Hair Follicle Germ Research Articles

Large-Scale Preparation of Hair Follicle Germs Using a Microfluidic Device.

Hair follicle morphogenesis during embryonic development is driven by the formation of hair follicle germs (HFGs) via interactions between epithelial and mesenchymal cells. Bioengineered HFGs are potential tissue grafts for hair regenerative medicine because they can replicate interactions and hair follicle morphogenesis after transplantation. However, a mass preparation approach for HFGs is necessary for clinical applications, given that thousands of de novo hair follicles are required to improve the appearance of a single patient with alopecia. In this study, we developed a microfluidics-based approach for the large-scale preparation of HFGs. A simple flow-focusing microfluidic device allowed collagen solutions containing epithelial and mesenchymal cells to flow and generate collagen microbeads with distinct Janus structures. During the 3 days of culture, the collagen beads contracted owing to cellular traction forces, resulting in collagen- and cell-dense HFGs. The transplantation of HFGs into nude mice resulted in highly efficient de novo hair follicle regeneration. This method provides a scalable and robust tissue graft preparation approach for hair regeneration.

Cryopreservation of engineered hair follicle germs for hair regenerative medicine

Hair regenerative medicine must involve practical procedures, such as cryopreservation of tissue grafts. This can aid in evaluating tissue safety and quality, as well as transportation to a clinic and multiple transplants. Hair follicle germs (HFGs), identified during invivo development, are considered effective tissue grafts for hair regenerative medicine. However, to the best of our knowledge, methods for cryopreserving HFGs have not been explored yet. This study investigated the efficacy of slow vitrification methods for freezing HFGs. Cryoprotectants such as dimethyl sulfoxide (DMSO) and carboxylated poly-l-lysine were used for vitrification. The results indicate that DMSO vitrification yielded the most efficient de novo hair regeneration in mouse skin, comparable to that of non-cryoprotected HFGs. A microfinger was fabricated to scale up the cryopreservation method, considering that thousands of tissue grafts were required per patient in clinical practice. The microfinger can be used for a series of processes, holding the HFG, replacing it with a cryopreservation solution, freezing it in liquid nitrogen, thawing it in a warm medium, and transplanting it into the skin. Although de novo hair regeneration by HFGs cryopreserved using microfingers was reduced by approximately 20 % compared to those cryopreserved using flat plates for fertilized eggs, it exceeded 50 %. These findings demonstrate that vitrification with DMSO and microfingers could be a useful approach for the cryopreservation of tissue grafts in hair regenerative medicine for hair loss.

Bioprinting of hair follicle germs for hair regenerative medicine.

Hair regenerative medicine is a promising approach to treat hair loss. The replication of in vivo tissue configurations and microenvironments, such as hair follicle germs, has been studied to prepare tissue grafts for hair regenerative medicine. However, such approaches should be scalable, because a single patient with alopecia requires thousands of tissue grafts. In this paper, we propose an approach for the scalable and automated preparation of highly hair-inductive tissue grafts using a bioprinter. Two collagen droplets (2µL each) containing mesenchymal and epithelial cells were placed adjacent to each other to fabricate hair-follicle-germ-like grafts. During three days of culture, the pairs of microgel beads were spontaneously contracted by cell traction forces, whereas the two cell types remained separated, where the densities of the cells and collagen were enriched more than 10 times. This approach allowed us to fabricate submillimeter objects printed with millimeter-order accuracy, facilitating scalable and automated tissue graft preparation. Because of mesenchymal-epithelial interactions, hair microgels (HMGs, i.e., collagen- and cell-enriched microgels) efficiently regenerate hair follicles and shafts when transplanted into the back skin of mice. However, the generated hair shafts mostly remain under the skin. Therefore, we printed microgel beads onto surgical suture guides arrayed on a stage. The microgel beads were contracted along with the suture guides in culture prior to transplantation. The guide-inserted HMGs significantly improved hair-shaft sprouting through the skin, owing to the control of the orientation of the HMGs transplanted into the skin. This approach is a promising strategy to advance hair regenerative medicine. STATEMENT OF SIGNIFICANCE: This study proposes an approach for the scalable and automated preparation of highly hair-inductive grafts using a bioprinter. Two collagen droplets containing mesenchymal and epithelial cells were placed adjacently. Cell traction forces caused the pairs of microgel beads to spontaneously contract in culture. Because of mesenchymal-epithelial interactions, hair microgels (HMGs) efficiently regenerated hair follicles on the back skin of mice. However, the generated hair shafts remained mostly beneath the skin. Therefore, we printed microgel beads onto surgical suture guides arrayed on a stage. The guide-inserted HMGs significantly improved hair-shaft sprouting through the skin owing to the control of the orientation of the HMGs in the skin. This approach represents a promising strategy for advancing hair regenerative medicine.

Art’s Work in the Age of Biotechnology: Shaping Our Genetic Futures

Art’s Work in the Age of Biotechnology: Shaping Our Genetic Futures

Bioprinting of Hair Follicle Germs for Hair Regenerative Medicine

Bioprinting of Hair Follicle Germs for Hair Regenerative Medicine

Trichoblastoma and trichoblastic carcinoma in the head and neck region

Trichoblastomas are benign cutaneous adnexal tumours which originate in the hair follicle germs. They are considered to be extremely rare and usually present in the head and neck region. Clinically, they present as flesh-coloured nodular growths mimicking the more commonly found basal cell carcinoma (Stanoszek et al., 2017 Nov) [1]. These tumours can potentially undergo malignant transformation which again is extremely infrequent.

Impact of adipose-derived stem cells on engineering hair follicle germ-like tissue grafts for hair regenerative medicine

Hair regenerative medicine has emerged as a promising treatment strategy for severe hair loss, such as end-stage androgenetic alopecia. Various approaches to engineering three-dimensional tissue grafts have been explored since they drive the ability to regenerate hair follicles when transplanted. In the present study, we demonstrated the assembly of human adipose-derived stem cells (hASCs) into hair follicle germ (HFG)-like aggregates for de novo hair regeneration. We mixed human dermal papilla cells (hDPCs), murine embryonic epithelial cells, and hASCs in suspension, and allowed them to form aggregates. During three days of culture, cells initially formed a single aggregate with a random distribution of the three cell types, but the epithelial and dermal papilla cells subsequently separated from each other and formed a dumbbell-shaped HFG, with hASCs localized on the hDPC aggregate side. The involvement of hASCs significantly increased gene expression associated with hair morphogenesis compared to HFGs without hASCs. The self-organization of the three cell types was observed in our scalable lab-made chip device. HFGs containing hASCs efficiently generated hair shafts upon transplantation to nude mice, while only a few shafts were generated with HFGs without hASCs. This approach may be a promising strategy for fabricating tissue grafts for hair regenerative medicine.

Hair follicle germs containing vascular endothelial cells for hair regenerative medicine

Hair regenerative medicine has emerged as a promising approach for the treatment of severe hair loss. Recent advances in three-dimensional tissue engineering, such as formation of hair follicle germs (HFGs), have considerably improved hair regeneration after transplantation in animal models. Here, we proposed an approach for fabricating HFGs containing vascular endothelial cells. Epithelial, dermal papilla, and vascular endothelial cells initially formed a single aggregate, which subsequently became a dumbbell-shaped HFG, wherein the vascular endothelial cells localized in the region of dermal papilla cells. The HFGs containing vascular endothelial cells exhibited higher expression of hair morphogenesis-related genes in vitro, along with higher levels of hair shaft regeneration upon transplantation to the dorsal side of nude mice, than those without vascular endothelial cells. The generated hair follicles represented functional characteristics, such as piloerection, as well as morphological characteristics comparable to those of natural hair shafts. This approach may provide a promising strategy for fabricating tissue grafts with higher hair inductivity for hair regenerative medicine.

Effects of platelet-rich plasma on in vitro hair follicle germ preparation for hair regenerative medicine

Hair regenerative medicine is a promising approach for the treatment of hair loss and involves the transplantation of follicular stem cells into bald spots to regenerate hair. Various approaches have been investigated to engineer tissue grafts for use in hair regenerative medicine. Tissue-like three-dimensional aggregates, such as bioengineered hair follicle germs (HFGs), have shown great promise for hair regeneration, with normal tissue morphology and hair cycles. However, these approaches have not yet been applied in clinical settings, and further studies are needed to improve hair generation efficiency. The biological molecules in invivo microenvironments around HFGs may provide cues for the invitro preparation of HFGs with higher trichogenic functionalities. Activated platelet-rich plasma releasate (PRPr) is an autologous source of signaling molecules including growth factors and cytokines. In this study, we investigated the effects of PRPr on the preparation of HFGs invitro. The presence of PRPr did not hinder the spontaneous formation of dumbbell-like HFGs from a suspension of embryonic skin-derived epithelial and mesenchymal cells in a custom-designed HFG culture plate. HFGs prepared with PRPr displayed greater levels of follicular gene expression compared to those prepared in the absence of PRPr. Moreover, the hair regeneration ability upon intracutaneous transplantation was significantly improved in the presence of PRPr. These results suggest that PRPr is beneficial for engineering HFGs for autologous hair regenerative medicine.

Generation of Hair Follicle Germs In Vitro Using Human Postnatal Skin Cells.

Modeling organoids with hair follicle germ-like properties provides an opportunity for developing strategies for alopecia drug discovery and replacement therapy, as well as investigating themolecular mechanisms underlying human hair follicle regeneration in vitro. Hair follicle germ reconstruction in vitro is based on dermal papilla hair-inducing abilities and the plasticity of skin epidermal keratinocytes. The current protocol describes a highly efficient approach suitable for adult human skin cell applications. This method allows to obtain hair follicle germs using tissues from one donor. Isolated and cultured for 2weeks, adult hair follicle dermal papilla cells and skin epidermal keratinocytes self-organize in hanging drop cultures generating organoids that exhibit the features of folliculogenesis onset.

Preparation of hair beads and hair follicle germs for regenerative medicine

Hair regenerative medicine is a promising approach for hair loss, during which autologous follicular stem cells are transplanted into regions of hair loss to regenerate hairs. Because cells transplanted as a single cell suspension scarcely generate hairs, the engineering of three-dimensional (3D) tissues before transplantation has been explored to improve this process. Here, we propose an approach to fabricate collagen-enriched cell aggregates, named hair beads (HBs), through the spontaneous constriction of cell-encapsulated collagen drops. Mouse embryonic mesenchymal cells or human dermal papilla cells were encapsulated in 2-μl collagen microgels, which were concentrated >10-fold in volume during 3 days of culture. Interestingly, HB constriction was attributed to attraction forces driven by myosin II and involved the upregulation of follicular genes. Single HBs with epithelial cells seeded in U-shaped microwells formed dumbbell-like structures comprising respective aggregates (named bead-based hair follicle germs, bbHFGs), during 3 days of culture. bbHFGs efficiently generated hair follicles upon intracutaneous transplantation into the backs of nude mice. Using an automated spotter, this approach was scalable to prepare a large number of bbHFGs, which is important for clinical applications. Therefore, this could represent a robust and practical approach for the preparation of germ-like tissues for hair regenerative medicine.

Spontaneous hair follicle germ (HFG) formation in vitro, enabling the large-scale production of HFGs for regenerative medicine

Hair follicle morphogenesis is triggered by reciprocal interactions between hair follicle germ (HFG) epithelial and mesenchymal layers. Here, we developed a method for large-scale preparation of HFGs in vitro via self-organization of cells. We mixed mouse epidermal and mouse/human mesenchymal cells in suspension and seeded them in microwells of a custom-designed array plate. Over a 3-day culture period, cells initially formed a randomly distributed single cell aggregate and then spatially separated from each other, exhibiting typical HFG morphological features. These self-sorted hair follicle germs (ssHFGs) were shown to be capable of efficient hair-follicle and shaft generation upon intracutaneous transplantation into the backs of nude mice. This finding facilitated the large-scale preparation of approximately 5000 ssHFGs in a microwell-array chip made of oxygen-permeable silicone. We demonstrated that the integrity of the oxygen supply through the bottom of the silicone chip was crucial to enabling both ssHFG formation and subsequent hair shaft generation. Finally, spatially aligned ssHFGs on the chip were encapsulated into a hydrogel and simultaneously transplanted into the back skin of nude mice to preserve their intervening spaces, resulting in spatially aligned hair follicle generation. This simple ssHFG preparation approach is a promising strategy for improving current hair-regenerative medicine techniques.

Functional Hair Follicle Regeneration by the Rearrangement of Stem Cells.

Hair follicles develop from the ectoderm in embryos and cyclically regenerate using proper spatiotemporal signaling molecules, which are conserved in organogenesis during adulthood. Previously, we demonstrated that bioengineered hair follicle germs could regenerate functional hair follicles via a three-dimensional cell manipulation technique, which we named the "organ germ method ." We could also regulate the type of hair follicle and pigmentation with correct structures by rearranging the source of the cells. In this article, we describe a detailed protocol for the regeneration of functional hair follicles and their stem cell niches by the rearrangement of embryonic or adult hair follicle-derived epithelial and mesenchymal cells.

Hair Follicle Regeneration by Transplantation of a Bioengineered Hair Follicle Germ.

Hair follicle morphogenesis is first induced by epithelial-mesenchymal interactions in the developing embryo. In the hair follicle, various stem-cell populations are maintained in specialized niches to promote repetitive hair follicle-morphogenesis, which is observed in the variable lower region of the hair follicle as a postnatal hair cycle. In contrast, the genesis of most organs is induced only once during embryogenesis. We developed a novel bioengineering technique, the Organ Germ Method, that employs three-dimensional stem cell culture for regenerating various organs and reproducing embryonic organogenesis. In this chapter, we describe a protocol for hair follicle germ reconstitution using adult follicle-derived epithelial stem cells and dermal papilla cells with intracutaneous transplantation of the bioengineered hair-follicle organ germ. This protocol can be useful not only for the clinical study of hair regeneration but also for studies of stem cell biology and organogenesis.

Hair organ regeneration via the bioengineered hair follicular unit transplantation

Organ regenerative therapy aims to reproduce fully functional organs to replace organs that have been lost or damaged as a result of disease, injury, or aging. For the fully functional regeneration of ectodermal organs, a concept has been proposed in which a bioengineered organ is developed by reproducing the embryonic processes of organogenesis. Here, we show that a bioengineered hair follicle germ, which was reconstituted with embryonic skin-derived epithelial and mesenchymal cells and ectopically transplanted, was able to develop histologically correct hair follicles. The bioengineered hair follicles properly connected to the host skin epithelium by intracutaneous transplantation and reproduced the stem cell niche and hair cycles. The bioengineered hair follicles also autonomously connected with nerves and the arrector pili muscle at the permanent region and exhibited piloerection ability. Our findings indicate that the bioengineered hair follicles could restore physiological hair functions and could be applicable to surgical treatments for alopecia.

Abstract SY37-01: Cilia and wounding cooperate with Hedgehog signaling to initiate basal cell carcinoma

Abstract Primary cilia are microtubule-based projections found on many human cells. These cilia do not move, but function as cellular antennae to receive and interpret signals. For example, olfactory cilia sense odorants in the environment. Other cilia sense cues from other cells, such as Hedgehog proteins, to coordinate tissue patterning and growth. We have identified mechanisms of ciliary signaling, cilium formation, and the regulation of ciliary composition. Defects in ciliary signaling cause some forms of birth defects, cancer, polycystic kidney disease, and other disorders collectively called ciliopathies. To examine whether cilia can participate in disorders beyond recognized ciliopathies, we assessed the prevalence of cilia in human tumors and the role of cilia in cancer. Human basal cell carcinomas (BCCs) are associated with misactivation of Hedgehog signaling, commonly through acquisition of mutations in Smoothened. We found that human BCCs are frequently ciliated. We tested the role of cilia in BCC by conditionally deleting genes required for ciliogenesis in a Hedgehog pathway-dependent mouse model of BCC. Ciliary ablation strongly inhibited BCC-like tumors induced by misactivation of the Hedgehog pathway at the level of Smoothened, revealing that BCC can be addicted to cilia. Skin tumors, like many other cancers, are associated with injury. Although stem cells participate in tissue regeneration after wounding, it is unclear whether these cells also contribute to epithelial tumors. Surprisingly, expression of an activated form of Smoothened by stem cells of the hair-follicle bulge and secondary hair germ does not induce robust Hedgehog signaling or produce BCCs. However, wounding recruits these cells from the follicle to the wound site, where downstream Hedgehog signal transduction is derepressed, giving rise to superficial BCC-like tumors. Thus, BCCs can originate from follicular stem cells and wounding can promote tumorigenesis by moving oncogene-expressing stem cells out of a suppressive niche. As we found that oncogenic Smoothened requires the cilium to promote tumorigenesis, we investigated how Smoothened moves to the cilium. We found that proteins of the Tectonic family form part of the transition zone, a region at the base of the cilium. Tectonic1 is an extracytosolic glycoprotein that interacts with transmembrane components of the transition zone to connect to a cytosolic transition zone complex comprised of the products of genes associated with two ciliopathies, Joubert and Meckel syndromes. Loss of components of the Tectonic transition zone complex in mice compromise ciliogenesis in some tissues, but in others, it is required for Smoothened and select other membrane proteins to localize to cilia. A complementary small molecule screen is identifying new chemicals that also prevent Smoothened localization to cilia and block oncogenic Hedgehog signaling. Interfering with the cellular machinery that moves Smoothened to cilia may be a complementary means of treating Hedgehog pathway-associated cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY37-01. doi:1538-7445.AM2012-SY37-01

Shh is required for Tabby hair follicle development

In embryonic Eda mutant ("Tabby") mice, the development of one of the two major types of hair, "primary" hair fails, but other "secondary" hairs develop in normal numbers, though shorter and slightly aberrant. In Tabby mice, Shh is undetectable in skin early on, but is activated during secondary hair formation. We inferred that Shh may be involved in primary hair formation, activated normally by Eda, and also possibly in secondary hair formation, activated by an Eda-independent pathway. Varying the dosage of Shh now supports these inferences. In Shh knockout mice, mice were totally hairless: primary and secondary hair follicle germs were formed, but further progression failed. Consistent with these findings, when Shh loss was restricted to the skin, secondary hair follicle germs were initiated on time in Tabby mice, but their subsequent development (down-growth) failed. An Shh transgene expressed in Tabby skin could not restore induction of primary hair follicles, but restored normal length to the somewhat aberrant secondary hair that was formed and prolonged the anagen phase of hair cycling. Thus, Shh is required for primary and secondary hair down-growth and full secondary hair length, but is not itself sufficient to replace Eda or make fully normal secondary hair.

Receptor activator of NF-κB (RANK) stimulates the proliferation of epithelial cells of the epidermo-pilosebaceous unit

Receptor activator of NF-κB (RANK), known for controlling bone mass, has been recognized for its role in epithelial cell activation of the mammary gland. Because bone and the epidermo-pilosebaceous unit of the skin share a lifelong renewal activity where similar molecular players operate, and because mammary glands and hair follicles are both skin appendages, we have addressed the function of RANK in the hair follicle and the epidermis. Here, we show that mice deficient in RANK ligand (RANKL) are unable to initiate a new growth phase of the hair cycle and display arrested epidermal homeostasis. However, transgenic mice overexpressing RANK in the hair follicle or administration of recombinant RANKL both activate the hair cycle and epidermal growth. RANK is expressed by the hair follicle germ and bulge stem cells and the epidermal basal cells, cell types implicated in the renewal of the epidermo-pilosebaceous unit. RANK signaling is dispensable for the formation of the stem cell compartment and the inductive hair follicle mesenchyme, and the hair cycle can be rescued by Rankl knockout skin transplantation onto nude mice. RANKL is actively transcribed by the hair follicle at initiation of its growth phase, providing a mechanism for stem cell RANK engagement and hair-cycle entry. Thus, RANK-RANKL regulates hair renewal and epidermal homeostasis and provides a link between these two activities.

Beta-catenin-dependent and -independent effects of DeltaN-plakoglobin on epidermal growth and differentiation.

Both beta-catenin and plakoglobin can stimulate the expression of Lef/Tcf target genes in vitro. beta-Catenin is known to associate with Lef/Tcf factors and to participate directly in transactivation in vivo, whereas the role of plakoglobin in transcriptional regulation has been less studied. To analyze the functions of plakoglobin in vivo, we generated transgenic mice expressing in the epidermis N-terminally truncated plakoglobin (DeltaN122-PG) lacking the glycogen synthase kinase 3beta phosphorylation sites and therefore protected against degradation (transgenic line K5-DeltaN122-PG). The expression of DeltaN122-PG led to the formation of additional hair germs, hyperplastic hair follicles, and noninvasive hair follicle tumors, a phenotype reminiscent of that induced by expression of N-terminally truncated beta-catenin. However, if expressed in beta-catenin-null epidermis, DeltaN122-PG did not induce new hair follicle germs and follicular tumors. Thus, DeltaN122-PG cannot substitute for beta-catenin in its signaling functions in vivo and the phenotype observed in K5-DeltaN122-PG mouse skin must be due to the aberrant activation of beta-catenin signaling. On the other hand, the expression of DeltaN122-PG in beta-catenin-null skin significantly increased the survival rate of mutant mice, rescued differentiation, and limited excessive proliferation in the interfollicular epidermis, suggesting that plakoglobin may be involved in the intracellular signaling events essential for epidermal differentiation.

Keratinocyte stem cells, label-retaining cells and possible genome protection mechanisms.

Stem cells are the crucial cells upon which the entire tissue is dependent. Here we define and discuss what is meant by and known about keratinocyte stem cells. One way in which these cells have been studied is by their ability to retain radioactivity labelled thymidine for long periods of time (label retaining cells, LRCs). The underlying mechanism has been assumed in the past to be slow cycling but a more likely explanation is the selective segregation of old and new DNA strands (Cairns's hypothesis). Experiments in the small intestine indicate that the stem cells here are selectively sorting their DNA and becoming LRCs. A possible role for p53 in stem cell biology is presented.