Follicular Stem Cells Research Articles

Ectopic expression of c-Myc in the skin affects the hair growth cycle and causes an enlargement of the sebaceous gland

The hair follicle continually undergoes dynamic remodelling in a cyclical manner involving tightly coordinated patterns of cell proliferation, differentiation and apoptosis. The oncoprotein c-Myc is a key regulator of these events in epidermal keratinocytes, but its importance in the hair growth cycle has not previously been determined. To determine the role of c-Myc in the hair growth cycle. We characterized the hair follicle phenotype of transgenic mice that permit expression of a switchable form of c-Myc (c-Myc-ER) in the suprabasal epithelial layers of the epidermis and hair follicle. c-Myc activation increased epithelial cell proliferation in the outer root sheath and distal hair follicle, without any substantial alteration in levels of apoptosis. Moreover, chronic c-Myc activation resulted in marked desynchronization of the murine hair growth cycle, uncoupling of hair cycle-related skin thickness and enlargement of the sebaceous gland. These data implicate c-Myc in the control of hair growth cycling and hair cycle-related epidermal and sebaceous gland homeostasis. We suggest that c-Myc may be activating follicular stem cells either directly or indirectly and that this has important implications for control of the 'hair cycle clock', hair growth and epidermal maintenance.

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

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

Nascent blood vessels in the skin arise from nestin-expressing hair-follicle cells.

Besides forming hair shafts, the highly organized, metabolically vigorous hair follicle plays several crucial roles in skin architecture. The follicle contains a distinct population of presumptive follicular stem cells that express nestin, also a marker for neural stem cells. These nestin-expressing follicle cells are located principally in the follicular bulge region. Nestin-driven GFP (ND-GFP), transfected into mice, principally labels cells in the bulge region, which is consistent with the cells' being the stem cells of the hair follicle. We report here that ND-GFP also labels developing skin blood vessels that appear to originate from hair follicles and form a follicle-linking network. This is seen most clearly by transplanting ND-GFP-labeled vibrissa (whisker) hair follicles to unlabeled nude mice. New vessels grow from the transplanted follicle, and these vessels increase when the local recipient skin is wounded. The ND-GFP-expressing structures are blood vessels, because they display the characteristic endothelial-cell-specific markers CD31 and von Willebrand factor. This model displays very early events in skin angiogenesis and can serve for rapid antiangiogenesis drug screening.

Versican expression by dermal papilla‐regenerated hair follicles – a promising tool for hair‐regrowth products

Hair follicle stem cells and dermal papilla cells play essential roles in the cyclical hair regeneration process. However, compared with the recent progress of follicular stem cell research, detailed analysis of dermal papilla cells is virtually unknown. We have focused on a large chondroitin sulfate proteoglycan molecule, versican, and prove its pivotal role in hair follicle induction both in vitro and in vivo. First, we examined versican expression with hair cycling by in situ hybridization (mRNA) and immunohistochemistry (protein). Results clearly showed specific versican expression in anagen onset of dermal papilla cells implying a function role of versican in hair induction. We then generated transgenic lines with LacZ or green fluorescent protein reporter genes under a versican regulatory element (promoter) control, and confirmed dermal papilla‐specific activation of the reporter. These transgenic lines were utilized to isolate the dermal papilla cell population by means of a fluorescent activated cell sorter.While freshly isolated dermal papilla cells were able to induce hair growth in a skin reconstitution assay when grafted with undifferentiated epidermal cells, this ability and versican expression were rapidly lost during passages in culture. In order to examine the functional role of versican in hair growth induction, versican full‐length cDNAs were transfected into inactive passaged dermal papilla cells. Impressively, this forced expression of versican molecules partially restored hair inductivity of dermal papilla cells in a skin reconstitution assay. These results demonstrated the pivotal role of versican in hair induction both in vitro and in vivo and will connect the missing link between the signal from dermal papilla cells to follicular stem cells, and initiation of hair regeneration. We have also shown that the hair growth‐promoting compound, cyclosporin A, can upregulate versican expression implying the application of a novel screening approach for hair growth‐promoting compounds by monitoring versican expression as an indicator.

Human follicular stem cells: their presence in plucked hair and follicular cell culture.

A considerable portion of the hair follicle remains attached to plucked hair and can be used for follicle cell culture. In this study we have phenotyped these cells in an attempt to identify the stem cell fraction. Reports in the literature have indicated that this cell population may be positive for cytokeratin (CK) 19. Because stem cells in general need to be protected from apoptosis, the presence of the apoptosis-suppressing Bcl-2 protein, together with the absence of the apoptosis-promoting Bax and the CK profile may be used as an indicator of the stem cell population in the hair follicle, and in cultures of hair follicle cells. Hair follicles from skin biopsies and plucked hair were derived from the scalps of healthy volunteers. Follicular cells were cultured from the plucked hairs. These hair follicles, plucked hairs and cultured cells were examined for their CK profiles, which are indicative of the type of cell (basal/stem cells) and for their status with respect to the proliferation marker Ki-67, Bax and Bcl-2. We found coexpression for CK19 and Bcl-2, but not Bax in two distinct areas, localized in the upper and lower third of the follicle from both skin biopsies and plucked hairs, while proliferation markers were negative in these areas. CK19 and Bcl-2 were also coexpressed in combination in a fraction of the follicular cell culture. The skin basal cell marker CK14 could be found throughout the outer root sheath of the hair follicle from both skin biopsies and plucked hairs, as well as in the follicular cell culture. Thus, CK19/Bcl-2-positive and Bax-negative cells can be obtained from cells derived from plucked hair and are retained in cultures made from these cells. If this phenotype represents follicular stem cells, our finding endorses the assumption that stem cells are located in the bulge area of the hair follicle, as we did not find them in or near the dermal papilla.

Hair follicle stem cells.

The workshop on Hair Follicle Stem Cells brought together investigators who have used a variety of approaches to try to understand the biology of follicular epithelial stem cells, and the role that these cells play in regulating the hair cycle. One of the main concepts to emerge from this workshop is that follicular epithelial stem cells are multipotent, capable of giving rise not only to all the cell types of the hair, but also to the epidermis and the sebaceous gland. Furthermore, such multipotent stem cells may represent the ultimate epidermal stem cell. Another example of epithelial stem cell and transit amplifying cell plasticity, was the demonstration that adult corneal epithelium, under the influence of embryonic skin dermis could form an epidermis as well as hair follicles. With regards to the location of follicular epithelial stem cells, immunohistochemical and ultrastructural data was presented, indicating that cells with stem cell attributes were localized to the prominent bulge region of developing human fetal hair follicles. Finally, a new notion was put forth concerning the roles that the bulge-located stem cells and the hair germ cells played with respect to the hair cycle.

Label-retaining cells (presumptive stem cells) of mice vibrissae do not express gap junction protein connexin 43.

We investigated whether connexin 43, a gap junction protein present in human epidermis and mouse hair follicle, can serve as a negative marker for keratinocyte stem cells. Experiments carried out in mouse pelage and vibrissae hair follicles demonstrated that most of the slowly cycling cells, detected as label-retaining cells, do not express connexin 43. In humans, cells with immunohistochemically undetectable levels of connexin 43 are found in the epidermal basal layer of neonatal foreskin, and in the follicular bulge region. About 10% of the basal keratinocytes are connexin 43 negative, as determined by flow cytometry. These cells are uniformly small and low in granularity suggesting that presumptive keratinocyte stem cells can be identified and separated based on connexin 43 expression.

An actinic keratosis is neither malignant nor premalignant: It is an initiated tumor

An actinic keratosis is neither malignant nor premalignant: It is an initiated tumor

Notch/RBP-J Signaling Regulates Epidermis/Hair Fate Determination of Hair Follicular Stem Cells

Notch signaling is involved in the cell fate determination of various cell lineages [1–9]. Notch interaction with its ligand induces the cleavage of its intracellular domain (IC) [10], and the Notch IC translocates to the nucleus and binds to RBP-J [11] to transactivate transcription of target genes [10, 12]. All four Notches in mammals [13–17] bind to RBP-J to exert their transactivation activities. Notch is expressed in developing or differentiating epidermis and hairs [13–15, 18–20], inhibits the terminal differentiation of the epidermis [21], and regulates hair differentiation [22]. The common stem cells that reside in the upper portion of hair follicles (the bulge) contribute to epidermal and hair cell formation [23, 24]. However, it is unknown what determines whether hair follicular stem cells will become hairs or epidermis. Here we report that conditionally disrupting the mouse RBP-J gene in a mosaic pattern to avoid embryonic lethality of RBP-J-deficiency [25] caused hair loss, epidermal hyperkeratinization, and epidermal cyst formation. Cyst formation is probably due to a combination of the aberrant fate determination of RBP-J-deficient stem cells to epidermal progenitors and their accelerated differentiation into epidermis. These results suggest that Notch/RBP-J signaling regulates the cell fate determination of hair follicular stem cells at the bulge region.

Label-retaining Cells in the Bulge Region are Directed to Cell Death after Plucking, Followed by Healing from the Surviving Hair Germ

Hair plucking is the most frequently used method of anagen induction within hair follicles. In this study, we found that plucking leads to the entire renewal of the follicular stem cell region of the mouse pelage follicle. Comparative histochemical analysis revealed that S100A4 protein was specifically distributed in the outer layer of the epithelial sac, which has been identified as the stem cell region of the pelage follicle, whereas the slow cycling cells that retained 5-bromo-2'-deoxyuridine label for 8 wk were located in the epithelial sac and also in the hair germ. Combined terminal deoxynucleotide transferase deoxyuridine triphosphate fluorescein nick end labeling method and immunohistochemistry revealed that positive cells were detected in the outer layer of the epithelial sac possessing both bromo-2'-deoxyuridine and S100A4 labels 4.5 h after plucking. No terminal deoxynucleotide transferase deoxyuridine triphosphate fluorescein nick end labeling signal, however, was observed in the hair germ. Serial inspection of the plucked follicle revealed that almost all regions of the epithelial sac became terminal deoxynucleotide transferase deoxyuridine triphosphate fluorescein nick end labeling positive 12 h after plucking. Terminal deoxynucleotide transferase deoxyuridine triphosphate fluorescein nick end labeling-positive cells ultimately degenerated without forming apoptotic bodies. Subsequently, the surviving label-retaining cells in the hair germ migrated upward to re-epithelialize the damaged portion. These results indicate that follicular stem cells in the epithelial sac underwent cell death after plucking. It is likely that the hair germ is responsible for the reconstruction of the stem cell region of the hair follicle.

Spatial relationship between Merkel cells and Langerhans cells in human hair follicles

The distributions of Merkel cells and Langerhans cells within human hair follicles have been reported. However, there has been no description of the relationship between Merkel cells and Langerhans cells, which were discovered by 19th century German pathologists. Merkel cells and Langerhans cells share some similar characteristics such as the localization of human hair follicles, a close association with peripheral nerves and the expression of several neuropeptides. Merkel cells were stained with CK20 or CAM5.2, while Langerhans cells were stained with CD1a or S-100 protein. We thus immunohistochemically confirmed the preferential localization of Merkel cells and Langerhans cells in normal human hair follicles. Using a double staining technique, two- and three-dimensional observations demonstrated that a small proportion of Merkel cells were closely contacted with Langerhans cells below the sebaceous gland level, presumably indicating the bulge area. Merkel cells and Langerhans cells connected directly or approached each dendrite within the basal layer of the outer root sheath. For the first time, we demonstrated a close anatomical relationship between Merkel cells and Langerhans cells within the bulge area of human hair follicles where follicular stem cells may be present. These morphological observations suggest a functional interaction between follicular Merkel cells and Langerhans cells. We herein hypothesize that Merkel cells communicate with Langerhans cells by characteristic dendrites in which some neuropeptides or cytokines may be stored.

Targeted elimination of the follicular label-retaining cells by photo-induced cell killing caused a defect on follicular renewal on mice.

One of the most well-known ways to identify stem cells is to search for the slow-cycling cells (label-retaining cells; LRCs), by labelling their DNA. The bulge of hair follicle is assumed to identify the location of the follicular stem cells. While this assumption was supported by previous analyses of cell tracing, it is still not clear whether the LRCs are critical for follicular renewal. To solve this problem, we tried to selectively eliminate the bromodeoxyuridine (BrdU)-labelled follicular LRCs by a photo-induced cell killing method, in combination with Hoechst 33258 treatment. We labelled the slow-cycling cells in the follicular bulge by repeated administration of the BrdU, followed by a chase period. When the labelled mice skin was irradiated at the telogen phase after the Hoechst 33258 treatment, we observed apoptotic cells in the bulge area 18 h after the irradiation and a defect on the follicular renewal at the next anagen phase. The time course of appearance of hair defect suggests that only the late telogen follicle is sensitive to irradiation. This finding suggests that the LRCs contribute to the renewal of the hair follicle, and they might proliferate at a restricted point in the hair cycle to supply hair germinative cells as previously predicted.

Arrector pili muscle: evidence of proximal attachment variant in terminal follicles of the scalp

There is general agreement that the proximal attachment site of the arrector pili (AP) muscle, via elastic tendons, is the bulge region, where follicular stem cells are situated. A variant of this insertion, in which the AP muscle surrounds the follicle circumference, has been described in human facial vellus hair follicles. To investigate whether terminal hair follicles of the human scalp may also present this variant of proximal insertion of the AP muscle. Histological and immunohistochemical methods were used. At the bulge level, bundles of the AP muscle were seen to encircle the entire hair follicle. Terminal follicles of the human scalp may also present this variant of proximal insertion of the AP muscle.

Androgenic Alopecia and Stress-Induced Premature Senescence by Cumulative Ultraviolet Light Exposure

Background: Ultraviolet light radiations (UVR) may be responsible for hair cycle synchronization and telogen effluvium in the animal and humans. The effect if any of cumulative UVR on chronic hair shedding and androgenic alopecia (AGA) is largely unknown. Objective: To compare the severity of AGA with the extent of solar elastosis. Method: The present study combining immunohistochemistry and computerized image analysis was undertaken in 140 men with AGA and 50 non-alopecic men. Solar elastosis was identified using the antibody to lysozyme, and collagen was revealed by Sirius red staining. The number and diameter of hair shafts were also assessed. Results: The scalp dermis was significantly thicker in AGA than in unaffected subjects. The difference was mainly due to a severer elastosis in baldness. The earliest signs of solar elastosis preceded hair thinning. When elastosis was thicker than 0.2 mm, a negative exponential correlation was found between hair diameter and severity of solar elastosis. Conclusion: Chronic UVR exposure of the scalp may affect the hair cycle and be one of the exogenous factors influencing negatively the progression of AGA. The induction of stress-induced premature senescence by reactive oxygen species and micro-inflammation might be operative at the level of the follicular stem cells.

Expression of Calcium-Binding S100 Proteins A4 and A6 in Regions of the Epithelial Sac Associated with the Onset of Hair Follicle Regeneration

In order to elucidate the onset mechanisms of hair regrowth, we characterized the expression of metastasis and cell-growth-associated calcium-binding S100 proteins in the regenerating follicular epithelium. Hair-cycle-dependent expression of S100A4 and S100A6 was found in the epithelial sac regions (bulge area and hair germ) of mouse pelage follicles. Protein localization of S100A4 was confined to the bulge area, the region where the presumed follicular stem cells reside, during the catagen-telogen-anagen transitional periods, whereas S100A6 protein was distributed throughout the epithelial sac regions during anagen phase. Prior to entering anagen phase, however, both S100 mRNAs were upregulated in the epithelial sac. Despite the induction of extensive cell death in the bulge region after plucking, a new hair cycle was initiated following transcription of S100A6 in the hair germ. Upon wounding stimuli, both the outer root sheath and the basal layer of epidermis expressed S100A6 mRNA prior to hyper-proliferation. Once the epithelial sac was induced to transcribe both S100 genes, resting follicles were concomitantly rejuvenated. These results suggest that S100A4 and S100A6 might play important roles in the activation of stem cells at the onset of follicle regeneration.

Lasers in dermatology--a critical update.

Lasers are accepted for treating nevus of Ota, other pigmented lesions, hair removal, vascular lesions, leg veins, tattoos, and for skin resurfacing. These are photothermal treatments, in which certain skin "targets" are heated, followed by selective wound healing. Small pigmented targets such as the dermal melanocytes in nevus of Ota, are best treated with short (< 1 microsecond) laser pulses. Large targets, such as hair follicles, have long thermal relaxation times and are best treated with longer pulses. In general, the ideal pulse duration is about equal to the thermal relaxation time for pigmented targets. However, sometimes the actual target is not pigmented and is at some distance from a pigmented structure. For example the follicular stem cells, which are not pigmented, line the outer root sheath far away from the pigmented hair shaft. These cells appear to be an important target for permanent hair destruction. Pulses longer than the thermal relaxation time of the hair shaft allow heat conduction and better damage of follicular stem cells. Epidermal cooling works far better with pulses longer than about 10 ms, delivered through a cold medium (e.g., cold sapphire in contact with the skin). Thus, the combination of cooling and long near-infrared laser pulses allows safe and effective pigmented hair removal in all skin types. In contrast, epidermal protection from short pulses is best with dynamic pre-cooling (e.g., cryogen spray), for example during portwine stain treatment. A major challenge for the future of photothermal laser treatments is to develop ways of treating non-pigmented skin "targets". New uses for lasers are emerging. Diagnostic laser imaging and spectroscopy will soon emerge in dermatology. A near-infrared laser confocal microscope provides histology-like images of human skin. Imaging is painless and takes only a few minutes. Lesions including melanoma, basal cell and squamous cell carcinoma, microvascular and inflammatory lesions, dermatophytes, verrucae, etc, have distinct appearances. However, sensitivity and specificity of laser-based diagnostic imaging has not yet been compared with histopathology. Laser phototherapy is also emerging in dermatology. The 308 nm excimer laser has recently been shown to clear psoriasis faster than conventional phototherapy. Scalp psoriasis may soon be treated by fiber-optic delivery of this UV laser. The variety and utility of lasers in dermatology will probably continue to grow.

Hair Follicular Stem Cells Give Rise to Both Hair and Skin

Hair Follicular Stem Cells Give Rise to Both Hair and Skin

Involvement of Follicular Stem Cells in Forming Not Only the Follicle but Also the Epidermis

The location of follicular and epidermal stem cells in mammalian skin is a crucial issue in cutaneous biology. We demonstrate that hair follicular stem cells, located in the bulge region, can give rise to several cell types of the hair follicle as well as upper follicular cells. Moreover, we devised a double-label technique to show that upper follicular keratinocytes emigrate into the epidermis in normal newborn mouse skin, and in adult mouse skin in response to a penetrating wound. These findings indicate that the hair follicle represents a major repository of keratinocyte stem cells in mouse skin, and that follicular bulge stem cells are potentially bipotent as they can give rise to not only the hair follicle, but also the epidermis.

Epithelial stem cells in the skin: definition, markers, localization and functions.

In recent years, cutaneous epithelial stem cells have attained a genuine celebrity status. They are considered the key resource for epidermal and skin appendage regeneration, and are proposed as a preferential target of cutaneous gene therapy. Follicular epithelial stem cells may also give rise to a large variety of epithelial tumors, and cutaneous epithelial stem cells likely are crucial targets for physical or chemical agents (including carcinogens) that damage the skin and its appendages. However, as this Controversies feature illustrates, few experts can agree on how exactly to define and identify these elusive cells, or on where precisely in the skin they are localized. Given their potential importance in skin biology, pathology and future dermatological therapy, it is, therefore, timely to carefully reconsider the basic questions: What exactly is a stem cell, and how can we reliably identify epithelial stem cells? How many different kinds are there, and how do they differ functionally? Where exactly in the skin epithelium is each of the putative stem cell subpopulations located, and can we selectively manipulate any of them?

Radiation Sensitivity of Human Hair Follicles in SCID-hu Mice

We developed an experimental model for studying the growth and epilation of the human hair follicle by implanting human scalp tissue onto immunodeficient C.B-17 scid/scid mice. The skin grafts showed continuous growth of black human hairs for at least 1 year and maintained the normal histological structure of a human hair follicle and other tissues associated with the skin. Using this in vivo model, we evaluated the effect of irradiation on the function of human hair follicles. Localized X irradiation (1 to 6 Gy) induced hair loss dose-dependently and synchronously in the third week after irradiation. The hairs undergoing epilation showed a gradual decrease in width toward the root. The minimum width at the thinnest portion of the surviving hair 4 weeks after irradiation suggested that epilation resulted from the breaking of hairs when the hair width decreased to less than 20 microm. After the highest-dose irradiation, the normal structure of the hair bulb was totally abrogated, and long and narrow epithelial tissues associated with regressed papillary cells remained. The surviving epithelia were morphologically similar to the outer epithelial sheath of the follicle associated with palisadic basal cell layers. In the third week some cells in the basal layers of the surviving epithelium in each follicle expressed proliferating cell nuclear antigen. By about 9 weeks after irradiation, the complete structure of the follicle regenerated, with hair growth activity even in the grafts irradiated at the highest dose, although about 30% of the hairs did not regrow. These findings suggest that follicular stem cells that survive high-dose exposure in the sheath-like epithelial tissue can reproduce the complete follicle structure. This animal model can be used to assess the effects of radiation exposure on human skin and to identify and characterize human follicular stem cells.