The aim of the present study was to accomplish de novo generation of reconstituted human skin with enriched hair follicles. Dermal papillae (DP) are known to play a crucial organizing role in hair follicle induction. However, generation of enriched human hair follicles using cultured DP cells has not been accomplished because DP cells easily lose their hair-inducing ability with culturing. To enhance the hair-inducing ability of DP cells, Wnt signaling pathway activation or three-dimensional (3D) spheroid culture methods were employed in previous studies. Herein, we assessed effects of the canonical Wnt/beta-catenin signaling activator CHIR99021 and found that it enhanced the expression of DP signature genes associated with hair-inducing ability. Further comparison of three different 3D culture methods revealed the highest expression of DP signature genes in spheroids generated by a floating drop method compared with other methods. CHIR99021 synergistically increased expression of DP signature genes in combination with floating drop culture. "Reconstituted skin assay" prepared using the most promising CHIR99021-stimulated 3D spheroids showed enrichment for human hair follicles. Labeled DP spheroids and derived cells were primarily found to be DP and dermal sheath cup (DSC) cells, implying organization of hair formation by DP spheroids. Finally, to evaluate the functional features of generated human skin and hair follicles, we injected human DSC cells, which reportedly show DP precursor behavior, and exhibit hair-inducing ability through incorporation into hair follicles, into mice. Histological studies revealed injected DSC cells in dermal sheath of hair follicles, consistent with a previous report, thus verifying the functionality of generated skin and hair follicles. Collectively, our findings demonstrate that DP spheroids synergistically stimulated by CHIR99021 and 3D culture contributed to hair follicle formation, thus making it possible to generate reconstituted hair follicle-enriched human skin with functional features. (C) 2019 Elsevier Inc. All rights reserved.