Shizu Takeda

Interaction of antiproliferative protein Tob with the CCR4-NOT deadenylase complex.

Tob protein, when overexpressed, suppresses growth of NIH3T3 cells, presumably by regulating expression of various growth‐related genes. However, the molecular mechanisms underlying Tob‐mediated regulation of gene expression have been obscure. To address this issue we established stable Tob‐expressing cell lines and used a proteomics approach to identify Tob‐interacting proteins. We found that Tob associates with the CCR4‐NOT complex. The carboxyl‐terminal half of Tob interacted with Cnot1, a core protein of the CCR4‐NOT complex. We further showed that the deadenylase activity associated with the complex was suppressed in vitro by Tob. These results suggest that the antiproliferative activity of Tob is shown post‐transcriptionally by controlling the stability of the target mRNAs in addition to its involvement in transcriptional regulation, reported previously. (Cancer Sci 2008; 99: 755–761)

Fabrication of transplantable corneal epithelial and oral mucosal epithelial cell sheets using a novel temperature-responsive closed culture device

Temperature‐responsive culture surfaces make it possible to harvest transplantable carrier‐free cell sheets. Here, we applied temperature‐responsive polymer for polycarbonate surfaces with previously developed closed culture devices for an automated culture system in order to fabricate transplantable stratified epithelial cell sheets. Histological and immunohistochemical analyses and colony‐forming assays revealed that corneal epithelial and oral mucosal epithelial cell sheets could be harvested with the temperature‐responsive closed culture devices. The results were similar to those obtained using temperature‐responsive culture inserts. These results indicate that the novel temperature‐responsive closed culture device is useful for fabricating transplantable stratified epithelial cell sheets. Copyright

A novel closed cell culture device for fabrication of corneal epithelial cell sheets

Automation technology for cell sheet‐based tissue engineering would need to optimize the cell sheet fabrication process, stabilize cell sheet quality and reduce biological contamination risks. Biological contamination must be avoided in clinical settings. A closed culture system provides a solution for this. In the present study, we developed a closed culture device called a cell cartridge, to be used in a closed cell culture system for fabricating corneal epithelial cell sheets. Rabbit limbal epithelial cells were cultured on the surface of a porous membrane with 3T3 feeder cells, which are separate from the epithelial cells in the cell cartridges and in the cell‐culture inserts as a control. To fabricate the stratified cell sheets, five different thicknesses of the membranes which were welded to the cell cartridge, were examined. Multilayered corneal epithelial cell sheets were fabricated in cell cartridges that were welded to a 25 µm‐thick gas‐permeable membrane, which was similar to the results with the cell‐culture inserts. However, stratification of corneal epithelial cell sheets did not occur with cell cartridges that were welded to 100–300 µm‐thick gas‐permeable membranes. The fabricated cell sheets were evaluated by histological analyses to examine the expression of corneal epithelial‐specific markers. Immunohistochemical analyses showed that a putative stem cell marker, p63, a corneal epithelial differentiation maker, CK3, and a barrier function marker, Claudin‐1, were expressed in the appropriate position in the cell sheets. These results suggest that the cell cartridge is effective for fabricating corneal epithelial cell sheets. Copyright

The efficient fabrication of corneal epithelial cell sheets by controlling oxygen concentration.

We have developed a novel method to accelerate the fabrication of epithelial cell sheets by controlling oxygen concentration. Rabbit limbal epithelial cells were proliferated efficiently under hypoxia (2% O2) in comparison to those proliferated under normoxia (20% O2), but were not stratified completely under 2% O2. In contrast, corneal limbal epithelial cells cultured under hypoxia were stratified by re-oxygenation after reaching confluence. Histological and immunofluorescence analyses and colony-forming assays showed that it was possible to fabricate the corneal epithelial cell sheets efficiently by controlling the oxygen concentration. These results indicate that this novel method can be a cost-effective tool for fabricating stratified epithelial cell sheets for corneal regenerative medicine.

Fabrication of corneal epithelial cell sheets maintaining colony-forming cells without feeder cells by oxygen-controlled method

The use of murine 3T3 feeder cells needs to be avoided when fabricating corneal epithelial cell sheets for use in treating ocular surface diseases. However, the expression level of the epithelial stem/progenitor cell marker, p63, is down-regulated in feeder-free culture systems. In this study, in order to fabricate corneal epithelial cell sheets that maintain colony-forming cells without using any feeder cells, we investigated the use of an oxygen-controlled method that was developed previously to fabricate cell sheets efficiently. Rabbit limbal epithelial cells were cultured under hypoxia (1-10% O2) and under normoxia during stratification after reaching confluence. Multilayered corneal epithelial cell sheets were fabricated using an oxygen-controlled method, and immunofluorescence analysis showed that cytokeratin 3 and p63 was expressed in appropriate localization in the cell sheets. The colony-forming efficiency of the cell sheets fabricated by the oxygen-controlled method without feeder cells was significantly higher than that of cell sheets fabricated under 20% O2 without feeder cells. These results indicate that the oxygen-controlled method has the potential to achieve a feeder-free culture system for fabricating corneal epithelial cell sheets for corneal regeneration.

Efficient fabrication of epidermal cell sheets using γ-secretase inhibitor

BACKGROUND Epidermal cell sheets have been utilized for regeneration of skin when skin defects occur and prevention of esophageal stricture after endoscopic submucosal dissection. To reduce the cost of cultivation, a novel culture method to shorten a culture process needs to be developed. OBJECTIVES To shorten a culture process of epidermal cell sheets, we developed a novel culture method to accelerate the fabrication of epidermal cell sheets using γ-secretase inhibitor. METHODS Normal human epidermal keratinocytes (NHEKs) were cultured using γ-secretase inhibitor, DAPT, during expansion of the cells to confluence and culture without DAPT during stratification. The cell growth, quantitative gene expression of stem/progenitor or differentiation markers, and protein expression of these markers were analyzed to verify the effectiveness of the novel method. RESULTS The proliferation of NHEKs on cell-culture inserts was promoted using DAPT. However, NHEKs were not stratified completely in the presence of DAPT. In contrast, NHEKs cultured using DAPT were stratified and differentiated by eliminating the inhibitor after the cells reached confluence. Real-time PCR analyses showed that the gene expressions of putative epithelial stem/progenitor cell markers and epidermis differentiation markers in the cell sheets fabricated using this novel method were significantly higher than those in the cell sheets fabricated without DAPT. Histological and immunofluorescence analyses revealed that it was possible to fabricate well-differentiated epidermal cell sheets efficiently by the novel culture method. The culture period was shortened to 67% of the time required for the control group. In feeder-free conditions, stratified epidermal cell sheets were also fabricated using DAPT. CONCLUSIONS The novel culture method using γ-secretase inhibitor, DAPT, was found to be effective for fabricating epidermal cell sheets.

A novel, flexible and automated manufacturing facility for cell-based health care products: Tissue Factory

Introduction Current production facilities for Cell-Based Health care Products (CBHPs), also referred as Advanced-Therapy Medicinal Products or Regenerative Medicine Products, are still dependent on manual work performed by skilled workers. A more robust, safer and efficient manufacturing system will be necessary to meet the expected expansion of this industrial field in the future. Thus, the ‘flexible Modular Platform (fMP)’ was newly designed to be a true “factory” utilizing the state-of-the-art technology to replace conventional “laboratory-like” manufacturing methods. Then, we built the Tissue Factory as the first actual entity of the fMP. Methods The Tissue Factory was designed based on the fMP in which several automated modules are combined to perform various culture processes. Each module has a biologically sealed chamber that can be decontaminated by hydrogen peroxide. The asepticity of the processing environment was tested according to a pharmaceutical sterility method. Then, three procedures, production of multi-layered skeletal myoblast sheets, expansion of human articular chondrocytes and passage culture of human induced pluripotent stem cells, were conducted by the system to confirm its ability to manufacture CHBPs. Results Falling or adhered microorganisms were not detected either just after decontamination or during the cell culture processes. In cell culture tests, multi-layered skeletal myoblast sheets were successfully manufactured using the method optimized for automatic processing. In addition, human articular chondrocytes and human induced-pluripotent stem cells could be propagated through three passages by the system at a yield comparable to manual operations. Conclusions The Tissue Factory, based on the fMP, successfully reproduced three tentative manufacturing processes of CBHPs without any microbial contamination. The platform will improve the manufacturability in terms of lower production cost, improved quality variance and reduced contamination risks. Moreover, its flexibility has the potential to adapt to the modern challenges in the business environment including employment issues, low operational rates, and relocation of facilities. The fMP is expected to become the standard design basis of future manufacturing facilities for CBHPs.

Evaluating Protein Sequence Signatures Inferred from Protein-Protein Interaction Data by Gene Ontology Annotations

We propose a systematic method to find sequence signatures of proteins which share a common interacting partner, using a protein similarity measure based on gene ontology (GO) annotations. In a computational experiment on our original human data set of protein-protein interactions determined by Y2H assays, we have succeeded in discovering convincing candidates for interacting sites and other functional regions.