Epithelial stem cells: stepping out of their niche

In this issue of Cell, have shown that two subpopulations of cells exist within the hair follicle stem cell niche. Despite being partially differentiated, clonal populations of suprabasal bulge region cells can regenerate skin and hair follicles as well as a new stem cell niche. The findings suggest that early lineage commitments of epithelial cells in the hair follicle may be reversible.     

Estrogen leads to reversible hair cycle retardation through inducing premature catagen and maintaining telogen

Estrogen dysregulation causes hair disorder. Clinical observations have demonstrated that estrogen raises the telogen/anagen ratio and inhibits hair shaft elongation of female scalp hair follicles. In spite of these clinical insights, the properties of estrogen on hair follicles are poorly dissected. In the present study, we show that estrogen induced apoptosis of precortex cells and caused premature catagen by up-regulation of TGF β2. Immediately after the premature catagen, the expression of anagen chalone BMP4 increased. The up-regulation of BMP4 may further function to prevent anagen transition and maintain telogen. Interestingly, the hair follicle stem cell niche was not destructed during these drastic structural changes caused by estrogen. Additionally, dermal papilla cells, the estrogen target cells in hair follicles, kept their signature gene expressions as well as their hair inductive potential after estrogen treatment. Retention of the characteristics of both hair follicle stem cells and dermal papilla cells determined the reversibility of the hair cycle suppression. These results indicated that estrogen causes reversible hair cycle retardation by inducing premature catagen and maintaining telogen.     

Melanocyte stem cells: a melanocyte reservoir in hair follicles for hair and skin pigmentation

Most mammals are coated with pigmented hair. Melanocytes in each hair follicle produce melanin pigments for the hair during each hair cycle. The key to understanding the mechanism of cyclic melanin production is the melanocyte stem cell (MelSC) population, previously known as 'amelanotic melanocytes'. The MelSCs directly adhere to hair follicle stem cells, the niche cells for MelSCs and reside in the hair follicle bulge-subbulge area, the lower permanent portion of the hair follicle, to serve as a melanocyte reservoir for skin and hair pigmentation. MelSCs form a stem cell system within individual hair follicles and provide a 'hair pigmentary unit' for each cycle of hair pigmentation. This review focuses on the identification of MelSCs and their characteristics and explains the importance of the MelSC population in the mechanisms of hair pigmentation, hair greying, and skin repigmentation.     

Co-factors of LIM domains (Clims/Ldb/Nli) regulate corneal homeostasis and maintenance of hair follicle stem cells

The homeostasis of both cornea and hair follicles depends on a constant supply of progeny cells produced by populations of keratin (K) 14-expressing stem cells localized in specific niches. To investigate the potential role of Co-factors of LIM domains (Clims) in epithelial tissues, we generated transgenic mice expressing a dominant-negative Clim molecule (DN-Clim) under the control of the K14 promoter. As expected, the K14 promoter directed high level expression of the transgene to the basal cells of cornea and epidermis, as well as the outer root sheath of hair follicles. In corneal epithelium, the transgene expression causes decreased expression of adhesion molecule BP180 and defective hemidesmosomes, leading to detachment of corneal epithelium from the underlying stroma, which in turn causes blisters, wounds and an inflammatory response. After a period of epithelial thinning, the corneal epithelium undergoes differentiation to an epidermis-like structure. The K14-DN-Clim mice also develop progressive hair loss due to dysfunctional hair follicles that fail to generate hair shafts. The number of hair follicle stem cells is decreased by at least 60% in K14-DN-Clim mice, indicating that Clims are required for hair follicle stem cell maintenance. In addition, Clim2 interacts with Lhx2 in vivo, suggesting that Clim2 is an essential co-factor for the LIM homeodomain factor Lhx2, which was previously shown to play a role in hair follicle stem cell maintenance. Together, these data indicate that Clim proteins play important roles in the homeostasis of corneal epithelium and hair follicles.     

Bioengineering the hair follicle: fringe benefits of stem cell technology

Recent advances in epithelial stem cell biology have resulted in the isolation of hair follicle stem cells, which generate hair follicles when injected into immunodeficient mice. These isolated hair follicle epithelial stem cells must be combined with 'inductive' dermal cells to produce new hair follicles. The advent of techniques for cultivating inductive dermal cells and competent epithelial stem cells creates the opportunity to bioengineer hair follicles for the treatment of hair loss.     

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.     

Platelet sonicates activate hair follicle stem cells and mediate enhanced hair follicle regeneration

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.     

Beta-catenin and Hedgehog signal strength can specify number and location of hair follicles in adult epidermis without recruitment of bulge stem cells

Using K14deltaNbeta-cateninER transgenic mice, we show that short-term, low-level beta-catenin activation stimulates de novo hair follicle formation from sebaceous glands and interfollicular epidermis, while only sustained, high-level activation induces new follicles from preexisting follicles. The Hedgehog pathway is upregulated by beta-catenin activation, and inhibition of Hedgehog signaling converts the low beta-catenin phenotype to wild-type epidermis and the high phenotype to low. beta-catenin-induced follicles contain clonogenic keratinocytes that express bulge markers; the follicles induce dermal papillae and provide a niche for melanocytes, and they undergo 4OHT-dependent cycles of growth and regression. New follicles induced in interfollicular epidermis are derived from that cellular compartment and not through bulge stem cell migration or division. These results demonstrate the remarkable capacity of adult epidermis to be reprogrammed by titrating beta-catenin and Hedgehog signal strength and establish that cells from interfollicular epidermis can acquire certain characteristics of bulge stem cells.     

A dermal niche for multipotent adult skin-derived precursor cells

A fundamental question in stem cell research is whether cultured multipotent adult stem cells represent endogenous multipotent precursor cells. Here we address this question, focusing on SKPs, a cultured adult stem cell from the dermis that generates both neural and mesodermal progeny. We show that SKPs derive from endogenous adult dermal precursors that exhibit properties similar to embryonic neural-crest stem cells. We demonstrate that these endogenous SKPs can first be isolated from skin during embryogenesis and that they persist into adulthood, with a niche in the papillae of hair and whisker follicles. Furthermore, lineage analysis indicates that both hair and whisker follicle dermal papillae contain neural-crest-derived cells, and that SKPs from the whisker pad are of neural-crest origin. We propose that SKPs represent an endogenous embryonic precursor cell that arises in peripheral tissues such as skin during development and maintains multipotency into adulthood.     

Hair follicle stem cells in the lower bulge form the secondary germ, a biochemically distinct but functionally equivalent progenitor cell population, at the termination of catagen

The lowermost portion of the resting (telogen) follicle consists of the bulge and secondary hair germ. We previously showed that the progeny of stem cells in the bulge form the lower follicle and hair, but the relationship of the bulge cells with the secondary hair germ cells, which are also involved in the generation of the new hair at the onset of the hair growth cycle (anagen), remains unclear. Here we address whether secondary hair germ cells are derived directly from epithelial stem cells in the adjacent bulge or whether they arise from cells within the lower follicle that survive the degenerative phase of the hair cycle (catagen). We use 5-bromo-2'-deoxyuridine to label bulge cells at anagen onset, and demonstrate that the lowermost portion of the bulge collapses around the hair and forms the secondary hair germ during late catagen. During the first six days of anagen onset bulge cells proliferate and self-renew. Bulge cell proliferation at this time also generates cells that form the future secondary germ. As bulge cells form the secondary germ cells at the end of catagen, they lose expression of a biochemical marker, S100A6. Remarkably, however, following injury of bulge cells by hair depilation, progenitor cells in the secondary hair germ repopulate the bulge and re-express bulge cell markers. These findings support the notion that keratinocytes can "dedifferentiate" to a stem cell state in response to wounding, perhaps related to signals from the stem cell niche. Finally, we also present evidence that quiescent bulge cells undergo apoptosis during follicle remodeling in catagen, indicating that a subpopulation of bulge cells is not permanent.     

Targeted inactivation of integrin-linked kinase in hair follicle stem cells reveals an important modulatory role in skin repair after injury

Integrin-linked kinase (ILK) is key for normal epidermal morphogenesis, but little is known about its role in hair follicle stem cells and epidermal regeneration. Hair follicle stem cells are important contributors to newly formed epidermis following injury. We inactivated the Ilk gene in the keratin 15--expressing stem cell population of the mouse hair follicle bulge. Loss of ILK expression in these cells resulted in impaired cutaneous wound healing, with substantially decreased wound closure rates. ILK-deficient stem cells produced very few descendants that moved toward the epidermal surface and into the advancing epithelium that covers the wound. Furthermore, those few mutant cells that homed in the regenerated epidermis exhibited a reduced residence time. Paradoxically, ILK-deficient bulge stem cells responded to anagen growth signals and contributed to newly regenerated hair follicles during this phase of hair follicle growth. Thus ILK plays an important modulatory role in the normal contribution of hair follicle stem cell progeny to the regenerating epidermis following injury.     

The human hair follicle contains two distinct K19 positive compartments in the outer root sheath: a unifying hypothesis for stem cell reservoir?

Up to now, the localization of stem cells in human anagen hair follicle relied on three complementary approaches; namely, detection of slow cycling cells, detection of high colony forming cells, and differential immunohistochemical staining. These techniques, however, gave conflicting results since stem cells were localized either as long label retaining cells in the so-called bulge area or as high colony forming cells in the lower third of the follicle. In the present study we investigated the expression of cytokeratin 19, a marker for putative stem cell-containing epithelial compartments, in order to characterize stem cell distribution in the human hair follicle throughout the hair cycle. We found that anagen human hair follicles contain two distinct reservoirs for stem cells located in the upper and lower thirds of the follicle. These two reservoirs fuse during the catagentelogen transition phase and individualize again in the newly forming anagen hair follicle.     

干细胞巢研究进展

干细胞巢即干细胞周围的微环境构成,一般包括干细胞的相邻细胞、粘附分子及基质等,但不同的干细胞有不同的巢结构。干细胞巢通过不同信号途径调控着干细胞的行为,使干细胞的自我更新和分化处于平衡状态。根据近年来有关干细胞巢的研究,本文从果蝇生殖系干细胞巢、哺乳动物造血干细胞巢、肠干细胞巢、毛囊表皮干细胞巢和神经干细胞巢等五个系统分别综述了干细胞巢的构成及其对干细胞的调节作用,探讨了干细胞巢作用于干细胞的内在机制。     

Extensive Hair-Shaft Elongation by Isolated Mouse Whisker Follicles in Very Long-Term Gelfoam? Histoculture

We have previously studied mouse whisker follicles in Gelfoam® histoculture to determine the role of nestin-expressing plutipotent stem cells, located within the follicle, in the growth of the follicular sensory nerve. Long-term Gelfoam® whisker histoculture enabled hair follicle nestin-expressing stem cells to promote the extensive elongation of the whisker sensory nerve, which contained axon fibers. Transgenic mice in which the nestin promoter drives green fluorescent protein (ND-GFP) were used as the source of the whiskers allowing imaging of the nestin-expressing stem cells as they formed the follicular sensory nerve. In the present report, we show that Gelfoam®-histocultured whisker follicles produced growing pigmented and unpigmented hair shafts. Hair-shaft length increased rapidly by day-4 and continued growing until at least day-12 after which the hair-shaft length was constant. By day-63 in histoculture, the number of ND-GFP hair follicle stem cells increased significantly and the follicles were intact. The present study shows that Gelfoam® histoculture can support extensive hair-shaft growth as well as hair follicle sensory-nerve growth from isolated hair follicles which were maintained over very long periods of time. Gelfoam® histoculture of hair follicles can provide a very long-term period for evaluating novel agents to promote hair growth.     

Human hair follicle-derived mesenchymal stem cells: Isolation,expansion, and differentiation

Hair follicles are easily accessible skin appendages that protect against cold and potential injuries. Hair follicles contain various pools of stem cells, such as epithelial, melanocyte, and mesenchymal stem cells (MSCs) that continuously self-renew, differentiate, regulate hair growth, and maintain skin homeostasis. Recently, MSCs derived from the dermal papilla or dermal sheath of the human hair follicle have received attention because of their accessibility and broad differentiation potential. In this review, we describe the applications of human hair follicle-derived MSCs (hHF-MSCs) in tissue engineering and regenerative medicine. We have described protocols for isolating hHF-MSCs from human hair follicles and their culture condition in detail. We also summarize strategies for maintaining hHF-MSCs in a highly proliferative but undifferentiated state after repeated in vitro passages, including supplementation of growth factors, 3D suspension culture technology, and 3D aggregates of MSCs. In addition, we report the potential of hHF-MSCs in obtaining induced smooth muscle cells and tissue-engineered blood vessels, regenerated hair follicles, induced red blood cells, and induced pluripotent stem cells. In summary, the abundance, convenient accessibility, and broad differentiation potential make hHF-MSCs an ideal seed cell source of regenerative medical and cell therapy.     

角蛋白15在大鼠皮肤发育中表达的研究

目的研究角蛋白15(K15)在大鼠皮肤发育中的表达状况,定位表皮干细胞.方法以不同年龄大鼠背部皮肤为标本,用组织学方法,观察出生后大鼠皮肤的形态发育变化;以K15单克隆抗体为一抗,进行免疫组织化学染色,观察K15在大鼠皮肤中的表达状况.结果(1)组织学方法显示,随着年龄的增长,大鼠背部表皮细胞层数逐渐变少;在毛囊的生长周期中,以隆突区为界,毛囊上段为恒定区,下段呈周期性变化(2)免疫组化染色显示,毛囊隆突区细胞胞浆表达K15,随年龄的增长,K15阳性细胞出现在毛母质细胞区、毛囊外根鞘和表皮基底层.结论表皮干细胞位于毛囊隆突区,与表皮的更新和毛囊的周期性变化有关.     

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.