International Investigative Dermatology (iid) 2018 Meeting Abstracts

[InBeforeTheCure]

JID is holding a meeting in Orlando from May 16-19. You can find abstracts for this here.

Here are a bunch of A.G.A/hair biology-related abstracts.

Causes of Androgenetic Alopecia

1381 Molecular mechanisms of donor dominance in androgenetic alopecia
Y. Doucet, J. Chen, H. Erbil Abaci, C. Jahoda, A. Christiano

Androgenetic alopecia (Androgenetic Alopecia) is a complex genetic trait that is characterized by regional hair follicle miniaturization in response to androgens. While female-pattern hair loss is characterized by a diffuse thinning of the scalp, male pattern can be induced upon elevation of testosterone levels. What confers regional susceptibility vs. refractivity on different regions of the scalp is unknown. Donor Dominance refers to the phenomenon by which hair follicles retain the characteristics of the donor site when transplanted to a recipient site. This property forms the basis for the success of hair transplantation. Since the calvarium begins to develop shortly prior to hair follicle induction, this suggests that the craniofacial dermis epigenetically/differentially influences hair follicle patterning and development. We noticed that the hair pattern in Androgenetic Alopecia overlaps precisely with the demarcations of scalp dermis/underlying bones, which have a dual origin (neuroectoderm for parietal bone), vs. mesoderm (for occipital bone). Computational analyses of RNA seq from parietal and occipital scalp of matching control and Androgenetic Alopecia affected volunteers revealed a striking differential gene expression profile along the cranial-caudal axis defining two distinct biosignatures that reflect: 1) the developmental origins of the skin and 2) the susceptibility to develop Androgenetic Alopecia. Functional annotation of the differentially expressed genes shows enriched pathways in Androgenetic Alopecia samples, including genes implicated in cartilage-ECM interaction (ADAMTS4), in immunity (CD300c, FCGR1A), and epigenetic factors. Using the ARACNe algorithm, we identified transcription factors or master regulators (MRs) that govern the molecular mechanisms of Androgenetic Alopecia. This list of MRs was used to perform functional studies. Altogether, we present novel insights into the genetic, epigenetic, and developmental factors required for temporal specification of the skin and the interdependence of hair follicle, skull and craniofacial development.

Human Hair Growth/Treatments

411 The efficacy of 1550-nm erbium-glass fractional laser treatment and its effect on the expression of insulin-like growth factor 1 and Wnt/β-catenin in androgenetic alopecia
J. Meephansan, U. Sabpayasarn, P. Phadungsaksawasdi, N. Ungpraphakorn

A new and effective treatment for androgenetic alopecia (Androgenetic Alopecia) is 1550-nm erbium-glass (Er:Glass) fractional laser treatment. The wound healing process associated with this treatment is believed to be due to the stimulation of hair regrowth, but the mechanism of healing is still unclear. Both the Wingless-related integration site (Wnt) proteins and insulin-like growth factor 1 (IGF-1) are important molecules that promote new hair growth. The aim of this study was to evaluate the efficacy of 1550-nm Er:Glass fractional laser treatment and determine the gene expression of IGF-1 and Wnt/β-catenin in patients with Androgenetic Alopecia. Twenty-three male and female patients with Androgenetic Alopecia Hamilton-Norwood stages III-IV (including type III vertex) or FPHL Ludwig types I-II were enrolled. They received 12 1550-nm Er:Glass fractional laser treatments at 2-week intervals. A scalp biopsy was performed on each patient at baseline and 24 hours after the 3rd laser treatment to evaluate mRNA levels of Wnt10A and IGF-1. Global and target photographs were collected monthly. Histopathological samples were collected at baseline, and during the 1st, 2nd, and 3rd monthsfollowing laser treatment. All adverse effects were reported during the study. Significant increases in hair count and shaft diameter that occurred from month 4 until the end of the study were observed (p=0.001). Histological results showed increases in the follicular unit, anagen hair count, and the anagen:telogen ratio.No up-regulation of Wnt10A and IGF-1 mRNA was observed. We concluded that 1550-nm Er:Glass fractional laser treatment can increase hair density and shaft diameter in patients with Androgenetic Alopecia. The mechanisms by which 1550-nm Er:Glass laser treatment induces new hair growth may not be limited to Wnt10A/β-catenin or IGF-1 expression.

573 A review of the use of platelet rich plasma for the treatment of androgenic alopecia
C. Stamatiou, S. Daunert, J. Jimenez

Androgenic Alopecia (Androgenetic Alopecia) commonly affects both men and women. Men are afflicted at rates as high as 80%, while 13% of premenopausal and close to 50% of postmenopausal women also demonstrate this form of hair loss. Available treatments include Minoxidil, a vasodilator, Finasteride, an androgen steroid hormone analogue, and hair transplantation. Minoxidil can cause headaches and weight gain and Finasteride can cause sexual dysfunction. Hair transplantation, although effective, has a very high cost and thus, is not a highly accessible treatment. A therapy currently being used in clinics is Platelet Rich Plasma (PRP), for the stimulation of hair growth. PRP is rich in growth factors, which are believed to help facilitate hair follicle restoration. PRP has shown to have minimal to no side effects. This review explores recently published studies on the use of PRP as a treatment for Androgenetic Alopecia. A literature search was conducted on the electronic databases: Pubmed, Google Scholar, and TRIP to identify novel studies on the effectiveness of PRP on Androgenetic Alopecia. We excluded articles before 2015 and those with other forms of Alopecia. The reviewed papers include a total number of 180 participants, 115 men and 65 women, and measured the effects of PRP by accounting for; (i) total number of hairs, (ii) the number of hairs in the anagen or telogen phases, (iii) hair thickness, (iv) hair density, (v) hair root strength, and (vi) post-treatment comparative before and after photos to measure the stage of Androgenetic Alopecia based on Eblings classification system. The results of these studies varied, 2 of 6 having no improvement, and 4 of 6 having statistically significant improvement (p<0.5). The varying results of each study and inconsistent methods indicates the need for more controlled studies with a larger number of participants. PRP is a promising treatment for Androgenetic Alopecia, but further experiments must be conducted to establish a standard for PRP preparation, mode of application, and frequency of treatment.

822 The proteomic profiling of hair anchorage from hair plucks across the human hair cycle
L.A. Elenich, P. Dwivedi, T. Chaudhary, J. Winget, B. Fisher, M. Davis

To identify cycle-specific protein biomarkers from hair plucks, we performed a global open-ended scanning experiment of the proteomic landscape from three stages of the hair growth cycle (anagen, telogen and exogen) using hair plucks from healthy, untreated subjects. Results from label-free quantitation (LFQ) and isobaric tagging experiments (iTRAQ) identified a pool of around 65 proteins that exhibited similar expression patterns between these two independent experiments. These proteins were further analyzed to tease out a picture of the biology of the two transition states (anagen to telogen and telogen to exogen) to better understand the human hair cycle. Based on the experimental findings, it is evident that when hair follicles cycle from anagen to telogen, metabolic changes coincide with structural modifications. During the telogen to exogen transition, not only is there a loss of proteins in terms of the overall population, but several proteins also display lower expression levels.

878 Individual variation in balance between platelet-secreted growth factors causing contradictory effects on hair follicle could potentially impact response to PRP therapy in patients with scalp hair loss
J. Mohammed, M. Abedin, R. Farah, A. Wipf, M. Hordinsky

Platelets contain α-granules that are reservoirs of critical growth factors (GFs) regulating cellular proliferation, migration, differentiation and angiogenesis. Due to the ability of platelets to secrete GFs that play critical roles in the natural healing process, platelet-rich plasma (PRP) prepared from the blood is injected to sites of injury to deliver high concentrations of autologous GFs. Several studies published over the past few years have evaluated the effectiveness of PRP to treat hair loss disorders such as Androgenetic Alopecia. It is widely believed that GFs released from platelets upon PRP injection act on skin and hair follicle stem cells thereby promoting neovascularization and most likely, follicle differentiation. However, despite several promising results reported from clinical trials on the therapeutic potential of PRP, the response has been inconsistent and in some cases conflicting. Its likely that patient response to PRP depends on the balance between platelet-secreted GFs known to promote (PDGF-A, PDGF-B, VEGF, FGFb, EGF, IGF and HGF) versus inhibit hair follicle growth (TGFβ1). As PRP injection results in 300% to 700% enrichment of platelets secreting large quantities of GFs that can have contradictory roles in hair follicle differentiation, we analyzed platelet expression of GFs in PRP samples prepared from patient volunteers by quantitative PCR. Expression of TGFβ1 was highest followed by PDGF-A and PDGF-B while low to undetectable transcript levels were noticed for IGF and HGF. Variability in platelet expression between patients was highest for TGFβ1, PDGF-A and PDGF-B and lowest for VEGF. Consistently, we detected high TGFβ1 levels in PRP compared to other growth factors and its concentration relative to PDGF-BB in PRP varied highly between patients. Thus, balance between platelet-secreted TGFβ1 and other GFs in PRP that promote hair follicle differentiation could determine patient response to PRP therapy.

1303 Insights gained from a chickens rapid hair development during hatching lead to discovery of hair growth peptide derived from egg yolk
C. Pereira, T. Nakamura, K. Shin, K. Park, N. Horie, S. Itami, Y. Uchida, M. Kim

Stimulation of VEGF-mediated vascularization improves hair growth in broad types of alopecia, including female pattern hair loss (FPHL), whose pathogenic mechanism remains unresolved. We noticed that hair mostly grows in a precocial bird, including the chicken, before birth. Pertinently, hairs are developed within one day in the middle stage of hatching. Hence, we hypothesized that: 1) the chicken egg contains a key hair growth factor, and 2) this key hair growth factor improves hair growth in mammals. We prepared our water-soluble peptide by serine protease treatment of chicken egg yolk and egg white. In vitro studies using cultured human hair follicle dermal papilla cells revealed that the water-soluble peptide of the egg yolk (but not of the egg white) stimulates VEGF production and cell growth. We next orally administered the water-soluble egg yolk peptide to mice. In parallel, as a positive control, we topically applied minoxidil on murine dorsal skin. Both our egg yolk peptide and minoxidil enhances murine hair growth. Moreover, when orally-administered, our egg yolk peptide improves hair growth in FPHL. Finally, we showed that VEGF expression is increased through IGF-1 receptor activation-induced HIF-1α transcription pathway and that our water-soluble egg yolk peptide increased IGF-1 production. Taken together, our water-soluble egg yolk peptide improves hair growth through its activation of a pathway that increases IGF-1 and VEGF production; and we have given the name, Hair Growth Peptide (HGPTM) to this water-soluble egg yolk peptide.

1307 Evaluating the effect of a combined biofunctional on a 3D-model of dermal papilla cells and its relevance to hair density
A. Perrin, C. Gondran, R. Zhao, X. Qu, K. Cucumel

Hair density reflects hair beauty and is a main concern in personal appearance. This feature is controlled by specialized fibroblasts located in the mesenchymal compartment at the base of the hair follicle, called the dermal papilla. These cells determine the hair follicle entrance in the anagen phase, as well as its maintenance, by expressing several markers that influence the keratinocytes of the matrix compartment, giving rise to the growing hair shaft. In this study, we targeted markers associated with the anagen phase and introduced a method to evaluate a combination of pea extract and hair boosters, on a 3D-model of Human Dermal Papilla Cells (HDPC) in spheroid culture. Moreover, ex vivo cultures of microdissected human hair follicles were used to analyze beta1-integrin expression and hair shaft elongation.Our results showed that, in the presence of the biofunctional, versican and noggin were increased in HDPC spheroids as well as beta-1 integrin staining in the hair follicle and hair shaft elongation. In addition, an in vivo study was performed on 40 volunteers, who received a placebo or a hair serum containing the biofunctional, over a period of 12 weeks. A significant decrease in scalp sebum was observed, as well as an improvement in scalp hydration and reduced TEWL. Moreover, after 3 month application, the biofunctional seems to be associated with an increased in the A/T ratio, with an increased number of hair in anagen phase. This study takes advantage of using 3D-cultures of HDPC to evaluate the effect of a biofunctional on hair inductivity markers. Moreover, our in vivo study revealed a healthier scalp with less visible oi lines and more hydration, a visible maintenance of the anagen phase and visible improvement of hair density.

1328 HIF1A stabilisation in the human hair follicle promotes glycolysis
KFatima Shah, M. Davis, M. Philpott

HIF1A is considered the master transcriptional regulator of cellular response to hypoxia and regulates glucose metabolism by promoting glycolysis. The epidermis and hair follicles (HF) are moderately to severely hypoxic. In particular, the HF is known to express markers of hypoxia and also preferentially engage in aerobic glycolysis which is the preferential metabolism of glucose to lactate despite the presence of oxygen. In this study we aim to determine whether stabilization of HIF1A is able to regulate HF aerobic glycolysis and what impact this has on HF signalling pathways. We investigated the localisation of HIF family members in human skin and HFs by immunostaining and following HIF stabilisation. To study the functional role of HIF1A in the HF we investigated the impact of HIF1A stabilisation on glycolysis in primary cultured ORS keratinocytes using a Seahorse metabolic analyser. HIF1A, HIF1B and HIF2A expression was observed within the epidermis, sebaceous glands and HFs. HIF1B, a non-oxygen dependent protein, showed the strongest staining within the HF and epidermis. Staining of isolated HFs showed strong positive nuclear staining for HIF1A, HIF1B and HIF2A in the outer root sheath (ORS) and this increased in follicles treated with the HIF stabilizer desferrioxamine (DFO), a chemical hydroxylase inhibitor which promotes HIF1A stabilization. Downstream of HIF1A stabilization, OCT4 stem like TF was expressed in these HIF1A stabilized ORS cells suggesting the HIF pathway has a role in stem cell activation. Metabolic analysis showed cells treated with DFO exhibited a shift to a more glycolytic phenotype compared to untreated cells. These data suggest HF and especially the ORS is hypoxic. HIF1A stabilisation promoted glycolysis over oxidative phosphorylation in ORS keratinocytes and we propose this may reduce oxidative stress and promote hair growth.

1332 Understanding the role of glycogen metabolism in human hair follicle biology
K. Figlak, R. Paus, M. Philpott

Human hair follicles (HF) present high demand for energy and biosynthesis precursors fulfilled mainly by aerobic glycolysis. HF contain high levels of glycogen, the main energy storage site in the human body. The Cori cycle describes a metabolic process in which surplus lactate is converted via gluconeogenesis into glucose, which can then be stored as glycogen. Since HF contain glycogen and most glucose metabolised by HF is converted to lactate, we have investigated the functional role of glycogen and the possibility of the HF operating their own Cori cycle. High levels of glycogen were found in the outer root sheath (ORS) and the cuticle of anagen HF with significant decrease in catagen and absence in telogen. Crucial glycogen metabolism enzymes presented distinctive expression patterns: glucose transporter 1 (Glut1) and glycogen phosphorylase (PYGL) in the ORS, phosphoglucomutase (PGM1) was expressed in the basal ORS, cuticle and hair matrix, whereas glycogen synthase 1 (GYS1) was expressed in the basal ORS and the IRS (Inner Root Sheath). Glycogen metabolism was further investigated by following the glucose route within whole HF using fluorescent glucose derivative. Treatment of primary ORS keratinocytes with PYGL inhibitor caused glycogen accumulation, and upon glucose starvation, inhibiton of PYGL diminished glycogen breakdown. The cytoplasmic isoform of phosphoenolpyruvate carboxykinase (PCK1), an essential enzyme for gluconeogenesis was found in the ORS suggesting HF are capable of synthesis of glucose from lactate. Experiments carried out in ORS keratinocytes showed glycogen synthesis after induction of lactic acidosis along with upregulation of GYS1 and PCK1. The identification of key glycogen metabolism enzymes and the stimulation of glycogen synthesis following lactic acidosis suggests a HF Cori cycle allowing the hair follicle to synthesise glycogen. Further experiments will test if glycogen serves as a source of energy and substrates for proliferation and keratinisation or apoptosis in catagen.

 

[InBeforeTheCure]

1355 Mapping of Wnt/β-catenin signals during the telogen-to-anagen transition of human hair follicles
N.J. Hawkshaw, J. Hardman, M. Alam, I. Hernandez, F. Jimenez-Acosta, R. Paus

The Wnt/β-catenin pathway is essential for epithelial hair follicle stem cell (eHFSC) activation and anagen induction in murine hair follicles (HFs). Despite this central role in the HF cycle, these mechanistic concepts have not been translated to humans. Using human telogen and early-anagen HFs obtained during hair transplant surgery, individual Wnt/β-catenin pathway mRNA transcripts in the human secondary hair germ (SHG) and bulge eHFSCs were localised by in situ hybridisation. This showed the direct β-catenin target genes, AXIN2 and LEF1, are upregulated in the SHG of early-anagen HFs. Also the gene required for Wnt ligand secretion, WLS, is elevated in the SHG. Of 7 Wnt ligands assessed, WNT3, WNT10A and WNT10B were detected in the SHG of early-anagen HFs. We then analysed β-catenin activity in the bulge region, AXIN2 and WLS were detected in basal eHFSCs of telogen and early-anagen HFs, suggesting human eHFSCs can self-regulate Wnt. Whilst this matched murine data, we identified fundamental differences i.e. Wnt1 and Wnt4 are transcribed in murine, but not in human telogen HFs. Conversely, upon anagen induction human eHFSCs switch on WNT4 and WNT10A mRNA, suggesting that these ligands activate human eHFSCs in situ. Finally we identified the Wnt inhibitor, SFRP1, to be downregulated in the SHG and eHFSCs upon anagen induction; this coincides with increased WNT signalling. This suggests that SFRP1 may suppress Wnt signalling in telogen HFs. Collectively we report the first evidence that, despite distinct differences to the murine hair cycle, core components of the Wnt pathway operate also during the human telogen-anagen transition, including the activation of adult human eHFSCs. Modulation of these specific Wnt ligands or antagonising SFRP1 may be a novel therapeutic strategy to induce anagen in human telogen HFs and thus counteract hair loss.

1365 Interest of Biotrinine®, a mix of Nasturtium officinale and Tropaeolum majus extracts, for chronic hair loss treatment
M. Lévêque, B. Sandrine, N. Castex-Rizzi

The hair follicle (HF) undergoes cycles of growth (anagen), regression (catagen) and resting (telogen) phases. Chronic hair loss is caused by the shortening of the active growing phase (anagen) and the extension of the quiescence phase (telogen) of the HF, which occurs mainly under the control of androgens that activate the expression of hair growth cycles inhibitors in dermal papilla cells. This imbalance leads first to the miniaturization of HFs, then to the loose of their ability to generate new hair shafts. The aim of this study was to evaluate the effect of Biotrinine® dry extract on signals and molecular markers of human dermal papilla cells (HDPCs) that regulate anagen phases onset, HF growth maintenance and the transition of anagen to catagen phase. Potential synergistic activities with adenosine triphosphate (ATP) were also investigated. Wnt/b-catenin pathway activation was measured using a gene reporter assay strategy (transfection of HDPCs with a lentivirus expressing luciferase gene under the control of TCF/LEF promoter). Keratinocyte Growth Factor (KGF), Hepatocyte Growth Factor (HGF) and Dickkopf-related protein 1 (DKK1) protein expression levels were measured in HDPCs culture supernatants using ELISA and Luminex technology. Aromatase expression in HDPCs was analysed at mRNA level (Q-PCR analysis) and proteic level (western blot analysis) following respectively 24 and 48 hours of incubation with Biotrinine® dry extract. We found that Biotrinine® dry extract activated Wnt/b-catenin pathway (+38%), enhanced KGF and HGF secretion when tested in association with ATP with synergistic activities (respectively +72% and +133%), and inhibited DKK1 production and release (-65%). Aromatase expression was also induced by Biotrinine® dry extract (+48%). The results of this study indicate that Biotrinine® is a good candidate for chronic hair loss treatment and could be associated with ATP to potentiate their respective effects.

1369 Blue light stimulatory effect on hair growth mediated by opsin and cryptochrome photoreceptors
S. Buscone, N.E. Uzunbajakava, A. Mardaryev, N. Botchkareva

The therapeutic approach of photobiomodulation shows encouraging efficacy in hair loss treatment: more than 20 light-based devices have been safety-cleared to market by the FDA for the hair growth management. Yet lack of knowledge of the complexity of the molecular pathways that can be modulated by light hampers further progress in this field. We have recently identified that 1) light-sensitive receptors Cryptochrome 1, Opsins 2 and 3 (with absorption bands in the blue-to-green region) are expressed in the human hair follicle, and 2) treatment with 3.2J/cm2 blue light (453nm) prolongs anagen phase in hair follicle ex vivo. In this study, we demonstrated the siRNA-mediated inhibition of either CRY1 or OPN3 in the ORS keratinocytes resulted in the reduced proliferation of the outer root sheath (ORS) keratinocytes in vitro. Microarray analysis revealed that either CRY1 or OPN3siRNA causes alterations in the expression of genes controlling proliferation and apoptosis in the ORS keratinocytes. CRY1 siRNA suppressed the expression of CDK6, PLEK2, TMBIM6, and KDELR1, while the expression of ULBP1, PAK2, FSTL1 and UBR5 was downregulated by OPN3 siRNA. The link between CRY1 and CDK6 was also confirmed in the hair follicle ex vivo model. Treatment of the ORS keratinocytes with 3.2J/cm2blue light (453nm) stimulated CRY1 protein synthesis in both nuclear and cytoplasmic fractions, suggesting that CRY1 can indeed act as a mediator of blue lightdependent effect on hair growth. In addition, 3.2J/cm2 blue light significantly promoted metabolic activity and proliferation of the ORS in vitro that was abrogated by OPN3knockdown. Thus, our data suggest a 453nm blue light at low radiant exposure exerts a positive effect on hair growth, potentially via interaction with CRY1 and OPN3.

1370 17β-estradiol may control human HF growth also via up-regulating the expression of cannabinoid receptor type1 expression
S. Togo, K. Sugawara, R. Paus, D. Tsuruta



The stimulation of two distinct isoforms of the estrogen receptor (ER), ERα and ERβ, is mainly responsible for the cutaneous effects of the key ER ligand, 17β-estradiol (E2), including the impact of E2 on ERβ-expressing human scalp hair follicles (HFs). While murine and canine hair growth is strongly inhibited by E2, in fronto-temporal male scalp HFs, E2 slightly prolongs anagen and stimulates hair shaft production ex vivo. However, E2 can also promote the dystrophic catagen pathway of the HF response to chemotherapy-induced damage. Interestingly, the endocannabinoid, anandamide (AEA), inhibits human HF growth via cannabinoid receptor type 1 (CB1), while estrogens may increase AEA concentrations by decreasing the activity of the AEA-degrading enzyme, fatty acid amide hydrolase (FAAH). Therefore, we have explored in human HF organ culture and in cultured human outer root sheath (ORS) keratinocytes whether ER stimulation affects intrafollicular CB1 expression. CB1 expression was significantly upregulated within the ORS of E2-treated (10-7 M, 24hrs) human HFs ex vivo, compared to vehicle-treated HFs by quantitative immunohistomorphometry. Furthermore, E2 significantly increased CB1 immunoreactivity in ORS keratinocytes (10-7 M, 24hrs). Finally, CB1 protein expression within the ORS of early anagen HFs was decreased in the back skin of ovariectomized mice compared to sham-operated mice. Taken together, the current pilot study reveals that E2 may render human HFs more sensitive to the catagen-promoting impact of endocannabinoids via increasing CB1 expression and suggests a novel (neuro-)endocrine cross-regulatory mechanism that need to be considered in future human hair therapy with E2. Specifically, our study raises the question whether topical E2 may be made more efficient in FPHL management if it is co-administered with a topical CB1 antagonist.

1376 Dermal white adipose tissue enhances proliferation, pigmentation and hair shaft elongation in human hair follicles ex vivo
C. Nicu, J. Pople, R. Bhogal, D. Ansell, R. Paus

In the past decade, the crosstalk between dermal white adipose tissue (DWAT) and hair follicles (HFs) has become of increasing interest. Murine studies have revealed that pathways involving BMP2, PDGFA, SHH and leptin are involved in the HF-DWAT axis. Despite such progress on murine models, human HF-DWAT communication remains virtually unexplored. In this study, we cultured micro-dissected HFs versus HFs surrounded by the immediate 3-4 layers of dermal adipocytes (HF+DWAT) from human scalp skin for 48hr ex vivo. Interestingly, quantitative immuno-histomorphometry of Ki67+ cells below Aubers line reveals that DWAT significantly enhances cell proliferation in the HF matrix, as well as increasing the number of DAPI+ nuclei. Furthermore, Masson Fontana staining shows a significant upregulation of melanin content within HFs cultured with the surrounding DWAT compared to HFs cultured on their own. We also carried out ex vivo cultures of a) HFs on their own b) HFs with the surrounding DWAT (HF+DWAT), and c) HFs with dissected subcutaneous scalp fat (SWAT) in the same well (HF+SWAT). Interestingly, hair shaft elongation is significantly higher in hair follicles grown together with the surrounding DWAT compared to HFs alone and HFs grown with dissected SWAT. Overall, our results suggest that DWAT-derived factors act upon human HFs via paracrine signalling to modulate key processes within the HF matrix. For the first time, we show that human DWAT influences scalp HFs ex vivo via enhancing proliferation, pigmentation and hair shaft elongation. Our results hold translational promise, and may point to the notion that deregulation of dermal adipocytes surrounding human HFs may play a more significant role in promoting hair disorders than previously imagined.

Cell-Based Therapies

1324 Impairment of hair-inducing capacity of 3D-cultured human dermal papilla cells by the ablation of STAT5
C. Seo, M. Kwack, M. Kim, J. Kim, Y. Sung



STAT5 is a signal transducer and activator of transcription (STAT) and its levels are specifically upregulated within the dermal papilla (DP) and the phosphorylated active form of STAT5 (phospho-STAT5; P-STAT5) is restricted to the DP cells. Recently, Harel et al. reported that the treatment with tofacitinib, a Janus kinase (JAK)-STAT signaling inhibitor, enhances the hair inductivity of human DP spheres. In contrast, Legrand et al. reported that reported that when the adenoviral overexpression of STAT5A or STAT5B was induced in mouse skin-derived precursors (SKPs), multipotent stem cells derived from the DP, their ability to form de novo hair follicles was significantly enhanced, while the overall number of induced hair follicles was significantly reduced in STAT5-deleted SKPs. These controversial reports prompted us to investigate role of STAT5 in trichogenicity of human DP cells using a small interfering RNA (siRNA)-mediated gene knock-down approach in DP spheres in combination with a patch hair reconstitution assay. We observed that expression of STAT5 and its target genes is elevated when non-trichogenic 2D-cultured human DP cells are induced to form in vivo-like trichogenic DP spheroids. In addition, we observed that hair follicle induction was significantly impaired when STAT5 (A or B) knock-down DP spheres were implanted together with newborn mouse epidermal cells. Our data demonstrate that enhanced DP trichogenicity in DP spheres, is at least partly due to enhanced STAT5 levels.

1333 Spheroids cell cultures as a promising study model for hair follice regeneration
F. Oppenheimer, J. Ceruti, G. Leirós, M. Balañá



Dermal Papilla Cells (DPC) induction on Hair Follicle Stem Cells (HFSC) is necessary for embryonic hair follicle neogenesis and adult hair cycling. As DPC inductive ability is lost when culturing in monolayer, 3D-culture strategies are necessary to regenerate hair follicles in skin substitutes. Our aim was to test different culture conditions to: a) generate DPC spheroids with improved inductive ability. b) generate heterotypic spheroids containing adult human DPC and HFSC to be used in complete skin regeneration. Wnt signaling is essential in maintaining the hair-inducing activity of DPC. We have previously shown that, Skin substitutes, containing DPC promoted early neovascularization resulting in a better healing process and graft take. Alkaline phosphatase activity (APa), the expression of Wnt ligands and angiogenic factors were evaluated comparing culture times and conditions. APa, which correlates with hair inductive potential of DPC, was 25-fold higher in spheroids than in monolayer cultured cells. VEGF and Angiogenin, which have been proved to promote skin angiogenesis and hair cycle, also showed an 8-fold higher expression in DPC spheroids. The effect of culture time of the spheroids on the expression of Wnt ligands agonists (Wnt5a, Wnt10b) and antagonist (Dkk1) was evaluated. After 48 hours, an 8-fold increase of Wnt agonists and a 4-fold decrease of Dkk1 expression was observed. Histological analyses of heterotypic spheroids of HFSC and DPC showed that in appropriate culture conditions, a core of DPC expressing α-Smooth Muscle Actin and Vimentin surrounded by an epithelium with a p63 positive basal layer. These results show the importance of the culture conditions in order to obtain DPC-spheroids with improved inductivity and heterotypic spheroids with histological features reminiscent of embryonic hair-buds.

1415 Induction of human hair growth using vascularized 3D hair follicle constructs
H. Abaci, A. Coffman, Y. Doucet, J. Chen, J. Jackow, E. Wang, Z. Guo, A. Christiano



Human skin constructs (HSCs) have the potential to provide an effective therapy for patients with significant skin injuries and to enable human-relevant drug screening. However, incorporation of engineered skin appendages, such as hair follicles (HFs), into HSCs has been a long-standing challenge. Employing 3D-printing technology, we initially developed a biomimetic approach for generation of human HFs within HSCs through guiding physiological 3D organization of cells in the HF microenvironment. Our method allows for restoration of dermal papilla cell (DPC) hair inductivity by controllable DPC spheroid formation and initiation of the crosstalk between epidermal-mesenchymal cells, leading to HF induction in HSCs in an entirely ex vivo context. However, engraftment of these HSCs onto immunodeficient mice did not efficiently induce de novo hair formation; instead, we found necrosis at the center of the grafts. Histological analyses showed that there was a lack of vasculature and blood supply in the grafts. To address this, we prevascularized the HSCs containing 255 HFs/cm2 by encapsulating GFP-tagged human umbilical vein endothelial cells (HUVECs) into the dermal compartment. The stimulation of HUVECs with growth factors induced spontaneous capillary formation in vitro, where the capillary networks surrounded the HFs. Engraftment of prevascularized HSCs onto mice led to human hair growth, confirmed by human-specific nuclear staining and PCR analyses using human vs. mouse-specific primers. We also found that HUVECs in the grafts formed an organized capillary network with the surrounding mouse vessels and promoted blood supply to the grafted skin/hair. The ability to regenerate an entire HF from cultured human cells will have a transformative impact on the medical management of different types of alopecia.

Wound-Induced Hair Follicle Neogenesis

1349 Stimulation of hair follicle stem cell proliferation through an IL-1α dependent activation of γδT-cells
R. Gund, P. Lee, A. Dutta, N. Pincha, I. Rana, S. Ghosh, D. Witherden, E. Kandyba, A. MacLeod, K. Kobielak, W. Havran, C. Jamora



The cutaneous wound healing program is a product of a complex interplay among diverse cell types within the skin. One fundamental process mediated by these reciprocal interactions is the mobilization of local stem cells pools to promote tissue regeneration and repair. Using the epidermis specific ablation of caspase-8 as a model of mouse wound healing, we analyzed the signaling components responsible for epithelial stem cell proliferation. We found that IL-1α and IL-7 secreted from keratinocytes function synergistically to expand the activated population of resident epidermal γδ T-cells. A downstream effect of activated γδ T-cells is the preferential proliferation of hair follicle stem cells. On the other hand, IL-1α dependent stimulation of dermal fibroblasts optimally stimulates epidermal stem cell proliferation. These findings provide new mechanistic insights into the regulation and function of epidermal-immune cell interactions and how components classically associated with inflammation can differentially influence distinct stem cell niches within a tissue.

 

[InBeforeTheCure]

1410 Non-coding double stranded RNA induces retinoic acid synthesis and retinoid signaling to control regeneration
D. Kim, R. Chen, S. Kim, A. Park, B. Evans, V. Yu, E. Oh, L.S. Miller, S. Kang, G. Ghiaur, J. Yu, W. Huang, M. Kane, L. Garza



Wound induced hair follicle neogensis (WIHN) is a model of tissue regeneration in adults where hair follicles develop de novo following deep wounding. dsRNA generated after wounding activates TLR3 to induce WIHN in mice but mechanisms involved and the translatability to humans is unclear. By transcriptome and proteomic analysis, we unexpectedly found a highly significant (p<3.6x10ˆ-15 and p<8.2x10ˆ-80, respectively) overlap of upregulated genes in keratinocytes following treatment with dsRNA or retinoic acid (RA). Further, mass spectrometry analysis of dsRNA stimulated cultured human keratinocytes indicated highly induced RA synthesis but not other retinoid metabolites (retinol and retinyl ester) (n=3, p<0.001). Similarly, in the skin of wild type mice, stimulation with dsRNA during wounding induced an RA morphogen gradient that correlated to future areas of WIHN (n=4, P<0.01). The induction of RA by dsRNA was dependent upon TLR3 as the skin of TLR3-deficient mice had lower baseline RA levels and failed to induce RA following wounding (n=4, p<0.05). In addition, mice with keratinocyte-specific deletion (n=4-6, p<0.01) or global deletion (n=6-11, p<0.001) of RA receptor alpha (RARa) had an almost entirely absent WIHN and were resistant to exogenous administration of dsRNA (Poly I:C). Finally, exogenous RA induced hair follicle stem cell markers such as KRT15 and KRT19 (n=3, p<0.05) and rescued the impaired WIHN in TLR3-deficient mice (n=6-11, p<0.05). In a preliminary human trial, ablative CO2 laser wounding combined with retinoic acid appeared to induce the formation of small vellous follicles. Taken together, these results define a novel mechanism involving dsRNA/TLR3 innate immune responses and RA nuclear hormone receptor pathways in tissue regeneration.

1419 Wound regeneration deficit in rats correlates with low morphogenetic potential and distinct transcriptome profile of epidermis
C.F. Guerrero-Juarez, M. Plikus

Large excisional wounds in mice prominently regenerate new hair follicles (HFs) and fat, yet humans are deficient for this regenerative behavior. Currently, wound-induced regeneration remains a clinically desirable, but only partially understood phenomenon. We show that large excisional wounds in rats, across seven strains fail to regenerate new HFs. We compared wound transcriptomes between mice and rats at the time of scab detachment, which coincides with the onset of HF regeneration in mice. In both species, wound dermis and epidermis share core dermal and epidermal transcriptional programs respectively, yet prominent inter-species differences exist. Compared to mice, rat epidermis expresses distinct transcriptional and epigenetic factors, markers of epidermal repair, hyperplasia, and inflammation, and lower levels of WNT signaling effectors and regulators. When recombined on the surface of excisional wounds with vibrissa dermal papillae, partial-thickness skin grafts containing distal pelage HF segments, but not interfollicular epidermis, readily regenerated new vibrissa-like HFs. Together, our findings establish rats as a non-regenerating rodent model for excisional wound healing and suggest that low epidermal competence and associated transcriptional profile may contribute to its regenerative deficiency. Future comparison between rat and mouse may lend further insight into the mechanism of wounding-induced regeneration and causes for its deficit.

1424 Mapping the establishment of fibroblast heterogeneity during skin development and wound repair
R.R. Driskell, L. Salz

The process of wound healing is complex and involves coordination of multiple cell types required to regenerate the 3D architecture of skin. In adults, upon completion of wound healing a scar is formed, which is composed of fibrous matrix generated by fibroblasts, and the healed skin lacks hair follicles. Skin fibroblasts arise from a homogenous population during early embryonic development but differentiate into heterogenous populations that have non-overlapping functions in development, skin maturation and wound healing. In mouse skin, we have identified two fibroblast subsets. Reticular fibroblasts construct the dermal architecture of the skin by secreting matrix and differentiating into adipocytes. Importantly, reticular fibroblasts are lineage restricted and do not contribute to hair follicle formation. Rather, a second fibroblast subset, consisting of papillary fibroblasts is central to hair follicle formation by differentiating into dermal papillae, the dermal sheath, and arrector pili. We have found that adult skin scarring is the result of tissue repair that utilizes a single type of lineage restricted fibroblast, rather than the coordinated functions of heterogenous fibroblast populations. To understanding how to control fibroblast heterogeneity in wounds we are mapping all fibroblast populations during skin development. In this regard, we have generated large single-cell-RNA-sequencing datasets consisting of tens of thousands of cells each from embryonic day 14.5, 17.5 and post-natal day 2, 5, and 21, which are key developmental times for the establishment of fibroblast heterogeneity. This has allowed us to map, using molecular signatures, the establishment of fibroblast heterogeneity. Our analyses have revealed that the canonical Wnt transcription factors, Lef1/Tcf1,3,4 are key regulators of fibroblast heterogeneity. In summary, we have developed invaluable datasets which will be an essential resource for defining factors that regulate fibroblast heterogeneity during development to investigate during wound healing.

1450 Regulation of skin regeneration by non-coding RNA sensing
N. Islam, R. Silverman, L. Garza

The molecular and physiological processes governing wound healing and regeneration have not yet been fully defined. Here we investigate the mechanisms of skin regeneration after injury through a rare event of mammalian regeneration known as Wound Induced Hair Neogenesis (WIHN). We have shown that non-coding dsRNA released by tissue damage stimulates TLR3 and β-catenin to promote WIHN. However, the RNases which either promote or inhibit dsRNA biogenesis remain unresolved. Following injury or exogenous dsRNA (polyIolyC) treatment, we find significant overlap and upregulation of OAS transcripts (≤15/19,234 transcripts, p=8.3*10-13) in microarray data. The antiviral dsRNA-activated OAS family is responsible for producing metabolites that activate the endoribonuclease RNase L. We hypothesized RNase L might normally promote the degradation of dsRNA containing transcripts by cleaving single-stranded regions in these RNAs in a negative feedback loop and effectively limit regeneration. To explore this, we show that an Rnasel-/- mouse model exhibits a significantly enhanced regenerative capacity and a 5-fold increase in WIHN (N=10, P<0.001). RNAi-mediated ablation of RNase L in keratinocytes significantly increases core morphogenetic markers in response to polyIolyC, including WNT7b, TLR3, Sonic hedgehog, and interferon-β (N=3, P<0.01). Furthermore, we show that a specific non-coding dsRNA, U1 RNA, is induced 10-fold after tissue injury (N=3, P<0.05) and even more so in Rnasel-/-mice. In vitro transcribed U1 RNA can potently stimulate morphogenesis and regeneration both in vitro and in vivo; WT mice treated with U1 RNA exhibit nearly a 4-fold increase in WIHN compared to vehicle (N=3, P<0.05). Keratinocytes treated with U1 RNA exhibit similar induction as polyIolyC of WNT7b, TLR3 and KRT15, a hair follicle stem cell marker. Altogether, these results suggest a novel role for RNase L in regulating non-coding dsRNA levels and sensing during regeneration.

General Mouse Studies

128 Analysis of mice skin distribution using MALDI-MSI after subcutaneous injections of a potent novel peptide hair growth promoter, FOL-005
D. Bonnel, R. Legouffe, M. Ekblad, F. Farcette, J. Stauber, J. Alenfall

Hair loss, such as androgenic alopecia or other forms of alopecia, often causes major psychological distress. Today only few and unsatisfactory therapeutic options are available and hence new treatment strategies need to be developed. The multifunctional, immunomodulatory glycoprotein, osteopontin, is expressed by hair follicles (HFs) and a newly generated, modified osteopontin-derived peptide, FOL-005, has been shown to highly and reproducibly induce hair growth in mice. In a recently completed clinical phase I/IIa study, subcutaneous injections of FOL-005 were shown to be a safe treatment that resulted in 8 % increase in hair growth at one of the doses tested. As part of the preclinical program for further development of a new formulation and an optimal treatment regimen with FOL-005, the distribution of subcutaneously injected FOL-005 into mice was followed in skin for up to 24 hours using the MALDI-MSI technology. MALDI-MSI, an advanced label free technique based on the combination of mass spectrometry imaging and histology, provides qualitative and quantitative data on the drug distribution and hence can add information on efficiency or potential toxicity. FOL-005 was found to be distributed exclusively in the treated skin after injection and the concentration was shown to decrease with time. No distribution was detected outside the skin indicating that FOL-005 is locally degraded. The obtained data identify only a local distribution of FOL-005 peptide and further supports the clinical development as a new, much-needed treatment principle for alopecia patients.

1298 External light activates hair follicle stem cells through eyes via the ipRGC-SCN-sympathetic neural circuit
S. Fan, Y. Chang, C. Chen, T. Chen, M. Plikus, S. Chen, S. Lin

Changes in external light patterns can alter stem cell activities in peripheral tissues and result in both daily and seasonal cycles of tissue regeneration. Daily oscillation of cellular activities requires hierarchical and slow entrainment of cell-autonomous clocks by the central clock in the suprachiasmatic nucleus (SCN). It remains unclear whether stem cells in otherwise photo-insensitive tissues can bypass circadian clock to achieve rapid responses to changes in external light. Here we show that light stimulation of animals eyes results in rapid activation of hair follicle stem cells to initiate a new anagen. The light signals are interpreted by intrinsically photosensitive retinal ganglion cells (ipRGCs) in the retina and these cells signal via the photoreceptor molecule melanopsin through optic nerves to SCN. Subsequently, efferent sympathetic nerves are directly activated and intact sympathetic innervation to hair follicles is required for the regenerative response of hair follicles. Local increase of norepinephrine release in skin activates hair follicle stem cells by modulating hedgehog signaling. Thus, eyes are a gateway that allows immediate communication between the external megaenvironment with the internal stem cell microenvironment. External light can directly regulate tissue stem cells via an ipRGC-SCN-autonomic nervous system circuit. Since the ativation of sympathetic nerves are not limited to skin only, this circuit may also facilitate rapid adaptive responses to external light in other tissues.

1308 A microtubule catastrophe factor regulates stem cell position and number in the skin
R. Moreci, J. Underwood, T. Lechler

Asymmetric cell divisions drive cell fate specification and the formation of complex tissue architectures. This often requires orienting the mitotic spindle to position daughter cells and/or segregate cell fate determinants. Asymmetric cell divisions drive the stratification and differentiation of the interfollicular epidermis during embryogenesis. Oriented divisions have also been observed during the production of hair follicle stem cell progenitors. However, whether spindle orientation is required for the production of these stem cells and how this is coupled to their cell fate remains unknown. During spindle positioning, astral microtubules of the mitotic spindle interact with specific regions of the cell cortex. To determine whether regulated dynamics of these astral microtubules are important for spindle orientation, we focused on kinesin family members known to promote microtubule disassembly. We found that loss of Kinesin Family Member 18B (KIF18B) in cultured keratinocytes resulted in an increase in astral microtubule length and density, and disrupted spindle orientation. However, mitosis proceeded normally in these cells. Loss of Kif18b in mice resulted in phenotypes consistent with loss of spindle orientation in the interfollicular epidermis. Additionally, KIF18B was required for hair follicle morphogenesis. We traced this defect back to the earliest stage of hair follicle formation, the placode. In KIF18B mutant skin, placodes were initially specified but stem cells were mislocalized and expanded at the expense of the morphogenetically active basal cell population. These data identify a new regulator of spindle orientation and directly demonstrate the importance of orienting spindles in hair follicle cell fate specification.

1313 Fzd2 controls multiple aspects of epidermal development through distinct signaling mechanisms
D.M. Brennan-Crispi, M. Xu, J. Horrell, M. Frankfurter, Y. Zhang, E.E. Morrisey, S.E. MIllar

Wnt ligands bind to Frizzled (FZD) receptors to activate both β-catenin-dependent (canonical) and β-catenin-independent (non-canonical) signaling. Non-canonical signaling includes planar cell polarity (PCP) and Wnt/calcium pathways. In the skin, canonical Wnt signaling is required for hair follicle development and regenerative growth, while PCP signaling controls hair follicle orientation, and in vivo roles for Wnt/calcium signaling have not been described. Limited information is available regarding the functions of individual FZD receptors in these processes. Here, using constitutive and inducible epidermal deletion mouse models, we show that the FZD family member FZD2 is required for hair follicle placode formation and normal postnatal hair growth, suggesting that it mediates canonical signaling in hair follicles. In addition, we find that early deletion of Fzd2 in embryonic epidermis unexpectedly causes defective stratification, cornification and barrier formation, a phenotype that has not been described previously upon loss of either canonical or PCP signaling. Fzd2 mutant epidermis displays a striking reduction in expression of the desmosomal component plakophilin 1 (PKP1). Loss of function mutations of PKP1 in humans and mice cause ectodermal dysplasia and skin fragility, phenotypes that overlap with those observed in epidermal Fzd2 mutants but do not include defective epidermal stratification. These data indicate that FZD2 plays multiple roles in skin epithelial development and homeostasis: mediating canonical Wnt signaling in hair follicles; controlling expression of PKP1; and a novel early function in regulating epidermal stratification, cornification and barrier formation independent of PKP1.

1319 Claudin-1 and -3 maintain proper hair follicle structure and regulate telogen effluvium
K. Yamaga, R. Tokumasu, A. Yamamoto, A. Tamura, I. Katayama, H. Murota, S. Tsukita

Claudins (CLDNs) are major transmembrane proteins of tight junctions (TJs), which seal the intercellular space of epithelial cells. Patients with a nonsense mutation in CLDN1 gene develop ichthyosis and scalp hair loss. The importance of TJs in epidermal barrier formation is well-known; however, little is known about how TJs affect the homeostasis and structure of hair follicles (HFs). To tackle this issue, we investigated the impact of TJs on retaining hairs.In this study, murine back skin samples were immunohistochemically stained for CLDN1 and CLDN3 to detect their localization in HFs. Additionally, we conducted daily skin observation and histological analysis in wild-type (WT), Cldn1 knockdown (1KD), Cldn3 knockout (3KO) and double mutant with 1KD and 3KO (1KD3KO) mice. Both CLDN1 and CLDN3 were localized in inner bulge cell layer, isthmus, infundibulm and sebaceous gland. Although 1KD or 3KO single mutant mice showed normal hair coat, 1KD3KO double mutant mice lost almost all hairs in their first telogen between postnatal day 20 (P20) and P25. As for the structure of HFs, the bulge region, whose inner layer cells sustain telogen hairs, in WT mice was composed of well-organized two cell layers and was localized below sebaceous glands. On the other hand, 1KD3KO bulge region was enlarged with disorganized three or four cell layers and was localized closer to the skin surface, adjacent to sebaceous glands. Furthermore, hair canals of isthmus and infundibulm in 1KD3KO mice were widened. Taken together, our findings indicate that CLDN1 and CLDN3 are critical for proper HF structure, especially cell alignment and localization of bulge region, thus preventing telogen hair loss. Induction or maintenance of CLDN1 and CLDN3 may become a new treatment target for patients with alopecia.

1311 Efficacy of topical tofacitinib in promoting hair growth in non-scarring alopecia
J. Meephansan, J. Thummakriengkrai, T. Pongcharoensuk, P. Suchonwanit



Tofacitinib is a janus kinase 3 (JAK3) inhibitor that promotes hair growth; however, the efficacy and mechanism of this effect are not yet understood. This study aimed to evaluate the efficacy and mechanism of topical tofacitinib on hair growth in mice. Eight-week-old male C57BL/6 mice were divided equally into four groups and treated topically with tofacitinib, minoxidil, or vehicle once daily for 21 days. Weekly photographs were taken to determine the area and rate of hair growth, and tissue samples were collected for histopathological evaluation. mRNA and protein expression of anagen-maintaining growth factors, including vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1), were determined via RT-PCR and ELISA, respectively. Tofacitinib-treated mice exhibited more hair regrowth than either minoxidil-treated or control mice did between days 7 and 21 (P<0.05). Topical tofacitinib also promoted more rapid hair growth rate than topical minoxidil or control did (P<0.001). Histopathology showed a distinct increase in the number of hair follicles, mostly in the anagen phase, in the tofacitinib-treated group. Hair follicles in the minoxidil- and vehicle-treated groups were more often classified as catagen and anagen. VEGF mRNA and protein expression in the tofacitinib-treated group was significantly greater than those in the other groups (P<0.05). IGF-1 mRNA expression was not upregulated in tofacitinib-treated mice. Topical tofacitinib is effective in promoting hair growth, and the possible mechanism involves increased VEGF levels and lowered inflammation. This study will help develop a new therapeutic option for non-scarring alopecia.

 

[InBeforeTheCure]

1326 Hes1 regulates anagen initiation and hair follicle regeneration through modulation of hedgehog signaling
H. Lin, P. Lin, L. Yang



Adult hair follicles undergo repeated cycling of regression (catagen), resting (telogen), and regeneration (anagen), which is maintained by hair follicle stem cells (HFSCs). The initiation of the regenerative cycle and maintenance of HFSC self-renewal capability is not fully understood. Here, by epithelial deletion of Hes1, a major Notch downstream transcriptional repressor, we found that hair growth is retarded but the hair cycle progresses normally. Remarkably, activation of the secondary hair germ (HG) is delayed and the anagen phase is shortened in Hes1-deficient hair follicles. This developmental delay is not associated with a change in follicular lineages. Importantly, an in vivo repetitive depilation assay indicated that Hes1 is required for HFSC self-renewal and long-term hair regeneration. The underlying mechanism involves Hes1-mediated modulation of Shh responsiveness in anagen, during which Hes1 is specifically upregulated in the lower bulge/HG. Without Hes1, ciliogenesis is affected and Shh signaling is compromised. We reveal a critical function of Hes1 in potentiating Shh signaling in anagen initiation, which allows sufficient signaling strength to expand the HG and replenish HFSCs to maintain the hair cycle.

1330 Flexible fate determination ensures robust differentiation in the skin hair follicle
T. Xin, V. Greco



Tissue regeneration is sustained by stem cell self-renewal and differentiation. How stem cells coordinately differentiate into multiple cell types is largely unclear. Recent studies underline the intrinsic heterogeneity among stem cells or common progenitors, suggesting orchestration at the stem cell/progenitor level. Here, by tracking and manipulating the same stem cells and their progeny in live mice, we uncover an unanticipated flexibility during homeostatic stem cell differentiation in hair follicle. Though stem cells appear primed through spatial regulation, we find they retain full potency to establish all the differentiation lineages. Furthermore, hair progenitors previously thought to be unipotent, were found flexibly changing differentiation outcomes as a consequence of dynamic relocation. Finally, differentiation and tissue architecture were maintained normal despite ectopic differentiation stimulus. These show a flexible cell fate determination mechanism that contributes to differentiation robustness. This work supports a model of continually fate priming and late commitment to achieve coordinated differentiation.

1334 Beyond goosebumps: Interactions between the hair follicle, the arrector pili muscle, and the sympathetic nerve during development and hair follicle regeneration
Y. Shwartz, M. Gonzalez Celeiro, C. Chen, S. Lin, Y. Hsu



Piloerection, commonly known as goosebumps, involves three interconnected cell types: the hair follicle, the arrector pili muscle (APM), and the sympathetic nerve. The interactions between these three cell types during development and adult tissue maintenance remains poorly understood. Here, we identify a central role of the developing hair follicle in regulating the formation of APMs, which then attract sympathetic innervation to the hair follicle stem cells. Although dispensable for hair follicle development, impulses from the sympathetic nerves are crucial for regulating hair follicle stem cell activity during hair follicle regeneration. Formation of the APMs requires Sonic Hedgehog secreted from the developing hair follicles. Once developed, APMs do not undergo turnover, providing a stable anchor that maintains sympathetic innervations to the hair follicle stem cells. APM ablation leads to concurrent loss of sympathetic nerve innervation to the hair follicles. Our results uncover a novel function of APM in bridging the bodys sympathetic modulations to influence hair follicle stem cell activity, and illustrate an example for how a developing tissue regulates the establishment of the niche to modulate its regeneration in adulthood. Our results may also explain why hair loss is a common side effect of beta-blockers, which suppress the sympathetic tones, and why loss of APMs is commonly associated with permanent hair loss conditions such as in androgenic alopecia.

1338 Dermal Wnt/β-catenin activation tunably controls hair follicle initiation
K. Gupta, D. Chen, J. Levinsohn, K. Choate, M. Taketo, P. Myung



Hair follicles (HFs) initially form from the undifferentiated epidermis through epithelial-mesenchymal interactions. However, the cellular and molecular basis and hierarchical relationships of these interactions remain poorly delineated. Previous work showed that Wnt/β-catenin signaling is uniformly active in the upper dermis prior to HF development and is essential for HF initiation, but it is unknown how dermal Wnt activity functions to impact this early event. A major challenge in addressing this question is rooted in our lack of ability to dissect the initial cellular events that control HF initiation. Using in vivo genetic mouse models, we show that modulating the number of β-catenin-activated (Wnt+) dermal cells prior to HF initiation tunably determines the number of HF epithelial cells and associated HF dermal niche (dermal condensate, DC) cells that form. To begin to address how dermal Wnt controls HF initiation, single-cell RNA sequencing analysis of embryonic skin was performed, which showed that non-DC Wnt-active dermal cells lie on a differentiation trajectory toward a DC cell fate. Consistent with this, genetic lineage tracing confirmed that Wnt-active dermal cells present prior to HF initiation give rise to DC cells. Further, DCs of mutant HFs were composed selectively of wildtype Wnt-active dermal cells, indicating a cell-autonomous requirement for Wnt signaling in DC formation. By live imaging of embryonic skin explants, we examined how Wnt regulates dermal cell behaviors and found that Wnt-active dermal cells migrate to form the DC and that dermal Wnt regulates actomyosin activity. These data suggest that dermal Wnt activation tunably controls DC formation by regulating dermal cell migration. Moreover, this study provides evidence that an early patterned pre-DC population may direct HF epithelial cell fate specification.

1357 β-catenin regulation in Krox20 lineage hair follicle epithelial cells
C. Liao, L. Le



The in vivo exploration of genetic regulations in skin and hair follicle development has been successfully achieved by taking advantage of multiple epithelial cell specific promoter-driven Cre alleles in mice. Commonly used epithelial Cre can be classified into two major categories; the first type labels both skin and hair follicle (e.g. K14-Cre and K5-Cre), and the second type mainly marks hair follicle (e.g. K15-Cre and K19-Cre). Recently, Krox20-Cre has been shown as a new class of tool which identifies a subset of hair follicle epithelial cells differentiating toward hair shaft components during hair follicle morphogenesis. β-catenin is a key regulator of epithelial homeostasis; multiple hair and skin phenotypes have been reported on aberrant β-catenin expressions in several epithelial lineages. However, its specific function in the hair shaft progenitors is not known. Here we report the employment Krox20-Cre to address the contribution of β-catenin signaling in Krox20 lineage cells. We generate mice with loss-of-function or gain-of-function β-catenin in KROX20 cells and characterize their phenotypes. By comparing the similarity and discrepancy with previously reported β-catenin phenotypes in other epithelial Crelines, we are able to distinguish the contribution of β-catenin in epithelial cells from KROX20 and non- KROX20 epithelial cells in the development of skin and its appendices. Our results suggest the potential transcriptional regulation between KROX20 and β-catenin, and show that Krox20-Cre can serve as a new useful tool to delineate the genetic contribution from different subtypes of epithelial cells.

1359 Hair growth is induced by blockade of macrophage-derived oncostatin M and downstream JAK-STAT5 signaling in hair follicle stem cells
E. Wang, A. Christiano

Our lab recently demonstrated that blockade of JAK-STAT signaling using topical JAK inhibitors was sufficient to induce hair growth (anagen) in resting (telogen) in C57BL/6 mouse hair follicles, raising the possibility that JAK-STAT signaling is required for maintaining hair follicle stem cells (HFSC) in their quiescent state. To define the mechanism by which this occurs, here, we demonstrate that the IL-6 family cytokine Oncostatin M (OSM) is a negative regulator of hair growth that maintains HFSC quiescence via JAK-STAT5 signaling in vivo. We found that the OSM receptor (OSMRβ), co- receptor gp130 and activated pSTAT5 are co-expressed in telogen HFSCs, and that OSM is produced in the telogen dermis. Conditional epidermal ablation of OSMRβ or STAT5 during early- and mid-telogen (P42 – P60) shortens the telogen phase significantly, and promotes activation of HFSCs both in vivo and in vitro. Unexpectedly, we identified that the endogenous source of OSM is not intrinsic to the HF dermal cells, but rather, emanates from a distinct subset of TREM2+ dermal macrophages we identified using single-cell RNA sequencing of dermal CD45+ immune cells across murine telogen skin. Furthermore, we found that depletion of macrophages using three independent approaches, including neutralizing antibodies (anti-CSF1R), topical small-molecule CSF1R inhibitors (pexidartinib/PLX3397, BLZ945 and GW2580), and genetic ablation (with Csf1r-CreER::R26-iDTR mice) during telogen, promotes hair growth by removing the endogenous source of OSM. Hair growth and proliferation of HFSCs were associated with depletion of this subset of TREM2+ macrophages, which were found to be spatially, temporally and functionally relevant for HFSC quiescence during telogen. Our findings highlight the role of immune cells in establishing a quiescent niche for HFSCs, and invites future clinical investigation into treating human hair disorders characterized by arrested telogen follicles by targeting a cell type outside the HF itself.

1362 The role of Fgf20 in establishing the hair follicle dermal condensate
L.C. Biggs, O.J.M. Mäkelä, S. Myllymäki, R. Das Roy, K. Närhi, M.L. Mikkola



Hair follicle (HF) morphogenesis depends on reciprocal signalling between the epidermis and the underlying mesenchyme. The epithelial placode forms first and is accompanied by condensation of the adjacent dermis, known as the dermal condensate (DC). The DC is the precursor to the permanent, mesenchymal component of the HF, the dermal papilla (DP), which regulates the cycles of hair growth and rest throughout life, and is capable of generating a new hair follicle upon transplantation under epidermis. Thus, elucidation of the cellular and molecular mechanisms of DC development would be of great utility for regenerative purposes. Recently, our lab established the first molecular clue in DC development with the discovery that epithelial factor Fgf20 is necessary for DC induction. In the current study, we use a multifaceted approach including RNAseq and organ culture in combination with live tissue imaging to uncover the molecular and cellular responses to Fgf20 signalling as well as to determine the cellular events preceding and during DC development. We report that Fgf20 facilitates fibroblast migration and in short-term experiments can induce expression of some but not all DC markers. These cells, identified by Sox2 expression, do not arise from a predetermined population of Sox2+ cells. Further, as DC development progresses, these cells exit the cell cycle and migrate to form the DC. Additionally, DC cells adopt a convex shape early during HF morphogenesis, a phenotype that is recapitulated by local administration of FGF20 ex vivo, thus providing evidence that this is Fgf20-dependent. We show that modulation of FGF signalling during DC morphogenesis not only recapitulates the epidermal phenotype of Fgf20-/-, but it is also critical for DC cell attraction and maintenance. Together, these data suggest a role for Fgf20 in the cellular and molecular regulation of DC morphogenesis.

1387 Identifying the key niche signals for hair follicle formation
K. Mok, Z. Wang, R. Sennett, A. Rezza, N. Heitman, A. Ma'ayan, M. Rendl

Specialized mesenchymal cells in dermal condensates (DC) play a crucial role in regulating hair follicle (HF) progenitors in epidermal placodes (Pc) to orchestrate HF morphogenesis. Nevertheless, to date the inductive signals during DC specification and how DC cells interplay with Pc progenitors to facilitate HF development remain unknown. Here we identify precursor cells of the DC (pre-DC) before it appears as specialized cell cluster. With fluorescence-activated cell sorting we co-isolate pre-DC and the DC as it matures, together with the Pc at stage 0 HFs, and other lineage-related populations. We then define the gene expression patterns in each population with next-generation RNA sequencing. Through cross-comparisons we define a molecular time-lapse of dynamically changing gene signatures in consecutive developmental stages during the three earliest HF formation stages. Within the stage-specific pre-DC and DC molecular signatures we uncover several putative key hair inductive signals. With genetic manipulation in live embryonic skin cultures we aim to effectively test the role of newly identified signals in their interplay between the niche and placode progenitors that govern hair follicle formation.

1390 Lineage-committed fibroblast populations are more efficient at regenerating hair follicles in chamber grafting assays when compared to undifferentiated embryonic fibroblasts
L. Salz, R.R. Driskell

Hair follicle formation during skin development requires interactions between epithelial progenitors and a progressively differentiating dermal fibroblast population. Heterogenous fibroblasts originate from a homogenous mesenchyme and become lineage committed after E16.5 days. Hair follicle formation begins at ∼E14.5, which is prior to fibroblast lineage commitment, suggesting that this time point holds the highest potential for fibroblasts to become dermal papilla. We tested the hypothesis that undifferentiated embryonic fibroblasts (E14.5) possess an enhanced ability to support de novo hair follicle formation, utilizing the chamber grafting assay. We performed chamber grafting assays, containing E14.5, E17.5, or P5 skin fibroblasts. Our results revealed that E17.5 and P5 fibroblasts produced robust hair follicle formation, while E14.5 skin lacked the ability to form hair de novo, contradicting the original hypothesis. Our result supports the idea that there are two critical developmental states of dermal fibroblasts. The early embryonic state is undifferentiated and uncommitted fibroblasts that requires the embryonic environment to fully mature. The uncommitted state makes it susceptible to external perturbations that will result in drastic deviation from its functional differentiation program. In contrast, differentiated and committed fibroblasts, found after E16.5, are already intrinsically defined, therefore less sensitive to external perturbation. To support our hypothesis, that E14.5 fibroblasts are undifferentiated while E17.5 and P5 fibroblasts are differentiated we generated single-cell RNA sequencing datasets from each time point. We found that skin fibroblasts from E14.5 dermis lacked heterogeneity, and revealed a molecular signature similar to pre-cursor fibroblasts. In contrast, E17.5 and P5 skin fibroblasts contained multiple fibroblast populations with clearly defined molecular signatures. Further work is now required to understand the mechanisms of fibroblast differentiation, which can be harnessed to induce regenerative pathways in adult injured skin.

1392 Activation of Id2 gene regulatory network ruling quiescence of hair follicle stem cells
X. Wang, G. Wang, R. Ramos, X. Wang, P. Daszczuk, P. Mazurek, K. Yun, M. Israel, M. Plikus, K. Kobielak

Previously, we revealed how the underlying molecular mechanism of Bone Morphogenetic Protein (BMP) signaling governs the homeostasis of hair follicle stem cells (hfSCs) in vivo. Here, we focused on testing role of Id2 gene, one of the target genes which we identified to be downregulated in hfSCs after inhibition of BMP pathway. To test the function of Id2 gene in hfSCs in vivo we used Id2 gain of function approach (GOF) by generating transgenic mouse line. Our data demonstrated that Id2 overexpression in hfSCs results in prolonged telogen and a delay in anagen activation, maintaining stem cells quiescence. Further, we performed hfSCs sorting and RNA-seq analysis of GOF-Id2 hfSCs at first postnatal cycle. By comparison with common signature genes in quiescent bulge we demonstrated that approximately 10% of the genes were affected by overexpression of Id2 in hfSCs. Interestingly approximately half of those genes overlapped with common signature genes which previously we published to be affected by BMP pathway, thus working synergistically with that pathway. On the other hand our data also suggest that although Id2 is a direct target and an effector of BMP pathway in hfSCs in vivo, second half of those genes work at least partially independent from BMP signaling on gene regulatory network of quiescent bulge.

 

[lemoncloak]

Look at all those goodies

 

[razzmatazz91]

Holy sh*t! You really have put in some work into this haven’t you?

This will take some time to go through...

 

[dermrafok]

Incredible work! Especially in the section: Wound-Induced Hair Follicle Neogenesis. AWESOME!

 

[itsjustsimon]

747384852 new studies, yet 0 new treatments in the last 25 years.

Anyway, thanks for this @InBeforeTheCure

 

[SheddieMurphy]

747384852 new studies, yet 0 new treatments in the last 25 years.

Anyway, thanks for this @InBeforeTheCure

this. makes you think.

 

[Noisette]

Wound-Induced Hair Follicle Neogenesis

1410 Non-coding double stranded RNA induces retinoic acid synthesis and retinoid signaling to control regeneration
D. Kim, R. Chen, S. Kim, A. Park, B. Evans, V. Yu, E. Oh, L.S. Miller, S. Kang, G. Ghiaur, J. Yu, W. Huang, M. Kane, L. Garza

Wound induced hair follicle neogensis (WIHN) is a model of tissue regeneration in adults where hair follicles develop de novo following deep wounding. dsRNA generated after wounding activates TLR3 to induce WIHN in mice but mechanisms involved and the translatability to humans is unclear. By transcriptome and proteomic analysis, we unexpectedly found a highly significant (p<3.6x10ˆ-15 and p<8.2x10ˆ-80, respectively) overlap of upregulated genes in keratinocytes following treatment with dsRNA or retinoic acid (RA). Further, mass spectrometry analysis of dsRNA stimulated cultured human keratinocytes indicated highly induced RA synthesis but not other retinoid metabolites (retinol and retinyl ester) (n=3, p<0.001). Similarly, in the skin of wild type mice, stimulation with dsRNA during wounding induced an RA morphogen gradient that correlated to future areas of WIHN (n=4, P<0.01). The induction of RA by dsRNA was dependent upon TLR3 as the skin of TLR3-deficient mice had lower baseline RA levels and failed to induce RA following wounding (n=4, p<0.05). In addition, mice with keratinocyte-specific deletion (n=4-6, p<0.01) or global deletion (n=6-11, p<0.001) of RA receptor alpha (RARa) had an almost entirely absent WIHN and were resistant to exogenous administration of dsRNA (Poly I:C). Finally, exogenous RA induced hair follicle stem cell markers such as KRT15 and KRT19 (n=3, p<0.05) and rescued the impaired WIHN in TLR3-deficient mice (n=6-11, p<0.05). In a preliminary human trial, ablative CO2 laser wounding combined with retinoic acid appeared to induce the formation of small vellous follicles. Taken together, these results define a novel mechanism involving dsRNA/TLR3 innate immune responses and RA nuclear hormone receptor pathways in tissue regeneration.

Hey thank you very much @InBeforeTheCure

I found this clinical trial. We can see that Doctor Luis Garza is the principal investigator. Just to remember, he worked with Doctor Cotsarelis for many years (they have found together the role of PGD2)

Clinical trial began in January 2018 and ending in 2025 (Early Phase 1)

"Study to Determine Effect of Gentle Wounding to Stimulate Hair Follicle Neogenesis"
​

Brief Summary:
The investigators have extensive evidence in mouse that wounding leads to the generation of new hair follicles in the skin. This can be an important new therapy for patients with scarring, but especially those with alopecia.

The question is whether gentle wounding in human subjects can cause the generation of a new hair follicle.

The plan is to first carefully map a small area of the scalp without hair follicles. Investigators will then try various modalities of gentle wounding (including fractionated Carbon Dioxide (CO2) laser, mild curetting) of the surface epithelium in the presence and absence of FDA approved topical medications (including retinoids). Investigators will then prospectively monitor the area for hair growth both by noninvasive visual monitoring (including photographs and dermoscopy) and biopsies.

The outcomes of this study hopefully will allow new therapies for especially scarring alopecia conditions where hair follicles are completely lost and there are no current therapies.

source: https://clinicaltrials.gov/ct2/show/NCT03491267

 

[Ikkaku]

We need follica asap

 

[InBeforeTheCure]

Holy sh*t! You really have put in some work into this haven’t you?

Pretty tedious of course, but didn't take me all that long tbh.

 

[razzmatazz91]

Hey thank you very much @InBeforeTheCure

I found this clinical trial. We can see that Doctor Luis Garza is the principal investigator. Just to remember, he worked with Doctor Cotsarelis for many years (they have found together the role of PGD2)

Clinical trial began in January 2018 and ending in 2025 (Early Phase 1)

"Study to Determine Effect of Gentle Wounding to Stimulate Hair Follicle Neogenesis"
​

Brief Summary:
The investigators have extensive evidence in mouse that wounding leads to the generation of new hair follicles in the skin. This can be an important new therapy for patients with scarring, but especially those with alopecia.

The question is whether gentle wounding in human subjects can cause the generation of a new hair follicle.

The plan is to first carefully map a small area of the scalp without hair follicles. Investigators will then try various modalities of gentle wounding (including fractionated Carbon Dioxide (CO2) laser, mild curetting) of the surface epithelium in the presence and absence of FDA approved topical medications (including retinoids). Investigators will then prospectively monitor the area for hair growth both by noninvasive visual monitoring (including photographs and dermoscopy) and biopsies.

The outcomes of this study hopefully will allow new therapies for especially scarring alopecia conditions where hair follicles are completely lost and there are no current therapies.

View attachment 86468

source: https://clinicaltrials.gov/show/NCT03491267

Holy sh*t! Phase 1 trials will end in 2025?
That’s depressing.

Anyway, thanks for the info.

 

[dermrafok]

Holy sh*t! Phase 1 trials will end in 2025?
That’s depressing.

Anyway, thanks for the info.

Depressing... Why?

 

[hanginginthewire]

Depressing... Why?

2025 for the end of only Phase One? Yeah that’s depressing.

 

[Javell]

2025 for the end of only Phase One? Yeah that’s depressing.

Yeah, this means that that treatment is at least 20 years away.
I guess this is the new generation of coumpounds Follica talked about.

 

[itsjustsimon]

Depressing... Why?

I immediately get the picture in my head of someone reading this 20 years from now thinking to himself: “Damn this balding fuckers actually believed something would be out soon” yet he goes to another room to drop another pill of Propecia down his throat

 

[dermrafok]

Nobody knows what will happen ...

 

[lemoncloak]

We need follica asap

Shiseido will sh*t on follica. Maybe I'm wrong but calling it now.

 

[sktboiboi]

Hey inbeforethecure:

if u can tell me what the associated genes for these IncRNAs are, it's a major step towards figuring out what is primarily causing Androgenetic Alopecia- molecularly.


Most upregulated incRNAs in alopecic tissue when compared to normal tissue:

ASHGV40018997 239.8588454 Up T131417 G030772 RNA-seq 16 ND ND ND <== upregulated a whooping 239 folds in alopecic tissue

ASHGV40008202 74.2665386 Up T056221 G012997 RNA-seq 11 ND ND ND

ASHGV40005196 71.7819478 Up T008148 G001747 RNA-seq 1 ND ND ND

Most downregulated incRNAs in alopecic tissue when compared to normal tissue:

ASHGV40059635 44.8525426 Down TCONS_ 00020279 XLOC_ 009624 RNA-seq 12 Intergenic ND ND

ASHGV40041683 33.8073415 Down ENST0000 0565748 CTD2636A23.2 GENCODE 5 Natural antisense HMGCS1 Hydroxy methylglutarylCoA synthase <== about cholesterol biosynthesis and cellular responses to cholesterol

ASHGV40059356 29.3693894 Down TCONS_ 00006918 XLOC_ 002730 RNA-seq 3 Intergenic ND ND









Most upregulated mRNAs in alopecic scalp(so yes, Prostate cancer is indeed related to Androgenetic Alopecia by the same mechanisms)- but i cant figure out what the 2nd and 3rd does:

ASHGV40021887 69.7530454 Up NM_001282275 PRAC2 17 Hs.236557 Prostate cancer susceptibility candidate 2

ASHGV40032572 66.2385552 Up NM_181615 KRTAP20-1 21 Hs.553697 Keratin-associated protein 20-1

ASHGV40017687 30.3940994 Up NM_015161 ARL6IP1 16 Hs.634882 ADP ribosylation factor-like GTPase 6 interacting protein 1


Most downregulated mRNAs in alopecic tissue compared to normal tissue::

SPDYE4 17 Hs.368429 Speedy/RINGO cell cycle regulator family member E4

STARD9 15 Hs.122061 StAR-related lipid transfer domain-containing 9

CDH26 20 Hs.729046 Cadherin 26