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Label-retaining Cells Reside in the Bulge Area of Pilosebaceous Unit: Implications for Follicular Stem Cells, Hair Cycle, and Skin Carcinogenesis
Abstract
Inconsistent with the view that hair follicle stem cells reside in the matrix area of the hair bulb, we found that label-retaining cells exist exclusively in the bulge area of the mouse hair follicle. The bulge consists of a subpopulation of outer root sheath cells located in the midportion of the follicle at the arrector pili muscle attachment site. Keratinocytes in the bulge area are relatively undifferentiated ultrastructurally. They are normally slow cycling, but can be stimulated to proliferate transiently by TPA. Located in a well-protected and nourished environment, these cells mark the lower end of the "permanent" portion of the follicle. Our findings, plus a reevaluation of the literature, suggest that follicular stem cells reside in the bulge region, instead of the lower bulb. This new view provides insights into hair cycle control and the possible involvement of hair follicle stem cells in skin carcinogenesis.
- ... Unwanted hair loss can pose psychosocial distress to affected individuals [1]. Hair regeneration depends on the activation of hair follicle stem cells (HFSCs) [2][3][4]. As the hair follicle (HF) is an integral part of skin [5], its growth and the activity of HFSCs are regulated by various nearby cells of the HFSC niche in the skin [6,7]. ...... Over the past 3 decades, progress has been made in understanding how the growth of HFs is regulated, particularly due to the discovery of HFSCs [2][3][4][20][21][22]. HFSCs are first identified as slow-cycling label-retaining cells located in the bulge epithelium [2,22]. ...... Over the past 3 decades, progress has been made in understanding how the growth of HFs is regulated, particularly due to the discovery of HFSCs [2][3][4][20][21][22]. HFSCs are first identified as slow-cycling label-retaining cells located in the bulge epithelium [2,22]. In addition to this population of relatively quiescent stem cells, HFs harbor another population of primed stem cells with faster activation dynamics in the secondary hair germ of telogen HFs [3,5,23]. ...Abstract Stem cell activity is subject to non-cell-autonomous regulation from the local microenvironment, or niche. In adaption to varying physiological conditions and the ever-changing external environment, the stem cell niche has evolved with multifunctionality that enables stem cells to detect these changes and to communicate with remote cells/tissues to tailor their activity for organismal needs. The cyclic growth of hair follicles is powered by hair follicle stem cells (HFSCs). Using HFSCs as a model, we categorize niche cells into 3 functional modules, including signaling, sensing and message-relaying. Signaling modules, such as dermal papilla cells, immune cells and adipocytes, regulate HFSC activity through short-range cell-cell contact or paracrine effects. Macrophages capacitate the HFSC niche to sense tissue injury and mechanical cues and adipocytes seem to modulate HFSC activity in response to systemic nutritional states. Sympathetic nerves implement the message-relaying function by transmitting external light signals through an ipRGC-SCN-sympathetic circuit to facilitate hair regeneration. Hair growth can be disrupted by niche pathology, e.g. dysfunction of dermal papilla cells in androgenetic alopecia and influx of auto-reacting T cells in alopecia areata and lichen planopilaris. Understanding the functions and pathological changes of the HFSC niche can provide new insight for the treatment of hair loss.
- ... Hair follicle stem cells reside in a specialized structure, called the bulge, which was originally identified as a non-cycling, quiescent cell cluster in the resting hair follicles (Blanpain, Lowry, Geoghegan, Polak, & Fuchs, 2004;Cotsarelis, Sun, & Lavker, 1990;Tumbar et al., 2004). The bulge stem cells show several characteristic properties: infrequent cell divisions (Cotsarelis et al., 1990;Tumbar et al., 2004); unique abilities of self-renewal and multipotent differentiation ; distinct molecular signature, including specific surface expression of α6-integrin and CD34 (Trempus et al., 2003), K19 and transcription factors Sox9, Lhx2 and Tcf3/4 Morris et al., 2004;Tumbar et al., 2004) ( Fig. 1). ...... Hair follicle stem cells reside in a specialized structure, called the bulge, which was originally identified as a non-cycling, quiescent cell cluster in the resting hair follicles (Blanpain, Lowry, Geoghegan, Polak, & Fuchs, 2004;Cotsarelis, Sun, & Lavker, 1990;Tumbar et al., 2004). The bulge stem cells show several characteristic properties: infrequent cell divisions (Cotsarelis et al., 1990;Tumbar et al., 2004); unique abilities of self-renewal and multipotent differentiation ; distinct molecular signature, including specific surface expression of α6-integrin and CD34 (Trempus et al., 2003), K19 and transcription factors Sox9, Lhx2 and Tcf3/4 Morris et al., 2004;Tumbar et al., 2004) ( Fig. 1). In addition, several promoters have been identified to mark distinct populations of bulge cells and confirmed their long-term contributions to the entire hair lineages by lineage tracing experiments : Gli1 (Brownell, Guevara, Bai, Loomis, & Joyner, 2011), K15 (Liu, Lyle, Yang, & Cotsarelis, 2003;Morris et al., 2004) and Lgr5 (Jaks et al., 2008) preferentially marks upper-bulge, mid-bulge, lower-bulge/hair germ, respectively ( Fig. 1). ...... After the nucleotide analogs are withdrawn (¼ chase), over a period of time, cells dilute the label by divisions or to be shed out from the tissue by differentiation. In contrast, slower-cycling cells retain the label even after a long-term chase and can be detected as LRCs (Cotsarelis et al., 1990). ...
- ... Epidermal homeostasis is maintained by skin epithelial adult stem cells which are a population of cells featured by their quiescent and slowcycling characteristics (Morrison & Spradling, 2008). In the early 90', stem cells in the skin were successfully identified by their label retaining ability due to their slow mitotic rate (Cotsarelis, Sun, & Lavker, 1990). These labeled stem cells are predominantly located in the bulge region of hair follicle (Cotsarelis et al., 1990). ...... In the early 90', stem cells in the skin were successfully identified by their label retaining ability due to their slow mitotic rate (Cotsarelis, Sun, & Lavker, 1990). These labeled stem cells are predominantly located in the bulge region of hair follicle (Cotsarelis et al., 1990). A later experiment grafted LacZ+ bugle tissue to replace another normal bulge hair follicle resulting in the entire newly synthesized hair follicle with LacZ positive cells. ...... However, the bulge stem cells appear to only give rise to the hair follicle but not the interfollicular epithelium surrounding the hair follicle. In the labeling experiment mentioned above, label retaining cells were not observed in the interfollicular epithelium (Cotsarelis et al., 1990). This finding revealed an open question about the location of stem cells for the interfollicular epithelium. ...
- ... In human skin, p73 was strongly co-expressed with p63α in the stem cell compartment of the HF, termed the bulge (Fig 1B, top panel). The bulge is a specific area of the ORS containing HF stem cells that is located between the attachment site of the arrector pili muscle and the opening of the sebaceous gland [32]. We also saw expression of p73 in the bulge region of murine HFs. ...... Most cells (>85%) that expressed Trp73 also expressed Trp63 ( Fig 3C). The percentage of cells with Trp73 expression (p73+/p63-and p73+/p63+) was highest in the outer bulge cluster (Fig 3C), the stem cell compartment of the HF [32]. Trp73 was expressed by a subset of cells in all of the basal IFE clusters (Fig 3C), the stem cell compartment of the epidermis. ...p63 is a transcriptional regulator of ectodermal development that is required for basal cell proliferation and stem cell maintenance. p73 is a closely related p53 family member that is expressed in select p63-positive basal cells and can heterodimerize with p63. p73-/- mice lack multiciliated cells and have reduced numbers of basal epithelial cells in select tissues; however, the role of p73 in basal epithelial cells is unknown. Herein, we show that p73-deficient mice exhibit delayed wound healing despite morphologically normal-appearing skin. The delay in wound healing is accompanied by decreased proliferation and increased levels of biomarkers of the DNA damage response in basal keratinocytes at the epidermal wound edge. In wild-type mice, this same cell population exhibited increased p73 expression after wounding. Analyzing single-cell transcriptomic data, we found that p73 was expressed by epidermal and hair follicle stem cells, cell types required for wound healing. Moreover, we discovered that p73 isoforms expressed in the skin (ΔNp73) enhance p63-mediated expression of keratinocyte genes during cellular reprogramming from a mesenchymal to basal keratinocyte-like cell. We identified a set of 44 genes directly or indirectly regulated by ΔNp73 that are involved in skin development, cell junctions, cornification, proliferation, and wound healing. Our results establish a role for p73 in cutaneous wound healing through regulation of basal keratinocyte function.
- ... C'est la raison pour laquelle les cellules souches sont appelées Label-Retaining Cells (ou LRCs). Elles ont été identifiées, chez la souris au niveau de la région permanente du follicule pileux (bulge), par Cotsarelis et ses collaborateurs(Cotsarelis et al., 1990). Fuchs et ses collaborateurs ont adapté cette dernière technique afin de visualiser l'histone H2B couplé au fluorochrome GFP (H2BGFP)(Tumbar, 2004).Depuis plusieurs années, différentes populations de cellules souches ont été identifiées dans différentes niches. ...... Cependant, alors que les cellules souches du bulge (K15+) ne permettent de donner que des cellules TA, qui ne vivront pas longtemps, les cellules souches de l'isthme et de l'infundibulum (Lrig1+ et Lgr6+) génèrent des clones de cellules souches. Ceci a pour conséquence la perte progressive des HFSCs du bulge si le besoin de cicatrisation s'avère être long.Après la mise au point de la technique des LCRs par Cotsarelis et ses collaborateurs(Cotsarelis et al., 1990), de nouvelles méthodes ont été développées afin d'isoler spécifiquement les cellules souches du follicule pileux (HFSCs) en couplant l'utilisation de marqueurs spécifiques avec la technologie du FACS (cytrométrie en flux). Le Cluster de différentiation 34 (CD34) est l'une des premières protéines identifiées qui est exprimée à la surface membranaire des cellules souches hématopoïétiques. ...L’histone variante H2A.Z, histone de la famille H2A est enrichie dans certaines régions non transcrites de la chromatine, telles que la chromatine péricentromérique, centromérique et télomérique. Elle existe sous la forme de deux isoformes, H2A.Z-1 et H2A.Z-2, qui diffèrent par seulement 3 acides aminés et sont codés par deux gènes distincts, H2afz et H2afv. L’histone H2A.Z apparait impliquée dans divers évènements cellulaires tels que la transcription, la réparation de l’ADN ainsi que la prolifération et la différenciation cellulaire. La fonction de H2A.Z a été, jusqu’ici, analysée surtout grâce à la culture cellulaire in-vitro. Peu d’informations sont disponibles concernant le rôle de H2A.Z in-vivo dans différents organes, reflétant le manque de modèles animaux permettant l’invalidation génique de H2A.Z. Nous avons créé un modèle de souris transgénique permettant de réaliser in-vivo le double knock-out conditionnel (KI/cKO) des gènes H2afz et H2afv de manière tissu-spécifique dans les kératinocytes de la peau. Ce modèle d’étude in-vivo est unique car le seul à ce jour permettant d’éliminer complètement l’expression de H2A.Z. L’histone variante est physiologiquement présente dans toutes les cellules wild-type. Si les deux gènes codant pour H2A.Z sont délétés, la concentration de l’histone diminue au fur et à mesure des mitoses successives et finit par disparaître.L’épiderme en constante prolifération (tissu mitotique) mais aussi en constante différenciation (tissu post-mitotique), ainsi que le follicule pileux où ces deux processus intervenent de manière cyclique lors de la formation du poil, constituent un excellent modèle afin de disséquer le rôle spécifique de H2A.Z dans les processus de prolifération et de différenciation.L’induction par le tamoxifène du transgène K14CreERT2 invalidant les gènes H2A.Z (H2afz et H2afv) dans les kératinocytes, a tout d’abord été réalisée chez la souris adulte âgée de 6-8 mois. Elle entraine progressivement la perte totale de l’histone variante H2A.Z dans les cellules d’amplification transitoire (TA) qui se multiplient activement : au niveau du follicule pileux en phase de croissance (anagène) et au niveau des cellules situées de place en place au niveau de l’assise basale de l’épiderme. Le blocage en phase G2/M de ces cellules, perturbe l’homéostasie de la peau et appelle en retour une migration des cellules souches du follicule pileux vers l’épiderme, entrainant un épaississement de l’épiderme et une alopécie dans la région ventrale thoracique.Lors de la mise en place de la peau embryonnaire la délétion des deux gènes H2afz et H2afv, par l’induction du transgène K14CreERT2 suite à l’injection de tamoxifène ou l’utilisation du transgène K5Cre dont l’activité est constitutive, entraine la perte progressive de l’histone H2A.Z dans toutes les cellules épidermiques et les cellules des bourgeons pileux, qui toutes ont un fort indice de prolifération. La perte de H2A.Z entrainant le blocage des cellules en phase G2/M, ces cellules se différencient et s’accumulent dans la couche cornée.En conclusion, les différents phénotypes développés après le knock-out de H2A.Z dans les kératinocytes chez l’adulte ainsi qu’au cours de l’embryogénèse de la peau, nous ont permis de montrer l’implication de H2A.Z dans la progression de la mitose, et par la même directement son implication dans la régulation de l’homéostasie de l’épiderme.
- ... The mesenchymal is composed of specialized fibroblasts divided into the dermal papilla (DP), located at the pro-ximal end of the HF and surrounded by matrix cells, and the dermal sheath (DS), considered as a reservoir of DP cells [4]. The DP plays an important role in the induction and maintenance of the hair growth cycle by acting as inductive signal while driving the differentiation of epithelial stem cells residing in the bulge area of the HF, and generating the complex follicular product, the shaft, and the sheath [5][6][7][8]. In the adult hair, the HF undergo continuous self-renewal and cycling, dividing in three phases anagen, catagen, and telogen [3]. ...The hair follicle is a complex structure that goes through a cyclic period of growth (anagen), regression (catagen), and rest (telogen) under the regulation of several signaling pathways, including Wnt/ β-catenin, FGF, Shh, and Notch. The Wnt/β-catenin signaling is specifically involved in hair follicle morphogenesis, regeneration, and growth. β-catenin is expressed in the dermal papilla and promotes anagen induction and duration, as well as keratinocyte regulation and differentiation. In this study, we demonstrated the activation of β-catenin by a polyphenolic compound 3,4,5-tri-O-caffeoylquinic acid (TCQA) in mice model and in human dermal papilla cells to promote hair growth cycle. A complete regrowth of the shaved area of C3H mice was observed upon treatment with TCQA. Global gene expression analysis using microarray showed an upregulation in hair growth-associated genes. Moreover, the expression of β-catenin was remarkably upregulated in vivo and in vitro. These findings suggest that β-catenin activation by TCQA promoted the initiation of the anagen phase of the hair cycle.
- ... The maintenance and repair of these appendages is enabled by resident adult SCs that have the capacity for both prolonged self-renewal and generation of differentiated cells. Adult tissue homeostasis and repair in these organs have been thought to involve a few long-lived quiescent SCs that generate rapidly dividing, short-lived transit-amplifying proliferating progenitors, which then produce differentiated cells [4][5][6] . However, recent studies in several organs have used lineage tracing in combination with mathematical modelling and single-cell approaches to demonstrate that renewal capacity can be distributed over a large population of actively dividing progenitors or SCs 7 . ...The classical model of tissue renewal posits that small numbers of quiescent stem cells (SCs) give rise to proliferating transit-amplifying cells before terminal differentiation. However, many organs house pools of SCs with proliferative and differentiation potentials that diverge from this template. Resolving SC identity and organization is therefore central to understanding tissue renewal. Here, using a combination of single-cell RNA sequencing (scRNA-seq), mouse genetics and tissue injury approaches, we uncover cellular hierarchies and mechanisms that underlie the maintenance and repair of the continuously growing mouse incisor. Our results reveal that, during homeostasis, a group of actively cycling epithelial progenitors generates enamel-producing ameloblasts and adjacent layers of non-ameloblast cells. After injury, tissue repair was achieved through transient increases in progenitor-cell proliferation and through direct conversion of Notch1-expressing cells to ameloblasts. We elucidate epithelial SC identity, position and function, providing a mechanistic basis for the homeostasis and repair of a fast-turnover ectodermal appendage.
- ... The cyclic regeneration of HF involves phases of growth (Anagen) via regression (Catagen) to relative quiescence (Telogen; Geyfman et al, 2015). The entry of resting HF into Anagen requires the activation of HFSC located in the HF bulge (Cotsarelis et al, 1990;Tumbar et al, 2004), and the expansion of their progenitors found in the secondary hair germ, giving rise to a new Anagen HF (Tumbar et al, 2004;Greco et al, 2009;Rompolas et al, 2012). Anagen HF grows until other instructive signals promote their regression giving rise to a new HF cycle. ...Lymphatic vessels are essential for skin fluid homeostasis and immune cell trafficking. Whether the lymphatic vasculature is associated with hair follicle regeneration is, however, unknown. Here, using steady and live imaging approaches in mouse skin, we show that lymphatic vessels distribute to the anterior permanent region of individual hair follicles, starting from development through all cycle stages and interconnecting neighboring follicles at the bulge level, in a stem cell-dependent manner. Lymphatic vessels further connect hair follicles in triads and dynamically flow across the skin. At the onset of the physiological stem cell activation, or upon pharmacological or genetic induction of hair follicle growth, lymphatic vessels transiently expand their caliber suggesting an increased tissue drainage capacity. Interestingly, the physiological caliber increase is associated with a distinct gene expression correlated with lymphatic vessel reorganization. Using mouse genetics, we show that lymphatic vessel depletion blocks hair follicle growth. Our findings point toward the lymphatic vasculature being important for hair follicle development, cycling, and organization, and define lymphatic vessels as stem cell niche components, coordinating connections at tissue-level, thus provide insight into their functional contribution to skin regeneration.
- ... Hair biologists however consider the hair follicle an "end organ, " with its own complex microenvironment (109). The hair producing segment of the hair follicle is constantly being renewed from a stem cell pool (110)(111)(112). The hair follicle is mainly composed of keratinocytes that make up the hair shaft as well as the inner and outer root sheaths. ...The traditional diagnostic gold standard for inflammatory skin lesions of unclear aetiology is dermato-histopathology. As this approach requires an invasive skin biopsy, biopsy processing and analysis by specialised histologists, it is a resource intensive approach requiring trained healthcare professionals. In many health care settings access to this diagnostic approach can be difficult and outside emergency cases will usually take several weeks. This scenario leads to delayed or inappropriate treatment given to patients. With dramatically increased sensitivity of a range of analysis systems including mass spectrometry, high sensitivity, multiplex ELISA based systems and PCR approaches we are now able to “measure” samples with unprecedented sensitivity and accuracy. Other important developments include the long-term monitoring of parameters using microneedle approaches and the improvement in imaging systems such as optical coherence tomography. In this review we will focus on recent achievements regarding measurements from non-invasive sampling, in particular from plucked hair and skin tape-strips which seem well suited for the diagnosis of lupus erythematosus and psoriatic inflammation, respectively. While these approaches will not replace clinical observation – they can contribute to improved subgroup diagnosis, stratified therapeutic approaches and have great potential for providing molecular and mechanistic insight in to inflammatory skin diseases
- ... Once a HF and the first hair are formed during morphogenesis, the entire base of the HF, the cycling portion, undergoes programmed cell death during a period known as catagen. The HF then enters a "resting" phase, telogen, before the anagen period [14,15] during which the entire lower portion of the HF is repopulated from epithelial and melanocyte stem cells located in the bulge region [16,17]. Initiation of melanogenesis is tightly coupled to anagen progression [18], with the first melanin granules typically visible in the HF during the anagen IIIa stage, when the hair follicle bulb extends to the border of the dermis and subcutis [19]. ...Background: Copy number gain of the D-3-phosphoglycerate dehydrogenase (PHGDH) gene, which encodes the first enzyme in serine biosynthesis, is found in some human cancers including a subset of melanomas. Methods: In order to study the effect of increased PHGDH expression in tissues in vivo, we generated mice harboring a PHGDHtetO allele that allows tissue-specific, doxycycline-inducible PHGDH expression, and we analyzed the phenotype of mice with a ubiquitous increase in PHGDH expression. Results: Tissues and cells derived from PHGDHtetO mice exhibit increased serine biosynthesis. Histological examination of skin tissue from PHGDHtetO mice reveals the presence of melanin granules in early anagen hair follicles, despite the fact that melanin synthesis is closely coupled to the hair follicle cycle and does not normally begin until later in the cycle. This phenotype occurs in the absence of any global change in hair follicle cycle timing. The aberrant presence of melanin early in the hair follicle cycle following PHGDH expression is also accompanied by increased melanocyte abundance in early anagen skin. Conclusions: These data suggest increased PHGDH expression impacts normal melanocyte biology, but PHGDH expression alone is not sufficient to cause cancer.
- ... Elaine Fuchs and colleagues found that the majority of LRCs expressed the SC marker CD34 (Fig. 1c). The sorted and cultured CD34+ bulge cells were shown to regenerate the entire hair follicle after transplantation [20,21]. However, the bulge SCs were found to not directly generate TA cells but rather give rise to the Lgr5+ cell population located at the hair germ, and the hair germ cells in turn generated TA cells that further differentiated into the hair shaft in the hair matrix [22,23]. ...Stem cells give rise to all cells and build the tissue structures in our body, and heterogeneity and plasticity are the hallmarks of stem cells. Epigenetic modification, which is associated with niche signals, determines stem cell differentiation and somatic cell reprogramming. Stem cells play a critical role in the development of tumors and are capable of generating 3D organoids. Understanding the properties of stem cells will improve our capacity to maintain tissue homeostasis. Dissecting epigenetic regulation could be helpful for achieving efficient cell reprograming and for developing new drugs for cancer treatment. Stem cell-derived organoids open up new avenues for modeling human diseases and for regenerative medicine. Nevertheless, in addition to the achievements in stem cell research, many challenges still need to be overcome for stem cells to have versatile application in clinics.
- ... HFs are known to contain a well-characterized niche for grown-up SCs: the lump, which contains ESCs and melanocytic SCs [18]. SCs in the hair lump, an obviously differentiated structure inside the lower permanent portion of HFs, can generate the inter-follicular epidermis, HFs structures, and sebaceous glands [19,20]. The lump ESCs can likewise reconstitute in a simulated in vivo framework to a new HF [21,22]. ...Hair bio-engineering has risen at the crossing point of various manipulations to meet a clinical requirement for innovations to advance hair growth. The authors reported the microscopic and trichoscopic results of an autologous cell biological technique to compare, through histological, immunocytochemistry, and cytospin analysis, hair re-growth obtained by micro-grafts from scalp tissue containing Human Intra- and Extra-Dermal Adipose Tissue-Derived Hair Follicle Stem Cells (HD-AFSCs) versus placebo (saline solution). An autologous solution of micro-grafts was obtained from mechanical fragmentation and centrifugation of scalp biopsy’s (2 × 2 mm) using “Gentile protocol”. The micro-grafts solution was mechanically infiltrated on half of the selected patients’ scalps with Androgenic Alopecia (Norwood–Hamilton 2–5 and Ludwig 1–2). The other half was infiltrated with saline solution. Three injections were performed to each patient at 45-day intervals. Of the 35 patients who were enrolled, 1 was excluded and 1 was rejected. 23 and 44 weeks after the last micro graft’s injections, the patients displayed a hair density improvement, with a mean increment of 33% ± 7.5% and 27% ± 3.5% respectively, contrasted with baseline values, for the treated region. Microscopic assessment appeared, in scalp biopsies, to show an expansion in the number of hair follicles per mm2 following 11 months from the last micro-grafts application compared with baseline (1.4 + 0.27 versus 0.46 + 0.15, respectively; p < 0.05). HD-AFSCs contained in micro-grafts may represent a safe and effective alternative therapy option against hair loss.
- ... The quiescent state of telogen HFSCs is maintained by factors including BMP6 and FGF-18 from inner bulge cells [9], BMP4 from dermal fibroblasts and BMP2 from subcutaneous adipocytes [10]. In telogen, the lower HF component consists of two regions, the bulge/LPP [11] and secondary hair germ (SHG) [12], an epithelial extension at the base of the telogen HF. These compartments can be distinguished using region-specific markers, with the bulge compartment expressing the CD34 membrane glycoprotein [13] and the SHG selectively expressing the intracellular adhesion protein P-cadherin/ Cdh3 [14]. ...Melanocyte stem cells (McSCs) are the undifferentiated melanocytic cells of the mammalian hair follicle (HF) responsible for recurrent generation of a large number of differentiated melanocytes during each HF cycle. HF McSCs reside in both the CD34+ bulge/lower permanent portion (LPP) and the CD34- secondary hair germ (SHG) regions of the HF during telogen. Using Dct-H2BGFP mice, we separate bulge/LPP and SHG McSCs using FACS with GFP and anti-CD34 to show that these two subsets of McSCs are functionally distinct. Genome-wide expression profiling results support the distinct nature of these populations, with CD34- McSCs exhibiting higher expression of melanocyte differentiation genes and with CD34+ McSCs demonstrating a profile more consistent with a neural crest stem cell. In culture and in vivo, CD34- McSCs regenerate pigmentation more efficiently whereas CD34+ McSCs selectively exhibit the ability to myelinate neurons. CD34+ McSCs, and their counterparts in human skin, may be useful for myelinating neurons in vivo, leading to new therapeutic opportunities for demyelinating diseases and traumatic nerve injury.
- ... Hair-follicle bulge stem cells were originally shown to have the capacity to form hair-follicle cells, sebaceous-gland basal cells, and epidermis [1][2][3][4][5] . In addition to normal tissues homeostasis, these stem cells are involved in the regeneration of hair follicles and acute epithelial wound repair 6,7 . ...The bulge area of the hair follicle contains hair-follicle-associated pluripotent (HAP) stem cells. Here, we present effective cryopreservation procedures of the human hair follicle that preserve the differentiation potential of HAP stem cells. Whole hair follicles isolated from human scalp were cryopreserved by a slow-rate cooling medium and stored in liquid nitrogen. A careful thawing method was used to collect the upper parts of the human hair follicles which were cultured for four weeks in a Dulbecco’s Modified Eagle’s Medium with fetal bovine serum (FBS). Proliferating hair follicle cells were then shifted to DMEM/Ham’s Nutrient Mixture F-12 medium without FBS and allowed to grow for one week. These proliferating cells were able to produce HAP stem cell colonies with multilineage differentiation capacity. They produced keratinocytes, smooth muscle cells, cardiac muscle cells, neurons and glial cells. Interestingly, these cryopreserved hair follicles produced pluripotent HAP stem cell colonies similar to fresh follicles. These findings suggest that the cryopreserved whole human hair follicle preserves the ability to produce HAP stem cells, which will enable any individual to preserve a bank of these stem cells for personalized regenerative medicine.
- ... The hair follicle is dynamic, cycling between growth (anagen), regression (catagen), and resting (telogen) phases throughout life (Cotsarelis et al., 1990;Hoffman, 2000;Oshima et al., 2001). Geneexpression analysis identified several markers including nestin, nanog, OCT4 and Sox-2 that expressed in hair-follicle associated pluripotent (HAP) stem cells (Yu et al., 2010). ...Hair-follicle-associated pluripotent (HAP) stem cells reside in the upper part of the bulge area of the the hair follicle. HAP stem cells are nestin-positive and keratin 15-negative and have the capacity to differentiate into various types of cells in vitro. HAP stem cells are also involved in nerve and spinal cord regeneration in mouse models. Recently, it was shown that the DNA-damage response in non-HAP hair follicle stem cells induces proteolysis of type-XVII collagen (COL17A1/BP180), which is involved in hair-follicle stem-cell maintenance. COL17A1 proteolysis stimulated hair-follicle stem-cell aging, characterized by the loss of stemness signatures and hair-follicle miniaturization associated with androgenic alopecia. In the present study, we demonstrate that HAP stem cells co-express nestin and COL17A1 in vitro and in vivo. The expression of HAP stem cell markers (nestin and SSEA1) increased after HAP stem-cell colonies were formed, then decreased after differentiation to epidermal keratinocytes. In contrast COL17A1 increased after differentiation to epidermal keratinocytes. These results suggest that COL17A1 is important in differentiation of HAP stem cells.
- ... It is sometimes possible to obtain a nuclear DNA (nuDNA) profile from a hair, most often when root sheath cells adhere to the shaft [3]. However, hairs obtained at crime scenes are most often in the telogen (i.e., resting) phase because telogen hairs are no longer attached to the dermal papillae at the root of the hair follicle and fall out easily [4,5]. Telogen phase hairs are also fully keratinized, and the keratinization process results in very little or degraded nuDNA detectable [2,4,6]. ...Hair evidence is commonly found at crime scenes and is first analyzed using microscopy techniques. Hair can be processed for DNA analysis, but nuclear DNA analysis may result in a partial or no profile, and mitochondrial DNA analysis is less discriminatory. Single amino acid polymorphisms (SAPs) in hair shaft keratin proteins that result from non-synonymous single nucleotide polymorphisms (nsSNPs) in the genome are being studied as a method of supplementing microscopic comparison of questioned and known hair evidence. Most studies, however, use large amounts of hair (on the order of hundreds of centimeters of hair shaft length), not representative of operational practice in typical forensic casework analyses. Using a recently developed method of hair shaft protein extraction, this study determines how decreasing hair shaft sample length (i.e., 2 cm to 0.12 cm) affects the identification of hair proteins. For example, in 2 cm hair shaft samples, 16 hair shaft keratin proteins, KRT31-40 and KRT80-86, were high-abundant proteins identified with ˜65% average sequence coverage and 44 peptides on average per protein. When the hair shaft samples were decreased to 0.12 cm, this method still identified 15 hair shaft keratin proteins (i.e., except for KRT40) with ˜47% average sequence coverage and 26 peptides on average per protein. This study demonstrates that even with samples as small as 0.12 cm, hair shaft keratin proteins can still be reliably identified and potentially used forensically. Additionally, using the protein extraction technique described in this study, the adequate hair shaft length required for analysis should be in the range of 0.5 cm to 2 cm. Thus, peptide sequencing for SAP identification can be compatible with forensic casework sample sizes.
- ... They are not only involved in the construction of the interfollicular epithelium, but also in the formation of hair follicles and other skin appendages [9,10]. These cells express cytokeratin 15 (Krt15) [11,12], CD200, and integrin A6 (ITGA6) [13], are located in the niche of the hair follicle (bulge), and maintain a quiescent state (slow-cycling cells) before activation [14]. Some skin substitutes based on hair follicle stem cells have been studied [15][16][17]. ...Background: Induced pluripotent stem cells (iPSCs) can generate epithelial stem cells (EpSCs) as seed cells for skin substitutes to repair skin defects. Here, we investigated the effects of a human acellular amniotic membrane (hAAM) combined with iPSC-derived CD200+/ITGA6+ EpSCs as a skin substitute on repairing skin defects in nude mice. Methods: Human urinary cells isolated from a healthy donor were reprogrammed into iPSCs and then induced into CD200+/ITGA6+ epithelial stem cells. Immunocytochemistry and RT-PCR were used to examine the characteristics of the induced epithelial stem cells. iPSC-derived EpSCs were cultured on a hAAM, and cytocompatibility of the composite was analyzed by CCK8 assays and scanning electron microscopy. Then, hAAMs combined with iPSC-derived EpSCs were transplanted onto skin defects of mice. The effects of this composite on skin repair were evaluated by immunohistochemistry. Results: The results showed that CD200+/ITGA6+ epithelial stem cells induced from iPSCs displayed the phenotypes of hair follicle stem cells. After seeding on the hAAM, iPSC-derived epithelial stem cells had the ability to proliferate. After transplantation, CD200+/ITGA6+ epithelial stem cells on the hAAM promoted the construction of hair follicles and interfollicular epidermis. Conclusions: These results indicated that transplantation of a hAAM combined with iPS-derived EpSCs is feasible to reconstruct skin and skin appendages, and may be a substantial reference for iPSC-based therapy for skin defects.
- ... NSCs in the V-SVZ include slowly-dividing quiescent populations that are able to retain Sphase labels such as BrdU, referred to as label-retaining cells (LRC), for extended periods (Cotsarelis, Sun and Lavker, 1990;Codega et al., 2014). To exclude the possibility that BrdU+ cells in the dorsal V-SVZ are label-retaining quiescent NSCs (qNSC), we examined the proportion of cells that remained proliferative by immunostaining with the mitotic marker Phospho-Histone H3 (Ph-H3) following BrdU labeling ( Fig. 2A). ...The ventricular-subventricular zone (V-SVZ) of the forebrain is the source of neurogenic stem/precursor cells for adaptive and homeostatic needs throughout the life of most mammals. Here, we report that Suppressor of Fused (Sufu) plays a critical role in the establishment of the V-SVZ at early neonatal stages by controlling the proliferation of distinct subpopulations of stem/precursor cells. Conditional deletion of Sufu in radial glial progenitor cells (RGCs) at E13.5 resulted in a dramatic increase in the proliferation of Sox2+ Type B1 cells. In contrast, we found a significant decrease in Gsx2+ and a more dramatic decrease in Tbr2+ transit amplifying cells (TACs) indicating that innate differences between dorsal and ventral forebrain derived Type B1 cells influence Sufu function. However, many precursors accumulated in the dorsal V-SVZ or failed to survive, demonstrating that despite the over-proliferation of Type B1 cells, they are unable to transition into functional differentiated progenies. These defects were accompanied by reduced Gli3 expression and surprisingly, a significant downregulation of Sonic hedgehog (Shh) signaling. Therefore, these findings indicate a potential role of the Sufu-Gli3 regulatory axis in the neonatal dorsal V-SVZ independent of Shh signaling in the establishment and survival of functional stem/precursor cells in the postnatal dorsal V-SVZ.
- ... The H&E staining showed that the size of DPs in secondary HFs changed dramatically between anagen and telogen phases, and that HF undergo a structural change that brings DP closer to HFSCs in the telogen phase ( Figure 2F). Current evidence suggests that the proximity between DP and HFSCs is necessary for HFSCs activation and generation of a new hair [42][43][44]. Taken together, these results indicated that DPCs migrate close to the bulge where HFSCs are located during the telogen phase and may regulate HFSCs by deriving exosomes to generate new hair germ cells. ...Recent studies have demonstrated that dermal papilla cell-derived exosomes (DPC-Exos) promote the anagen stage of hair follicle (HF) growth and delay the catagen stage. However, the roles of DPC-Exos in regulating hair follicle stem cell (HFSC) quiescence and activation remain unknown. Here, we found that HFSC differentiation was induced by co-culture with DPCs, and that DPC-Exos attached to the surface of HFSCs. Using micro RNA (miRNA) high-throughput sequencing, we identified 111 miRNAs that were significantly differentially expressed between DPC-Exos and DPCs, and the predicted target genes of the top 34 differentially expressed miRNAs indicated that DPC-Exos regulate HFSCs proliferation and differentiation via genes involved in cellular signal transduction, fatty acid expression regulation, and cellular communication. The overexpression of miR-22-5p indicated that it negatively regulates HFSC proliferation and LEF1 was revealed as the direct target gene of miR-22-5p. We therefore propose the miR-22-5p-LEF1 axis as a novel pathway regulating HFSC proliferation.
- ... HFs are known to have a well-characterized niche for grown-up SCs-the bulge, which contains ESCs and melanocytic SCs [119]. SCs in the hair bulge, an obviously-differentiated compartment inside the lower portion of hair follicles, can produce inter-follicular epidermis, HF structures, and sebaceous glands [120,121]. The bulge ESCs can also reconstitute in a simulated in vivo framework to a new HF [122,123]. ...The use of stem cells has been reported to improve hair regrowth in several therapeutic strategies, including reversing the pathological mechanisms, that contribute to hair loss, regeneration of hair follicles, or creating hair using the tissue-engineering approach. Although various promising stem cell approaches are progressing via pre-clinical models to clinical trials, intraoperative stem cell treatments with a one-step procedure offer a quicker result by incorporating an autologous cell source without manipulation, which may be injected by surgeons through a well-established clinical practice. Many authors have concentrated on adipose-derived stromal vascular cells due to their ability to separate into numerous cell genealogies, platelet-rich plasma for its ability to enhance cell multiplication and neo-angiogenesis, as well as human follicle mesenchymal stem cells. In this paper, the significant improvements in intraoperative stem cell approaches, from in vivo models to clinical investigations, are reviewed. The potential regenerative instruments and functions of various cell populaces in the hair regrowth process are discussed. The addition of Wnt signaling in dermal papilla cells is considered a key factor in stimulating hair growth. Mesenchymal stem cell-derived signaling and growth factors obtained by platelets influence hair growth through cellular proliferation to prolong the anagen phase (FGF-7), induce cell growth (ERK activation), stimulate hair follicle development (β-catenin), and suppress apoptotic cues (Bcl-2 release and Akt activation).
- ... Hair follicle (HF) development is a complex morphogenetic process that relies on a variety of signaling systems, and on interactions between mesenchymal and epithelial tissues (Hardy, 1992;Oro and Scott, 1998). Under the biological regulation of stem cells, mature HFs undergo a cycling and continuous self-renewal process, with periods of active growth (anagen), followed by regression (catagen), and rest (telogen) (Cotsarelis et al., 1990;Paus and Cotsarelis, 1999;Fuchs and Segre, 2000;Oshima et al., 2001). In murine HF cycling, key parameters for the recognition of distinct stages have been defined in many studies (Chase et al., 1951;Chase, 1954;Straile et al., 2010). ...The hair follicle (HF) cycle is a complicated and dynamic process in mammals, associated with various signaling pathways and gene expression patterns. Non-coding RNAs (ncRNAs) are RNA molecules that are not translated into proteins but are involved in the regulation of various cellular and biological processes. This study explored the relationship between ncRNAs and the HF cycle by developing a synchronization model in Angora rabbits. Transcriptome analysis was performed to investigate ncRNAs and mRNAs associated with the various stages of the HF cycle. One hundred and eleven long non-coding RNAs (lncRNAs), 247 circular RNAs (circRNAs), 97 microRNAs (miRNAs), and 1,168 mRNAs were differentially expressed during the three HF growth stages. Quantitative real-time PCR was used to validate the ncRNA transcriptome analysis results. Gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses provided information on the possible roles of ncRNAs and mRNAs during the HF cycle. In addition, lncRNA–miRNA–mRNA and circRNA–miRNA–mRNA ceRNA networks were constructed to investigate the underlying relationships between ncRNAs and mRNAs. LNC_002919 and novel_circ_0026326 were found to act as ceRNAs and participated in the regulation of the HF cycle as miR-320-3p sponges. This research comprehensively identified candidate regulatory ncRNAs during the HF cycle by transcriptome analysis, highlighting the possible association between ncRNAs and the regulation of hair growth. This study provides a basis for systematic further research and new insights on the regulation of the HF cycle.
- ... Vital dye labeling was the first physical approach used for cell lineage tracing in the early 20 th century. This method employed agar chips impregnated with vital dyes and lipid-soluble carbocyanine dyes, such as octadecyl (C18) indocarbocyanines and oxacarbocyanine, that were integrated into the plasma membrane (4) (Fig. 1A); substrate activated horseradish peroxidase (HRP)conjugated lipid-soluble fluorescence (5,6); or DNA/histone level labeling (7)(8)(9). When cells migrate and divide, the fate of their descendants can be tracked by cell labeling. ...Site-specific recombinases, such as Cre, are a widely used tool for genetic lineage tracing in the fields of developmental biology, neural science, stem cell biology, and regenerative medicine. However, unspecific cell labeling by some genetic Cre tools remains a technical limitation of this recombination system, which has resulted in data misinterpretation and led to many controversies in the scientific community. In the past decade, to enhance the specificity and precision of genetic targeting, researchers have used two or more orthogonal recombinases simultaneously for labeling cell lineages. Here, we review the history of cell tracing strategies and then elaborate on the working principle and application of a recently developed dual genetic lineage tracing approach for cell fate studies. We place an emphasis on discussing the technical strengths and caveats of different methods, with the goal to develop more specific and efficient tracing technologies for cell fate mapping. Our review also provides several examples for how to use different types of DNA recombinase–mediated lineage tracing strategies to improve the resolution of the cell fate mapping in order to probe and explore cell fate–related biological phenomena in the life sciences.
- ... The matrix seems to be organized by two systems working in orthogonal dimensions and controlling two key operations of hair follicle morphogenesis, notably cell diversification and cell behavior [17]. The bulge cell progeny located in the upper follicle has been demonstrated to emigrate into the epidermis and to proliferate, thus contributing to the long-term maintenance of the epidermis [18,19]. Based on these observations, it has been proposed that the bulge is a major repository of skin keratinocyte stem cells, which may thus be regarded as the ultimate epidermal stem cell [20,21]. ...The aim of the study was the objective assessment of the effectiveness of a microfragmented dermal extract obtained with Rigenera™ technology in promoting the wound healing process in an in vivo homogeneous experimental human acute surgical wound model. The study included 20 patients with 24 acute postsurgical soft tissue loss and a planned sequential two-stage repair with a dermal substitute and an autologous split-thickness skin graft. Each acute postsurgical soft tissue loss was randomized to be treated either with an Integra® dermal substitute enriched with the autologous dermal micrografts obtained with Rigenera™ technology (group A—Rigenera™ protocol) or with an Integra® dermal substitute only (group B—control). The reepithelialization rate in the wounds was assessed in both groups at 4 weeks through digital photography with the software “ImageJ.” The dermal cell suspension enrichment with the Rigenera™ technology was considered effective if the reepithelialized area was higher than 25% of the total wound surface as this threshold was considered far beyond the expected spontaneous reepithelialization rate. In the Rigenera™ protocol group, the statistical analysis failed to demonstrate any significant difference vs. the controls. The old age of the patients likely influenced the outcome as the stem cell regenerative potential is reduced in the elderly. A further explanation for the unsatisfying results of our trial might be the inadequate amount of dermal stem cells used to enrich the dermal substitutes. In our study, we used a 1 : 200 donor/recipient site ratio to minimize donor site morbidity. The gross dimensional disparity between the donor and recipient sites and the low concentration of dermal mesenchymal stromal stem cells might explain the poor epithelial proliferative boost observed in our study. A potential option in the future might be preconditioning of the dermal stem cell harvest with senolytic active principles that would fully enhance their regenerative potential. This trial is registered with trial protocol number NCT03912675 .
- ... In DKO skin, both Gli1 and Gli2 transcriptional regulators in the hedgehog pathway were upregulated. Hair cycles are fueled by HF stem cells that reside in the "bulge" niche located at the base of the telogen phase HF [48]. In addition to Gli1, which is expressed in the hair bulge and germ [49,50], several other markers of the bulge stem cell compartment were upregulated in DKO skin. ...Retinol dehydrogenases catalyze the rate-limiting step in the biosynthesis of retinoic acid, a bioactive lipid molecule that regulates the expression of hundreds of genes by binding to nuclear transcription factors, the retinoic acid receptors. Several enzymes exhibit retinol dehydrogenase activities in vitro; however, their physiological relevance for retinoic acid biosynthesis in vivo remains unclear. Here, we present evidence that two murine epidermal retinol dehydrogenases, short-chain dehydrogenase/reductase family 16C member 5 (SDR16C5) and SDR16C6, contribute to retinoic acid biosynthesis in living cells and are also essential for the oxidation of retinol to retinaldehyde in vivo Mice with targeted knockout of the more catalytically active SDR16C6 enzyme have no obvious phenotype, possibly due to functional redundancy, because Sdr16c5 and Sdr16c6 exhibit an overlapping expression pattern during later developmental stages and in adulthood. Mice that lack both enzymes are viable and fertile, but display accelerated hair growth after shaving and also enlarged meibomian glands, consistent with a nearly 80% reduction in the retinol dehydrogenase activities of skin membrane fractions from the Sdr16c5/Sdr16c6 double-knockout mice. The up-regulation of hair-follicle stem cell genes is consistent with reduced retinoic acid signaling in the skin of the double-knockout mice. These results indicate that the retinol dehydrogenase activities of murine SDR16C5 and SDR16C6 enzymes are not critical for survival, but are responsible for most of the retinol dehydrogenase activity in skin, essential for the regulation of the hair-follicle cycle, and required for the maintenance of both sebaceous and meibomian glands.
- ... This apoptotic-driven regression causes DP to move upward, bringing it closer to the epithelial bulge. 21 Following complete regression, HF enters a quiescent phase (telogen), which can last several months. The replacement of the old hair shaft fiber by the forming club fiber at the end of telogen is called "exogen." ...The demand for an efficient therapy for alopecia disease has fueled the hair research field in recent decades. However, despite significant improvements in the knowledge of key processes of hair follicle biology such as genesis and cycling, translation into hair follicle replacement therapies has not occurred. Great expectation has been recently put on hair follicle bioengineering, which is based on the development of fully functional hair follicles with cycling activity from an expanded population of hair‐inductive (trichogenic) cells. Most bioengineering approaches focus on in vitro reconstruction of folliculogenesis by manipulating key regulatory molecular/physical features of hair follicle growth/cycling in vivo. Despite their great potential, no cell‐based product is clinically available for hair regeneration therapy to date. This is mainly due to demanding issues that still hinder the functionality of cultured human hair cells. The present review comprehensively compares emergent strategies using different cell sources and tissue engineering approaches, aiming to successfully achieve a clinical cure for hair loss. The hurdles of these strategies are discussed, as well as the future directions to overcome the obstacles and fulfill the promise of a “hairy” feat. Steps toward human hair follicle regeneration. Cell isolation: cell sources for bioengineering can be follicular (bulge stem cells, dermal papilla, and dermal sheath cells) and non‐follicular (keratinocytes, skin‐derived progenitors, and mesenchymal stem cells). Cell expansion: mesenchymal and epithelial cell sources are cultured in vitro. Bioengineering: cell clustering in 3D instructive hair bulbs. Implantation: bulbs could generate functional hair follicles.
- ... In the skin, SCs reside in the basal compartment of the interfollicular epidermis (IFE) and in discrete compartments within ectodermal skin appendages -namely the pilosebaceous unit and sweat gland (Lu et al., 2012). Decades of work primarily from mouse studies have demonstrated the presence of multiple SC pools residing in various compartments of the pilosebaceous unit, including the bulge Cotsarelis et al., 1990;Liu et al., 2003;Lyle et al., 1998;Morris et al., 2004;Trempus et al., 2003;Tumbar et al., 2004), hair germ (Greco et al., 2009;Ito and Kizawa, 2001;Ito et al., 2002;Rompolas et al., 2012), isthmus (Jensen et al., 2008;Nijhof et al., 2006;Snippert et al., 2010), junctional zone (Page et al., 2013), upper portion of the infundibulum (Jensen et al., 2008), and sebaceous gland (Cottle et al., 2013;Horsley et al., 2006;Jensen et al., 2009). In contrast to the pilosebaceous unit, basal SCs in the IFE are considered more homogenous and are thought to have one or two distinct subpopulations depending on the body site in mouse or human. ...How stem cells give rise to human interfollicular epidermis is unclear despite the crucial role the epidermis plays in barrier and appendage formation. Here we use single cell-RNA sequencing to interrogate basal stem cell heterogeneity of human interfollicular epidermis and find at least four spatially distinct stem cell populations that decorate the top and bottom of rete ridge architecture and hold transitional positions between the basal and suprabasal epidermal layers. Cell-cell communication modeling through co-variance of cognate ligand-receptor pairs indicate that the basal cell populations distinctly serve as critical signaling hubs that maintain epidermal communication. Combining pseudotime, RNA velocity, and cellular entropy analyses point to a hierarchical differentiation lineage supporting multi-stem cell interfollicular epidermal homeostasis models and suggest the transitional basal stem cells are stable states essential for proper stratification. Finally, alterations in differentially expressed transitional basal stem cell genes result in severe thinning of human skin equivalents, validating their essential role in epidermal homeostasis and reinforcing the critical nature of basal stem cell heterogeneity.
- ... In adult skin, HFSCs reside in an anatomical niche called the bulge (17), where they fuel the synchronized, cyclical bouts of quiescence (telogen), active hair growth (anagen), and destruction (catagen). Just above the bulge is the attachment site of the arrector pili muscle (APM), which thermoregulates by controlling the positioning of the hairs relative to the body surface. ...Aging manifests with architectural alteration and functional decline of multiple organs throughout an organism. In mammals, aged skin is accompanied by a marked reduction in hair cycling and appearance of bald patches, leading researchers to propose that hair follicle stem cells (HFSCs) are either lost, differentiate, or change to an epidermal fate during aging. Here, we employed single-cell RNA-sequencing to interrogate aging-related changes in the HFSCs. Surprisingly, although numbers declined, aging HFSCs were present, maintained their identity, and showed no overt signs of shifting to an epidermal fate. However, they did exhibit prevalent transcriptional changes particularly in extracellular matrix genes, and this was accompanied by profound structural perturbations in the aging SC niche. Moreover, marked age-related changes occurred in many nonepithelial cell types, including resident immune cells, sensory neurons, and arrector pili muscles. Each of these SC niche components has been shown to influence HF regeneration. When we performed skin injuries that are known to mobilize young HFSCs to exit their niche and regenerate HFs, we discovered that aged skin is defective at doing so. Interestingly, however, in transplantation assays in vivo, aged HFSCs regenerated HFs when supported with young dermis, while young HFSCs failed to regenerate HFs when combined with aged dermis. Together, our findings highlight the importance of SC:niche interactions and favor a model where youthfulness of the niche microenvironment plays a dominant role in dictating the properties of its SCs and tissue health and fitness.
- ... Hair follicle stem cells (HFSCs) can be obtained easily and isolated from the skin. HFSCs are located in a specialised niche within the outer root sheath of the hair follicles, known as the bulge [17,18]. HFSCs can differentiate not only into intact hair follicles but also into sebaceous glands, epidermal keratinocytes and other cells [19,20]. ...Tissue-engineered skin (TES), as an analogue of native skin, is promising for wound repair and regeneration. However, a major drawback of TES products is a lack of skin appendages and nerves to enhance skin healing, structural integrity and skin vitality. Skin appendages and nerves are important constituents for fully functional skin. To date, many studies have yielded remarkable results in the field of skin appendages reconstruction and nerve regeneration. However, patients often complain about a loss of skin sensation and even cutaneous chronic pain. Restoration of pain, temperature, and touch perceptions should now be a major challenge to solve in order to improve patients' quality of life. Current strategies to create skin appendages and sensory nerve regeneration are mainly based on different types of seeding cells, scaffold materials, bioactive factors and involved signaling pathways. This article provides a comprehensive overview of different strategies for, and advances in, skin appendages and sensory nerve regeneration, which is an important issue in the field of tissue engineering and regenerative medicine.
- ... Nevertheless, to ensure that Tyr + McSCs were responsible for interfollicular epidermal expansion, we injected BrdU during anagen onset to specifically label cycling McSCs but not quiescent dermal Tyr + cells and then followed with TAMinduction ( Supplementary Fig. 3k) 27,41 . BrdU + /Tomato + label retaining epidermal melanocytes were observed, verifying the origin of melanoma Mcs as follicular McSCs (Supplementary Fig. 3l-n). ...Melanoma, the deadliest skin cancer, remains largely incurable at advanced stages. Currently, there is a lack of animal models that resemble human melanoma initiation and progression. Recent studies using a Tyr-CreER driven mouse model have drawn contradictory conclusions about the potential of melanocyte stem cells (McSCs) to form melanoma. Here, we employ a c-Kit-CreER-driven model that specifically targets McSCs to show that oncogenic McSCs are a bona fide source of melanoma that expand in the niche, and then establish epidermal melanomas that invade into the underlying dermis. Further, normal Wnt and Endothelin niche signals during hair anagen onset are hijacked to promote McSC malignant transformation during melanoma induction. Finally, molecular profiling reveals strong resemblance of murine McSC-derived melanoma to human melanoma in heterogeneity and gene signatures. These findings provide experimental validation of the human melanoma progression model and key insights into the transformation and heterogeneity of McSC-derived melanoma.
- ... In AGA, the miniaturization of the follicles is determined by diminishment of anagen and with an improvement in the percentage of resting hair follicles (HFs), telogen, containing microscopic hairs in a hairless scalp [5]. Moreover, invading lymphocytes and mast cells have been seen around the miniaturizing follicle [6], detailed in the stem cell-rich lump zone [7]. ...Tissue engineering in hair regrowth aims to develop innovative and not-invasive procedures to advance the hair regrowth. A placebo-controlled, randomized, evaluator-blinded, half-head group study to compare hair regrowth with micrografts containing human hair follicle mesenchymal stem cells (HF-MSCs) vs. placebo was reported. After 58 weeks, 27 patients displayed in the targeted area an increase of hair count and hair density, respectively, of 18.0 hairs per 0.65 cm2 and 23.3 hairs per cm2 compared with baseline, while the control area displayed a mean decrease of 1.1 hairs per 0.65 cm2 and 0.7 hairs per cm2 (control vs. treatment: P
- ... In the classical model of tissue renewal, a small number of quiescent stem cells act as the starting point for renewal, being responsible for the formation of rapidly dividing transit-amplifying progenitors, which later differentiate into multiple lineages (Cotsarelis et al., 1990;Fuchs, 2009). Based on this model, a population of longlived, label-retaining cells residing in the OEE and the adjacent SR region in the proximal part of the adult mouse laCL were proposed to function as dental epithelial stem cells, (Fig. 5A) (Harada et al., 1999;Biehs et al., 2013). ...The tooth provides an excellent system for deciphering the molecular mechanisms of organogenesis, and has thus been of longstanding interest to developmental and stem cell biologists studying embryonic morphogenesis and adult tissue renewal. In recent years, analyses of molecular signaling networks, together with new insights into cellular heterogeneity, have greatly improved our knowledge of the dynamic epithelial-mesenchymal interactions that take place during tooth development and homeostasis. Here, we review recent progress in the field of mammalian tooth morphogenesis and also discuss the mechanisms regulating stem cell-based dental tissue homeostasis, regeneration and repair. These exciting findings help to lay a foundation that will ultimately enable the application of fundamental research discoveries toward therapies to improve oral health.
- ... Some non-quiescent cell types which are replenished include epithelial lining of the small intestine and colon [60] [61], epithelial lining of the breast duct [62], epihelial lining of the airways (e.g. lung) [63], epithelial lining of the the prostate [64], the haematopoietic system [65] and epidermal cells of the skin [66]. Cell differentiation through a lineage and tissue organization is tightly regulated by programmed cell death with diverse mechanisms [67], of which the most common and important is apoptosis [68]. ...Cancer is a disease of aberrant cell proliferation and tumour growth arising from the perturbation of the epigenetically defined, regulated and maintained cell identity by genetic mutations. It is a leading cause of death worldwide and most cancer types remain incurable. Omics technologies are quantitative analytical assays that allow high-quality and high-throughput measurements of different aspects of cellular regulation including genomics, transcriptomics, epigenomics, proteomics and metabolomics. These high-throughput technologies transformed the way cancer research is done, leading to tremendous advances in our understanding of cancer biology and modern targeted therapies. Integrative analysis of multi-omics datasets in cancer research requires use of dedicated algorithms, data analysis and visualization tools. These are developed and applied in interdisciplinary teams of scientists and clinicians working on collaborative projects. Both the technical complexities of data analysis and their integration, and the efficient independent exploration of the observations by all project partners are contemporary research challenges. This dissertation presents results addressing a broad spectrum of these questions. Chapter 1, Replacing the CNS-PNET Superentity with Four Novel Molecularly Defined Entities Driven by Structural Variants: Central nervous system primitive neuroectodermal tumours (CNS-PNETs) were a heterogeneous family of paediatric brain tumours with no histopathological markers, challenging diagnosis and poor prognosis. My work as a computational biologist contributed to the comprehensive description of this entity. In this study, we applied an integrative omics data analysis of methylomes, transcriptomes and genomes revealing that CNS-PNETs are a combination of a large group of misdiagnosed cases from other entities and four novel molecularly defined entities. I showed that these novel entities are driven by distinct and recurrent molecular drivers altered by different mechanisms of structural variants: the FOXR2 oncogene and MN1, CIC and BCOR tumour suppressors. Our results contributed to the elimination of CNS-PNETs as an officially recognized cancer entity and the recognition of four novel paediatric brain tumour entities in the World Health Organization classification of brain tumours. Chapter 2, SOPHIA, Structural Rearrangement Detection Based on Supplementary Alignments and a Population Background Model: Building on my work on structural variation in our study of CNS-PNETs, I developed the SOPHIA algorithm for detecting SVs in cancer genomes based on a large population background database and a corresponding bioinformatics tool written allowing fast detection of SVs from short read cancer genome sequencing datasets. SOPHIA later became the standard tool for structural variant detection in the DKFZ’s cancer genome analysis workflow. Chapter 3, EPISTEME, an Interactive and Integrative Platform for Analysing, Interpreting and Sharing Multi-Omics Data: During the development of SOPHIA and my research in projects analysing and interpreting structural variant data, I developed experiences analysing structural variant data detected by SOPHIA, integrating them with different omics layers such as gene expressions, interpreting, visualizing and sharing them with collaborators who were not computational scientists. Based on these experiences and using modern tools of interactive data visualization, I developed an interactive platform for integrative omics data analysis and visualization named EPISTEME, with the aim of facilitating omics data analysis by scientists with conceptual knowledge of cancer omics but no programming skills. EPISTEME is a comprehensive tool integrating genome, transcriptome, methylome and proteome data with clinical metadata in a user-friendly web-based system with in-browser statistical analyses and publication-quality vector graphics output. Chapter 4, SOPHIA-EPISTEME integration in DKFZ Cancer Genomics Projects Reveals Novel Disease Subtypes and Insights Across Cancer Types: With the integration of SOPHIA and EPISTEME in an integrative omics data analysis setting, my work identified novel oncogenes activated by enhancer hijacking and revealed novel molecularly defined subtypes in refractory multiple myeloma (MYCN enhancer hijacking via immunoglobulin rearrangements as a MYC replacement), adult acute myeloid leukaemia (MNX1 activation via enhancer hijacking putatively acting as a differentiation block mechanism) and paediatric neuroblastoma (ATOH1 activation via enhancer hijacking putatively acting as a MYCN replacement) in projects supported by the DKFZ Heidelberg Center for Personalized Oncology (DKFZ-HIPO) and the German Society for Paediatric Oncology and Haematology (GPOH) cancer research programmes.
- ... The conventional method to release growth factors from the platelets is to induce platelet contraction by calcium which activate thrombin. 4,31 In this study, we found that sonication of PRP which broke platelets into tiny pieces better released growth factors, as evidenced by growth factor array analysis. Sixteen growth factors of 41 growth factors analysed showed higher levels in sonicated PRP than in calcium-treated PRP, and several of them have been known to regulate hair follicle cell activities such as FGF7, VEGF, PDGF-BB, HB-EGF and TGF-β. ...An increasing number of studies show that platelet-rich plasma (PRP) is effective for androgenic alopecia (AGA). However, the underlying cellular and molecular mechanisms along with its effect on hair follicle stem cells are poorly understood. In this study, we designed to induce platelets in PRP to release factors by calcium chloride (PC) or by sonication where platelet lysates (PS) or the supernatants of platelet lysate (PSS) were used to evaluate their effect on the hair follicle activation and regeneration. We found that PSS and PS exhibited a superior effect in activating telogen hair follicles than PC. In addition, PSS injection into the skin activated quiescent hair follicles and induced K15+ hair follicle stem cell proliferation in K14-H2B-GFP mice. Moreover, PSS promoted skin-derived precursor (SKP) survival in vitro and enhanced hair follicle formation in vivo. In consistence, protein array analysis of different PRP preparations revealed that PSS contained higher levels of 16 growth factors (out of 41 factors analysed) than PC, many of them have been known to promote hair follicle regeneration. Thus, our data indicate that sonicated PRP promotes hair follicle stem cell activation and de novo hair follicle regeneration.
- ... To assess the status of long-term label retaining stem cells (LRCs) of the HFs we performed ethynyldeoxyuridine (EdU) pulse chase experiments. The retention of the EdU label in the bulge is a read out of the presence of the quiescent stem cells (Cotsarelis et al., 1990;Morgner et al., 2015). The expression of EdU was significantly reduced in the first telogen follicles, and completely absent from the second telogen bulge compartment, suggesting a loss of these quiescent pools of stem cells in the vinculin KO animals ( Fig 2C, C', D, D'). ...Vinculin, a mechanotransducer associated with both adherens junctions (AJ) and focal adhesions (FA) plays a central role in force transmission through these cell-cell and cell-substratum contacts. Here we describe the conditional knock out (KO) of vinculin in murine skin. Remarkably, we find that the loss of vinculin function results in the loss of bulge stem cell (BuSC) quiescence. We demonstrate that vinculin KO cells are impaired in force generation resulting in mechanically weak AJs. Mechanistically, vinculin functions by keeping α-catenin in a stretched conformation, which in turn regulates the retention of YAP1, another potent mechanotransducer and regulator of cell proliferation, to the junctions. Conditional KO of α-catenin specifically in the BuSCs further corroborates the importance of stable AJs in the maintenance of quiescence and stemness. Altogether, our data provides definitive mechanistic insights into the hitherto unexplored regulatory link between the mechanical stability of cell-junctions and the maintenance of BuSC quiescence.
- ... To further evaluate their dormancy, we performed a BrdU label-retaining assay, a method for detection of dormant cells in various tissues (Arai et al., 2004;Cotsarelis et al., 1990;Potten et al., 1978). After 2 weeks of BrdU treatment, 71.3% ± 2.3% of G 0 M low LT-HSCs were labeled, whereas only 39.4% ± 4.8% of G 0 M high LT-HSCs were labeled ( Figure 1H). ...Quiescent hematopoietic stem cells (HSCs) are typically dormant, and only a few quiescent HSCs are active. The relationship between “dormant” and “active” HSCs remains unresolved. Here we generate a G0 marker (G0M) mouse line that visualizes quiescent cells and identify a small population of active HSCs (G0Mlow), which are distinct from dormant HSCs (G0Mhigh), within the conventional quiescent HSC fraction. Single-cell RNA-seq analyses show that the gene expression profiles of these populations are nearly identical but differ in their Cdk4/6 activity. Furthermore, high-throughput small-molecule screening reveals that high concentrations of cytoplasmic calcium ([Ca2+]c) are linked to dormancy of HSCs. These findings indicate that G0M separates dormant and active adult HSCs, which are regulated by Cdk4/6 and [Ca2+]c. This G0M mouse line represents a useful resource for investigating physiologically important stem cell subpopulations. : Fukushima et al. show that G0 marker (G0M) discriminates between dormant and active HSCs within the conventional quiescent HSC fraction. Small-molecule screening reveals that high [Ca2+]c is linked to dormancy of HSCs. Moreover, upregulation of [Ca2+]c by thapsigargin enhances the bone marrow reconstitution ability of HSCs. Keywords: hematopoietic stem cell, HSC, cell cycle, G0 phase, p27, dormancy, CDK, calcium
- ... To reboot the progenitor pool throughout life, repeated cycles of follicle regression (catagen), rest (telogen), and regrowth (anagen) (7) (fig. S1B) derive new cells from the stem cell reservoir located in the bulge and germ of the upper follicle (8)(9)(10)(11)(12). At the beginning of regression, matrix progenitors stop proliferating and terminally differentiate (fig. ...Tissue homeostasis requires the balance of growth by cell production and regression through cell loss. In the hair cycle during follicle regression, the niche traverses the skin through an unknown mechanism to reach the stem cell reservoir and trigger new growth. Here we identify the dermal sheath that lines the follicle as the key driver of tissue regression and niche relocation via the smooth muscle contractile machinery that generates centripetal constriction force. We reveal that the calcium/calmodulin/myosin light chain kinase pathway controls sheath contraction. When this pathway is blocked, sheath contraction is inhibited, impeding follicle regression and niche relocation. Thus, our study identifies the dermal sheath as smooth muscle that drives follicle regression for reuniting niche and stem cells in order to regenerate tissue structure during homeostasis.
- Objectives: A retrospective case-series study comparing autologous activated platelet-rich plasma (AA-PRP) versus autologous non-activated platelet-rich plasma (A-PRP) in hair re-growth was reported. Methods: 90 patients, 63 males showing AGA in stage I–V by the Norwood–Hamilton scale and 27 females with AGA in stage I–III by the Ludwig scale, treated since 2013, were analyzed. 57 patients were treated with A-PRP injections and 33 patients were treated with AA-PRP in three sessions spaced 30 days average. Assessment of hair re-growth was evaluated in different weeks (Ws) after the treatment, summarized in four phases: T0, before the first infusion, T1 - 12 Ws, T2 - 23 Ws, T3 - 44 Ws, T4 - 58 Ws after the last treatment. Results: 12 Ws, 23 Ws, 44 Ws, and 58Ws after the last treatment, hair density measurements for patients treated with A-PRP and AA-PRP were 65 ± 5 and 28 ± 4 hairs/cm2 at T1, 28 ± 2 and 15 ± 3 hairs/cm2 at T2, 25 ± 3 and 14 ± 3 hairs/cm2 at T3, 23 ± 3 and 13 ± 3 hairs/cm² at T4. Conclusion: The effects of A-PRP and AA-PRP in hair re-growth during a long-term follow-up, was demonstrated.
- Tracking the fate of cells in murine epidermis in vivo has revealed that a committed progenitor cell population can maintain normal adult tissue in the long term without support from a long-lived, self-renewing population of stem cells. Here, we argue that these results challenge the dogma that stem-cell proliferation is required for the cellular homeostasis of the epidermis and other adult tissues, with important implications for tissue physiology and disease.
- The purpose of wound healing is to repair the skin to prevent infection and to restore tissue integrity and function. Unfortunately, in adults, this process is geared toward faster rates of healing, to prevent infection, which ultimately leads to a compromise in the quality of healing. This compromise results in scarring, where the architecture of the skin is distinct from the original tissue, significantly affecting function and overall quality of life. The ideal for future treatments is to increase the rates of healing while improving the quality of healing resulting in more of a regenerative process rather than a repair-orientated one.
- Stem cells are required for lifelong homeostasis and regeneration of tissues and organs in mammals, but the function of stem cells declines during aging. To preserve stem cells during life, they are kept in a quiescent state with low metabolic and low proliferative activity. However, activation of quiescent stem cells – an essential process for organ homeostasis/regeneration – requires concerted and faithful regulation of multiple molecular circuits controlling biosynthetic processes, repair mechanisms, and metabolic activity. Thus, while protecting stem cell maintenance, quiescence comes at the cost of vulnerability during the process of stem cell activation. Here we discuss molecular and biochemical processes regulating stem cells’ maintenance in and exit from quiescence and how age-related failures of these circuits can contribute to organism aging.
- Background: Postacne scars are still a challenge in its management. Microneedling is a popular minimally invasive technique in treatment of such scars. However, the addition of topical stem cell products after microneedling is considered a new treatment regimen for these scars. Objective: To compare efficacy of amniotic fluid-derived mesenchymal stem cell-conditioned media (AF-MSC-CM) and microneedling vs microneedling alone in management of atrophic acne scars. Methods: Ten cases with atrophic postacne scars received five sessions of microneedling, with 2-week interval on both sides of the face. Then, AF-MSC-CM was topically applied to right side of the face after microneedling. Clinical examination with histopathological and computerized histometric analysis was done 1 month after the sessions. Results: There was significant increase in the improvement percentage of acne scars on right side (dermaroller and AF-MSC-CM) vs left side of face (dermaroller; P < 0.001). Histologically, improvement of character of collagen and elastic fibers was noticed, especially on right side. Meanwhile, significant increase in epidermal thickness on both sides of face was detected. Conclusion: Amniotic fluid-derived mesenchymal stem cell-conditioned media combined with microneedling is more effective in management of atrophic postacne scars than microneedling alone.
- Demand for long-term hair reduction treatments has increased dramatically around the world in recent years, due to traditional factors, fashion, sport, wellbeing and health requirements. Some of the most popular options include laser and intense pulsed light (IPL) treatments, which are often administered by aesthetic practitioners. This article will discuss the hair growth cycle, as well as treatment options, parameters and outcomes.
- The skin is a large and complex organ. While skin development begins during early embryogenesis, full development is not complete until well into the postnatal years. Studies of skin development can shed light on a number of basic problems in contemporary biology: epithelial–mesenchymal interactions that establish organs (in skin, these tissue interactions occur in follicle, sweat gland and nail formation); cell–cell interactions through soluble mediators; gene regulation; apoptosis; differentiation (structural, biochemical and functional); and certain longstanding basic phenomena of development such as induction, pattern formation and differentiation. Rigorous understanding of embryogenesis allows definition of critical periods when the skin may be more vulnerable to developmental errors. Further understanding of these critical processes advances the study of developmental disorders of the skin with the promise of improving therapeutic options for these disorders.
- Stem cells can reside in a state of reversible growth arrest, or quiescence, for prolonged periods of time. Although quiescence has long been viewed as a dormant, low-activity state, increasing evidence suggests that quiescence represents states of poised potential and active restraint, as stem cells "idle" in anticipation of activation, proliferation, and differentiation. Improved understanding of quiescent stem cell dynamics is leading to novel approaches to enhance maintenance and repair of aged or diseased tissues. In this Review, we discuss recent advances in our understanding of stem cell quiescence and techniques enabling more refined analyses of quiescence in vivo.
- Tissue renewal requires proliferative progenitors with long-lasting potential. Designated stem cells within specialized niches are considered to be the primary mechanism for this requirement. Recent studies show that dispersed equipotent progenitors are sufficient to account for fast-paced cellular dynamics in skin oil glands and foetal gut epithelium.
- Stem cells support tissue maintenance, but the mechanisms that balance the rate of stem cell self-renewal with differentiation at a population level remain uncharacterized. Through investigating the regulation of germline stem cells by two PUF family RNA-binding proteins FBF-1 and FBF-2 in C. elegans , we find that FBF-1 restricts differentiation, while FBF-2 promotes both proliferation and differentiation. FBFs act on a shared set of target mRNAs; however, FBF-1 destabilizes target transcripts, while FBF-2 promotes their accumulation. These regulatory differences result in complementary effects of FBFs on stem cells. We identify a mitotic cyclin as one of the targets affecting stem cell homeostasis. FBF-1-mediated translational control requires the activity of CCR4-NOT deadenylase. Distinct abilities of FBFs to cooperate with CCR4-NOT depend on protein sequences outside of the conserved PUF family RNA-binding domain. We propose that the combination of FBF activities regulates the dynamics of germline stem cell proliferation and differentiation.
- Wound healing consists of multiple structured mechanism and is influenced by various factors. Epithelialization is one of the major aspect in wound healing and inhibition of this mechanism will greatly impair wound healing. Epithelialization is a process where epithelial cells migrate upwards and repair the wounded area. This process is the most essential part in wound healing and occurs in proliferative phase of wound healing. Skin stem cells which reside in several locations of epidermis contribute in the re-epithelialization when the skin is damaged. Epithelialization process is activated by inflammatory signal and then keratinocyte migrate, differentiate and stratify to close the defect in the skin. Several theories of epithelialization model in wound healing have been proposed for decades and have shown the mechanism of epidermal cell migration during epithelialization even though the exact mechanism is still controversial. This process is known to be influenced by the wound environment where moist wound environment is preferred rather than dry wound environment. In dry wound environment, epithelialization is known to be inhibited because of scab or crust which is formed from dehydrated and dead cells. Moist wound environment enhances the epithelialization process by easier migration of epidermal cells, faster epithelialization, and prolonged presence of proteinases and growth factors. This article focuses on the epithelialization process in wound healing, epithelialization models, effects of wound environment on epithelialization and epithelialization as the basis for products that enhance wound healing.
- Introduction: Felix Pinkus' concept of the vitreous membrane (VM) published in 1927 describes circular folds protruding into the outer root sheath (ORS), which, in his opinion, serve as interdigitations between the outer root sheath (ORS) and the VM. This concept currently seems to have fallen into oblivion. Objective: To determine the origin and possible function of the VM in the proliferation and vascularization of the hair follicle (HF). Methods: Serial investigation of healthy skin probes with histological (hematoxylin & eosin and periodic acid-Schiff) and immunohistochemical examination (Ki67, CD56, CD8, and collagen IV) were performed. Results: Morphological variations of the VM in various HFs such as protrusions and folds, the latter unilateral, bilateral or circular, some acute-angled, were found. Similarly, protrusions of the VM into the ORS were observed, that consisted of capillary tissue together with perifollicular tissue and VM mimicking minimal variants of the dermal papilla. Conclusions: Pinkus' concept of the VM is revisited, reproduced and possible functions are proposed. Since these structures are found in a HF region with a high metabolic dynamism, they may be involved in differentiation or nutrition, or else be formed as a result of pressure arising from outgrowing hair shafts.
- Wound-induced hair follicle neogenesis (WIHN) is a phenomenon that occurs in adult mammalian skin, where fully functional hair follicles are regenerated in the center of large full-thickness excisional wounds. Although originally discovered over 50 years ago in mice and rabbits, within the last decade it has received renewed interest, as the molecular mechanism has begun to be defined. This de novo regeneration of hair follicles largely recapitulates embryonic hair development, requiring canonical Wnt signaling in the epidermis, however, important differences between the two are beginning to come to light. TLR3 mediated double stranded RNA sensing is critical for the regeneration, activating retinoic acid signaling following wounding. Inflammatory cells, including Fgf9-producing γ-δ T cells and macrophages, are also emerging as important mediators of WIHN. Additionally, while dispensable in embryonic hair follicle development, Shh signaling plays a major role in WIHN and may be able to redirect cells fated to scarring wounds into a regenerative phenotype. The cellular basis of WIHN is also becoming clearer, with increasing evidence suggesting an incredible level of cellular plasticity. Multiple stem cell populations, along with lineage switching of differentiated cells all contribute towards the regeneration present in WIHN. Further study of WIHN will uncover key steps in mammalian development and regeneration, potentially leading to new clinical treatments for hair-related disorders or fibrotic scarring.
- Long considered both physiologic and inevitable, skin ageing is a degenerative phenomenon whereby both intrinsic and environmental factors conspire to produce an authentic disease. The consequences of this disorder are many and varied, ranging from atrophy and fragility to defective repair to deficient immunity and vulnerability to certain infections. The pathobiologic basis for skin ageing remains poorly understood. At a cellular level, stem cell dysfunction and attrition appear to be key events, and both genetic and epigenetic factors are involved in a complex interplay that over time results in deterioration of our main protective interface with the external environment. Past and current understanding of the cellular and molecular intricacies of skin ageing provide a foundation for future approaches designed to thwart the ageing phenotype. Herein, we provide a review of current insights into skin ageing, including the mechanisms of skin ageing, the role of stem cells in skin ageing and the implications of skin ageing for the microbiome and for the development of cancer. Conquest of the oft overlooked ‘disease’ of skin ageing should have broad implications that transcend the integument and inform novel approaches to retarding ageing and age-related dysfunction in those internal organs that youthful skin was designed to envelop and safeguard.
- The mammalian hair-fibre, together with the “inner root-sheath” (i.e. the axial layers of the follicle wall), grows upward by a proximal addition of cells. Changes in the inner root-sheath are responsible for the final shape and surface sculpture of the fibre. At its distal limit the inner root-sheath disintegrates owing to the effect of a de-keratinizing chemical agent.A bent, undulated, or “crimped” fibre is due to a differentiated progress of the changes leading to keratinization of the “fibre cortex”. The cells which give rise to a “medulla” are different chemically and less compressible than cortical cells.
- This electron-microscopic study of the catagen phase shows that the first alteration of regression of the follicle is localized in the papilla, where the cells withdraw their offshoots and break the contact with the basal lamina. Both at the level of the papilla and of the bulb structures appear that increase the cell cohesion. Under the influence of the outer root sheath an upward migration occurs. This is followed by plication and thickening of the basal lamina. The alterations in the connective tissue sheath occur in a further stage. The first signs of autolysis occur in the center of the epithelial column. At the end of the catagen stage macrophages take care of the clearing-up.
- The Journal of Investigative Dermatology publishes basic and clinical research in cutaneous biology and skin disease.
- What fraction of the proliferative pool cells in epithelial tissues functions as stem cells is still uncertain. Earlier models, based on little or no good evidence, have assumed that this fraction is close to one. Recently there have been developments suggesting that the fraction of stem cells is low, with considerable cell production being attributable to division in short-lived transit proliferative cells. This brings epithelial tissues into line with haematopoiesis and spermatogenesis. This review considers these newer developments and emphasises the similarities between three epithelial regions (skin, tongue and intestine) and bone marrow and testis. Some of the models currently under discussion relate cell position, division polarity, protection of stem genome and hence carcinogenesis. Some of the implications of these models are discussed.
- Stem cells are by definition present in all self-renewing tissues and are believed to play a central role in cell growth and differentiation. Existing evidence suggests that a subpopulation of epidermal basal keratinocytes represents stem cells; however, these cells have never been positively identified. In this paper we review evidence that in monkey palm epidermis there exist two morphologically distinct subpopulations of basal keratinocytes that are spatially segregated. One population, located in the shallow rete ridges, is characterized by a cytoplasm filled with tonofilaments and a highly convoluted ("serrated") dermal-epidermal junction; these cells may play a role in anchoring the epidermis to the dermis. In contrast, the other population, located at the tips of deep rete ridges, is characterized by a "primitive" cytoplasm containing abundant melanosomes and a relatively flattened ("nonserrated") dermal-epidermal junction. Tritiated thymidine labeling experiments suggest that the nonserrated basal keratinocytes are slow-cycling; however, a highly proliferative population of keratinocytes can be identified immediately above these basal cells. These findings are consistent with the concept that the nonserrated basal keratinocytes may represent stem cells that give rise to suprabasally located, transient amplifying cells before undergoing terminal differentiation. Monkey palm epidermis provides a model system for further studies of primate epidermal stem cells.
- Fourteen years have elapsed since publication of the second edition of this highly regarded book and in that time a vast new knowledge relating to the biology of skin has accumulated. Nevertheless, the third edition remains the same size as the second and covers the same basic subject matter, but here the similarity ends, for the book has been totally rewritten and the content only superficially resembles that of the earlier edition. The book presents a systematic discussion of the epidermis, dermis, and their modifications and constituents such as blood supply, cutaneous nerves, hair apparatus, nails, and glands. These topics are presented with discussions of gross, light microscopic, and electron microscopic morphology; embryology; function; cell kinetics and metabolism; and histochemistry. The picture of skin that evolves is that of a highly complex and dynamic organ for which the authors have a deep affection. The style is easily readable, and the
- What fraction of the proliferative pool cells in epithelial tissues functions as stem cells is still uncertain. Earlier models, based on little or no good evidence, have assumed that this fraction is close to one. Recently there have been developments suggesting that the fraction of stem cells is low, with considerable cell production being attributable to division in short-lived transit proliferative cells. This brings epithelial tissues into line with haematopoiesis and spermatogenesis. This review considers these newer developments and emphasises the similarities between three epithelial regions (skin, tongue and intestine) and bone marrow and testis. Some of the models currently under discussion relate cell position, division polarity, protection of stem genome and hence carcinogenesis. Some of the implications of these models are discussed.
- The formation of new hair follicles was quantitatively demonstrated in the tail skin of adult mice in the course of a two-stage carcinogenesis experiment with 7,12-dimethylbenz(a)anthracene as an initiator and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate as a promoter, as well as in experiments with 12-O-tetradecanoylphorbol-13-acetate alone. Two kinds of follicular neogenesis could be distinguished. The most frequently encountered type was characterized by the organization of new follicles from the upper neck and orifice regions of already existing follicles. During their development, these new follicles remained in close apposition to the original follicles but, after having reached a critical size, split off to form fully independent follicles. In the second, type of follicular neogenesis, which occurred very rarely, the new follicles seemed to arise directly from the epidermis between two sets of hair triads; however, these follicles never reached their final stage and did not produce hairs. The formation of new hair follicles may be explained by a "dedifferentiation" of epidermal cells caused by the tumor promoter. Because of the paucity and advanced stage of the papillomas formed in tail skin after long-term treatment with 12-O-tetradecanoylphorbol-13-acetate, no reliable comment as to whether the papillomas derive from the hair follicles can be made.
- It was observed that after subcutaneous tissue shaving for the radical therapy of hircismus and hyperhidrosis axillary hair often regrew. Histologic study of this phenomenon showed that hair bulb and most of the follicle up to a level near the sebaceous duct opening had been removed. Hair regrows from remnant outer root sheath, but only when sebaceous glands are preserved, that is when the upper portion of the follicular isthmus is intact. One or several solid epithelial pegs grow downward from the cut end of the trichilemma, and inner root sheath and new young hair are formed in its center. In hair peg stage, the lower tip of the hair follicle descends while new hair is growing in its center through the mitotic activity is growing in its center through the mitotic activity of hair germ cells and is prevented from pushing toward the skin surface by interlocking fusion between hair cuticula and sheath cuticula. Eventually, the epithelial cells wrap around a mass of mesenchymal cells and form a new bulb from which the terminal hair grows upward. The new matrix acquires a new complement of functioning melanocytes.
- A study was made on the stem cell concepts. When we are talking about stem cells, we do not necessarily, and certainly not always talk about hemopoietic stem cells. There are many kinds of stem cells in the adult organism and there is one definition only which can apply to all these stem cells, and that is: a self-maintaining capacity. This means that stem cells can maintain their numbers for at least one life span of the organism. This means that 'stemness' implies an extensive self-maintaining capacity, and we mean self-maintaining capacity for many cell cycles, which in the mouse in most cases of 200 cell cycles, and in man - especially with a hemopoietic or intestinal stem cell system - well in excess of a thousand cell cycles. Every cell in the body, after the zygote and the first few cleavage divisions, is already a differentiated cell. That is: stem cells in the late embryonic crypt stem cells, skin epithelial stem cells or liver stem cell, are already differentiated cells. It is true that there are some stem cells which can give rise to further differentiated products, but not all stem cells do so. The author has given in his paper some of his ideas, about G(o) and genetic housekeeping, proliferation control and cell interaction, which like all concepts are pure fiction until such time that appropriate experimental evidence corroborates them. But at least, with the hemopoietic stem cells assay methods are available and with a little bit of clear thinking, clear definition - according to the author - and proper use of the available assays, the ideas can be tested. (E. Szirmai - Stuttgart) - Stuttgart, GFR)
- The psychologic importance of hair to man is in inverse ratio to its physical function. Except for scalp hair and desultory areas of sexual hair, most of man's hair follicles are vestigial. Three problems of hair growth remain to be solved: (1) how the intermittent activity of hair follicles in both animals and man is controlled; (2) how the male hormone alters the hair cycle in human skin; and (3) why larger hairs are produced by testosterone in some areas of the body when in some individuals the hair follicles in the scalp regress. Studies in which skin grafts from rats of different ages were exchanged showed that hair follicles are innately programmed but can be slowly influenced by systemic factors. Steroid hormones, especially estrogens, slow down the moult cycle whereas thyroid hormones accelerate it. What establishes the innate rhythm remains problematical. The fact that plucking out the club hair initiates activity in resting follicles has been explained by the hypothesis that the mitotic inhibitor which accumulates during anagen is normally used up or dispersed during telogen or by wounding. However, contrary to this theory, follicular activity is not prolonged by epilation during anagen. Moreover, if rats are epilated within one or two days of eruption, only club hairs are removed since forceps cannot grasp the tips of the new hairs. Such epilation does not affect the anagen in progress, but remarkedly enough the subsequent resting phase is shortened. Both sexual hair and male-pattern baldness depend on androgenic hormones. Target organs of testosterone convert the hormone to active metabolites, chiefly 5alpha-dihydrotestosterone. In skin, however, 5alpha-dihydrotestosterone may not be the only active tissue androgen. The major metabolite of testosterone incubated with hair roots in androstenedione, and hirsute women without other obvious endocrine abnormality sometimes excrete high levels of androstanediol. Both steroids stimulated the sebaceous glands of hypophysectomized-castrated rats, which, however, showed only a limited response to testosterone. The androgenic steroids, the enzymes that convert them to their active metabolites, and the proteins that bind them are undoubtedly very important to the problems of the growth of sexual hair and male-pattern baldness.
- During dormancy there is very little incorporation of [3H]uridine in cells of hair germ and dermal papilla. During activation (both spontaneous and experimental) there is a marked increase in this parameter, expecially in the cells of the germ. After experimental activation by plucking, the increase in label in the germ is evident by 6 hr, reaching an initial maximum by 24 hr. The initial increase in [3H]uridine incorporation after activation precedes the increase in [3H]thymidine incorporation found previously, by about 6 hr. No evidence was found that the increase in [3H]uridine of papilla cells precedes the increase in [3H]uridine of germ cells. This finding militates against the possibility that the dermal papilla is triggered into an inductive role (vis-á-vis the germ cells) by means of circulating steroid hormones.
- Earlier studies of the vascular patterns around hair follicles revealed that actively growing follicles have an extensive microvasculature relative to that around quiescent follicles. In order to determine whether the establishment of the active perifollicular vascular pattern involves replication of endothelial and perivascular cells, we studied the 3H-thymidine labeling indices of these cells in autoradiographs of rat skin from regions containing either actively growing follicles or recently inactive follicles. In skin containing inactive follicles, the endothelial labeling index was 0.22%, a figure similar to those reported for the endothelium of small blood vessels in several other non-stimulated adult tissues. However, skin containing actively growing follicles displayed an endothelial labeling index of 2.35%. The labeling indices of perivascular cells paralleled those of the endothelial cells. Thus, endothelial and perivascular cells in vessels associated with actively growing follicles undergo significantly more DNA synthesis than those in vessels associated with inactive follicles. Replication of these cells apparently contributes to the extension of the microvascular network around growing follicles.
- Interaction between connective tissue cells and hair growth in the rat has been studied by radioautography after in vivo and in vitro pulse labelling with thymidine, uridine, histidine, leucine and proline. The connective tissue, which surrounds and integrates the hair bulbs as a unit, contains cells of various types which have a cyclic metabolic activity. This cyclic activity is coordinated with the hair growth cycle. The number of nuclei which actively synthesize DNA in the dermal cells, mainly those of endothelial and migratory cells, significantly increases during the short and transient anagen 4 substage. RNA and protein synthesizing activities are also present in all cell types and seem modulated by the hair cycle although to a lesser degree. This data provides an important basis for the interpretation of similar studies in alopecia areata.
- The morphology of mouse hair follicles in the catagen stage was examined under an electron microscope. In the early and late catagen stage, compact fine fibrils (120–130 Å in diameter) with a hollow profile and a beaded pattern along their long axis were found throughout the dilated intercellular spaces in dermal papilla. These fibrils were not only contiguous to papilla cell membranes but were also attached, and prepared to be inserted into the basal lamina-like structure that protruded from the basal lamina into the dilated intercellular spaces at the upper portion of the papilla. In the late catagen stage, hemi-desmosomes were found along the keratinocyte membranes adjacent to the basal lamina at the papilla—epithelial junction. Both the fine fibrils and hemidesmosomes, which were characteristic structures in hair follicles in the catagen stage, seem to act mechanically, in cooperation with the basal lamina, to form a firm adhesion structure between the papilla and the epithelial column during this stage.
- The sensitivity of hairless mice to cutaneous chemical carcinogenesis has been compared with that of normal mice of the same strain with hair. A single application of 125 mug methylcholanthrene in benzene was given to 48 hairless male mice (hr/hr Oslo strain) and to 96 male mice of the same strain with hair. Among hairless mice there were 94% papilloma-bearing animals with a total of 5.9 tumors per animal after 18 months of observation, compared to 22% papilloma-bearing animals with an average of 0.3 tumors per animal among the mice with hair. The hairless mice included 31% carcinoma-bearing and 23% sarcoma-bearing animals, whereas only 1% of the mice with hair were carcinoma bearing and 3% were sarcoma bearing. Hairless mice of the hr/hr Oslo strain are thus not refractory to chemical carcinogenesis, but under the experimental conditions used in this study they are significantly more sensitive than are mice from the same strain with hair. Giovanella et al. reported almost opposite results in 1970 and came to the general conclusion that hairless mice are refractory to chemical carcinogenesis due to lack of hair follicles. Since hairless mice always have some hair follicles and rudimentary pilosebaceous appendages, comparisons between chemical carcinogenesis in hairless mice and mice with hair can neither strengthen nor weaken any theory about the hair follicle origin of epidermoid carcinomas of mouse skin.
- In this paper we present keratin expression data that lend strong support to a model of corneal epithelial maturation in which the stem cells are located in the limbus, the transitional zone between cornea and conjunctiva. Using a new monoclonal antibody, AE5, which is highly specific for a 64,000-mol-wt corneal keratin, designated RK3, we demonstrate that this keratin is localized in all cell layers of rabbit corneal epithelium, but only in the suprabasal layers of the limbal epithelium. Analysis of cultured corneal keratinocytes showed that they express sequentially three major keratin pairs. Early cultures consisting of a monolayer of "basal" cells express mainly the 50/58K keratins, exponentially growing cells synthesize additional 48/56K keratins, and postconfluent, heavily stratified cultures begin to express the 55/64K corneal keratins. Cell separation experiments showed that basal cells isolated from postconfluent cultures contain predominantly the 50/58K pair, whereas suprabasal cells contain additional 55/64K and 48/56K pairs. Basal cells of the older, postconfluent cultures, however, can become AE5 positive, indicating that suprabasal location is not a prerequisite for the expression of the 64K keratin. Taken together, these results suggest that the acidic 55K and basic 64K keratins represent markers for an advanced stage of corneal epithelial differentiation. The fact that epithelial basal cells of central cornea but not those of the limbus possess the 64K keratin therefore indicates that corneal basal cells are in a more differentiated state than limbal basal cells. These findings, coupled with the known centripetal migration of corneal epithelial cells, strongly suggest that corneal epithelial stem cells are located in the limbus, and that corneal basal cells correspond to "transient amplifying cells" in the scheme of "stem cells----transient amplifying cells----terminally differentiated cells."
- A simple experimental technique was developed to provide an in vitro model for the study of human follicular keratinocytes. Anagen-phase human hairs were plucked from the scalp of healthy individuals; the follicles were separated, plated on coverslips coated with collagen G, and cultivated in McCoy 5A Medium in a CO2-incubator at 37 degrees C. Light and electron microscopy after 1, 2, 3, and 6 weeks showed selective and progressive cell growth with keratinocyte differentiation, producing multilayered cultures of cells joined with fully developed desmosomes. Three distinct patterns of differentiation, leading to the formation of an incomplete horny layer, were seen. The particular arrangement of tonofilaments, the considerable amounts of cytoplasmic glycogen, and the absence of malpighian differentiation were ultrastructural indicators of the follicular origin of the cultured cell population, which most likely grew from the outer root sheath of the hair. This technique may provide a promising model on which to base further studies of hair biologic processes and hair growth.
- As the hair follicle is one of the most rapidly growing tissues in the body, it must be nourished by a rich blood supply. Histological studies have indicated that the number of vessels about a growing follicle exceeds that about a resting follicle, so we postulated that the hair follicle might provide its own angiogenic stimulus during certain phases of its growth. Reported here are experiments testing the angiogenic properties of the growing (anagen) hair follicle. Using the rabbit corneal pocket angiogenesis assay and cycled anagen rat vibrissae hair follicles, we found that the mesenchymal dermal papilla had no angiogenic properties, but the anagen bulb was angiogenic. These findings suggest a mechanism for the cycling of hair follicles and an example of normal epithelium to mesenchyme interactions.
- Despite the obvious importance of epithelial stem cells in tissue homeostasis and tumorigenesis, little is known about their specific location or biological characteristics. Using 3H-thymidine labeling, we have identified a subpopulation of corneal epithelial basal cells, located in the peripheral cornea in a region called limbus, that are normally slow cycling, but can be stimulated to proliferate in response to wounding and to a tumor promotor, TPA. No such cells can be detected in the central corneal epithelium, suggesting that corneal epithelial stem cells are located in the limbus. A comparison of various types of epithelial stem cells revealed a common set of features, including their preferred location, pigment protection, and growth properties, which presumably play a crucial role in epithelial stem cell function.
- The effects of mechanical plucking on the anatomy of human anagen hair bulbs were studied histologically in biopsy specimens taken from scalp areas of ten volunteers immediately after plucking. Anagen hair bulbs were shown to tear off not arbitrarily but in reproducible patterns which include, apart from the "typical" break conically surrounding the dermal papilla, three additional break forms: (1) rupture of the hair around the upper third of the papilla resulting in so-called dysplastic anagen hairs of the trichogram, (2) rupture of the hair well above the dermal papilla resulting in "broken" anagen hairs, (3) total removal of the proximal follicle epithelium with removal of the dermal papilla resulting in so-called papilla hairs of the trichogram. Plucking also gives rise to alterations of the mesenchymal sheath of the hair follicle mainly leading to hemorrhages and a distinct edema entailing an increase in the volume of both the dermal papilla and the underlying "papilla cushion" of Pinkus. The different break types can be due to inappropriate plucking techniques or may depend on different subphases of the anagen stage.
- Techniques are described for the isolation and cultivation of functionally intact mouse hair follicles. Follicles were isolated by collagenase digestion of dermis from 5-day-old mice and purified by differential centrifugation and filtration. Purified follicles were cultured in a Type 1 collagen matrix using Medium 199 and 8% fetal calf serum as the basic nutrient. Viability of follicles was maintained in culture since the cultures incorporated thymidine into DNA and methionine into proteins for at least 7 days. Furthermore, follicles isolated from the collagen matrix after 7 days could reattach to a plastic culture substrate or be further cultivated in a fresh collagen matrix. Functional integrity of cultured follicles was maintained since some follicle-specific cytoskeletal proteins were synthesized in vitro, and follicles isolated from the collagen matrix after 7 days formed a haired skin when recombined with dermal fibroblasts and grafted to a skin site on nude mice. Only a minority of follicles appeared to produce a mature hair shaft in vitro by morphologic criteria, however, and synthesis of the total complement of hair proteins was not observed. Cholera toxin was a strong mitogen for cultured follicles, whereas epidermal growth factor was slightly mitogenic. Epidermal growth factor stimulated the release of a Type 1 collagenase by follicle cells, however. This model system provides an opportunity for the systematic analysis of factors required for the induction of hair growth and the underlying physiology of hair follicle development. This model should also be useful for studying the role of the hair follicle in skin carcinogenesis.
- This review is designed to inform the clinician of current concepts regarding the pathogenesis of chemically induced skin cancer. Chemicals induce cutaneous cancers in a wide variety of experimental animals and in humans. The most common benign experimental tumors are papillomas and keratoacanthomas, whereas common malignancies are squamous cell carcinomas and melanomas. Carcinoma development is a multistage process that involves at least three mechanistically distinct steps. Initiation is the earliest change in an epidermal cell exposed to carcinogens such as polycyclic aromatic hydrocarbons, alkylating agents, or nitrosamines. This stage appears to result from carcinogen-induced deoxyribonucleic acid damage leading to a mutation-like genetic change. Only a limited number of epidermal genes may be changed to yield the initiated cell, and one has been identified as the Harvey ras gene, a gene involved in epidermal proliferation. Initiated epidermal cells are not malignant but are insensitive to the normal signals for terminal differentiation in the epidermis. The second stage, tumor promotion, results from repeated exposure of initiated skin to one of a variety of noncarcinogenic promoting agents such as phorbol esters, benzoyl peroxide, anthralin, or certain halogenated aromatic hydrocarbons. Some promoters require specific cellular receptors and produce transient changes in the growth or differentiation of the epidermis. Collectively these agents produce a tissue environment that is conducive to the selective clonal outgrowth of the initiated cell population, resulting in a clinically apparent premalignant tumor. During the third stage of carcinogenesis, premalignant cells are converted to malignancy. This step may occur spontaneously, but the frequency is greatly enhanced by exposure to mutagens, including several initiating agents. Thus malignant conversion is likely due to additional mutations in a benign tumor cell. The Harvey ras gene may also be a potential target in the conversion step. Several agents, such as corticosteroids and retinoids, have been identified as anticarcinogens for skin. They appear to be primarily antipromoting agents and could important clinical applications. Melanomas can be induced in several species by repeated exposure to initiators or by exposure to an initiator and a tumor promoter. the experimental pathogenesis of this tumor is unclear. It is proposed that intermediates in the synthesis of melanin pigment could act as endogenous carcinogens or promoters in melanoma development. Increased awareness of the mechanisms of chemical carcinogenesis in skin will enhance cancer prevention in this tissue. Furthermore, astute
- The distribution and persistence of radioactively labeled benzo(a)pyrene [B(a)P] in the skin of adult female SENCAR mice were investigated by autoradiography of epidermal whole mounts and cross-sections at intervals following a single initiating application of 200 nmol of either [3H]B(a)P (2 mCi) or [14C]B(a)P (23 muCi). One day after treatment, the entire thickness of the skin was labeled; the grain density was greatest over hair follicles, sebaceous glands, and interfollicular epidermis. At 1 and 2 weeks, decreases in the nuclear grain density were consistent with the overall pattern of epidermal renewal. One month after treatment, carcinogen label-retaining cells made up approximately 2% of the interfollicular basal cells. They were also present in the hair follicles, approximately 4 and 5% of basal cells in the infundibulum and external root sheath, respectively. They were rare in the germ region and dermal papilla. Carcinogen label-retaining cells were compared with slowly cycling [3H]thymidine label-retaining cells and "maturing" basal cells, two distinct proliferative subsets of adult murine epidermis. Carcinogen label-retaining cells were found to have characteristics of the slowly cycling cells: (a) most of the carcinogen labeled nuclei were found in the central regions of the epidermal proliferative units; (b) treatment of the carcinogen label-retaining cells with 2 micrograms of 12-O-tetradecanoylphorbol-13-acetate elicited labeled mitoses within 1 day, and a general decrease in grain density over basal nuclei. In contrast, maturing basal cells 4 days after a single injection of [3H]thymidine were found at the periphery of the epidermal proliferative units. Within 1 day after treatment with 2 micrograms of 12-O-tetradecanoylphorbol-13-acetate, maturing basal cells were displaced to the suprabasal layers. Double isotope-double emulsion autoradiographs demonstrated doubly labeled cells 1 month after continuous labeling with [3H]thymidine and [14C]B(a)P and provide evidence that the radioactive carcinogen is retained by the slowly cycling [3H]thymidine label-retaining cells. These observations suggest that a slowly cycling population of epidermal cells may be relevant to the initiation phase of two-stage carcinogenesis.
- 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.
- The amount and distribution of fibronectin associated with hair follicles was found to vary during the hair growth cycle in the rat. Immunocytochemical staining of follicles in mid-late anagen (the growth stage) revealed the presence of fibronectin in the dermal papilla matrix, in the basement membrane separating this from the epithelial cells of the hair bulb, and in the basement membrane and connective tissue sheath which underly the cells of the outer root sheath. Early in catagen, the transitional stage, staining of the dermal papilla matrix disappeared. Fibronectin persisted in the basement membrane and connective tissue sheath, which undergo corrugation and apparent thickening in catagen. After follicle shortening, the telogen (resting) stage is reached, at which point fibronectin staining was found to be minimal, being restricted to the basement membrane around the secondary germ. The onset of anagen, involving cell division and follicle elongation, was associated with a great increase in the amount of fibronectin in this zone and in and around the dermal papilla. Analysis of entry into anagen by [3H]thymidine incorporation and autoradiography revealed that growth could be detected before the increase in fibronectin expression. However, growing cells, even in a suprabasal position, always had some fibronectin at their surface. Immunoelectron microscopy of early anagen follicles confirmed the light microscopic findings and also showed that fibronectin was present in small vesicles close to the surface of dermal papilla and some epithelial cells. Increased deposition of laminin and type IV collagen in early anagen follicles was also noted, emphasizing the importance of basement membrane components during morphogenetic events in vivo.
- The purpose of this investigation was to characterize the [3H]thymidine label-retaining and the "maturing" classes of basal cells from the dorsal epidermis of adult SENCAR mice and to compare their early cellular kinetic responses to topical application of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Autoradiography of epidermal whole mounts and cross sections demonstrated that injection of [3H]thymidine every 6 h for 1 week labeled 95% of the basal nuclei, including those in the central region of the epidermal proliferative units. One month later, the labeling index was reduced to 2%; 90% of the label-retaining cells were within a nuclear diameter of the central suprabasal column of the proliferative units. When mice were treated with 2 micrograms of TPA 1 month after labeling, mitotic label-retaining cells were found within 22 h after treatment. Seventy-five percent of the label-retaining cells remained on the basal layer through the 28-h experimental period. In contrast, the basal labeling index following a 1-h pulse of [3H]thymidine was 5%. Eighty-five percent of the labeled cells were found in the periphery of the proliferative units. By 4 days after pulse labeling, most of the originally labeled cells had divided, although vertical cross sections indicated that 92% remained on the basal layer. When mice were treated with TPA on day 4, labeled cells were rarely found in mitosis. Instead, about 60% of the labeled cells were displaced to the suprabasal layers. These observations suggest that 2 classes of epidermal basal cells have different early responses to TPA treatment: the label-retaining cells proliferate, and most of the "maturing" cells continue to differentiate.
- The possibility that epidermis which has never been exposed to carcinogen can give rise to carcinomas if it is transplanted onto carcinogen-treated dermis was raised by the experiment reported by Billingham, Orr, and Woodhouse in 1951. They treated mice topically with methylcholanthrene for 12 weeks, left them untreated for 2 weeks to allow the methylcholanthrene to disappear, and then transplanted autografts of untreated epidermis obtained by trypsinization of tail skin to superficial beds prepared in the treated areas on the thorax. Tumors apparently developed from the untreated surface epidermis at 62% of the graft sites. However, in preparation of the graft beds the bases of surviving hair follicles were left in situ. Consequently, it was possible that the tumors arose from residual treated epithelium and not from the untreated epidermal grafts. To test this possibility, the original experiment was repeated in the present investigation except that F! hybrid mice were used as the methylcholanthrene-treated hosts and inbred parent strain mice as the donors of the untreated epidermal grafts. This provided a strong (H-2) histocompatibility marker for determining the origin of the tumors by their differential transplantability to preimmunized parent strain versus F! hybrid recipients. Of 14 carcinomas which arose at the graft sites in the primary hosts, none grew progressively in the parent strain whereas all grew in the F) hybrids indicating that they indeed arose from F! host cells. These results suggest that the interpretation of the original experiment as evidence of indirect epidermal carcinogenesis is incorrect.
- Hair growth from the vertex, temple, mustache, finger, arm and leg of three Japanese men (60, 30, and 21 years of age) was measured by direct observation from October, 1966 to November 1968. Detailed information regarding hair growth cycles in the regions tested is presented.
- Changes in the basement membrane (multistratification or thickening) have been observed in three zones of the human hair follicle: in the upper portion of the outer root sheath, around the tip of the papilla and within the papilla. The most striking finding is the presence within the papilla of a connecting system between capillaries, components of the dermis and epithelial cells of the follicle, consisting of multilayered, reticular structures in all respects similar to the limiting membranes of epithelium and endotheliu. These peculiar features of the basement membrane are discussed in the light of the latest data in the literature.
- Chemotropic morphogenesis of new blood vessel patterns with active haemodynamics in the skin of rabbits was induced by intradermal injection of highly purified monocyto-angiotropin (MAT), one of the low-molecular-weight (4,500) polypeptide monokines exuded by activated mammalian (porcine) monocytes (macrophage type) on their culture in serum-free, fully synthetic media. Evidence is presented that neohypervascularization of the skin is associated with enhanced tissue function as represented by hair growth. Exclusively in the area of MAT injection, numerous capillary-like vessel formations arose from preexisting vessels, and the quiescent hair follicles (telogen) were activated to growing ones (anagen) within 1-2 weeks.
