## 4 Discussion
### Decrease of Nanog Expression and Increase of EB Size Depend on Oxygen Tension  
The size of EBs increased over time from Day 2 to Day 10. Nanog, which is an index of the undifferentiated state, was expressed on Days 2 and 5 but was not expressed on  
Figure 8: ICF staining for collagen type 1 (**a-c**) and Runx2 (**d-f**) proteins in OGCs on Day 26.  
Figure 9: Comparison of the intensity of fluorescence in ICF staining for collagen type 1 (**a**) and Runx2 (**b**) proteins on Day 26. One asterisk (*) indicates that 0.01 \(\leq\)\(p\)-value \(<\) 0.05. Two asterisks (***) indicate that the \(p\)-value < 0.01.  
Days 8 or 10, indicating that the undifferentiated characteristics of EBs were kept until Day 5. The results of this study supported the speculation of [PERSON] et al. [10], that the differentiation state of EBs differs before and after Day 5.  
The oxygen tension was changed on Day 5 in this study because the increases in EB size were insufficient on Day 2, and the state of differentiation of EBs had progressed by Day 10. As a result, the size of EBs cultured under hypoxia remained smaller than the size of EBs cultured under normoxia, and EBs grew even larger under hyperoxia. According to [PERSON] et al., when EBs were formed from mouse embryonic stem (ES) cells and were cultured under hypoxia (1%) for 5 days, they were found to grow equally [21]. However, the number of viable EBs decreased when the culture was continued for 5 days. That study did not comment on the size of the EBs, but the results suggested that hypoxia suppressed the increased number of cells constituting EBs. In addition, according to a study by [PERSON] et al., hypoxia (3%) at the initial stage of the re-programming strengthened the colonization when hiPS cells were produced from dental pulp cells [22]. However, hypoxia strongly inhibited the colonization and the increase in hiPS cells at a late stage. Furthermore, according to [PERSON] et al., when mouse ES cells were cultured under hypoxia (5%), normoxia (20%) or hyperoxia (40%) for 4 days, cells proliferated the most in hyperoxia and the least in hypoxia [23]. In this study, we changed the oxygen tension to hypoxia on Day 5 of EB formation. As a result, the increase in EBs was suppressed; thus, our results confirmed the above results. Cell death can result easily due to a lack of oxygen and nourishment within cell agglomerations, which suggests that this was why the proliferative rate decreased [24]. Hypoxia decreased the number of mitochondria in cells, and it was thought that a metabolism change, such as the citric acid cycle [25], might have influenced that. However, one study reported that no difference was seen in the proliferative rate of cells with hypoxia (2%) or normoxia in periodontal ligament stem cells or in dental pulp stem cells [26]. In addition, there was a report that hypoxia (5%) enhanced the generation of iPS cells [27]. In other words, these findings suggest that many factors may play important roles in regulating the proliferation and differentiation of iPS cells, such as the type of cell, the oxygen tension, the exposure state and the time. On the other hand, hyperoxia increased the size of EBs and the quantity of OGCs in this study. It has been reported that an increase in oxygen tension allows Wnt signal transmission to regulate an increase in differentiation and cell activation [28].