachment plates in serum-free media to generate embryoid bodies (EBs). After a 3-day culture, the EBs were transferred to a gelatin-coated dish supplemented with activin A alone or with basic fibroblast growth factor (induction groups). As a control, EBs were cultured without these growth factors (control group). Cultured tissues from all groups were histologically examined for 2 weeks. Results: In the induction groups, the presence of respiratory epithelium-like tissue was observed with hematoxylin and eosin staining after 14 days of culture. Conclusions: This study demonstrated the potential use of iPS cells in regeneration of the respiratory epithelium.

Generation of airway epithelial cells with native characteristics from mouse induced pluripotent stem cells

Airway epithelial cells derived from induced pluripotent stem (iPS) cells are expected to be a useful source for the regeneration of airway epithelium. Our preliminary study of embryoid body (EB) formation and the air-liquid interface (ALI) method suggested that mouse iPS cells can differentiate into airway epithelial cells. However, whether the cells generated from mouse iPS cells had the character and phenotype of native airway epithelial cells remained uninvestigated. In this study, we generated airway epithelial cells from EBs by culturing them under serum-free conditions supplemented with Activin and bFGF and by the ALI method and characterized the iPS cell-derived airway epithelial cells in terms of their gene expression, immunoreactivity, morphology, and function. Analysis by quantitative real-time reverse transcription-polymerase chain reaction(RT-PCR) revealed that the expression of the undifferentiated cell marker Nanog decreased time-dependently after the induction of differentiation, whereas definitive endoderm markers Foxa2 and Cxcr4 were transiently up-regulated. Thereafter, the expression of airway epithelium markers such as Tubb4a, Muc5ac, and Krt5 was detected by RT-PCR and immunostaining. The formation of tight junctions was also confirmed by immunostaining and permeability assay. Analysis by hematoxylin and eosin staining and scanning electron microscopy indicated that the cells generated from mouse iPS cells formed airway-epithelium-like tissue and had cilia, the movement of which was visualized and observed to be synchronized. These results demonstrate that the airway epithelial cells generated by our method have native characteristics and open new perspectives for the regeneration of injured airway epithelium.

Potential of laryngeal muscle regeneration using induced pluripotent stem cell-derived skeletal muscle cells

ABSTRACT Conclusion Induced pluripotent stem (iPS) cells may be a new potential cell source for laryngeal muscle regeneration in the treatment of vocal fold atrophy after recurrent laryngeal nerve paralysis. Objectives Unilateral vocal fold paralysis can lead to degeneration, atrophy, and loss of force of the thyroarytenoid muscle. At present, there are some treatments such as thyroplasty, arytenoid adduction, and vocal fold injection. However, such treatments cannot restore reduced mass of the thyroarytenoid muscle. iPS cells have been recognized as supplying a potential resource for cell transplantation. The aim of this study was to assess the effectiveness of the use of iPS cells for the regeneration of laryngeal muscle through the evaluation of both in vitro and in vivo experiments. Methods Skeletal muscle cells were generated from tdTomato-labeled iPS cells using embryoid body formation. Differentiation into skeletal muscle cells was analyzed by gene expression and immunocytochemistry. The tdTomato-labeled iPS cell-derived skeletal muscle cells were transplanted into the left atrophied thyroarytenoid muscle. To evaluate the engraftment of these cells after transplantation, immunohistochemistry was performed. Results The tdTomato-labeled iPS cells were successfully differentiated into skeletal muscle cells through an in vitro experiment. These cells survived in the atrophied thyroarytenoid muscle after transplantation.

Effective embryoid body formation from induced pluripotent stem cells for regeneration of respiratory epithelium.

We have previously demonstrated the potential use of induced pluripotent stem (iPS) cells for regeneration of respiratory epithelium by culturing embryoid bodies (EBs). The aim of the present study was to determine the most effective conditions for EB formation from iPS cells for regeneration of respiratory epithelium.

Visualization of mouse induced pluripotent stem cells for evaluation of tracheal regeneration

Abstract Conclusion: Visualization of mouse induced pluripotent stem (iPS) cells with the use of a fluorescent protein was successfully achieved for evaluation of tracheal regeneration. Objectives: Tracheal epithelial cells derived from iPS cells are expected to be a useful cell source for tracheal regeneration. Our previous study demonstrated that mouse iPS cells differentiated into tracheal epithelial cells. However, when they are implanted into tracheal defects in experimental animals, it is difficult to determine whether the regenerated tracheal epithelium is in fact derived from iPS cells. The purpose of this study was to develop a visualization technique for iPS cells for evaluation of tracheal regeneration. Methods: Fluorescent marker tdTomato was transfected into iPS cells. Tracheal epithelial cells were generated from tdTomato-labeled iPS cells using embryoid body formation to detect the expression of tdTomato. The artificial material with tdTomato-labeled iPS cells was implanted into tracheal defects in nude rats. The survival and distribution of tdTomato-labeled iPS cell-derived cells were examined using the IVIS Imaging System and immunostaining. Results: The expression of tdTomato was detected in both undifferentiated cells and tracheal epithelial cells in vitro. tdTomato-labeled iPS cell-derived cells were successfully detected in the tracheal defects by IVIS Imaging System and immunostaining.

Implantation site-dependent differences for tracheal regeneration with induced pluripotent stem cells (iPS cells)

Abstract Conclusion: The histological findings and quantitative measurements demonstrated that there were differences in teratoma formation according to the site of implantation. Elucidating the mechanisms of the teratoma formation caused by the site of implantation moves the field another step closer to clinical use of induced pluripotent stem (iPS) cells for tracheal regeneration. Objectives: Our previous study demonstrated the potential for iPS cells to be used as a new cell source for tracheal regeneration. However, teratoma formation remains a major problem. Implantation site-dependent differences in teratoma formation have been reported. In this study, the teratoma-forming propensity after implantation into tracheal defects and abdominal subcutaneous tissue was examined histologically and quantitatively. Methods: Mouse iPS cells were cultured in artificial material under various conditions. After cultivation in vitro, artificial materials with cultured iPS cells were then implanted into cervical tissue around tracheal defects and into abdominal subcutaneous tissue in nude rats. Teratoma formation was evaluated histologically and quantitatively with measurement of maximum diameter (MD). Results: Teratoma was observed in 10 of 11 rats with cervical tissue around tracheal defects and in 3 of 11 rats with abdominal subcutaneous tissue implants. The average MD was 5.36 mm in the trachea and 0.97 mm in the abdomen.

Implantation of Induced Pluripotent Stem Cell–Derived Tracheal Epithelial Cells

Objectives: Compared with using autologous tissue, the use of artificial materials in the regeneration of tracheal defects is minimally invasive. However, this technique requires early epithelialization on the inner side of the artificial trachea. After differentiation from induced pluripotent stem cells (iPSCs), tracheal epithelial tissues may be used to produce artificial tracheas. Herein, we aimed to demonstrate that after differentiation from fluorescent protein-labeled iPSCs, tracheal epithelial tissues survived in nude rats with tracheal defects. Methods: Red fluorescent tdTomato protein was electroporated into mouse iPSCs to produce tdTomato-labeled iPSCs. Embryoid bodies derived from these iPSCs were then cultured in differentiation medium supplemented with growth factors, followed by culture on air-liquid interfaces for further differentiation into tracheal epithelium. The cells were implanted with artificial tracheas into nude rats with tracheal defects on day 26 of cultivation. On day 7 after implantation, the tracheas were exposed and examined histologically. Results: Tracheal epithelial tissue derived from tdTomato-labeled iPSCs survived in the tracheal defects. Moreover, immunochemical analyses showed that differentiated tissues had epithelial structures similar to those of proximal tracheal tissues. Conclusions: After differentiation from iPSCs, tracheal epithelial tissues survived in rat bodies, warranting the use of iPSCs for epithelial regeneration in tracheal defects.

Regeneration of tracheal epithelium using mouse induced pluripotent stem cells

Abstract Conclusion The findings demonstrated the potential use of induced pluripotent stem cells for regeneration of tracheal epithelium. Objective Autologous tissue implantation techniques using skin or cartilage are often applied in cases of tracheal defects with laryngeal inflammatory lesions and malignant tumor invasion. However, these techniques are invasive with an unstable clinical outcome. The purpose of this study was to investigate regeneration in a tracheal defect site of nude rats after implantation of ciliated epithelium that was differentiated from induced pluripotent stem cells. Method Embryoid bodies were formed from mouse induced pluripotent stem cells. They were cultured with growth factors for 5 days, and then cultured at the air–liquid interface. The degree of differentiation achieved prior to implantation was determined by histological findings and the results of real-time polymerase chain reaction. Embryoid bodies including ciliated epithelium were embedded into collagen gel that served as an artificial scaffold, and then implanted into nude rats, creating an ‘air–liquid interface model’. Histological evaluation was performed 7 days after implantation. Results The ciliated epithelial structure survived on the lumen side of regenerated tissue. It was demonstrated histologically that the structure was composed of ciliated epithelial cells.