Embryonic development of hair follicle pluripotent stem (hfPS) cells

Our laboratory discovered nestin-expressing hair follicle stem cells and demonstrated their pluripotency. We have shown that nestin-positive and K15-negative multipotent hair follicle stem cells are located above the hair follicle bulge, and we termed these cells hair follicle pluripotent stem (hfPS) cells. We have previously shown that hair follicle stem cells can regenerate peripheral nerve and spinal cord. In the present study, we describe the embryonic development of the hair follicle stem cell area (hfPSCA), which is located above the bulge and below the sebaceous glands in the adult mouse. At embryonic day 16.5 (E16.5) of nestindriven GFP (ND-GFP) transgenic mice, which express nestin in hfPS cells, the ND-GFP hair follicle stem cells are located in mesenchymal condensates. At postnatal day 0 (P0), the ND-GFP-expressing cells are migrating to the upper part of the hair follicle from the dermal papilla. At P3, keratin 15 (K15)-positive cells, derived from ND-GFP dermal papilla cells, are located in the outer-root sheath and basal layer of the epidermis. By P10, the ND-GFP have formed the K15-positive outer-root sheath as well as the ND-GFP hfPSA. These results suggest that ND-GFP hfPS cells in the dermal papilla form nestin-expressing hair follicle stem cells in the first hair cycle. These observations provide new insight into the origins of hfPS cells and the hfPSCA.     

Neural Crest Stem Cell-specific Deletion of the Pygopus2 Gene Modulates Hair Follicle Development

We show that neural crest stem cells affect mouse hair follicle development. During embryogenesis hair follicle induction is regulated by complex reciprocal and functionally redundant signals between epidermis and dermis, which remain to be fully understood. Canonical Wnt signalling is a hallmark of neural crest cells and also a prerequisite for hair follicle induction prior to hair placode formation in the epidermis. As neural crest stem cells invade the epidermis during early embryonic development we aimed at determining whether neural crest cells affect hair follicle development. To attenuate, but not silence, canonical Wnt signalling specifically in neural crest cells, we analyzed Wnt1-cre(+/?)::Pygo2(?/?) mice in which the β-catenin co-activator gene, Pygopus 2 (Pygo2), is deleted specifically in neural crest cells. Both, hair density and hair thickness were reduced in mutant mice. Furthermore, hair development was delayed and the relative ratio of hair types was affected. There was a decrease in zig-zag hairs and an increase in awl hairs. Mouse neural crest stem cells expressed ectodysplasin, an essential effector in the formation of zig-zag hair. Taken together, our data support the novel notion that neural crest cells are involved in the earliest stages of hair follicle development.     

Lef1 Contributes to the Differentiation of Bulge Stem Cells by Nuclear Translocation and Cross-Talk with the Notch Signaling Pathway

Lymphoid enhancer binding factor-1 (Lef1) is an essential regulatory protein in the Wnt signal pathway, which controls cell growth and differentiation. Investigators in the field of skin biology have confirmed that multipotent bulge stem cells (BSCs) are responsible for hair follicle development and regeneration. However, the role of Lef1 remains poorly understood. In this study, we investigated the pattern of Lef1 expression at different stages of the hair growth cycle. Lef1 was strongly expressed during anagen but attenuated in both catagen- and telogen-phase hair follicles in vivo. When stem cells were induced to differentiate toward a hair fate in a co-culture system, Lef1 was notably up-regulated and accumulated in the nucleus, appearing to activate the target protein c-myc and jagged1. Simultaneously, the Wnt and Notch signaling pathways were co-activated, as confirmed by the increased expression of β-catenin and notch1. Plasmids expressing Lef1 and ΔNLef1, a construct in which the β-catenin-binding domain of Lef1 was deleted, were used to evaluate the effects of Lef1 on stem cell differentiation. Lef1 overexpression promoted bulge stem cell differentiation toward a hair fate, which was accompanied by the subsequent migration of β-catenin into the nucleus, whereas no changes were observed in the control group. Taken together, our results demonstrate that Lef1 plays an important role in bulge stem cell differentiation, promoting β-catenin translocation into the nucleus, activating downstream signaling molecules, eventually causing hair follicle bulge stem cells to adopt the hair fate.     

Epidermal stem cells

The clinical implications of understanding epidermal stem cell biology abound. Thousands of burns victims across the world have benefited from early research into the proliferation of epidermal keratinocytes in vitro. Advances now indicate there are a number of stem cell repositories within the epidermis, two of which, the interfollicular epidermis and the bulge region of the hair follicle, may supply each other when damaged. This review details the progress made in the identification and characterisation of stem cells within the epidermis and discusses the molecules involved in the epidermal stem cell's choice of fate. Finally, the skin, like bone marrow, could be a readily accessible source of stem cells for therapeutic intervention and evidence of skin stem cell plasticity is highlighted.     

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

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.     

毛囊干细胞的研究进展

【摘要】毛囊干细胞是毛囊组织维持自我更新的基础,它具有干细胞的一般特征,普遍认为定位于毛囊隆突部。毛囊干细胞的标记物是分离和鉴定细胞的重要依据,在多种信号通路调控下可以分化为毛囊、皮脂腺和表皮。至此,毛囊干细胞在组织工程皮肤中的作用引起人们的关注,就毛囊干细胞的研究进展作一阐述。     

Tcf3 and Lef1 regulate lineage differentiation of multipotent stem cells in skin

In skin, multipotent stem cells generate the keratinocytes of the epidermis, sebaceous gland, and hair follicles. In this paper, we show that Tcf3 and Lef1 control these differentiation lineages. In contrast to Lef1, which requires Wnt signaling and stabilized beta-catenin to express the hair-specific keratin genes and control hair differentiation, Tcf3 can act independently of its beta-catenin interacting domain to suppress features of epidermal terminal differentiation, in which Tcf3 is normally shut off, and promote features of the follicle outer root sheath (ORS) and multipotent stem cells (bulge), the compartments which naturally express Tcf3. These aspects of Tcf3's action are dependent on its DNA binding and Groucho repressor-binding domains. In the absence of its beta-catenin interacting domain, Lef1's behavior (Delta NLef1) seems to be markedly distinct from that of Delta NTcf3. Delta NLef1 does not suppress epidermal differentiation and promote ORS/bulge differentiation, but rather suppresses hair differentiation and gives rise to sebocyte differentiation. Taken together, these findings provide powerful evidence that the status of Tcf3/Lef complexes has a key role in controlling cell fate lineages in multipotent skin stem cells.     

Two- and Three-Dimensional Culture of Keratinocyte Stem and Precursor Cells Derived from Primary Murine Epidermal Cultures

In the skin, multipotent keratinocyte stem cells (KSC) are localised in the hair follicle bulge region. Although, KSC can be cultivated and grown in two-dimensional (2D) culture they rapidly lose stem cell markers when isolated from their niche. Currently, there is no KSC culture method available which recapitulates an environment similar to the KSC niche in the hair follicle. Here we describe the successful establishment of an in vitro 3D stem cell culture model developed from clonally growing keratinocyte lines derived from neonatal mice using culture conditions previously established for human keratinocytes. After 20 passages, keratinocyte lines showed a stable ratio of holoclones (stem cells), meroclones (stem and precursor cells) and paraclones (differentiating cells), with approximately 29% holoclones, 54% meroclones and 17% paraclones, and were thus termed keratinocyte stem and precursor cell (KSPC) cultures. In high calcium medium, KSPC cultures grown at the air-liquid interphase differentiated and formed epidermal equivalents. Notably, and in contrast to primary keratinocytes, keratinocytes from KSPC cultures were able to aggregate and form spherical clusters in hanging drops, a characteristic hallmark shared with other stem cell types. Similar to the in vivo situation in the hair follicle bulge, KSPC aggregates also showed low proliferation, down-regulation of keratin 6, absence of keratin 1, and expression of the KSC markers keratin 15, Sox9, NFATc1 and Zfp145. KSPC aggregates therefore provide an optimal in vitro 3D environment for the further characterisation and study of normal and genetically modified KSPC.     

Re-Assessing K15 as an Epidermal Stem Cell Marker

The intermediate filament keratin 15 (K15) is present in variable amounts in various stratified epithelia, but has also been reported to be a stem cell marker in the hair follicle. Using peptide specific antibodies, we evaluated the temporal and spatial distribution pattern of K15 expression/localization during normal epidermal development and initiation of hair follicle formation, and in the injured mature epidermis (e.g., during acute injury and repair and in tumorigenesis). During development, K15 expression is first localized to a subset of epidermal basal cells and the overlying periderm at E12.5, but its expression is seen throughout the basal layer by E15.5 and beyond. In hair follicle morphogenesis, initial peg formation occurs in a K15-null area at E14.5 and as peg elongation proceeds through to the mature hair follicle, K15 expression follows the leading edge with positive cells restricted to the outer root sheath. In an epidermal injury model, K15 is first up-regulated and associated with both the basal and suprabasal layers of the interfollicular epidermis then expression becomes sporadic and down-regulated before a basal layer-specific association is re-established in the repaired epidermis. During tumorigenesis, K15 is first mis-expressed, and is ultimately down-regulated. Our data suggest that K15 protein expression may reflect not only expression in a stem or progenitor cell subpopulation, but also reflects the activity and responsiveness of basal-like cells to loss of homeostasis of the epidermal differentiation program. Thus, the data suggest caution in using K15 alone to delineate epidermal stem cells, and underscore the need for further investigation of K15 and other markers in epidermal cell subpopulations.     

Age-associated inflammation inhibits epidermal stem cell function

Altered stem cell homeostasis is linked to organismal aging. However, the mechanisms involved remain poorly understood. Here we report novel alterations in hair follicle stem cells during skin aging, including increased numbers, decreased function, and an inability to tolerate stress. Performing high-throughput RNA sequencing on aging stem cells, cytokine arrays, and functional assays, we identify an age-associated imbalance in epidermal Jak–Stat signaling that inhibits stem cell function. Collectively, this study reveals a role for the aging epidermis in the disruption of cytokine and stem cell homeostasis, suggesting that stem cell decline during aging may be part of broader tumor-suppressive mechanisms.     

Immunohistochemical study of hair follicle stem cells in regenerated hair follicles induced by Wnt10b

The regulation of the periodic regeneration of hair follicles is complicated. Although Wnt10b has been reported to induce hair follicle regeneration, the characteristics of induced hair follicles, especially the target cells of Wnt10b, have not yet been clearly elucidated. Thus, we systematically evaluated the expression and proliferation patterns of Wnt10b-induced hair follicles. We found that Wnt10b promoted the proliferation of hair follicle stem cells from 24 hours after AdWnt10b injection. Seventy-two hours after AdWnt10b injection, cells outside of bulge area began to proliferate. When the induced hair follicle entered full anagen, although the hair follicle stem cells were normal, canonical Wnt signaling was maintained in the hair precortex cells. Our results reveal that the target cells that overexpressed Wnt10b included hair follicle stem cells, hair precortex cells, and matrix cells.     

Transient expression of keratin 19 is induced in originally negative interfollicular epidermal cells by adhesion of suspended cells

Keratin 19 and nuclear reactivity to an endogenous lectin, galectin-1, represent a potential marker of epidermal stem cells. We detected expression of keratin 19 and nuclear binding sites for galectin-1 in adult cells migrating from the hair follicle, where cells expressing keratin 19 are located in the bulge region. The results were compared with the expression of both markers in cells adhering from suspension prepared from the interfollicular epidermis without keratin-19-positive cells and with nuclear binding sites for galectin-1. The results were compared with data from basal cell carcinomas. All cells were analyzed concerning size, as it is known that cell diameter influences the clonogenic potential of keratinocytes. The major result of this study is the observation of transient expression of keratin 19 and nuclear galectin-1 binding sites in originally negative interfollicular epidermal cells induced by adhesion. These cells were very small in size, similar to basal cells of the interfollicular epidermis or the bulge region of the hair follicle. The influence of the suspension regimen on beta1-integrin expression, cell diameter and growth was also monitored. A population of cells highly positive for beta1 integrin of the same diameter as keratin-19-positive cells insensitive to induction of terminal differentiation by lack of anchorage was characterized. Cells of the same size were also observed in the keratin-19-positive cells of basal cell carcinomas. In conclusion, the expression of poor levels of differentiation induced by cell adhesion is transient. Also, keratin 19 expression should not be exclusively regarded as a marker of stem cell activity.     

Distinct epidermal stem cell compartments are maintained by independent niche microenvironments

The mammalian epidermis is a stratified, multilayered epithelium, consisting of the interfollicular epidermis and associated appendages, which extend into the dermis and include hair follicles, sebaceous glands, and sweat glands. Stem cells are essential for the maintenance of this tissue and are also potential sources of multipotent adult precursor cells. Stem cell populations occupying specific locations or niches have been identified in the interfollicular epidermis, the hair follicle and the sebaceous gland. Recent research has focused on how the stem cell niches provide specific sites where stem cells can reside indefinitely and undergo self-renewal or differentiation into specific cell lineages, as required for epidermal replenishment or hair follicle growth.     

Unveiling Hair Follicle Stem Cells

Adult mammalian skin consists of the epidermis, hair follicles (HFs), and sebaceous glands (SGs). Each of these three epithelial lineages contains its own stem cell (SC) population for normal tissue homeostasis, HF cycling, and repair of the epidermis following injury. Here, we provide an overview of the current knowledge on follicle SCs of the adult skin, including their essential features and, most importantly, the control of follicle SC fate. Wnt/β-catenin is required for follicle SC maintenance and niche biology, and β-catenin activation is essential for promoting quiescent follicle SCs to proliferate and terminally differentiate along the hair cell lineage. Further, β-catenin stabilization promotes de novo HF morphogenesis, and constitutively active β-catenin expression results in pilomatricoma. Both bone morphogenetic protein (BMP) and transforming growth factor-β (TGF-β) signals are required for quiescent niche maintenance: BMP deletion results in SC activation, whereas TGF-β may play a role in SC identity maintenance.     

Adenovirus-Mediated Wnt5a Expression Inhibits the Telogen-To-Anagen Transition of Hair Follicles in Mice

The canonical Wnt/β-catenin pathway plays an important role in hair cycle induction. Wnt5a is a non-canonical Wnt family member that generally antagonizes canonical Wnt signaling in other systems. In hair follicles, Wnt5a and canonical Wnt are both expressed in cells in the telogen stage. Wnt5a has been shown to be critical for controlling hair cell fate. However, the role that Wnt5a plays in the transition from the telogen to anagen stage is unknown. In this study, using whole-mount in situ hybridization, we show that Wnt5a is produced by several other cell types, excluding dermal papilla cells, throughout the hair cycle. For example, Wnt5a is expressed in bulge and secondary hair germ cells in the telogen stage. Our studies focused on the depilated 8-week-old mouse as a synchronized model of hair growth. Interestingly, overexpression of adenovirus Wnt5a in the dorsal skin of mice led to the elongation of the telogen stage and inhibition of the initiation of the anagen stage. However, following an extended period of time, four pelage hair types grew from hairless skin that was induced by Wnt5a, and the structure of these new hair shafts was normal. Using microarray analysis and quantitative arrays, we showed that the expression of β-catenin and some target genes of canonical Wnt signaling decreased after Wnt5a treatment. These data demonstrate that Wnt5a may inhibit the telogen stage to maintain a quiescent state of the hair follicle.