Long-Term Culture of Mouse Vibrissal Dermal Papilla Cells andDe NovoHair Follicle Induction

Abstract

We have succeeded in culturing dermal papilla (DP) cells long term and developed new techniques that enhance their hair follicle-inducing efficiency in a patch assay. The outgrowing DP cells from mouse vibrissae were markedly stimulated by 10% fetal bovine serum-Dulbecco's modified essential medium that included fibroblast growth factor-2 (FGF-2). Moreover, the potency of proliferation was maintained during serial cultivations (more than 30 passages). We combined these established DP cells with epidermal cells and implanted them subcutaneously into athymic mice to examine their hair follicle-inducing ability. New hair follicles were induced by dissociated DP cells at earlier passages (under passage 4), but the cells from later passages could not induce follicles. We next aggregated the DP cells to form spheres and then injected them with epidermal cells. Unlike the dissociated DP cells, the spheres made from the later passaged cells (more than 10 passages) did induce new hair follicles. We examined several genes specific for DP of anagen follicles and confirmed that their expression level was elevated in the spheres compared with their expression level in adherent DP cells. These results suggest that FGF-2 is essential for dermal papilla cell culture and that sphere formation partially models the intact DP, resulting in hair follicle induction, even by later passaged cells.

Restoration of the intrinsic properties of human dermal papilla in vitro

“…1A). Real-time PCR analysis of total RNA isolated from freshly isolated human DP (fDP), cultured DP (cDP) cells, and fibroblasts (Fibros) detected upregulation of genes that have been used to confirm successful DP cell isolation (Botchkarev and Kishimoto, 2003;Rendl et al, 2005;Ito et al, 2007;Osada et al, 2007;Driskell et al, 2009;Ohyama et al, 2010), such as alkaline phosphatase (ALPL), noggin (NOG), WNT inhibitory factor 1 (WIF1), and versican (VCAN), in fDP as compared with cDP cells and Fibros, demonstrating successful RNA recovery (Fig. 1B).…”

“…Previous studies reported various approaches to maintaining in vivo characteristics in cultured DP cells, including co-culture with keratinocytes Inamatsu et al, 1998;Kobayashi et al, 2011), cultivation on extracellular matrices, and sphere formation (Osada et al, 2007;Young et al, 2009;Higgins et al, 2010;Kobayashi et al, 2011). The hair inductive capacity of rodent and canine DP cells was maintained in cultures treated with keratinocyte-derived factors (Inamatsu et al, 1998;Kobayashi et al, 2011) and ligands in signaling pathways involved in hair follicle morphogenesis, including Wnt3a and Bmp6 (Kishimoto et al, 2000;Shimizu and Morgan, 2004;Rendl et al, 2008).…”

Hair follicle dermal papilla cells at a glance

“…their ability to induce hair follicles) (Ohyama et al, 2010;Yang and Cotsarelis, 2010;Horne et al, 1986;Kishimoto et al, 2000;Lichti et al, 1993;Rendl et al, 2008). Culture media have been developed that extend the time for which DP cells can be cultured (Limat et al, 1993;Osada et al, 2007;Roh et al, 2004), and activation of Wnt and Bmp signalling pathways in mouse DP cells can delay loss of trichogenicity (Kishimoto et al, 2000;Rendl et al, 2008). Other strategies to preserve the properties of DP cells are to grow them in three-dimensional aggregates (Osada et al, 2007;Higgins et al, 2010) or to culture them together with keratinocytes on extracellular matrix substrates in order to mimic the in vivo microenvironment (Havlickova et al, 2009).…”

Rapid Genetic Analysis of Epithelial-Mesenchymal Signaling During Hair Regeneration

“…On the other hand, a culture system that can preserve DP cell function is very useful as DP potency is gradually lost once the primary dermal cells are passaged. Potential culture systems include the use of biomaterials derived from artificial niche or aggregation cultures 2,7,8,13 . Another improvement will be to generate a reliable epidermal cell freezing method with a high cell recovery rate as this will reduce the use of the second set of mouse pups (step 7) to generate fresh keratinocytes in dermal-specific loss-or gain-of-function studies.…”