Mingxing Lei

Therapeutic strategy for hair regeneration: hair cycle activation, niche environment modulation, wound-induced follicle neogenesis, and stem cell engineering.

Introduction: There are major new advancements in the fields of stem cell biology, developmental biology, regenerative hair cycling, and tissue engineering. The time is ripe to integrate, translate, and apply these findings to tissue engineering and regenerative medicine. Readers will learn about new progress in cellular and molecular aspects of hair follicle development, regeneration, and potential therapeutic opportunities these advances may offer. Areas covered: Here, we use hair follicle formation to illustrate this progress and to identify targets for potential strategies in therapeutics. Hair regeneration is discussed in four different categories: i) Intra-follicle regeneration (or renewal) is the basic production of hair fibers from hair stem cells and dermal papillae in existing follicles. ii) Chimeric follicles via epithelial–mesenchymal recombination to identify stem cells and signaling centers. iii) Extra-follicular factors including local dermal and systemic factors can modulate the regenerative behavior of hair follicles, and may be relatively easy therapeutic targets. iv) Follicular neogenesis means the de novo formation of new follicles. In addition, scientists are working to engineer hair follicles, which require hair-forming competent epidermal cells and hair-inducing dermal cells. Expert opinion: Ideally self-organizing processes similar to those occurring during embryonic development should be elicited with some help from biomaterials.

Modulating hair follicle size with Wnt10b/DKK1 during hair regeneration.

Hair follicles have characteristic sizes corresponding to their cycle‐specific stage. However, how the anagen hair follicle specifies its size remains elusive. Here, we showed that in response to prolonged ectopic Wnt10b‐mediated β‐catenin activation, regenerating anagen hair follicles grew larger in size. In particular, the hair bulb, dermal papilla and hair shaft became enlarged, while the formation of different hair types (Guard, Awl, Auchene and Zigzag) was unaffected. Interestingly, we found that the effect of exogenous WNT10b was mainly on Zigzag and less on the other kinds of hairs. We observed dramatically enhanced proliferation within the matrix, DP and hair shaft of the enlarged AdWnt10b‐treated hair follicles compared with those of normal hair follicles at P98. Furthermore, expression of CD34, a specific hair stem cell marker, was increased in its number to the bulge region after AdWnt10b treatment. Ectopic expression of CD34 throughout the ORS region was also observed. Many CD34‐positive hair stem cells were actively proliferating in AdWnt10b‐induced hair follicles. Importantly, subsequent co‐treatment with the Wnt inhibitor, DKK1, reduced hair follicle enlargement and decreased proliferation and ectopic localization of hair stem cells. Moreover, injection of DKK1 during early anagen significantly reduced the width of prospective hairs. Together, these findings strongly suggest that Wnt10b/DKK1 can modulate hair follicle size during hair regeneration.

Tuning Wnt Signals for More or Fewer Hairs

Activation of β-catenin was shown to be of central importance for hair development and cycling. Recent progress brought more understanding to how Wnt signaling is regulated during hair follicle generation and regeneration, telogen-anagen reentry, and extra-follicular macro-environmental modulation. This new understanding presents multiple possibilities to fine tune Wnt signaling for desired hair growth.

Gsdma3 is a new factor needed for TNF-α-mediated apoptosis signal pathway in mouse skin keratinocytes

Gsdma3, a newly found gene, is expressed restrictedly in mouse skin keratinocytes and gastrointestinal tract. But until now, there is little information on the regulation and the function of Gsdma3 in skin keratinocytes. In our previous study, we found that Gsdma3 mutation resulted in a decrease in catagen-associated apoptosis of hair follicle keratinocytes. Apoptosis of skin keratinocytes is strictly regulated by a series of signal pathways, among of which, tumor necrosis factor (TNF)-α-induced signal pathway has been extensively studied. To further investigate the role and the pathway of Gsdma3 involved in skin keratinocyte apoptosis, using immunofluorescence, RT-PCR, western blot and TUNEL analysis, we showed here that accompanying TNF-α-induced apoptosis and Caspase-3 expression in mouse skin keratinocytes in vivo and in vitro, Gsdma3 expression was significantly upregulated. After Gsdma3 gene mutation, TNF-α-induced apoptosis and Caspase-3 expression in skin keratinocytes were reduced. The injection of Gsdma3 expression plasmid could directly enhance the apoptosis and Caspase-3 expression in skin keratinocytes. These results, taken together, indicated that in mouse skin keratinocytes, Gsdma3 expression could be regulated by TNF-α. Gsdma3 was not only involved in but also necessary for the TNF-α-induced apoptosis pathway by directly enhancing the Caspase3 expression as well as the apoptosis induction.

Gsdma3 gene is needed for the induction of apoptosis-driven catagen during mouse hair follicle cycle

Gsdm is a newly found gene family, which is restricted in its expression to the gastrointestinal tract and the skin epithelium. As a main member of the Gsdma subfamily, Gsdma3 is expressed specifically in the hair follicle of mouse skin, but its function remains largely unclear. By hematoxylin and eosin staining, we showed that Gsdma3 gene mutation caused an abnormal catagen phase with unshortened length and unshrunk structure of the hair follicle, in which the development of catagen phase was inhibited. TUNEL staining further revealed that the apoptosis of the hair follicle was obviously decreased in mutant mice. Caspase-3 downregulation was also detected by immunofluorescence, Western blot and RT-PCR in the hair follicle of the mutant mice. After intradermal injection of Gsdma3 gene expression plasmid, apoptosis as well as Caspase-3 expression in the hair follicle of mutant mice was enhanced, and so the catagen retardation of Gsdma3-mutant mice was rescued. Our results confirmed that Gsdma3 gene mutation interfered with catagen formation during mouse hair follicle cycle and, by upregulation of Caspase-3 expression and promotion of apoptosis, Gsdma3 gene could play an essential role in normal catagen induction.

Aging, alopecia, and stem cells

Intrinsic epigenetic status and extrinsic environmental factors affect hair follicle stem cells [Also see Research Article by Matsumura et al. and Report by Wang et al.] Many tissues turn over during adult life, and declines in this process are associated with the progression of aging. Whether renewal is continual (as in the intestinal villi) or episodic (as in hair follicles), it is mainly attributed to somatic stem cells. One fundamental question is whether a decline of tissue renewal reflects the lifelong accumulation of external insults or the internal progression of a clock within stem cells? On pages 613 and 575 of this issue, Wang et al. (1) and Matsumura et al. (2), respectively, gain insight into this phenomenon by examining hair follicle growth and aging.

Self-organization process in newborn skin organoid formation inspires strategy to restore hair regeneration of adult cells

Significance This study opens avenues to improve the ability of adult skin cells to form a fully functional skin, with clinical applications. Our investigation elucidates a relay of molecular events and biophysical processes at the core of the self-organization process during tissue morphogenesis. Molecules key to the multistage morphological transition are identified and can be added or inhibited to restore the stalled process in adult cells. The principles uncovered here are likely to function in other organ systems and will inspire us to view organoid morphogenesis, embryogenesis, and regeneration differently. The application of these findings will enable rescue of robust hair formation in adult skin cells, thus eventually helping patients in the context of regenerative medicine. Organoids made from dissociated progenitor cells undergo tissue-like organization. This in vitro self-organization process is not identical to embryonic organ formation, but it achieves a similar phenotype in vivo. This implies genetic codes do not specify morphology directly; instead, complex tissue architectures may be achieved through several intermediate layers of cross talk between genetic information and biophysical processes. Here we use newborn and adult skin organoids for analyses. Dissociated cells from newborn mouse skin form hair primordia-bearing organoids that grow hairs robustly in vivo after transplantation to nude mice. Detailed time-lapse imaging of 3D cultures revealed unexpected morphological transitions between six distinct phases: dissociated cells, cell aggregates, polarized cysts, cyst coalescence, planar skin, and hair-bearing skin. Transcriptome profiling reveals the sequential expression of adhesion molecules, growth factors, Wnts, and matrix metalloproteinases (MMPs). Functional perturbations at different times discern their roles in regulating the switch from one phase to another. In contrast, adult cells form small aggregates, but then development stalls in vitro. Comparative transcriptome analyses suggest suppressing epidermal differentiation in adult cells is critical. These results inspire a strategy that can restore morphological transitions and rescue the hair-forming ability of adult organoids: (i) continuous PKC inhibition and (ii) timely supply of growth factors (IGF, VEGF), Wnts, and MMPs. This comprehensive study demonstrates that alternating molecular events and physical processes are in action during organoid morphogenesis and that the self-organizing processes can be restored via environmental reprogramming. This tissue-level phase transition could drive self-organization behavior in organoid morphogenies beyond the skin.

Roles of GasderminA3 in Catagen-Telogen Transition during Hair Cycling

Hair follicles undergo cyclic behavior through regression (catagen), rest (telogen) and regeneration (anagen) during postnatal life. The hair cycle transition is strictly regulated by the autonomous and extrinsic molecular environment. However, whether there is a switch controlling catagen-telogen transition remains largely unknown. Here we show that hair follicles cycle from catagen to the next anagen without transitioning through a morphologically typical telogen after Gsdma3 mutation. This leaves an ESLS (epithelial strand-like structure) during the time period corresponding to telogen phase in WT mice. Molecularly, Wnt10b is upregulated in Gsdma3 mutant mice. Restoration of Gsdma3 expression in AE (alopecia and excoriation) mouse skin rescues hair follicle telogen entry and significantly decreases the Wnt10b-mediated Wnt/β-catenin signaling pathway. Overexpression of Wnt10b inhibits telogen entry by increasing epithelial strand cell proliferation. Subsequently, hair follicles with a Gsdma3 mutation enter the second anagen simultaneously as WT mice. Hair follicles cannot enter the second anagen with ectopic WT Gsdma3 overexpression. A luciferase reporter assay proves Gsdma3 directly suppresses Wnt signaling. Our findings suggest Gsdma3 plays an important role in catagen-telogen transition by balancing the Wnt signaling pathway, and that morphologically typical telogen is not essential for the initiation of a new hair cycle.

Upregulation of interfollicular epidermal and hair infundibulum β-catenin expression in Gsdma3 mutant mice

Skin hyperplasia associated with hair follicle abnormality can be seen in many skin diseases caused by gene mutations. Gsdma3 was reported to be a mutation hotpot gene whose mutation contributed to various skin hyperplasia phenotypes in Bsk, Dfl, Rco2, Fgn, Re (den), and Rim3 mice. However, the signaling molecules involved in these skin anomalies due to Gsdma3 mutations have not yet been addressed. In this study, using hematoxylin and eosin staining, we showed that Gsdma3 mutation gave rise to thickened skin and lengthened hair infundibula throughout the hair follicle cycle. Using immunofluoresence staining, we found that Gsdma3 had a spatial expression profile very similar to that of β-catenin in the epidermis and skin appendages. Furthermore, we showed that epidermal β-catenin expression was increased at all postnatal stages in Gsdma3 mutant mice. These results suggest that Gsdma3 may play a role in the proliferation and differentiation of epidermal cells and hair follicles through negatively regulating β-catenin expression.

Hoxc13 is a crucial regulator of murine hair cycle.

Hair follicles undergo cyclical growth and regression during postnatal life. Hair regression is an apoptosis-driven process strictly controlled by micro- and macro-environmental signals. However, how these signals are controlled remains largely unknown. Hoxc13, a member of the Hox gene family, is reported to play an important role in hair follicle differentiation. In the present study, we observed that Hoxc13 was highly expressed in the outer root sheath, matrix, medulla and inner root sheath of hair follicles in a hair cycle-dependent manner. We therefore investigated the role of Hoxc13 in hair follicle cycling. Injection of ShRNA (ShHoxc13) to suppress Hoxc13 in early anagen promoted premature catagen entry, shown by significantly decreased hair length and hair bulb size, increased percentage of catagen hair follicles, hair cycle score and TUNEL+ cells and inhibited proliferation. In contrast, local injection of recombinant Hoxc13 polypeptide (rhHoxc13) during the late anagen phase prolonged the anagen phase. Additionally, rhHoxc13 injections during the telogen phase significantly promoted hair growth and induced the anagen progression. At the molecular level, the expression of phosphorylated smad2 (p-smad2), a key factor of active TGF-β1 signaling, was up-regulated in the ShHoxc13-treated hair follicles and down-regulated in rhHoxc13-treated hair follicles, suggesting that Hoxc13 might block anagen–catagen transition by inhibiting the TGF-β1 signaling. Taken together, our data strongly suggest that Hoxc13 is a novel and crucial regulator of the hair cycle. This might also provide an understanding of the mechanism of the ‘hair cycle clock’ and the development of alopecia treatments.