Analysis of extracellular vesicles as a potential index for monitoring differentiation of neural lineage cells from induced pluripotent stem cells.

To improve cell production efficacy, it is important to evaluate cell conditions during culture. Extracellular vesicles (EVs) secreted from various cells are involved in stem cell differentiation. As EVs carry information about their source cells, we hypothesized that they may serve as a noninvasive index of cell conditions. We evaluated changes in EV morphology, concentration, and microRNA (miRNA) and protein expression in culture supernatants during the differentiation of induced pluripotent stem cells (iPSCs) into neural lineage cells, for application in regenerative medicine for Parkinson's disease. We observed EVs (50-150 nm) in culture supernatants of iPSCs and differentiated cells. The EVs expressed the exosome markers CD63, CD81, and CD9. Throughout differentiation, the EV concentration in the supernatants decreased, and EV miRNA and protein expression changed substantially. Especially, miR-106b, involved in neural stem cell differentiation and normal brain development, was considerably downregulated. CD63 expression correlated with the CORIN-positive cell rate, which is an index of differentiation. Thus, EV concentration and miRNA and protein expression may reflect the differentiation status of iPSCs. These findings pave the way for the development of novel and sensitive cell culture monitoring methods.

Fabrication of tissue-engineered cell sheets by automated cell culture equipment.

Most cells for regenerative medicine are currently cultured manually. In order to promote the widespread use of regenerative medicine, it will be necessary to develop automated culture techniques so that cells can be produced in greater quantities at lower cost and with more stable quality. In the field of regenerative medicine technology, cell sheet therapy is an effective tissue engineering technique whereby cells can be grafted by attaching them to a target site. We have developed automated cell culture equipment to promote the use of this cell sheet regenerative treatment. This equipment features a fully closed culture vessel and circuit system that avoids contamination with bacteria and the like from the external environment, and it was designed to allow 10 cell sheets to be simultaneously cultured in parallel. We used this equipment to fabricate 50 sheets of human oral mucosal epithelial cells in five automated culture tests in this trial. By analyzing these sheets, we confirmed that 49 of the 50 sheets satisfied the quality standards of clinical research. To compare the characteristics of automatically fabricated cell sheets with those of manually fabricated cell sheets, we performed histological analyses using immunostaining and transmission electron microscopy. The results confirmed that cell sheets fabricated with the automated cell culture are differentiated in the same way as cultures fabricated manually.

Fabricating retinal pigment epithelial cell sheets derived from human induced pluripotent stem cells in an automated closed culture system for regenerative medicine.

Regenerative medicine has received a lot of attention as a novel strategy for injuries and diseases that are difficult to cure using current techniques. Cell production, which is vital for regenerative medicine, has undergone remarkable progress via breakthroughs in developmental biology and tissue engineering; currently, cell production requires numerous experimental operators performing manual, small-scale cell cultures. Other major obstacles for cell production and regenerative medicine include the variable quality of products based on the experimental procedure, the skills of operators, the level of labor required for production, and costs. Technological developments are required to overcome this, including automation instead of manual culture. Age-related macular regeneration (AMD) is a refractory ocular disease that causes severe deterioration in central vision due to senescence in the retinal pigment epithelium (RPE). Recently, we performed an autologous transplantation of induced pluripotent stem (iPS) cell-derived RPE cell sheets and started clinical research on allografts from RPE cell suspensions differentiated from iPS cells. The use of regenerative therapies for AMD using iPS cell-derived RPE is expected to become more widespread. In the present study, human iPS cell-derived RPE cells were cultured to form RPE cell sheets using equipment with a closed culture module. The quality of the automated cultured RPE cell sheets was confirmed by comparing their morphological and biological properties with those of manually generated RPE cell sheets. As a result, machine-cultured RPE sheets displayed the same quality as manually cultured RPE sheets, showing that iPS cell-derived RPE cell sheets were successfully cultured by an automated process.