Hair Germ Model In Vitro via Human Postnatal Keratinocyte-Dermal Papilla Interactions: Impact of Hyaluronic Acid

Abstract

Hair follicle (HF) reconstruction in vitro is a promising field in alopecia treatment and human HF development research. Here, we combined postnatal human dermal papilla (DP) cells and skin epidermal keratinocytes (KCs) in a hanging drop culture to develop an artificial HF germ. The method is based on DP cell hair-inducing properties and KC self-organization. We evaluated two protocols of aggregate assembling. Mixed HF germ-like structures demonstrated the initiation of epithelial-mesenchymal interaction, including WNT pathway activation and expression of follicular markers. We analyzed the influence of possible DP cell niche components including soluble factors and extracellular matrix (ECM) molecules in the process of the organoid assembling and growth. Our results demonstrated that soluble factors had little impact on HF germ generation and Ki67+ cell score inside the organoids although BMP6 and VD3 maintained effectively the DP identity in the monolayer culture. Aggrecan, biglycan, fibronectin, and hyaluronic acid (HA) significantly stimulated cell proliferation in DP cell monolayer culture without any effect on DP cell identity. Most of ECM compounds prevented the formation of cell aggregates while HA promoted the formation of larger organoids. In conclusion, our model could be suitable to study cell-cell and cell-niche interactions during HF reconstruction in vitro.

Tissue engineering strategies for human hair follicle regeneration: How far from a hairy goal?

“…One approach consists in coculturing DPCs with keratinocytes or keratinocyte‐conditioned medium, shown to provide DPCs with molecular cues involved in EM crosstalk during hair growth . Also, distinct players in this signaling crosstalk (including Wnt, BMP and FGF) were reported to prolong the hair‐inducing ability of cultured DPCs . More recently, pharmacological modulation of JAK–STAT signaling pathway was shown to improve cultured human DPCs’ inductivity .…”

“…Considering the undoubted contribution of 3D culture for DPCs' fitness, numerous studies have explored the contribution of biomaterial‐based strategies to support DP hair inductive behavior in vitro . Biomaterials have been employed either (a) as supportive scaffold with known stiffness for 3D cell culture; or (b) as supportive matrix for the encapsulation of dissociated cells .…”

“…Importantly, as most studies have used rodent cell‐based assays (Table ), the actual contribution HFSCs for human HF bioengineering needs to be further validated. Although challenging, some researchers are now committed to establish in vitro human post‐natal cell‐based HF germs …”

“…Additionally, two recent studies have achieved growth of mouse hair in vivo after transplantation of in vitro formed structures, either consisting of mouse adult DP and epidermal cells, or of mouse iPSCs, encapsulated in hydrogel matrices. Different artificial 3D microenvironments composed of silk‐gelatin, hyaluronic acid, and collagen were shown to improve hair germs for hair regenerative medicine. In a similar approach, human HF resembling vellus hair were recreated in vitro from a mixture of DP cells, keratinocytes and melanocytes in a collagen matrix, and named “microfollicles.” Presently, researchers are joining efforts to develop more complex structures that can mimic the DP tridimensional morphology while providing a spatiotemporal delivery of molecular cues needed for human hair morphogenesis.…”

“…35,40 Also, distinct players in this signaling crosstalk (including Wnt, BMP and FGF) were reported to prolong the hair-inducing ability of cultured DPCs. 38,39,[41][42][43] More recently, pharmacological modulation of JAK-STAT signaling pathway was shown to improve cultured human DPCs' inductivity. 44 Another strategy to reproduce native DP trichogenicity is the establishment of three-dimensional (3D) sphere cultures, which has been pointed out as one of the most effective means to restore intact anagenic DP transcriptional signature.…”