Spatial patterns produced by a reaction-diffusion system in primary hair follicles

This paper is the third in a series examining the role of a reaction-diffusion (RD) system as the principal mechanism providing spatial information for cell differentiation during hair follicle initiation and development and hair fibre formation. A theoretical mechanism is described by which the RD system supplies positional information during hair follicle development. Solutions of the RD system within the primordial follicle are described as well as the sequence of spatial patterns provides the follicle/epidermis boundary conditions required to account for the density and grouping of follicles during initiation. At the same time the spatial patterns are also shown to be capable of providing the positional information which determines various geometrical aspects of follicle development; in particular the development of follicles at an angle to the skin surface and the initiation and location of sweat glands and sebaceous glands on the follicle.     

The role of a reaction-diffusion system in the initiation of primary hair follicles

A mechanism based on a reaction-diffusion system is proposed for the initiation of hair follicles in the epidermis during fetal development. It is demonstrated that initiation of primary follicles in a series of waves, within the proposed mechanism, is a consequence of the size and shape dependent properties of the reaction-diffusion system without the need for the propagation of signals through the skin. The observed trio grouping of follicles and variation of primary follicle density per unit skin area during development are also correctly predicted. An explanation, based on the reaction-diffusion system and the variation of its characteristic spatial wavelength with time during development, is suggested for the termination of both primary and secondary follicle initiation as well as follicle neogenesis. The proposed initiation mechanism is basically the same as that used to explain various spatial patterns observed in hair fibre formation (Nagorcka & Mooney, 1982).     

Study of the keratinization process in human hair follicle by X-ray microdiffraction.

Synchrotron X-ray micro-diffraction studies along the follicle and the hair fibre allowed us to follow the keratinization process and the progressive organization of the keratin: i) molecular organization appeared progressively in the follicle; the formation of alpha-helices was completed inside the follicle. ii) supramolecular organization appeared only outside the follicle, far from the bulb; filament structure was observed far from the follicle. Comparisons between structures observed for in vitro and in vivo grown hair, whatever in the bulb or in the fibre, indicate there is no evidence of any structural difference. More, no variation in the transition in vivo/in vitro zone has been observed. In vitro and in vivo fibres exhibited the same structure.     

Genetic mosaic analysis indicates that the bulb region of coat hair follicles contains a resident population of several active multipotent epithelial lineage progenitors.

The hair follicle represents an excellent model system for exploring the properties of lineage-forming units in a dynamic epithelium containing multiple cell types. During its growth (anagen) phase, the proximal-distal axis of the mouse coat hair (pelage) follicle provides a historical record of all epithelial lineages generated from its resident stem cell population. An unresolved question in the field is whether the bulb region of anagen pelage follicles contains multipotential progenitors and whether their individual contribution to cellular census fluctuates over time. To address this issue, chimeric follicles were harvested in midanagen from three types of genetic mosaic mouse models. Analysis of the distribution of genotypic markers, including digital three-dimensional reconstruction of serially sectioned chimeric follicles, revealed that on average the bulb contains four or fewer active progenitors, each capable of giving rise to all six follicular epithelial fates. Moreover, analysis of mosaic pelage, as well as cultured whisker follicles provided evidence that bulb-associated progenitors can give rise to expanding descendant clones during midanagen, leading to the conclusion that the bulb contains dormant or symmetrically dividing stem cells. This latter feature resembles the behavior of hematopoietic stem cells after bone marrow transplantation, and raises the question of whether this property may be shared by stem cells in other self-renewing epithelia.     

Hair follicle stem cells: walking the maze

The discovery of epithelial stem cells (eSCs) in the bulge region of the outer root sheath of hair follicles in mice and man has encouraged research into utilizing the hair follicle as a therapeutic source of stem cells (SCs) for regenerative medicine, and has called attention to the hair follicle as a highly instructive model system for SC biology. Under physiological circumstances, bulge eSCs serve as cell pool for the cyclic regeneration of the anagen hair bulb, while they can also regenerate the sebaceous gland and the epidermis after injury. More recently, melanocyte SCs, nestin+, mesenchymal and additional, as yet ill-defined "stem cell" populations, have also been identified in or immediately adjacent to the hair follicle epithelium, including in the specialized hair follicle mesenchyme (connective tissue sheath), which is crucial to wound healing. Thus the hair follicle and its adjacent tissue environment contain unipotent, multipotent, and possibly even pluripotent SC populations of different developmental origin. It provides an ideal model system for the study of central issues in SC biology such as plasticity and SC niches, and for the identification of reliable, specific SC markers, which distinguish them from their immediate progeny (e.g. transient amplifying cells). The current review attempts to provide some guidance in this growing maze of hair follicle-associated SCs and their progeny, critically reviews potential or claimed hair follicle SC markers, highlights related differences between murine and human hair follicles, and defines major unanswered questions in this rapidly advancing field.     

Migration of melanoblasts into the developing murine hair follicle is accompanied by transient c-Kit expression.

Disruption of the c-Kit/stem cell factor (SCF) signaling pathway interferes with the survival, migration, and differentiation of melanocytes during generation of the hair follicle pigmentary unit. We examined c-Kit, SCF, and S100 (a marker for precursor melanocytic cells) expression, as well as melanoblast/melanocyte ultrastructure, in perinatal C57BL/6 mouse skin. Before the onset of hair bulb melanogenesis (i.e., stages 0-4 of hair follicle morphogenesis), strong c-Kit immunoreactivity (IR) was seen in selected non-melanogenic cells in the developing hair placode and hair plug. Many of these cells were S100-IR and were ultrastructurally identified as melanoblasts with migratory appearance. During the subsequent stages (5 and 6), increasingly dendritic c-Kit-IR cells successively invaded the hair bulb, while S100-IR gradually disappeared from these cells. Towards the completion of hair follicle morphogenesis (stages 7 and 8), several distinct follicular melanocytic cell populations could be defined and consisted broadly of (a) undifferentiated, non-pigmented c-Kit-negative melanoblasts in the outer root sheath and bulge and (b) highly differentiated melanocytes adjacent to the hair follicle dermal papilla above Auber's line. Widespread epithelial SCF-IR was seen throughout hair follicle morphogenesis. These findings suggest that melanoblasts express c-Kit as a prerequisite for migration into the SCF-supplying hair follicle epithelium. In addition, differentiated c-Kit-IR melanocytes target the bulb, while non-c-Kit-IR melanoblasts invade the outer root sheath and bulge in fully developed hair follicles.     

Cell proliferation and cortical cell production in relation to wool growth

The relationship of wool growth to cell proliferation in the follicle bulb and to the subsequent migration and growth of the fibre cortical cells was investigated in 10 Peppin Merino sheep. These sheep had been maintained on a low, medium or high level of nutrient intake to ensure a wide range in wool growth. The number and mitotic activity of the germinal cells in the follicle bulb were determined after administration of colchicine. Cortical cell size was measured following isolation of the fibre cells by acid-treatment of wool. The average fibre production of the follicle varied from 4.1 x 10(4) to 13.2 x 10(4) micron3/day in these sheep. There were also substantial differences between sheep in the mitotic activity of the germinal cells in the bulb, the rate of cell proliferation being highly correlated with the average daily fibre production of the follicle (r = + 0.88, n = 10). However, the size of the germinal cell population differed from sheep to sheep and was not closely related to the level of fibre production (r = + 0.48, n = 10). The average turnover time of these cells was inversely related to fibre production and varied from 41.6 to 19.4 h (r = -0.82, n = 10). Multiple regression analysis of the data showed that the average daily fibre production of the follicle was largely determined by the number of germinal cells present in the bulb and their rate of proliferation (R = +0.95, n = 10). Variations in cell turnover time and in cortical cell size were not significant in influencing the rate of fibre production. In these sheep, the average cortical cell varied in size from 658 to 1279 micron 3 and the positive correlation (r = + 0.83, n = 10) found between cell size and fibre production is considered to merely reflect an allometric relationship. The proportion of germinal cells contributing to the fibre cortex was found to be small and variable, ranging from 9.4 to 17.8%. Furthermore, this proportion was not related to the nutritional level of these sheep, and it is thought that the variability in the distribution of cells to the fibre may be attributed to genetic differences between sheep.     

Multi-potentiality of a new immortalized epithelial stem cell line derived from human hair follicles

We previously demonstrated that keratin 15 expressing cells present in the bulge region of hair follicles exhibit properties of adult stem cells. We have now established and characterized an immortalized adult epithelial stem cell line derived from cells isolated from the human hair follicle bulge region. Telogen hair follicles from human skin were microdissected to obtain an enriched population of keratin 15 positive skin stem cells. By expressing human papillomavirus 16 E6/E7 genes in these stem cells, we have been able to culture the cells for >30 passages and maintain a stable phenotype after 12 mo of continuous passage. The cell line was compared to primary stem cells for expression of stem cell specific proteins, for in vitro stem cell properties, and for their capacity to differentiate into different cell lineages. This new cell line, named Tel-E6E7 showed similar expression patterns to normal skin stem cells and maintained in vitro properties of stem cells. The cells can differentiate into epidermal, sebaceous gland, and hair follicle lineages. Intact beta-catenin dependent signaling, which is known to control in vivo hair differentiation in rodents, is maintained in this cell line. The Tel-E6E7 cell line may provide the basis for valid, reproducible in vitro models for studies on stem cell lineage determination and differentiation.     

The role of hair follicle nestin‐expressing stem cells during whisker sensory‐nerve growth in long‐term 3D culture

We have previously reported that nestin‐expressing hair follicle stem cells can differentiate into neurons, Schwann cells, and other cell types. In the present study, vibrissa hair follicles, including their sensory nerve stump, were excised from transgenic mice in which the nestin promoter drives green fluorescent protein (ND‐GFP mice), and were placed in 3D histoculture supported by Gelfoam®. β‐III tubulin‐positive fibers, consisting of ND‐GFP‐expressing cells, extended up to 500 µm from the whisker nerve stump in histoculture. The growing fibers had growth cones on their tips expressing F‐actin. These findings indicate that β‐III tubulin‐positive fibers elongating from the whisker follicle sensory nerve stump were growing axons. The growing whisker sensory nerve was highly enriched in ND‐GFP cells which appeared to play a major role in its elongation and interaction with other nerves in 3D culture, including the sciatic nerve, the trigeminal nerve, and the trigeminal nerve ganglion. The results of the present report suggest a major function of the nestin‐expressing stem cells in the hair follicle is for growth of the follicle sensory nerve. J. Cell. Biochem. 114: 1674–1684, 2013. © 2013 Wiley Periodicals, Inc.     

Real-time confocal imaging of trafficking of nestin-expressing multipotent stem cells in mouse whiskers in long-term 3-D histoculture

We have previously demonstrated that nestin-expressing multipotent hair follicle stem cells are located above the hair follicle bulge and can differentiate into neurons and other cell types in vitro. The nestin-expressing hair follicle stem cells promoted the recovery of pre-existing axons when they were transplanted to the severed sciatic nerve or injured spinal cord. We have also previously demonstrated that the whisker hair follicle contains nestin-expressing stem cells in the dermal papilla (DP) as well as in the bulge area (BA), but that their origin is in the BA. In the present study, we established the technique of long-term Gelfoam? histoculture of whiskers isolated from transgenic mice in which nestin drives green fluorescent protein (ND-GFP). Confocal imaging was used to monitor ND-GFP-expressing stem cells trafficking in real time between the BA and DP to determine the fate of the stem cells. It was observed over a 2-week period that the stem cells trafficked from the BA toward the DP area and extensively grew out onto Gelfoam? forming nerve-like structures. This new method of long-term histoculture of whiskers from ND-GFP mice will enable the extensive study of the behavior of nestin-expressing multipotent stem cells of the hair follicle.     

Communication compartments in hair follicles and their implication in differentiative control.

Observations on hair follicles presented in this paper show that boundaries to junctional communication are formed between groups of cells following different pathways of differentiation. The patterns of junctional communication in the bulbs of rat vibrissa follicles and human hair follicles were studied by microinjection of the fluorescent tracer dye Lucifer Yellow CH. Dye spread was extensive between undifferentiated cells of the hair bulb matrix but communication boundaries were found between groups of morphologically distinct cells. For example, boundaries to dye spread were observed between undifferentiated matrix cells and cells in the early stage of differentiation into the inner root sheath, between Huxley's and Henle's layers in the early inner root sheath and between cells of the cuticle and cortex of the hair. Dye did not spread between epithelial cells of the hair bulb and mesenchymal cells of the connective tissue sheath or dermal papilla. The patterns of dye spread became more complex (increased boundary formation and subcompartmentation) as differentiation progressed in higher regions of the hair bulb. The observed communication can be related to previous ultrastructural studies by others on the distribution of gap junctions in the wool follicle. These results show that junctional communication, with its consequent intercellular spread of small ions and molecules, is associated with uniformity of expression and behaviour within cell populations and that interruption of communication through the formation of boundaries and communication compartments is temporally and spatially related to the production of subpopulations of cells committed to the expression of different phenotypes.     

Restoration of hair growth by surgical implantation of follicular dermal sheath.

The capacity of lower follicle dermal sheath to restore hair growth was tested by removing the lower halves of follicles, and then immediately implanting material containing dermal sheath cells from these bases, into the remaining upper epidermal follicle cavity. Over 60% of recipient follicles produced stout emergent vibrissa fibres and some operations resulted in multiple hair production from a single follicle. Histological examination revealed new dermal papillae within large bulb structures which were sited below the level of amputation--a feature that indicated that the new dermal papilla was derived from implanted material. For many follicles, the failure to produce emergent fibres could be accounted for after histological examination. These results provide clear evidence that lower follicle dermal sheath cells are capable of replacing those of the dermal papilla and it shows that they can do so in the context of the upper follicle. However, because elements of lower follicle epidermis were present in the implant material, the interactive sequence of events cannot be established. Dermal sheath cells have immense potential for papilla cell replacement: questions remain as to whether the distinction between sheath and papilla cells is one of context, or whether the transition requires specific external influences.     

Label-retaining cells reside in the bulge area of pilosebaceous unit: implications for follicular stem cells, hair cycle, and skin carcinogenesis

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.     

Multipotent nestin-expressing stem cells capable of forming neurons are located in the upper,middle and lower part of the vibrissa hair follicle

We have previously demonstrated that the neural stem-cell marker nestin is expressed in hair follicle stem cells. Nestin-expressing cells were initially identified in the hair follicle bulge area (BA) using a transgenic mouse model in which the nestin promoter drives the green fluorescent protein (ND-GFP). The hair-follicle ND-GFP-expressing cells are keratin 15-negative and CD34-positive and could differentiate to neurons, glia, keratinocytes, smooth muscle cells and melanocytes in vitro. Subsequently, we showed that the nestin-expressing stem cells could affect nerve and spinal cord regeneration after injection in mouse models. In the present study, we separated the mouse vibrissa hair follicle into three parts (upper, middle and lower). Each part of the follicle was cultured separately in DMEM-F12 containing B-27 and 1% methylcellulose supplemented with basic FGF. After 2 mo, the nestin-expressing cells from each of the separated parts of the hair follicle proliferated and formed spheres. Upon transfer of the spheres to RPMI 1640 medium containing 10% FBS, the nestin-expressing cells in the spheres differentiated to neurons, as well as glia, keratinocytes, smooth muscle cells and melanocytes. The differentiated cells were produced by spheres which formed from nestin-expressing cells from all segments of the hair follicle. However, the differentiation potential is greatest in the upper part of the follicle. This result is consistent with trafficking of nestin-expressing cells throughout the hair follicle from the bulge area to the dermal papilla that we previously observed. The nestin-expressing cells from the upper part of the follicle produced spheres in very large amounts, which in turn differentiated to neurons and other cell types. The results of the present study demonstrate that multipotent, nestin-expressing stem cells are present throughout the hair follicle and that the upper part of the follicle can produce the stem cells in large amounts that could be used for nerve and spinal cord repair.     

Multipotent nestin-expressing stem cells capable of forming neurons are located in the upper,middle and lower part of the vibrissa hair follicle

We have previously demonstrated that the neural stem-cell marker nestin is expressed in hair follicle stem cells. Nestin-expressing cells were initially identified in the hair follicle bulge area (BA) using a transgenic mouse model in which the nestin promoter drives the green fluorescent protein (ND-GFP). The hair-follicle ND-GFP-expressing cells are keratin 15-negative and CD34-positive and could differentiate to neurons, glia, keratinocytes, smooth muscle cells and melanocytes in vitro. Subsequently, we showed that the nestin-expressing stem cells could affect nerve and spinal cord regeneration after injection in mouse models. In the present study, we separated the mouse vibrissa hair follicle into three parts (upper, middle and lower). Each part of the follicle was cultured separately in DMEM-F12 containing B-27 and 1% methylcellulose supplemented with basic FGF. After 2 mo, the nestin-expressing cells from each of the separated parts of the hair follicle proliferated and formed spheres. Upon transfer of the spheres to RPMI 1640 medium containing 10% FBS, the nestin-expressing cells in the spheres differentiated to neurons, as well as glia, keratinocytes, smooth muscle cells and melanocytes. The differentiated cells were produced by spheres which formed from nestin-expressing cells from all segments of the hair follicle. However, the differentiation potential is greatest in the upper part of the follicle. This result is consistent with trafficking of nestin-expressing cells throughout the hair follicle from the bulge area to the dermal papilla that we previously observed. The nestin-expressing cells from the upper part of the follicle produced spheres in very large amounts, which in turn differentiated to neurons and other cell types. The results of the present study demonstrate that multipotent, nestin-expressing stem cells are present throughout the hair follicle and that the upper part of the follicle can produce the stem cells in large amounts that could be used for nerve and spinal cord repair.     

Wool fibre crimp is determined by mitotic asymmetry and position of final keratinisation and not ortho- and para-cortical cell segmentation

Crimp, a distinguishing feature of sheep fibres, significantly affects wool value, processing and final fabric attributes. Several explanations for fibre bending have been proposed. Most concentrate on relative differences in the physicochemical properties of the cortical cells, which comprise the bulk of the fibre. However, the associations between cortical properties and fibre crimp are not consistent and may not reflect the underlying causation of fibre curvature (FC). We have formulated a mechanistic model in which fibre shape is dictated primarily by the degree of asymmetry in cell supply from the follicle bulb, and the point at which keratinisation is completed within the follicle. If this hypothesis is correct, one would anticipate that most variations in fibre crimp would be accounted for by quantitative differences in both the degree of mitotic asymmetry in follicle bulbs and the distance from the bulb to the point at which keratinisation is completed. To test this hypothesis, we took skin biopsies from Merino sheep from sites producing wool differing widely in fibre crimp frequency and FC. Mitotic asymmetry in follicle bulbs was measured using a DNA-labelling technique and the site of final keratinisation was defined by picric acid staining of the fibre. The proportion of para- to ortho-cortical cell area was determined in the cross-sections of fibres within biopsy samples. Mitotic asymmetry in the follicle bulb accounted for 0.64 (P < 0.0001) of the total variance in objectively measured FC, while the point of final keratinisation of the fibre accounted for an additional 0.05 (P < 0.05) of the variance. There was no association between ortho- to para-cortical cell ratio and FC. FC was positively associated with a subjective follicle curvature score (P < 0.01). We conclude that fibre crimp is caused predominantly by asymmetric cell division in follicles that are highly curved. Differential pressures exerted by the subsequent asymmetric cell supply and cell hardening in the lower follicle cause fibre bending. The extent of bending is then modulated by the point at which keratinisation is completed; later hardening means the fibre remains pliable for longer, thereby reducing the pressure differential and reducing fibre bending. This means that even highly asymmetric follicles may produce a straight fibre if keratinisation is sufficiently delayed, as is the case in deficiencies of zinc and copper, or when keratinisation is perturbed by transgenesis. The model presented here can account for the many variations in fibre shape found in mammals.     

Inhibition of BMP signaling in P-Cadherin positive hair progenitor cells leads to trichofolliculoma-like hair follicle neoplasias

Background  

Skin stem cells contribute to all three major lineages of epidermal appendages, i.e., the epidermis, the hair follicle, and the sebaceous gland. In hair follicles, highly proliferative committed progenitor cells, called matrix cells, are located at the base of the follicle in the hair bulb. The differentiation of these early progenitor cells leads to specification of a central hair shaft surrounded by an inner root sheath (IRS) and a companion layer. Multiple signaling molecules, including bone morphogenetic proteins (BMPs), have been implicated in this process.     

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.