(W1030-05-25) CD133-Positive Dermal Papilla Cells are a Major Driver of Hair Follicle Neogenesis

Purpose: Generating normal appendages in skin equivalents for skin loss injuries have long been major clinical challenges to dermatologists and surgeons. Engineered skin substitutes (ESS) are examples of regenerative medicine that have been successfully used for wound closure for burns, burn scars, and chronic wounds. Nevertheless, an ESS only fulfills basic skin functions because it fails to produce hair follicles (HFs) and other adnexa, such as sweat glands. Therefore, cell-based bioengineering of HFs is emerging as a promising strategy to overcome the hurdles in skin tissue engineering. A major contributing factor to the failure of cell-based HF regeneration is the lack of a sufficient number of highly specialized and inductive mesenchymal fibroblasts, which are located in a unique niche at the base of the hair follicle, called the dermal papilla (DP). Due to DP heterogeneity, how DP fibroblasts drive epidermal keratinocytes to construct hair follicles remains unknown. The objectives of our study are to systematically characterize the specific hair-inducing DP fibroblast population during early hair growth stage and assess their ability to promote hair follicle formation from human keratinocytes in ESS.

Methods: We and other groups characterized a unique DP fibroblast subpopulation that can be identified by the cell surface marker CD133. Thus, we have developed a double fluorescent transgenic mouse strain, from which we are able to isolate CD133+ DP fibroblasts and CD133- DP fibroblasts from mouse anagen hair follicles for systematic comparison in in vitro and in vivo models. Cellular properties and DP characteristics of isolated CD133+ and CD133- DP fibroblasts were assessed and compared using both in vitro 2D, 3D spheroid, and hydrogel culture. Skin reconstitution assay was used to determine whether CD133+ and CD133- DP fibroblasts still possess the ability to initiate hair follicle neogenesis in vivo.

Results: In two-dimensional (2D) culture, both DP populations gradually lost the expression of the DP marker versican. After transferring the cells from 2D culture to 3D spheroid culture, versican expression was restored in both CD133+ and CD133- DP fibroblasts. While CD133+ DP continued to maintain CD133 expression, CD133- DP fibroblasts did not gain CD133+ expression. CD133+ DP spheroids appeared tighter and showed stronger AP staining- a well-recognized marker for hair follicle inductivity- than CD133- DP spheroids. Furthermore, CD133+ DP fibroblasts express a higher level of DP signature gene than CD133- DP fibroblasts. In addition, CD133+ DP fibroblasts have a strong ability to form 3D aggregates in hydrogel. Together, the data show that CD133+ DP fibroblasts possess increased DP characteristics compared to CD133- DP fibroblasts. To characterize the hair inductivity of CD133+ and CD133- DP fibroblasts, we performed skin reconstitution assays. Interestingly, mice grafted with CD133+ DP fibroblasts had dark hairs growing in healed wounds 35 days after grafting. Reconstituted skin with CD133- DP fibroblasts showed almost no newly formed hairs in healed wounds. Histological analysis confirmed the formation of ample hair follicle structures in healed wound inoculated with CD133+ DP fibroblasts.

Conclusion: In summary, our findings demonstrate that CD133+ DP fibroblasts are the driver of hair follicle neogenesis and could potentially be used to reprogram the dermal niche in human ESS and induce hair follicle morphogenesis.

References: 1. Zhou L, Xu M, Yang Y, Yang K, Wickett RR, Andl T, et al. Activation of beta-Catenin Signaling in CD133-Positive Dermal Papilla Cells Drives Postnatal Hair Growth. PLoS One 2016;11:e0160425
2. Zhou L, Yang K, Carpenter A, Lang RA, Andl T, Zhang Y. CD133-positive dermal papilla-derived Wnt ligands regulate postnatal hair growth. Biochem J 2016;473:3291-305
3. Zhou L, Yang K, Xu M, Andl T, Millar SE, Boyce S, et al. Activating beta-catenin signaling in CD133-positive dermal papilla cells increases hair inductivity. FEBS J 2016;283:2823-35

Acknowledgments: The Institutional Animal Care and Use Committee of the University of Cincinnati approved all experimental procedures involving mice. The study does not include human subjects.

Double labeling of DP fibroblasts to distinguish between CD133+ and CD133- cells in postnatal murine hair follicles in versican-GFP; CD133-CreERT2; CAG-tdTomato mouse strain.



3D culture of versican+; CD133+ and versican+; CD133- DP fibroblasts in spheroids.



CD133+ DP fibroblasts induce de novo hair follicle formation in reconstituted skin.