Hair Regeneration Methods Using Cells Derived from Human Hair Follicles and Challenges to Overcome

Abstract

The hair follicle is a complex of mesenchymal and epithelial cells acquiring different properties and characteristics responsible for fulfilling its inductive and regenerative role. The epidermal and dermal crosstalk induces morphogenesis and maintains hair follicle cycling properties. The hair follicle is enriched with pluripotent stem cells, where dermal papilla (DP) cells and dermal sheath (DS) cells constitute the dermal compartment and the epithelial stem cells existing in the bulge region exert their regenerative role by mediating the epithelial-mesenchymal interaction (EMI). Many studies have developed and focused on various methods to optimize the EMI through in vivo and in vitro approaches for hair regeneration. The culturing of human hair mesenchymal cells resulted in the loss of trichogenicity and inductive properties of DP cells, limiting their potential application in de novo hair follicle generation in vivo. Epithelial stem cells derived from human hair follicles are challenging to isolate and culture, making it difficult to obtain enough cells for hair regeneration purposes. Mesenchymal stem cells and epithelial stem cells derived from human hair follicles lose their ability to form hair follicles during culture, limiting the study of hair follicle formation in vivo. Therefore, many attempts and methods have been developed to overcome these limitations. Here, we review the possible and necessary cell methods and techniques used for human hair follicle regeneration and the restoration of hair follicle cell inductivity in culture.

Keywords: dermal papilla cells; dermal sheath cells; epithelial stem cells; human hair follicle regeneration.

Figure 1

Morphology and markers of cells isolated and cultured from human hair follicles. These are the results of staining with various markers after isolating and culturing cells from human hair follicles. The image shows dermal papilla (DP) cells after two weeks of culture, expressing a-SMA, versican, and ALP in cultured DP cells (passage 1). Outer root sheath (ORS) cells were isolated and cultured from the ORS derived from the epithelium, and the image shows the cells on day 10. Keratin 1–3 were strongly expressed, while keratin 17 and 19 were not expressed. Additionally, sebocytes were isolated and cultured from human sebaceous glands (SG), with the image showing the cells on day 10. These sebocytes strongly expressed keratin 17 and keratin 19, and lipid secretion was observed through Oil Red O and Nile Red staining, adapted with permission from Ref. [80].

Figure 2

Method to restore the trichogenicity of cultured human dermal papilla (DP) cells or epithelial stem cells (Epi stem cells). Cultured human epithelial stem cells (Epi stem cells) are induced into iPSCs, mixed with mouse dermal cells, and utilized in hair regeneration assays to promote hair follicle formation. Cultured dermal papilla cells (DP cells) recover their hair follicle regenerative ability through iPSC induction, 3D cultures, or treatment with small molecules, inhibitors, or exosomes. These treated DP cells are then combined with mouse epidermal cells and employed in hair regeneration assays to induce hair follicle formation.

Figure 3

Currently reported methods for hair regeneration using cultured cells. This describes the models used to evaluate hair regeneration with isolated cells reported to date. Patch Assay: In this model, epidermal and dermal cells are mixed and injected subcutaneously into a nude mouse. Hair follicle formation can be observed two weeks later. Hair follicles do not form when using mouse dermal or epidermal cells alone, but mixing both types of cells results in hair follicle formation after two weeks. Chamber Assay: A chamber is inserted into a cut in the skin of a nude mouse, and a mixture of mouse dermal and epidermal cells is added to the chamber. Two weeks later, the chamber is removed, revealing a layer formed by the two cell types. By four weeks, hair follicle formation can be observed. Sandwich Assay: Dermal cells are placed between the epidermis and dermis of the footpad or sole skin, and the tissue is xenografted into another mouse. Hair follicle formation potential is then evaluated. Hair Germ Assay: Dermal and epidermal cells are mixed into a collagen gel drop, which is then injected into a nude mouse. Hair follicle formation is observed. Bearding Skin Organoid: Human pluripotent stem cells (hPSCs) are induced into iPSCs and treated with various chemicals to induce hair follicles in vitro.