A Two-Stepped Culture Method for Efficient Production of Trichogenic Keratinocytes

Abstract

Successful hair follicle (HF) neogenesis in adult life depends on the existence of both capable dermal cells and competent epidermal keratinocytes that recapitulate embryonic organogenesis through epithelial-mesenchymal interaction. In tissue engineering, the maintenance of trichogenic potential of adult epidermal cells, while expanding them remains a challenging issue. We found that although HF outer root sheath keratinocytes could be expanded for more than 100 passages as clonogenic cells without losing the proliferative potential with a 3T3J2 fibroblast feeder layer, these keratinocytes were unable to form new HFs when combined with inductive HF dermal papilla (DP) cells. However, when these high-passage keratinocytes were cocultured with HF DP cells for 4 days in vitro, they regained the trichogenic ability to form new HFs after transplantation. We found that the short-term coculture with DP cells enhanced both Wnt/β-catenin signaling, a signaling cascade key to HF development, and upregulated the expression of HF-specific genes, including K6, K16, K17, and K75, in keratinocytes, indicating that these cells were poised toward a HF fate. Hence, efficient production of trichogenic keratinocytes can be obtained by a two-stepped procedure with initial cell expansion with a 3T3J2 fibroblast feeder followed by short-term coculture with DP cells.

FIG. 1.

High-passage keratinocytes maintained proliferative potential, but not hair follicle (HF)-forming capability. (a–f) Appearance of keratinocytes expanded by a 3T3J2 fibroblast feeder layer. The lethally irradiated 3T3J2 fibroblasts stayed around the colonies as supporting cells (yellow arrows). Rat keratinocytes at passage 2(a), passage 8(b), passage 18(c), passage 42(d), passage 51(e), and passage 87(f) (arrowheads indicate single colonies). (g) Magnified view of rat keratinocytes at passage 5. Cells often grew as expanding colonies. Two colonies composed of densely packed keratinocytes are shown here (arrowheads). At the periphery of the colonies were feeder fibroblasts (yellow arrows). (h) Patch assay. Passage 43 keratinocytes were unable to form new HFs when combined with passage 3 dermal papilla (DP) cells in patch assay. Only one amorphous mass underneath the skin of nude mice was revealed. (i) Histology of the mass in (h) demonstrated only calcified and fibrous tissue. No hairs or follicular structures were identified (scale bar: 100 μm). Color images available online at www.liebertpub.com/tec

FIG. 2.

Short-term coculturing of high-passage keratinocytes with DP cells enhanced HF-forming capability. (a) Appearance of passage 36 outer root sheath epidermal cells (keratinocytes) cocultured with DP cells for 4 days. Cocultured keratinocytes grew as colonies composed of tightly packed cells (arrowheads indicate single colony). DP cells stayed at the periphery of epidermal colonies like supporting feeder cells (yellow arrow). The morphology of keratinocytes and colonies remained the same in the coculture system as if they were in the standard 3T3J2 supportive system as in Figure 1a–g. (b) Result of hair regeneration patch assay. The cells in (a) were used in patch assay. Multiple hair fibers (white arrows) were observed in the hypodermis of nude mice. An amorphous mass (black arrow) was the residue of injected cells. (c) Scanning electron microscopic image showed overlapping shingle-like cuticles that are typical of hair shafts. (d) Histology of the induced hair showing multiserial ladder medullation typical of rodent hair. The inset showed a magnified view of the hair shaft. (e), (f) Demonstrating follicular structure in serial sections of patch model. Arrowheads in (e) indicating hair bulb and arrowheads in (f) indicating hair shaft laddering. SC: subcutaneous fat (scale bar: 100 μm). Color images available online at www.liebertpub.com/tec

FIG. 3.

Upregulation of hair differentiation genes of cocultured epidermal cells. Follicular differentiation gene expression for ORS (K6, K16, K17) and companion layer (K75/K6hf) demonstrated a synchronously increasing trend. The nonfollicular epidermal differentiation marker, K1, was also elevated during coculture. The basal keratinocyte marker of K14 was also upregulated. *p<0.05.

FIG. 4.

Enhancement β-catenin expression and Wnt/β-catenin signaling pathway by coculture with DP cells. (a) Quantitative β-catenin expression in keratinocytes during coculture with DP cells. Quantitative real-time polymerase chain reaction showed that expression of β-catenin mRNA progressively increased during coculture with DP cells. Data of fold change from five experiments (mean±SD) were shown. (b) Western blotting of nuclear and cytosolic β-catenin protein in epidermal cells from day 0 and 4 during coculture. Nuclear-specific TBP (TATA binding protein) and cytosolic GADPH were used as controls for nuclear and cytoplasmic fractions, respectively. N, nuclear fraction; C, cytoplasmic fraction. (c) Quantitative analysis from Western blotting in (b). Total β-catenin protein level increased after a 4-day coculture with DP cells. (d) Quantitative analysis of nuclear localization of β-catenin in (b). The nuclear-to-cytoplasmic (N/C) β-catenin ratio was increased after coculture. (e) Confocal image of β-catenin expression in keratinocytes. Colocalization of green (β-catenin) and blue (nucleus) stains was displayed in the bright sky color, indicating nuclear expression of β-catenin. As the coculture period increased, the nuclear localization of β-catenin also increased (scale bar: 20 μm). *p<0.05. Color images available online at www.liebertpub.com/tec

FIG. 5.

Two-step preparation of trichogenic cultured adult keratinocytes. Step 1 (left panel): population expansion by means of traditional feeder supporting cultivation from clonogenic ORS keratinocytes. Step 2 (right panel): reprogramming takes place, while cultured DP cells enable high-passage epidermal cells to adopt follicular fate with successful expression of follicular-specific genes. These educated and reprogrammed epidermal cells show their ability of folliculogenesis in hair regeneration models. Color images available online at www.liebertpub.com/tec