Hair-bearing human skin generated entirely from pluripotent stem cells.

The skin is a multilayered organ, equipped with appendages (that is, follicles and glands), that is critical for regulating body temperature and the retention of bodily fluids, guarding against external stresses and mediating the sensation of touch and pain1,2. Reconstructing appendage-bearing skin in cultures and in bioengineered grafts is a biomedical challenge that has yet to be met3-9. Here we report an organoid culture system that generates complex skin from human pluripotent stem cells. We use stepwise modulation of the transforming growth factor ß (TGFß) and fibroblast growth factor (FGF) signalling pathways to co-induce cranial epithelial cells and neural crest cells within a spherical cell aggregate. During an incubation period of 4-5 months, we observe the emergence of a cyst-like skin organoid composed of stratified epidermis, fat-rich dermis and pigmented hair follicles that are equipped with sebaceous glands. A network of sensory neurons and Schwann cells form nerve-like bundles that target Merkel cells in organoid hair follicles, mimicking the neural circuitry associated with human touch. Single-cell RNA sequencing and direct comparison to fetal specimens suggest that the skin organoids are equivalent to the facial skin of human fetuses in the second trimester of development. Moreover, we show that skin organoids form planar hair-bearing skin when grafted onto nude mice. Together, our results demonstrate that nearly complete skin can self-assemble in vitro and be used to reconstitute skin in vivo. We anticipate that our skin organoids will provide a foundation for future studies of human skin development, disease modelling and reconstructive surgery.

Hair follicle epidermal stem cells define a niche for tactile sensation.

The heterogeneity and compartmentalization of stem cells is a common principle in many epithelia, and is known to function in epithelial maintenance, but its other physiological roles remain elusive. Here we show transcriptional and anatomical contributions of compartmentalized epidermal stem cells in tactile sensory unit formation in the mouse hair follicle. Epidermal stem cells in the follicle upper-bulge, where mechanosensory lanceolate complexes innervate, express a unique set of extracellular matrix (ECM) and neurogenesis-related genes. These epidermal stem cells deposit an ECM protein called EGFL6 into the collar matrix, a novel ECM that tightly ensheathes lanceolate complexes. EGFL6 is required for the proper patterning, touch responses, and αv integrin-enrichment of lanceolate complexes. By maintaining a quiescent original epidermal stem cell niche, the old bulge, epidermal stem cells provide anatomically stable follicle-lanceolate complex interfaces, irrespective of the stage of follicle regeneration cycle. Thus, compartmentalized epidermal stem cells provide a niche linking the hair follicle and the nervous system throughout the hair cycle.

Basal cell carcinoma preferentially arises from stem cells within hair follicle and mechanosensory niches.

Basal cell carcinoma (BCC) is characterized by frequent loss of PTCH1, leading to constitutive activation of the Hedgehog pathway. Although the requirement for Hedgehog in BCC is well established, the identity of disease-initiating cells and the compartments in which they reside remain controversial. By using several inducible Cre drivers to delete Ptch1 in different cell compartments in mice, we show here that multiple hair follicle stem cell populations readily develop BCC-like tumors. In contrast, stem cells within the interfollicular epidermis do not efficiently form tumors. Notably, we observed that innervated Gli1-expressing progenitors within mechanosensory touch dome epithelia are highly tumorigenic. Sensory nerves activate Hedgehog signaling in normal touch domes, while denervation attenuates touch dome-derived tumors. Together, our studies identify varying tumor susceptibilities among different stem cell populations in the skin, highlight touch dome epithelia as "hot spots" for tumor formation, and implicate cutaneous nerves as mediators of tumorigenesis.

Different requirements for GFRα2-signaling in three populations of cutaneous sensory neurons.

Many primary sensory neurons in mouse dorsal root ganglia (DRG) express one or several GFRα's, the ligand-binding receptors of the GDNF family, and their common signaling receptor Ret. GFRα2, the principal receptor for neurturin, is expressed in most of the small nonpeptidergic DRG neurons, but also in some large DRG neurons that start to express Ret earlier. Previously, GFRα2 has been shown to be crucial for the soma size of small nonpeptidergic nociceptors and for their target innervation of glabrous epidermis. However, little is known about this receptor in other Ret-expressing DRG neuron populations. Here we have investigated two populations of Ret-positive low-threshold mechanoreceptors that innervate different types of hair follicles on mouse back skin: the small C-LTMRs and the large Aß-LTMRs. Using GFRα2-KO mice and immunohistochemistry we found that, similar to the nonpeptidergic nociceptors, GFRα2 controls the cell size but not the survival of both C-LTMRs and Aß-LTMRs. In contrast to the nonpeptidergic neurons, GFRα2 is not required for the target innervation of C-LTMRs and Aß-LTMRs in the back skin. These results suggest that different factors drive target innervation in these three populations of neurons. In addition, the observation that the large Ret-positive DRG neurons lack GFRα2 immunoreactivity in mature animals suggests that these neurons switch their GFRα signaling pathways during postnatal development.

Perivascular hair follicle stem cells associate with a venule annulus.

The perivascular microenvironment helps in maintaining stem cells in many tissues. We sought to determine whether there is a perivascular niche for hair follicle stem cells. The association of vessels and follicle progenitor cells began by embryonic day 14.5, when nascent hair placodes had blood vessels approaching them. By birth, a vascular annulus stereotypically surrounded the keratin 15 negative (K15-) stem cells in the upper bulge and remained associated with the K15- upper bulge throughout the hair cycle. The angiogenic factor Egfl6 was expressed by the K15- bulge and was localized adjacent to the vascular annulus, which comprised post-capillary venules. Although denervation altered the phenotype of upper bulge stem cells, the vascular annulus persisted in surgically denervated mouse skin. The importance of the perivascular niche was further suggested by the fact that vascular annuli formed around the upper bulge of de novo-reconstituted hair follicles before their innervation. Together, these findings demonstrate that the upper bulge is associated with a perivascular niche during the establishment and maintenance of this specialized region of hair follicle stem cells.

Non-competitive metabotropic glutamate 1 receptor antagonists block activity of slowly adapting type I mechanoreceptor units in the rat sinus hair follicle.

Previous studies suggested that Group I metabotropic glutamate (mGlu) receptors play a role in mechanotransduction processes of slowly adapting type I mechanoreceptors. Using an isolated rat sinus hair follicle preparation we tested a range of compounds. Surprisingly, only non-competitive mGlu1 receptor antagonists produced profound and long-lasting depression of mechanically evoked firing. 6-Amino-N-cyclohexyl-N,3-dimethylthiazolo[3,2-alpha]benzimidazole-2-carboxamide hydrochloride (YM-298198) had an IC(50) of 8.7 muM (95% CI 5.7 to 13.2 microM), representing the most potent known blocker of type I mechanoreceptors. The derivative 6-amino-N-cyclohexyl-3-methylthiazolo[3,2-alpha]benzimidazole-2-carboxamide hydrochloride (desmethyl YM-298198) had a comparable potency. Another compound 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) had a similar depressant effect, although it was less potent with an approximate IC(50) of 100 microM. Between three and seven times the concentration of CPCCOEt and YM-298198 respectively was required to produce similar depressions in slowly adapting type II units. No depression, and some weak excitatory effects, were observed using the following ligands: the competitive mGlu1 receptor antagonist alpha-amino-5-carboxy-3-methyl-2-thiopheneacetic acid (3-MATIDA) (300 microM), the phosphoserine phosphatase inhibitor dl-2-amino-3-phosphonopropionic acid (dl-AP3) (2 mM), non-competitive mGlu5 receptor antagonists 3-((2-methyl-1,3-thiazol-4-yl)ethynyl)pyridine; (S)-3,5-DHPG, (S)-3,5-dihydroxyphenylglycine (MTEP) (10 microM) and 2-methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) (100 microM), the mGlu1 receptor agonist (S)-3,5-dihydroxyphenylglycine ((S)-3,5-DHPG) (500 microM), and the mGlu5 receptor agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) (1 mM). The results suggest that the non-competitive mGlu1 receptor antagonists are not acting at conventional mGlu1 receptors but at other binding sites, possibly those directly associated with mechanogated channels or on any of a number of indirect biochemical pathways. YM-298198 and related compounds may prove to be useful ligands to identify mechanosensitive channel proteins. The selective interference of type I units may provide further evidence that Merkel cells are mechanotransducers. Finally such compounds may deliver insights or treatments for Merkel cell carcinoma.

Myelinated nerve endings in human skin.

We used immunohistochemical techniques and confocal microscopy to study the morphometry of myelinated nerve endings in glabrous and hairy skin. A total of 30 healthy volunteers took part in this study designed to assess the possibility of obtaining reliable information on myelinated fibers using samples of hairy skin and to determine whether differences exist between myelinated terminations from different sites. We obtained consistent information on cutaneous myelinated terminations using hairy as well as glabrous skin samples. Myelinated endings from hairy and glabrous skin differ in density and distribution. However, from a comparison of our findings with data from nerve biopsy studies, we conclude that all cutaneous myelinated terminations are thinner terminal branches of large myelinated A beta fibers, whereas cutaneous terminations of small myelinated A delta fibers lose their myelin before entering the dermis and become indistinguishable from C-fiber terminations. The classic criteria, based on fiber size, used to distinguish myelinated fiber subgroups in sensory nerves are therefore not suitable for identifying myelinated terminations in the skin.

Substance P as an immunomodulatory neuropeptide in a mouse model for autoimmune hair loss (alopecia areata).

Alopecia areata (AA) is an autoimmune disorder of the hair follicle characterized by inflammatory cell infiltrates around actively growing (anagen) hair follicles. Substance P (SP) plays a critical role in the cutaneous neuroimmune network and influences immune cell functions through the neurokinin-1 receptor (NK-1R). To better understand the role of SP as an immunomodulatory neuropeptide in AA, we studied its expression and effects on immune cells in a C3H/HeJ mouse model for AA. During early stages of AA development, the number of SP-immunoreactive nerve fibers in skin is increased, compared to non-affected mice. However, during advanced stages of AA, the number of SP-immunoreactive nerves and SP protein levels in skin are decreased, whereas the expression of the SP-degrading enzyme neutral endopeptidase (NEP) is increased, compared to control skin. In AA, NK-1R is expressed on CD8+ lymphocytes and macrophages accumulating around affected hair follicles. Additional SP supply to the skin of AA-affected mice leads to a significant increase of mast cell degranulation and to accelerated hair follicle regression (catagen), accompanied by an increase of CD8+ cells-expressing granzyme B. These data suggest that SP, NEP, and NK-1R serve as important regulators in the molecular signaling network modulating inflammatory response in autoimmune hair loss.

Reduction of postincisional allodynia by subcutaneous bupivacaine: findings with a new model in the hairy skin of the rat.

BACKGROUND: An incision of hairy skin of the rat's back provides a new model for postincisional pain to determine the importance of cutaneous anesthesia. METHODS: Male Sprague-Dawley rats were anesthetized with sevoflurane and given a 0.6-ml subcutaneous injection of bupivacaine (0.25%) under the incision site or the medial lumbar dorsum or at the nuchal midline, 30 min before a 1.0-cm skin incision. Mechanical stimuli (von Frey hairs, 18-250 mN) were applied to measure nociception, indicated by twitching of local subcutaneous muscles, the cutaneus trunci muscle reflex. A graded response score, averaging the twitches weighted by their vigor, or a population response score, measuring the fraction of rats that showed any response, was assessed for 3 days before and over 7 days after incision. von Frey hairs were applied 0.5 cm from the incision to test primary hyperalgesia and 2.0 cm contralateral to the incision for secondary hyperalgesia. RESULTS: Incision induced responses to stimuli that had no effect on intact skin (allodynia) and also enhanced responses to forces that normally gave less than the full reflex (hyperalgesia). Hyperalgesia was present 30 min after surgery, peaked at 3-6 h, and persisted through the week; allodynia had a similar onset but was briefer. Both changes were transiently reversed by subcutaneous morphine (2.5 mg/kg intraperitoneal). Subcutaneous bupivacaine (0.25%), injected preoperatively at the incision site and anesthetizing skin for 2-3 h, suppressed primary allodynia for 1 week but had no effect on hyperalgesia. Secondary allodynia was obliterated, and secondary hyperalgesia attenuated by this treatment. Bupivacaine injected subcutaneously at the nuchal midline before surgery was also effective in abbreviating primary and secondary allodynia, with no signs of sedation, ataxia, or preconvulsive behavior. CONCLUSIONS: Incision of rat hairy skin changes pain responses, similar to pain in humans. Preincisional subcutaneous bupivacaine selectively suppresses and shortens allodynia for times far outlasting its local anesthesia, an effect largely from systemic actions.

Immunohistochemical reactions of receptors to met-enkephalin, VIP, substance P, and CGRP located on Merkel cells in the rat sinus hair follicle.

The role of Merkel cells in type I cutaneous mechanoreceptors remains enigmatic though mechanical transduction or neuromodulation function has been proposed. It has been shown that mammalian Merkel cells express immunohistochemical reactions of met-enkephalin, VIP, substance P, and CGRP, though the reactivity differs between species. If any one of these peptides acts as a transmitter or modulator for Merkel nerve terminals, these structures must have a specific receptor for the substance. We therefore studied the immunohistochemical localization of the above-mentioned neuropeptides and their receptors in Merkel cell-nerve endings in rat whisker pads. Specimens were doubly stained with polyclonal antibodies to neuropeptides and their receptors combined with a monoclonal antibody to cytokeratin 20, which was used for the labeling of Merkel cells. Merkel cells in the rat sinus hair follicles showed positive immunoreactions for all peptides studied, whereas the immunoreactions of receptors to these peptides were localized on Merkel cell membranes but not on the axon terminals. These results suggest that neuropeptides released from Merkel cells act on Merkel cells themselves by an autocrine mechanism.

Three-dimensional imaging of human skin and mucosa by two-photon laser scanning microscopy.

BACKGROUND: Various structural components of human skin biopsy specimens are difficult to visualize using conventional histologic approaches. METHODS: We used two-photon microscopy and advanced imaging software to render three-dimensional (3D) images of in situ nerves, blood vessels, and hair follicles labeled with various fluorescent markers. Archived frozen human skin biopsy specimens were cryosectioned up to 150 micro m in thickness and fluorescently stained with rhodamine- or fluorescein-labeled antibodies or lectins. Optical sections were collected by two-photon microscopy and the resulting data sets were analyzed in three dimensions using Voxx software. RESULTS: Reconstructed image volumes demonstrated the complex 3D morphology of nerves, blood vessels and adnexal structures in normal mucocutaneous tissue. CONCLUSION: Two-photon microscopy and Voxx rendering software allow for detailed 3D visualization of structures within human mucocutaneous biopsy specimens, as they appear in situ, and facilitate objective interpretation of variations in their morphology. These techniques may be used to investigate disorders involving cutaneous structures that are difficult to visualize by means of traditional microscopy.

Hair-cycle-associated remodeling of the peptidergic innervation of murine skin, and hair growth modulation by neuropeptides.

As the neuropeptide substance P can manipulate murine hair growth in vivo, we here further studied the role of sensory neuropeptides in hair follicle biology by determining the distribution and hair-cycle-dependent remodeling of the sensory innervation in C57BL/6 mouse back skin. Calcitonin-gene-related peptide, substance P, and peptide histidine methionine (employed as vasoactive intestinal peptide marker) were identified by immunohistochemistry. All of these markers immunolocalized to bundles of nerve fibers and to single nerve fibers, with distinct distribution patterns and major hair-cycle-associated changes. In the epidermis and around the distal hair follicle and the arrector pili muscle, only calcitonin-gene-related peptide immunoreactive nerve fibers were visualized, whereas substance P and peptide histidine methionine immunoreactive nerve fibers were largely restricted to the dermis and subcutis. Compared to telogen skin, the number of calcitonin-gene-related peptide, substance P, and peptide histidine methionine immunoreactive single nerve fibers increased significantly (p < 0.01) during anagen, including around the bulge region (the seat of epithelial stem cells). Substance P significantly accelerated anagen progression in murine skin organ culture, whereas calcitonin-gene-related peptide and a substance-P-inhibitory peptide inhibited anagen (p < 0.05). The inhibitory effect of calcitonin-gene-related peptide could be antagonized by coadministrating substance P. In contrast to substance P, calcitonin-gene-related peptide failed to induce anagen when released from subcutaneous implants. This might reflect a differential functional assignment of the neuropeptides calcitonin-gene-related peptide and substance P in hair growth control, and invites the use of neuropeptide receptor agonists and antagonists as novel pharmacologic tools for therapeutic hair growth manipulation.

Regulation of NGF-family ligands and receptors in adulthood and senescence: correlation to degenerative and regenerative changes in cutaneous innervation.

During development, a highly differential neurotrophin dependency is reported for various types of nerve endings in the whisker follicle. To what extent these dependencies extend and play a role in adulthood is largely unresolved. We show here, using in situ hybridization and immunohistochemistry that the expression of neurotrophins and trk/p75 receptors persists in adulthood. As suggested by their expression profiles, many classes of cutaneous nerve endings disclose similar ligand-receptor dependencies in adult animals as during development, while other populations appear to switch their dependency. Furthermore, our data suggest that sensory endings that have a high turnover due to mechanical wear and tear, e. g. Merkel cell-neurite complexes at the level of ring sinus show a more complex ligand-receptor expression phenotype than do endings with a less vulnerable location, e.g. the Merkel cell-neurite complexes at the rete ridge collar. Thus, neurotrophin-3 (NT3)/trkA signalling is suggested to be important for a continuous terminal plasticity of Merkel cell-neurite complexes at the level of ring sinus in adulthood. Evidence supporting a role for neurotrophin signalling in maintaining the adult cutaneous innervation also comes from the close correlation between altered ligand-receptor expression(s) and axonal/terminal aberrations in senescence. Thus, an ageing-related decrease in target neurotrophin expression, notably NT3 and NT4, results in a site-specific loss of sensory terminals concomitant with an aberrant growth of regenerating/sprouting axons into new target fields. Ageing of the cutaneous innervation, manifested in degenerative and regenerative events, seems strongly associated with changes in neurotrophic interactions between sensory neurons and target tissues.

Vasoactive intestinal polypeptide (VIP) innervation of the human eyelid glands.

This study was conducted to obtain morphological proof of innervating nerve fibres in the glands of the human eyelid (accessory lacrimal glands of Wolfring, meibomian glands, goblet cells, glands of Zeis, glands of Moll, sweat glands, glands of lanugo hair follicles) and identification of the secretomotorically active neuropeptide vasoactive intestinal polypeptide (VIP) as a common transmitter. Epoxy-embedded ultrathin sections of tissue samples from human eyelids were studied using electron microscopy. Paraffin sections fixed in Bouin-Hollande solution were immunostained with rabbit antiserum against VIP. With the electron microscope we were able to identify nerves in the glandular stroma of all the glands examined with the exception of goblet cells. Intraepithelial single axons were only seen in the parenchyma of Wolfring glands. The morphological findings corresponded with the immunological finding of VIP-positive, nerve-like structures in the same locations, with the exception of lanugo hair follicle glands, and goblet cells. Our findings indicate that the glands of the eyelids and main lacrimal gland represent a functional unit with VIP as a possible common stimulating factor.

A rapid and dynamic regulation of GDNF-family ligands and receptors correlate with the developmental dependency of cutaneous sensory innervation.

Glial cell line-derived neurotrophic factor (GDNF) and neurturin (NTN) are members of the transforming growth factor-beta family and have been shown to elicit neurotrophic effects upon several classes of neurons including dopaminergic neurons, motoneurons, parasympathetic, sympathetic as well as primary sensory neurons. However, there is little information available on their roles in cutaneous innervation. Herein, we have studied the regulation of gdnf, ntn and the GDNF family receptors and examined their role in the development of facial cutaneous innervation in GDNF mutant mice. A dynamic spatial and temporal regulation of gdnf, ntn and their ligand binding receptors within the follicle-sinus complex correlate with development of distinct subclasses of sensory nerve endings. Furthermore, development of NGF-dependent myelinated mechanoreceptors, i.e. reticular and transverse lanceolate endings also require GDNF during ending formation and maintenance. In addition, ligand and receptor association seems to be intricately linked to a local Schwann cell-axon interaction essential for sensory terminal formation. Our results suggests that functionally specified nerve endings depend on different GDNF family members and that in contrast to neurotrophins, this family of neurotrophic factors may be acting at local sites of terminal Schwann cell-axon growth cone interactions and that they collaborate with neurotrophins by supporting the same populations of neurons but at different times in development.

Cytoplasmic Ca2+ concentrations in intact Merkel cells of an isolated, functioning rat sinus hair preparation.

An isolated, functioning sinus hair preparation was developed to investigate cytoplasmic Ca2+ concentrations in intact Merkel cells using microfluorimetric techniques. Intracellular Ca2+ levels were monitored by means of photon counters in small groups of Merkel cells loaded with the calcium fluorescent indicators fura-2 or fluo-3. Mechanical stimulation of Merkel cells with fine glass rods resulted in small transient increases in intracellular Ca2+ levels (by about 20%) in the group of Merkel cells around the stimulating probe. A rise in Ca2+ is presumed to be essential for the postulated synaptic transmission to the afferent nerve terminal. Depolarization with a high concentration of potassium chloride (100 mM) caused increases in intracellular Ca2+ concentrations in Merkel cells (by about 70%) only in the presence of extracellular Ca2+, indicating an influx of Ca2+ through voltage-gated channels. The Ca2+ response was abolished neither by (+)-BayK8644 nor omega-conotoxin, suggesting that the Ca2+ channels are different from the classical L- or N-type channels. Extracellular application of ATP (10 microM to 5 mM) caused dose-dependent increases in intracellular Ca2+ levels in Merkel cells of up to sevenfold from the basal level of about 100 nM. Similar responses to ATP were also measured during superfusion with Ca(2+)-free medium, suggesting intracellular stores as the main Ca2+ source. Pre-incubation of Merkel cells with the purinoceptor antagonist suramin (100 microM) for 30 min reduced the Ca2+ responses to ATP by about 50% compared with control conditions. In conclusion, the results have demonstrated that a rise in intracellular Ca2+ in Merkel cells can be evoked by mechanical stimulation, membrane depolarization and chemical stimulation by ATP. These observations strongly suggest a possible contribution of Ca2+ to the normal responsiveness of Merkel cell mechanoreceptors, in turn supporting the hypothesis that Merkel cells are involved in the mechano-electric transduction process in sinus hair type I mechanoreceptors.