These studies support a number of major conclusions. First, our studies identify a Sox2+ endogenous dermal precursor within the follicle DP and DS that, when prospectively isolated, homes back to a hair follicle niche, induces hair follicle morphogenesis, and displays neural and dermal potential. Second, we show that these endogenous precursors generate SKPs when cultured, and that these two cell populations are very similar with regard to their transcriptome and their functional properties when transplanted into the dermis. Third, we demonstrate that SKPs can clonally reconstitute the dermis and induce hair follicle morphogenesis, properties predicted of a dermal stem cell. Fourth, the serial reconstitution experiments show that SKPs maintain their multipotentiality and their ability to self-renew within their hair follicle niche, and that they can serially induce hair follicle formation. Finally, our follicle graft/lineage tracing studies argue that Sox2+ dermal precursors can be recruited out of their niche to contribute differentiated dermal cells. Together, these experiments provide evidence for a hair follicle-associated dermal precursor that functions to regulate hair morphogenesis, and to maintain the intact or injured dermis and that, when cultured, generate SKPs that display all the predicted properties of multipotent dermal stem cells.
On the basis of these findings, we propose that these adult dermal precursors derive from a subset of embryonic mesenchymal precursors within the presumptive dermis that interact with epidermal precursors and undergo a transition to a Sox2+ precursor state, as recently suggested (Driskell et al., 2009
). We also propose that hair follicles provide a niche for maintenance of the mesenchymal precursor phenotype, and that in adult life, these Sox2+ follicle cells both regulate follicle morphogenesis, and serve as a reservoir of dermal precursor activity, contributing dermal cells for the normal and injured dermis. The broader mesenchymal potential displayed by SKPs (Lavoie et al., 2009
) and whisker follicle papillae (Jahoda et al., 2003
; Wojciechowicz et al., 2008
) suggests that they may also serve as precursors for adipocytes and/or blood vessel smooth muscle cells.
That DP mesenchymal cells induce hair follicle morphogenesis is well-established (Jahoda et al., 1984
; Oliver, 1967
). A precursor function for DP cells has also been previously suggested based upon their ability to contribute cells to healing wounds upon transplantation, and to generate non-dermal cell types when cultured (Waters et al., 2007
). The data we present here provide strong evidence that this is indeed the case. Moreover, our finding that both DP and DS cells express Sox2 suggests that these anatomically-distinct cells may in fact represent a common precursor population. In support of this idea, previous studies showed that cells move between the DS and DP (Tobin et al., 2003
), and that lower DS cells can regenerate the DP (McElwee et al., 2003
). We suggest that since DP cells rarely divide, they may represent a quiescent stem cell population, with DS cells functioning as a transit amplifying population that ultimately provides differentiated cell types. Alternatively, DS cells may represent dermal stem cells, and provide cells both for dermal differentiation, and for the DP, where the cells acquire their inductive properties and function to regulate follicle morphogenesis.
One of the surprising findings reported here is the ability of transplanted Sox2:EGFP+ cells and the SKPs they generate to home back to their hair follicle niche. Interestingly, a hint of this activity was previously obtained in experiments transplanting GFP+ DP/DS cells into ear skin (McElwee et al., 2003
). These findings may reflect a previously-undescribed mechanism for recruiting DS cells into the DP and/or for allowing re-establishment of the DS/DP when hair follicles re-enter the hair growth cycle. Clearly, SKPs are attracted to cells that are similar, as seen in patch assays where single dissociated SKP cells aggregated to form structures that nucleated hair follicle formation. While the relevant attractive signal is unknown, evidence that it is cell-intrinsic comes from our data showing that more rat than mouse cells aggregate to form the DP, even within the same tissue environment.
A second surprising finding is that Sox2:EGFP expression was regulated during the hair cycle; DP and DS cells did not express Sox2 during catagen or telogen. While some cells exit the DP during hair follicle regression (Tobin et al., 2003
), many are retained, implying that at least some precursors regulate Sox2 expression in response to their external micro-environment. Since Sox2 is associated with maintenance of a stem cell state (Avilion et al., 2003
) then this may suggest that the niche regulates the “stemness” of these dermal precursors. However, a recent study also showed Sox2:EGFP expression in the DP of developing hair follicles, and provided evidence that the level of expression was associated with follicle type, with small zig zag hairs expressing low or no Sox2:EGFP (Driskell et al., 2009
). Thus, Sox2 expression may be associated with multiple functional properties, and it will be important to determine whether Sox2 is a readout for different functional states and/or whether it actually regulates the properties of these dermal precursors.
One key question in the stem cell field has been whether multipotent precursors like SKPs actually reflect endogenous precursor cells, or whether they are cells that dedifferentiate in culture. Data presented here showing that prospectively-isolated Sox2+ follicle DP and DS cells give rise to SKPs, and that these Sox2+ cells are very similar to SKPs with regard to their transcriptome and functional properties, argue that SKPs accurately reflect this endogenous precursor population. This finding, together with our data showing that SKPs display all of the properties predicted of dermal stem cells, provide strong support for the idea that the Sox2+ precursors we have identified here are endogenous dermal stem cells that serve to induce hair morphogenesis and maintain the dermis.