The long-term goal of the study is to generate human hair follicle (HF) germ-like structures in vitro. In this part of the work, we examined aggregates assembled using dermal papilla cells (DPCs) and keratinocytes (KCs) suspensions (mixed aggregates) and DPCs spheroids covered by KCs (coated aggregates) cultured in hanging drop model. RT qPCR analysis revealed upregulation of HF markers (Lef1, P cadherin, EdaR, CK75 and Wnt5a) in mixed aggregates in contradiction to coated ones. Lef1 and P cadherin positive KCs were identified at DPCs and KCs borderline. Any morphogenetic events had not been observed in coated aggregates. Downregulation of DPCs hair inducing abilities trough the passaging decreased the size of mixed aggregates and the number of proliferative cells within. Accordingly, DPCs and KCs interplay is essential for proper self-assembly initiation while DPCs hair inducing abilities controls the amount of cells involved in this process.
Mixed aggregates structure and morphology had been investigated. Versican positive DPCs formed round-shaped structures; KCs generated multilayered trabeculae embedded into DPCs spheroids. CK5 and CK6 positive KCs were found in an external layer of trabeculae, surrounding the epithelial compartment and adjacent to DPCs. We did not found any markers of epidermal differentiation including loricrin, CK 1, and CK10. DPCs and KCs were positive for CD44, hyaluronic acid (HA) receptor. We showed that addition of 0.5 mg/ml HA to cell suspension prior to aggregate formation increased the size of aggregates and the number of proliferative cells. However HA did not maintain DPCs specific markers during 2D cultivation. Therefore HA influences size of aggregates not by upregulation of DPCs hair inducing abilities. CD44 staining detected microvillus-type plasma membrane protrusions on the DPCs in mixed aggregates. Microvilli have been shown to be involved in HA metabolism: synthesis and digestion. Accordingly, the results demonstrated the considerable impact of HA in aggregate formation, self-organization and HF development.

Morphogenetic Events in Hair Follicle Heterotypic Spheroids