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Hair follicle morphogenesis is heavily dependent on reciprocal, sequential, and epithelial-mesenchymal interaction (EMI) between epidermal stem cells and the specialized cells of the underlying mesenchyme, which aggregate to form the dermal condensate (DC) and will later become the dermal papilla (DP). Similar models were developed with a co-culture of keratinocytes and DP cells. Previous studies have demonstrated that co-culture with keratinocytes maintains the in vivo characteristics of the DP. However, it is often challenging to develop three-dimensional (3D) DP and keratinocyte co-culture models for long term in vitro studies, due to the poor intercellular adherence between keratinocytes. Keratinocytes exhibit exfoliative behavior, and the integrity of the DP and keratinocyte co-cultured spheroids cannot be maintained over prolonged culture. Short durations of culture are unable to sufficiently allow the differentiation and re-programming of the keratinocytes into hair follicular fate by the DP. In this study, we explored a microgel array approach fabricated with two different hydrogel systems. Using poly (ethylene glycol) diacrylate (PEGDA) and gelatin methacrylate (GelMA), we compare their effects on maintaining the integrity of the cultures and their expression of important genes responsible for hair follicle morphogenesis, namely Wnt10A, Wnt10B, and Shh, over prolonged duration. We discovered that low attachment surfaces such as PEGDA result in the exfoliation of keratinocytes and were not suitable for long-term culture. GelMA, on the hand, was able to sustain the integrity of co-cultures and showed higher expression of the morphogens overtime.

Objectives Reproducing human hair follicles in vitro is often limited by various reasons such as the lack of a systematic approach to culture distinct hair follicle cell types to reproduce their spatial relationship. Here, we reproduce hair follicle‐like constructs resembling the spatial orientation of different cells in vivo, to study the role of keratinocytes in maintaining cellular compartmentalization among hair follicle‐related cells. Materials and methods Dermal papilla (DP) cells, HaCaT keratinocytes and human dermal fibroblast (HDF) cells were seeded sequentially into three‐dimensional (3D) microwells fabricated from polyethylene glycol diacrylate hydrogels. Quantitative polymerase chain reaction was used to compare inductive gene expression of 3D and two‐dimensional (2D) DP. DP and HaCaT cells were transfected with green fluorescent protein and red fluorescent protein lentivirus, respectively, to enable cell visualization using confocal microscopy. Results The 3D DP cultures showed significantly enhanced expression of essential DP genes as compared 2D cultures. Core‐shell configurations containing keratinocytes forming the outer shell and DP forming the core were observed. Migratory polarization was mediated by cell‐cell interaction between the keratinocytes and HDF cells, while preserving the aggregated state of the DP cells. Conclusions Keratinocytes may play a role in maintaining compartmentalization between the DP and the surrounding HDF residing in the dermis, and therefore maintains the aggregative state of the DP cells, necessary for hair follicle development and function. Cultures comprising of dermal papilla cells, keratinocytes, and human dermal fibroblasts were prepared from sequential seeding into hydrogel microwells, in order to reproduce the spatial orientation of hair follicular cells in vivo. This work demonstrated the importance of keratinocytes in maintaining cellular compartmentalization and dermal papilla aggregation, which is necessary for hair follicle development and function.

Androgenetic alopecia (AGA) is characterized by a progressive and patterned transformation of thick, pigmented terminal scalp hairs into short, hypo-pigmented vellus-like hairs. The use of Minoxidil and Finasteride to treat AGA are often associated with complications in safety and efficacy. However, herbal remedies are deemed to have lesser side effects in many societies. This study aims to identify potential hair growth properties of individual compounds from a Chinese proprietary medicine known as Yangxue Shengfa capsule (YSC), used in China for many years for improving AGA. Six marker compounds, including 2,3,5,4'-tetrahydroxystilbene-2-O-β-D-glucoside (TSG), Chlorogenic acid, Emodin, Ferulic acid, Isoimperatorin, and Paeoniflorin were used for simultaneous HPLC quantification and anti-AGA screening. Simultaneous quantification of these components was performed on 75% (v/v) methanol extracts of YSC, using a Welch Ultimate XB-C18 column and gradient elution. Five compounds significantly promoted cell proliferation in cultured immortalized human Dermal Papilla Cells (DPC). Multiple genes associated with the progression of AGA, including IGF-1, DKK-1, and TGF-β1, were found to be regulated by some of these compounds. Interestingly, Ferulic acid and Emodin demonstrated good pharmacological properties against AGA, thereby concluding the potential of these bioactives to be used in the treatment against AGA.