新鲜分离的人毛乳头（DP）能诱导毛囊再生，但在体外逐渐丧失这种功能，极大限制了临床上大量重建毛囊治疗患者。海藻盐是一种天然多糖，我们将其制备成海藻酸钠水凝胶并对其进行了初步改造，发现人DP细胞在其中3D培养后会聚集成球，毛囊诱导能力显著恢复。本研究中，我们首先将继续调整海藻酸钠水凝胶的性状，将不同代数人DP细胞培养在不同的海藻酸钠微球中，检测毛囊诱导相关分子的表达，同时与小鼠或人的角质形成细胞混合注入裸鼠皮下观察毛囊再生情况，以得到最佳的水凝胶制备条件；再在不同时间点取上述培养DP细胞的RNA行RNA-seq检测以寻找恢复诱导能力的重要分子，然后在不同培养方式培养的DP细胞中进行调节并在裸鼠体内验证其毛囊诱导能力；最后制备DP特异性敲除/敲入鼠以验证其功能。本研究的最终目的在于建立理想的DP体外培养条件并寻找维持和恢复其毛囊再生诱导能力的关键因子，为临床上脱发性疾病的治疗提供新思路。

Freshly isolated human dermal papilla (DP) has been shown to be able to induce de novo hair growth. However, it quickly loses its hair-inducing properties upon in vitro expansion, which greatly limits its clinical application for hair follicle reconstruction. In the preliminary study, we established a three-dimensional (3D) culture system with sodium alginate hydrogel mainly composed of alginate, a kind of natural polysaccharide extracted from brown seaweed, and found that human DP cells cultured in this system would aggregate into microspheres and the hair-inducing ability of DP cells was at least significantly recovered. In this study, to obtain the optimal conditions for DP culture, we will continue to adjust the preparation of sodium alginate hydrogels. The expression of hair-inductive markers will be analyzed in DP cells at different passages cultured in adjusted sodium alginate microspheres by quantitative RT-PCR, immunostaining and immunoblotting. Then, these DP cells will be co-implanted with newborn mouse epidermal cells or human low-passage keratinocytes into nude mice to evaluate their hair-inducing ability. In addition, to seek the key signals involved in the restoration of hair-inducing properties of DP cells cultured in the optimal 3D culture system, we intend to execute RNA-seq analysis of DP cells at monolayered culture (2D) and microspheres (3D) at various time points. Through gain- and loss-of function approaches in vitro, together with hair follicle reconstitution in vivo, we will explore the roles of the key genes from RNA-seq analysis in maintaining or restoring the hair-inducing properties of DP cells at 2D and 3D culture. Finally, by generating conditional transgenic mice, we will explore the roles of several indicated key genes in the DP in hair regeneration. Our ultimate goal is to establish an ideal culture system for DP cells in vitro and figure out the key signals during maintenance and restoration of inductive properties, which will provide new cues for hair loss treatment.