## 4 Discussion
Considering that the convergent number of cells in 200 \(\upmu\)m diameter alginate sphere is approximately around 10, while the cellular number of in vivo human DP ranges from 500 to 2000, in vivo human DP has much more cellular density than alginate sphere since [PERSON]'s line of human DP range from 80 \(\upmu\)m to 180 \(\upmu\)m in general depending on the body site of hair follicles [9]. There is a report that the cellular topology within dermal papilla and dermal sheath can have a different potential to regenerate [41]. Their group also reported the compartments above and below [PERSON]'s line have the heterogeneous hair inducing ability as well as their _in vitro_ cultivation properties [42]. How to maintain their dormant status under optimal density within alginate sphere as well as what kind of dormant cells can survive in alginate sphere would be the critical questions in further study. Our experiments have a limitation to elucidate why they do have their optimal density in alginate sphere with human dermal papilla cells. Specially to investigate whether they have any senescent characteristics would be an important question to address. It is also critical to note whether dermal sheath or dermal cup cell, which have more potential to be proliferative and considered as more mesenchymal stem cell property than dermal papilla cells, have similar characteristics in alginate sphere or not. To determine the long-term characteristics of the cells remaining in the alginate spheres, we investigated the gene expression pattern of the DPC markers [27]. Signature genes including SOX2, ALPL, WIF1, Noggin, BMP4 and VCAN were up-regulated after cultivation in alginate spheres compared with normal 2D cultivation. Interestingly, in this experiment, the explants from the cells seeded in the alginate spheres expressed the signature genes more highly than 2D cells. The cells at 80 days after cultivation in the alginate spheres had the highest expression level of interested genes,which means that the remaining cells after cellular convergence in the alginate sphere are similar in nature to in vivo DPCs. Compared with in vivo DPCs, DPCs in alginate spheres have distinct characteristics. We concluded that, in mature anagen stage, there are three main factors that make the dermal papilla functional: the dormancy factor, proliferative factor and maintenance factor (Figure 6). The dormancy factors cause DPCs to undergo dormancy to sustain stemness and the inductive characteristics of the cells. The proliferating factors, which in vivo DPCs do not have, cause the cells to grow. Normally, only during the short time between the telogen and anagen stages, the proliferating factors are enhanced in in vivo dermal papilla [12; 13]. The maintenance factors are from the epithelial cells around the dermal papilla and cause the DPCs to remain alive. Compared to _in vitro_ DPCs, DPCs in the alginate spheres had more of the dormancy factor and less of the proliferating factors giving the cells in the spheres more stem cell-like in vivo characteristics. We concluded that there are passive changes related to the dormant state and cellular death in 3D cultivation. The alginate sphere environment was found to maintain the dormancy of DPCs with increased trichogenicity. Our novel approach using alginate spheres demonstrates how DPCs behave inside the sphere microenvironment. The dynamics and the stem cell-like character of cells in alginate spheres are likely to be broadly applicable to hair biology as well as a number of complex model systems. **Supplementary Materials:** The following are available online at [[http://www.mdpi.com/2076-3417/8/10/1993/s1](http://www.mdpi.com/2076-3417/8/10/1993/s1)]([http://www.mdpi.com/2076-3417/8/10/1993/s1](http://www.mdpi.com/2076-3417/8/10/1993/s1)), Table S1: Design of the primers used this study. **Author Contributions:** [PERSON] and [PERSON] developed the concepts, designed the experimental methodology, analyzed the data, wrote, reviewed and approved the final paper. [PERSON] and [PERSON]-H.K.