Hypoxia, human hair follicle stem cells’ niche and alopecia

Ponente: Dr. Bruno A. Bernard

All hair follicles undergo a succession of different phases including a growth phase (anagen), a degradation phase (catagen), a resting phase (telogen) and a neomorphogenetic phase (neogen).

It was recently proposed that the hair follicle was a bistable organ, transiting from active steady state to dormant state under a stochastic way (B.A.Bernard, Exp.Dermatol.2012, 21:401-3). Of particular interest is the telogen phase, which includes a latency period called kenogen (A.Rebora & M.Guarrera, Dermatol. 2002, 205:108-10) during which the follicle remains empty after the hair shaft has been lost. Kenogen follicles thus appear as a potential reservoir for increased hair density. Neogen phase is also a key phase, during which all hair follicle compartments are regenerated from local reservoirs of stem cells. Both functional and immunohistological studies (CD29, K19) suggested the existence of at least two pools of pluripotent cells, one located in the upper ORS, the other one in the lower ORS of human hair follicle (S.Commo et al, Differentiation 2000, 66:157-64). The upper and lower reservoirs of K19+ cells are not identical, however, and can be distinguished by their differential expression of stem-cell associated markers K15 and CD200, more highly expressed in the upper reservoir, and CD271, CD29 and CD34 which appear more specific for the lower reservoir. This differential epitope mapping was not restricted to the ORS as the connective tissue sheath (CTS) in conjunction with these two reservoirs also harbors distinct compartments identified by differential chondroitin- and heparin-sulfate epitopes (S.Malgouries et al., Br.J.Dermatol. 2008, 158: 234-42).

Considering the essential role of the stem cell niche in stem cell maintenance and function, we further studied micro-environmental factors that may influence human hair follicle stem cell reservoirs. Two biological markers expressed by the putative stem cell reservoirs, namely K19 and CD271, have been described as being hypoxia-induced genes. Considering that hypoxia appears to be a feature of both embryonic and adult stem cell niches  we decided to further study the expression of two highly sensitive markers of hypoxia, namely carbonic anhydrase IX (CA IX) and glucose transporter 1 (GLUT1). ACIX and GLUT-1 expression is restricted to the lower ORS of anagen human hair follicle (B.A.Bernard et al., Ann. Dermatol.Venereol. 2013, 140:5-8; M.Rathman-Josserand et al., J.Invest.Dermatol. 2013, in press). Furthermore, double immune-fluorescence staining demonstrated that a subset of ACIX+ cells is indeed positive for K19  as well as CD34. This particular finding is of interest given that CD34hi cells were recently found to be depleted in bald scalps (L.A.Garza et al., J.Clin.Invest. 2011, 121:613-622). These results also highlight the fact that the upper CD200+ and lower CD34+ stem cell reservoirs of human hair follicle are subjected to very different microenvironments with only the latter bathing in a hypoxic environment. Altogether, we believe that the human hair follicle harbors two epithelial stem cell compartments that share a certain number of biological markers but that can be also distinguished by the differential expression of certain proteins on the one hand, and their niche-microenvironment on the other hand. Although the respective role of these two reservoirs has not been established in humans, the fact that the pool of CD34+ cells decreases in androgenetic alopecia (L.A.Garza et al., J.Clin.Invest. 2011, 121:613-622) strongly suggests that the lower reservoir is somehow involved in the control of hair cycle, and more specifically in hair regrowth, according to the hair follicle predetermination model (A.A.Panteleyev et al., J.Cell Sci. 2001, 114:3419-3431). Alternatively, the lower reservoir could represent a “primitive” stem cell population able to give rise to all hair follicle epithelial cells (V.Jaks et al., Nat.Genet. 2008, 40:1291-1299) and/or act as a signaling center in the telogen follicle stem cell niche, imposing a prolonged cross-talk between hair follicle stem cells and DP (Y.C.Hsu et al., Cell 2011, 144:92-105) and thus facilitating the neogen phase to proceed in an optimized fashion. Protecting this lower reservoir by maintaining the hypoxic signaling might thus appear as a new approach to sustain hair growth and cycling.

To investigate the potential impact of hypoxia on hair follicle stem and early progenitor cell populations, we measured the colony forming efficiency (CFE) of keratinocytes derived from the Outer Root Sheath (ORS) of human follicles, cultured under normoxic or hypoxic conditions. Although the absolute number of clones was not statistically different for the two conditions, cell density and differentiation were reduced under hypoxic culturing conditions, suggesting maintenance of cell immaturity. Based on these results, we hypothesized that induction of hypoxia signaling may be important in maintaining hair follicle stem cell functionality. Hypoxia signaling is mediated by the hypoxia-inducible transcription factor HIF1, the  subunit of which is degraded in an oxygen-dependent manner through prolyl-4-hydroxylase (P4H)-mediated hydroxylation. A potent P4H competitive inhibitor, named Stemoxydine, was tested for its ability to induce hypoxia-like signaling. Transcriptomic studies show that in vitro treatment of hair follicles with Stemoxydine in normoxic conditions modifies the expression of a panel of genes in the same manner as culturing under hypoxic conditions. In addition, when ORS-derived hair follicle cells were grown in normoxic conditions with Stemoxydine, CFE and clone morphology were similar to those observed in hypoxic conditions. These results suggested that hypoxia may be an important regulator of stem/progenitor cells function in the human hair follicle. We hypothesized that molecules that mimicked hypoxic signaling, such as Stemoxydine®, may figure as new approach to sustain hair growth and cycling. Dr.P.Reygagne (Centre Sabouraud, Saint-Louis hospital, Paris, France) performed double blind clinical studies versus placebo which indeed demonstrated that a 5% Stemoxydine® treatment for 3 months resulted in a significant increase of visible hair density (+4% vs placebo).