Bulge-Derived Epithelial Cells Isolated from Human Hair Follicles Using Enzymatic Digestion or Explants Result in Comparable Tissue-Engineered Skin

Abstract

Hair follicle stem cells, located in the bulge region of the outer root sheath, are multipotent epithelial stem cells capable of differentiating into epidermal, sebaceous gland, and hair shaft cells. Efficient culturing of these cells is crucial for advancements in dermatology, regenerative medicine, and skin model development. This investigation aimed to develop a protocol for isolating enriched bulge-derived epithelial cells from scalp specimens to produce tissue-engineered substitutes. The epithelium, including hair follicles, was separated from the dermis using thermolysin, followed by microdissection of the bulge region. Epithelial stem cells were isolated using enzymatic dissociation to create a single-cell suspension and compared with the direct explant culture and a benchmark method which isolates cells from the epidermis and pilosebaceous units. After 8 days of culture, the enzymatic digestion of microdissected bulges yielded 5.3 times more epithelial cells compared to explant cultures and proliferated faster than the benchmark method. Cells cultured from all methods exhibited comparable morphology and growth rates. The fully stratified epidermis of tissue-engineered skin was similar, indicating comparable differentiation potential. This enzymatic digestion method improved early-stage cell recovery and expansion while maintaining keratinocyte functionality, offering an efficient hair bulge cell-extraction technique for tissue engineering and regenerative medicine applications.

Keywords: cell culture technique; hair follicle; primary cell culture; regenerative medicine; stem cells; tissue-engineered skin.

Figure 1

Schematic representation of human skin and the pilosebaceous unit. The multilayered structure of the epidermis, dermis, and adipose tissue, along with the hair follicle and associated sebaceous gland, are illustrated. The epidermis is stratified into the basal, spinous, granular, and cornified layers. The hair follicle is divided into distinct anatomical regions: the infundibulum, isthmus, bulge, and bulb. The bulge region, known to harbor epithelial stem cells, is located at the insertion point of the arrector pili muscle. The sebaceous gland, connected to the hair follicle, plays a role in sebum production. The dermal papilla, situated at the base of the follicle within the bulb, is essential for hair follicle regeneration.

Figure 2

Flowchart of methodological approaches used to obtain epithelial cell populations from human scalp tissue. Scalp tissue was cut into small pieces and incubated overnight with thermolysin. After separating the epithelium from the dermis, epithelial cells from the epidermis and hair follicles were either extracted using the two-step thermolysin and trypsin keratinocyte-isolation procedure that served as the benchmark method (A) or microdissected to enrich for bulge-derived epithelial cells (B). After microdissection, bulge-derived cells were isolated using two different protocols; (B1) bulges were digested with trypsin to obtain a single-cell suspension, or (B2) bulges were cultivated as explants. For all conditions, cells were cultured on a human feeder layer (HFL) in keratinocyte medium. Created in BioRender. De koninck, H. (2025) https://BioRender.com/c48t805 (accessed on 29 December 2024).

Figure 3

Effect of the isolation method on the number of cells and colony-forming efficiency after the extraction and in primary culture. (A) Phase-contrast microscopy of epithelial cells derived from trypsin-digested bulges (A1), explanted bulges (A2), or trypsin-digested cells extracted from scalp (epidermis and hair follicles) (A3) after 6 days of culture. Pictures show cells from the same donor. (B) Number of bulge-derived epithelial cells obtained according to the isolation method used at the extraction step (B1) and extrapolated for 8 days of culture (B2). (C) Comparison of cells isolated by digestion from the bulge or from the scalp. Population doubling time of epithelial cells derived from digested bulge and from scalp after primary culture (C1) and number of epithelial cells per cm² extrapolated for 8 days of culture (C2). Colony-forming efficiency (CFE) after the primary culture. (C3). Scale bar = 1 cm (A,C3). Each point represents a different donor (N). Detailed information about the donors, identified by their corresponding symbols, is provided in the table in the Section 4.1. The quantification of CFE (%) was performed in at least three technical replicates (n). Data are presented as mean ± standard error of the mean (SEM). Statistical analysis: bilateral t-test (B2,C1,C3) or Wilcoxon test (C2) for paired samples. (*) p-value < 0.05, (**) p-value < 0.01.

Figure 4

Effect of the extraction method on the cell population doubling time and colony-forming efficiency over cell passages. (A) The population doubling time of epithelial cells derived from digested bulges, explanted bulges, or from scalps (epidermis and hair follicles) after 1 (A1), 2 (A2), and 5 (A3) passages. (B) Comparative colony-forming efficiency (CFE) of cells extracted with the 3 methods after 1 (B1), 2 (B2) and 5 (B3) passages. Each point represents a different donor (N). Detailed information about the donors, identified by their corresponding symbols, is provided in the table in Section 4.1. Data are presented as mean ± standard error of the mean (SEM). Statistical analysis: Friedman test with Dunn’s multiple comparisons (A1) and one-way ANOVA with Tuckey’s multiple comparisons (A2,A3,B1–B3). * p-value < 0.05.

Figure 5

Effect of the extraction method on the production of tissue-engineered skin substitutes (TESs). TESs were produced with scalp fibroblasts and keratinocytes obtained from digested bulges (TES-1, A1,B1,C1,D1), explanted bulges (TES-2, A2,B2,C2,D2), or from scalp (epidermis, hair follicles) (TES-3, A3,B3,C3,D3). (A1–A3) Masson’s Trichrome staining (colors the cells in pink and extracellular matrix in blue). (B1–B3) Immunostaining of Ki67 (red, white arrows). (C1–C3) Immunostaining of keratin 15 (K15, red). (D1–D3) Immunostaining of keratin 19 (red, white arrows). Pictures are representative of TESs made with cells from two different donors (see the table in Section 4.1—⬣,●). Nuclei are stained with Hoechst (blue). Scale bars = 50 µm.

Figure 6

Effect of the extraction method on the dermal–epidermal junction and epidermal differentiation (TES). TESs were produced with scalp fibroblasts and keratinocytes obtained from digested bulges (TES-1, A1,B1,C1), explanted bulges (TES-2, A2,B2,C2), or from scalp (epidermis, hair follicles) (TES-3, A3,B3,C3). (A1–A3) Immunostaining of filaggrin (red). (B1–B3) Immunostaining of collagen IV (red). (C1–C3) Immunostaining of β-catenin (red). Pictures are representative of TESs made with cells from two different donors (see the table in Section 4.1—⬣,●). Nuclei are stained with Hoechst (blue). Scale bars = 50 µm.