ABSTRACT
Hair follicle cells contribute to wound healing, skin circulation, and skin diseases including skin cancer, and hair transplantation is a useful technique to study the participation of hair follicle cells in skin homeostasis and wound healing. Although hair follicle transplantation is a well-established human hair-restoration procedure, follicular transplantation techniques in animals have a number of shortcomings and have not been well described or optimized. To facilitate the study of follicular stem and progenitor cells and their interaction with surrounding skin, we have established a new murine transplantation model, similar to follicular unit transplantation in humans. Vibrissae from GFP transgenic mice were harvested, flip-side microdissected, and implanted individually into needle hole incisions in the back skin of immune-deficient nude mice. Grafts were evaluated histologically and the growth of transplanted vibrissae was observed. Transplanted follicles cycled spontaneously and newly formed hair shafts emerged from the skin after 2 weeks. Ninety percent of grafted vibrissae produced a hair shaft at 6 weeks. After pluck-induced follicle cycling, growth rates were equivalent to ungrafted vibrissae. Transplanted vibrissae with GFP-positive cells were easily identified in histological sections. We established a follicular vibrissa transplantation method that recapitulates human follicular unit transplantation. This method has several advantages over current protocols for animal hair transplantation. The method requires no suturing and minimizes the damage to donor follicles and recipient skin. Vibrissae are easier to microdissect and transplant than pelage follicles and, once transplanted, are readily distinguished from host pelage hair. This facilitates measurement of hair growth. Flip-side hair follicle microdissection precisely separates donor follicular tissue from interfollicular tissue and donor cells remain confined to hair follicles. This makes it possible to differentiate migration of hair follicle cells from interfollicular epidermis in lineage tracing wound experiments using genetically labeled donor follicles.
Subject(s)
Hair Follicle/transplantation , Skin Transplantation/methods , Vibrissae/transplantation , Wound Healing/physiology , Animals , Cell Differentiation , Cell Movement , Hair Follicle/cytology , Hair Follicle/growth & development , Male , Mice , Mice, Nude , Mice, Transgenic , Microdissection/methods , Models, Animal , Skin Physiological Phenomena , Stem Cells/physiology , Vibrissae/cytology , Vibrissae/growth & developmentABSTRACT
Although mammals are notoriously poor at regeneration compared with many lower-order species, the hair follicle, particular to mammals, is capable of regeneration following partial amputation. The detailed internal mechanism of this phenomenon is still unclear. Development and regrowth of the hair follicle depends on dermal-epidermal interaction within the hair follicle. Previous studies have shown that Wnt/ß-catenin, Shh, Bmp, PDGF, TGF and Notch signals all take part in the development and growth of the hair follicle, and the Wnt/ß-catenin signaling additionally plays an indispensable role in hair follicle morphogenesis and regrowth. In this study, we investigated the localization, as well as, protein levels of Wnt/ß-catenin signaling molecules during amputated whisker follicle regeneration.
Subject(s)
Dermis/transplantation , Gene Expression Regulation , Hair Follicle/transplantation , Regeneration/genetics , Vibrissae/transplantation , Wnt Signaling Pathway/genetics , Animals , Bone Morphogenetic Protein 1/genetics , Bone Morphogenetic Protein 1/metabolism , Bone Morphogenetic Protein Receptors/genetics , Bone Morphogenetic Protein Receptors/metabolism , Dermis/metabolism , Dissection , Female , Hair Follicle/metabolism , Hedgehog Proteins/genetics , Hedgehog Proteins/metabolism , Mice , Mice, Nude , Platelet-Derived Growth Factor/genetics , Platelet-Derived Growth Factor/metabolism , Rats , Rats, Sprague-Dawley , Receptors, Notch/genetics , Receptors, Notch/metabolism , Vibrissae/metabolism , Wnt Proteins/genetics , Wnt Proteins/metabolism , beta Catenin/genetics , beta Catenin/metabolismABSTRACT
We previously demonstrated that whole hair follicles could be cryopreserved to maintain their stem-cells differentation potential. In the present study, we demonstrated that cryopreserved mouse whisker hair follicles maintain their hair growth potential. DMSO better cryopreserved mouse whisker follicles compared to glycerol. Cryopreserved hair follicles also maintained the hair follicle-associated-pluripotent (HAP) stem cells, evidenced by P75NTR expression. Subcutaneous transplantation of DMSO-cryopreserved hair follicles in nude mice resulted in extensive hair fiber growth over 8 weeks, indicating the functional recovery of hair shaft growth of cryopreserved hair follicles.
Subject(s)
Hair Follicle/transplantation , Hair/growth & development , Vibrissae/transplantation , Animals , Cryopreservation , Mice , Mice, Nude , Mice, Transgenic , Pluripotent Stem Cells/metabolismABSTRACT
Tissue reaction of the mollusk Planorbarius corneus to the introduction of a transplant (cat vibrissa) was examined. The transplant was introduced into mollusk tissues with the use of an injection needle. After a day, flattened haemocytes were found on the surface of the transplant. The wound channel formed by the needle was arrested by a capsule formed of 5-15 layers of flattened cells. The cavity of the wound channel and the core of the vibrissa were also filled with haemocytes. During incubation of the vibrissa in vitro, adhesion and sedimentation of haemocytes on its surface was observed.
Subject(s)
Hemocytes/immunology , Immunity, Innate , Mollusca/immunology , Animals , Cats , Cell Movement/immunology , Coculture Techniques , Hemocytes/cytology , Mollusca/cytology , Tissue Culture Techniques , Transplantation, Heterologous , Vibrissae/immunology , Vibrissae/transplantationABSTRACT
Organ replacement regenerative therapy is purported to enable the replacement of organs damaged by disease, injury or aging in the foreseeable future. Here we demonstrate fully functional hair organ regeneration via the intracutaneous transplantation of a bioengineered pelage and vibrissa follicle germ. The pelage and vibrissae are reconstituted with embryonic skin-derived cells and adult vibrissa stem cell region-derived cells, respectively. The bioengineered hair follicle develops the correct structures and forms proper connections with surrounding host tissues such as the epidermis, arrector pili muscle and nerve fibres. The bioengineered follicles also show restored hair cycles and piloerection through the rearrangement of follicular stem cells and their niches. This study thus reveals the potential applications of adult tissue-derived follicular stem cells as a bioengineered organ replacement therapy.
Subject(s)
Adult Stem Cells/cytology , Hair Follicle/cytology , Adult , Adult Stem Cells/transplantation , Animals , Bioengineering , Cell Differentiation , Cell Proliferation , Hair Follicle/transplantation , Humans , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Middle Aged , Vibrissae/cytology , Vibrissae/transplantationABSTRACT
Limbal stem cell deficiency (LSCD) leads to severe ocular surface abnormalities that can result in the loss of vision. The most successful therapy currently being used is transplantation of limbal epithelial cell sheets cultivated from a limbal biopsy obtained from the patient's healthy, contralateral eye or cadaveric tissue. In this study, we investigated the therapeutic potential of murine vibrissae hair follicle bulge-derived stem cells (HFSCs) as an autologous stem cell (SC) source for ocular surface reconstruction in patients bilaterally affected by LSCD. This study is an expansion of our previously published work showing transdifferentiation of HFSCs into cells of a corneal epithelial phenotype in an in vitro system. In this study, we used a transgenic mouse model, K12(rtTA/rtTA) /tetO-cre/ROSA(mTmG) , which allows for HFSCs to change color, from red to green, once differentiation to corneal epithelial cells occurs and Krt12, the corneal epithelial-specific differentiation marker, is expressed. HFSCs were isolated from transgenic mice, amplified by clonal expansion on a 3T3 feeder layer, and transplanted on a fibrin carrier to the eye of LSCD wild-type mice (n = 31). The HFSC transplant was able to reconstruct the ocular surface in 80% of the transplanted animals; differentiating into cells with a corneal epithelial phenotype, expressing Krt12, and repopulating the corneal SC pool while suppressing vascularization and conjunctival ingrowth. These data highlight the therapeutic properties of using HFSC to treat LSCD in a mouse model while demonstrating a strong translational potential and points to the niche as a key factor for determining stem cell differentiation.
Subject(s)
Corneal Diseases/surgery , Epithelium, Corneal/cytology , Eye Abnormalities/surgery , Hair Follicle/transplantation , Limbus Corneae/abnormalities , Limbus Corneae/surgery , Stem Cell Transplantation , Stem Cells/physiology , Animals , Cell Transdifferentiation , Cells, Cultured , Hair Follicle/cytology , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Vibrissae/cytology , Vibrissae/transplantationABSTRACT
BACKGROUND: In vitro-constructed grafts can be used for human bladder augmentation. There are many diseases in which autologous cells cannot be used for this purpose. The aim of the present study was to examine the potential of rat vibrissae hair follicle cells to form cultures, which could serve as a source for in vitro creation of urinary bladder wall grafts. METHODS: Two hundred vibrissae were excised from young Wistar male rats. Two different digestions were performed, in dispase and in collagenase. All follicles were additionally incubated in trypsin and ethylenediamine tetraacetic acid. Two different culture media based on DMEM (Dulbecco's Modified Eagle's Medium) were used: the first was supplemented with keratinocyte growth factor (KGF) and the second with epidermal growth factor. Immunocytochemical detection of cytokeratin, CD34, p63, Ki-67 (proliferation index), and HMB45 (Human Melanoma Black 45) was performed. RESULTS: Forty-eight primary cultures of rat follicle vibrissae cells were established from 200 hair follicles (24% successful rate). Twenty-four primary cultures were obtained after dispase digestion and 24 after collagenase treatment. Each group was cultured in 2 different media. A heterogeneity of primary cultures was observed. Cells formed a monolayer within a period of 2 to 4 weeks. The 24 primary cultures established after dispase treatment exhibited monolayers of small cuboid cells expressing cytokeratin and CD34. In the 40th passage 20%-40% of cells expressed p63; 85% of these cells from late passages were positive for Ki-67, indicating preserved mitotic potential. Epithelial-like phenotype was observed after dispase digestion and cultivation in KGF-supplemented medium. After 3 weeks, the morphology of these cells changed into fibroblast-like. These cultures were negative for CD34. Fibroblast-like cell growth was observed after collagenase treatment in both KGF- and EGF-supplemented media. These cells were positive for the melanocyte cell marker (HMB45). CONCLUSIONS: Culture media and isolation conditions influence hair follicle stem cell differentiation. The stem cell niche within the hair follicles is a reservoir of cells, which can be potentially used for in vitro creation of urinary bladder wall grafts.
Subject(s)
Urinary Bladder/surgery , Vibrissae/cytology , Vibrissae/transplantation , Animals , Antigens, CD34/metabolism , Cell Culture Techniques , Epidermal Growth Factor/pharmacology , Humans , Keratins/metabolism , Male , Mitosis , Rats , Rats, Wistar , Vibrissae/drug effectsABSTRACT
The antennal imaginal disc was transplanted between male larvae of two different heliothine moth species, Heliothis virescens (HV) and Helicoverpa zea (HZ). Males of these species respond to distinct pheromone blends, have different peripheral and central olfactory neuron specificities, as well as distinct arrangements of antennal lobe olfactory glomeruli, in the specialized male macroglomerular complex (MGC). After pupal development and adult eclosion, unilateral (with one antennal disc left intact) and bilateral antennal transplant males were assayed in a wind tunnel to both species' pheromone blends to determine their ability to discriminate between the two signals. The postmetamorphic developmental effects of interspecific transplantation upon the primary olfactory centers in the moth brain were then examined in these same individuals. Behavioral tests showed that both types of unilateral transplant continued to exhibit upwind anemotactic flight to the normal recipient blend with occasional flights to the donor blend. In contrast, bilateral transplants preferred the HV pheromone blend regardless of the direction of transplant, with some males of each type also responding to the HZ blend. Neuroanatomic evaluation of the MGC revealed that the donor arrangement of MGC glomeruli was induced in 73% HZ donor to HV recipient transplants and 56% of the reciprocal transplant. Surprisingly, several V-Z bilateral transplant males responded to both HV and HZ pheromone blends and had two HV MGC structures. This behavioral outcome was unexpected, because responses to the HV blend are mediated by inputs that are normally antagonistic to HZ males and the normal HV antenna lacks olfactory receptor neurons capable of responding to the essential minor pheromone component of the HZ blend. These data indicate a plasticity in developmental pathways regulating the expression of peripheral olfactory receptor neurons and in the glomerular processing of species-specific olfactory information.
Subject(s)
Discrimination, Psychological/physiology , Olfactory Nerve/anatomy & histology , Olfactory Nerve/physiology , Sex Attractants/physiology , Vibrissae/transplantation , Animals , Male , Moths , Species Specificity , Vibrissae/physiologyABSTRACT
The hair follicle is a source of epithelial stem cells and site of origin for several types of skin tumors. Although it is clear that follicles arise by way of a series of inductive tissue interactions, identification of the signaling molecules driving this process remains a major challenge in skin biology. In this study we report an obligatory role for the secreted morphogen Sonic hedgehog (Shh) during hair follicle development. Hair germs comprising epidermal placodes and associated dermal condensates were detected in both control and Shh -/- embryos, but progression through subsequent stages of follicle development was blocked in mutant skin. The expression of Gli1 and Ptc1 was reduced in Shh -/- dermal condensates and they failed to evolve into hair follicle papillae, suggesting that the adjacent mesenchyme is a critical target for placode-derived Shh. Despite the profound inhibition of hair follicle morphogenesis, late-stage follicle differentiation markers were detected in Shh -/- skin grafts, as well as cultured vibrissa explants treated with cyclopamine to block Shh signaling. Our findings reveal an essential role for Shh during hair follicle morphogenesis, where it is required for normal advancement beyond the hair germ stage of development.
Subject(s)
Proteins/physiology , Skin Transplantation/physiology , Skin/embryology , Trans-Activators , Vibrissae/embryology , Adipose Tissue/embryology , Animals , Embryonic Induction , Epidermis/embryology , Hedgehog Proteins , Mice , Mice, Knockout , Mice, Nude , Morphogenesis , Organ Culture Techniques , Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Sebaceous Glands/embryology , Veratrum Alkaloids/pharmacology , Vibrissae/drug effects , Vibrissae/transplantationABSTRACT
The dermal papilla is a discrete group of cells at the base of the hair follicle and is implicated in controlling the hair growth cycle. Early passage dermal papilla cells can induce hair growth in vivo, but, upon further culturing, this property is lost. In order to study the events occurring in hair induction, a representative dermal papilla cell line was required. We have transfected passage 1 rat vibrissa dermal papilla cells with a polyomavirus large T gene encoding a temperature-sensitive T antigen, and generated permanent cell lines in which the immortalizing function can be switched off by temperature shift. The cells established without crisis, resembled cells in the starting population, and retained the aggregative properties of early passage dermal papilla cells. Growth studies were performed on the immortalized cell lines, which showed that transferring the cells to the restrictive temperature for the large T gene product resulted in cell senescence or quiescence, and changes in morphology. Implantation of cell pellets into the ears of immunologically compatible rats showed that the immortal cells retained hair-inductive ability. Cytokines are believed to have an important role in the control of hair growth. The pattern of cytokine gene expression in the immortal cell lines was compared with early passage dermal papilla cells and a non-hair-inducing dermal papilla cell line, using reverse transcriptase-polymerase chain reaction. Epidermal growth factor, tumour necrosis factor, and interleukin-1a were detected in the immortalized and non-hair-inducing dermal papilla cell lines, but were absent in passage 2 dermal papilla cells. All other cytokines examined were detected in all the cell types under study. These results demonstrate that the polyomavirus large Ttsa-immortalized dermal papilla cell lines are very similar to passage 2 dermal papilla cells and thus provide a good model for hair growth studies. Cytokine expression profiles indicate that the expression of several cytokines may be implicated in hair induction. Further studies are under way to investigate the relationship between cytokine expression and the hair growth cycle.
Subject(s)
Antigens, Polyomavirus Transforming/genetics , Cytokines/biosynthesis , Gene Expression , Transfection , Vibrissae/metabolism , Animals , Antigens, Polyomavirus Transforming/biosynthesis , Base Sequence , Cell Division , Cell Line, Transformed , Cells, Cultured , Cellular Senescence , DNA Primers , Exons , Fluorescent Antibody Technique , Genetic Vectors , Interleukin-1/biosynthesis , Male , Molecular Sequence Data , Polymerase Chain Reaction , Rats , Rats, Inbred Strains , Restriction Mapping , Temperature , Tumor Necrosis Factor-alpha/biosynthesis , Vibrissae/cytology , Vibrissae/transplantationABSTRACT
Adult vibrissa follicle dermal papillae have the capacity to induce hair growth and follicle formation when associated with epidermis from various sources. However, the range of conditions under which hair follicle induction will take place has not been established. The question of whether or not the adult papilla carries information to impose fibre-type specificity has also not been fully answered. This study describes how the implantation of isolated papillae into small incisional cuts on the rat ear pinna resulted in the subsequent emergence of abnormally large hair fibres from the wound sites. Many of these hairs were found to display vibrissa-type characteristics. Histological observations indicated that the papillae had interacted with the edges of the wound epidermis to produce new, and particularly large follicles, while immunohistochemical staining revealed that early follicle construction was accompanied by a profusion of the basement membrane constituents laminin and type IV collagen in the subjacent dermis. These findings show that adult rat papillae retain the capacity, as displayed by embryonic dermis, to determine vibrissa specificity in induced follicles.
Subject(s)
Epidermis/pathology , Vibrissae/physiology , Wounds and Injuries/pathology , Animals , Cell Differentiation/physiology , Collagen/metabolism , Ear , Immunohistochemistry , Laminin/metabolism , Rats , Skin/metabolism , Vibrissae/anatomy & histology , Vibrissae/growth & development , Vibrissae/transplantationABSTRACT
A method for transplanting whole or partial whisker follicles from adult mice to a site beneath the kidney capsule of syngeneic mice is described. Follicles were removed from the upper lip and the growing whiskers plucked. The follicles were either left intact or divided into two parts by transection and implanted under the kidney capsule. The intact whole follicles remained viable and regenerated whiskers which were later shed. The lower one-half or one-third follicle implants reorganized their base and produced a short, curled whisker. On the other hand, none of the upper one-half or two-thirds implants regenerated a dermal papilla, and no whisker production was observed. However, when a single dermal papilla which had been dissected out from another follicle was introduced into each upper follicle cavity in contact with the cut edge, a bulbar region emerged, and subsequently, a long, thick, medullated whisker developed from the implants. This technique should be useful in studying the induction and regeneration of adult mouse whiskers.
Subject(s)
Kidney , Vibrissae/transplantation , Animals , Female , Kidney/pathology , Mice , Mice, Inbred C57BL , Regeneration , Time Factors , Vibrissae/growth & development , Vibrissae/physiologyABSTRACT
In the mouse whiskerpad there is a group of vibrissal follicles arranged in five rows, which are topologically represented in the contralateral somatosensory cortex by the barrelfield. Each vibrissal follicle is a specialized sensory organ containing a large number of receptors, mostly Merkel cells. In these experiments, the parts of the vibrissal follicles containing most of the receptors were transplanted to different regions of the whiskerpad of newborn mice, to know whether "new", supranumerary barrels could develop. The results confirm this hypothesis. However, the "new" barrels are not topologically represented in the barrelfield, as normal barrels do.
Subject(s)
Fetal Tissue Transplantation/physiology , Mechanoreceptors/physiology , Somatosensory Cortex/physiology , Vibrissae/transplantation , Animals , Animals, Newborn , Embryo, Mammalian , Mice , Mice, Inbred C3H , Microscopy, Electron, Scanning , Reference Values , Vibrissae/physiology , Vibrissae/ultrastructureABSTRACT
Retention of the capacity to induce the growth of hair by cultured adult rat vibrissa dermal papilla cells has been investigated. Small pellets of serially cultured papilla cells were implanted into the bases of the exposed follicular epidermis of amputated adult rat vibrissa follicles. Amputated follicles that received no cell implants or implants of cultured dorsal skin fibroblasts were used as controls. Over 50% of follicles implanted with cultured papilla cells in the passage range 1-3 grew hairs. In contrast none of the follicles that received late passage cells (range 6-15) produced hairs; and spontaneous regeneration of hair occurred in only 3% of the control follicles. These results demonstrate that cultured papilla cells of early passage numbers retain their ability to induce hair growth. Histological examination confirmed that the implanted papilla cells interacted with follicular epidermis to organize the development of new, hair-producing bulbs, each containing a discrete dermal papilla. An important observation was that aggregative behaviour leading to papilla formation was only manifested by early passage papilla cell implants. This persisting embryonic characteristic appears to be an essential functional component of papilla cell activity which operates to regulate the profound morphogenetic changes that occur during the hair growth cycle.
Subject(s)
Vibrissae/growth & development , Animals , Cells, Cultured , Hair/anatomy & histology , Hair/growth & development , Rats , Vibrissae/anatomy & histology , Vibrissae/transplantationABSTRACT
Rat vibrissa dermal papillae were transplanted between the epidermis and dermis of isolated embryonic mouse skin and then grafted onto nude mice. The papillae induced the formation of hair follicles which were larger than those of the host skin but smaller than vibrissa follicles. The potential of isolated dermal papillae to induce follicles with characteristics of those from which the papillae originated is discussed. One of the major factors affecting the sizes of induced follicles may have been related to the splitting of the papilla mass and dispersal of the cells by invading cords of epidermal cells from the host skin during induction.
Subject(s)
Cell Differentiation , Embryonic Induction , Hair/embryology , Skin/embryology , Vibrissae/transplantation , Animals , Mice , Mice, Nude , RatsABSTRACT
The barrelfield is the cortical "map" of the ensemble of vibrissal follicles on the mouse whiskerpad. Earlier, we had shown that the skin of the embryonic whiskerpad, when put in culture before having received its innervation, is capable of producing vibrissal follicles arranged in a pattern similar to that formed in vivo; we had also demonstrated that the destruction of vibrissal follicles, and of the terminals that innervate them, leads to important modifications in the architecture of the barrelfield. Here we report on the architecture of barrelfields made to differ from normal as a consequence of radical modifications produced in the corresponding whiskerpad during gestation and at birth: transplantation of additional whiskerpads; rotations (of 90 degrees and 180 degrees) of one whiskerpad; removal and reimplantation of one whiskerpad; removal of one whiskerpad; and division of the infraorbital nerve. The results of these experiments, in which only the morphological correlates of a sensory cortical map have been studied, strengthen the hypothesis that the role played by the sensory periphery in the establishment of such an entity is, indeed, an important one.