Polyclonal origin and hair induction ability of dermal papillae in neonatal and adult mouse back skin

Hair follicle development and growth are regulated by Wnt signalling and depend on interactions between epidermal cells and a population of fibroblasts at the base of the follicle, known as the dermal papilla (DP). DP cells have a distinct gene expression signature from non-DP dermal fibroblasts. However, their origins are largely unknown. By generating chimeric mice and performing skin reconstitution assays we show that, irrespective of whether DP form during development, are induced by epidermal Wnt activation in adult skin or assemble from disaggregated cells, they are polyclonal in origin. While fibroblast proliferation is necessary for hair follicle formation in skin reconstitution assays, mitotically inhibited cells readily contribute to DP. Although new hair follicles do not usually develop in adult skin, adult dermal fibroblasts are competent to contribute to DP during hair follicle neogenesis, irrespective of whether they originate from skin in the resting or growth phase of the hair cycle or skin with β-catenin-induced ectopic follicles. We propose that during skin reconstitution fibroblasts may be induced to become DP cells by interactions with hair follicle epidermal cells, rather than being derived from a distinct subpopulation of 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.     

NG2 proteoglycan expression in mouse skin: altered postnatal skin development in the NG2 null mouse.

In early postnatal mouse skin, the NG2 proteoglycan is expressed in the subcutis, the dermis, the outer root sheath of hair follicles, and the basal keratinocyte layer of the epidermis. With further development, NG2 is most prominently expressed by stem cells in the hair follicle bulge region, as also observed in adult human skin. During telogen and anagen phases of the adult hair cycle, NG2 is also found in stem cell populations that reside in dermal papillae and the outer root sheaths of hair follicles. Ablation of NG2 produces alterations in both the epidermis and subcutis layers of neonatal skin. Compared with wild type, the NG2 null epidermis does not achieve its full thickness due to reduced proliferation of basal keratinocytes that serve as the stem cell population in this layer. Thickening of the subcutis is also delayed in NG2 null skin due to deficiencies in the adipocyte population.     

Appearance of hair follicle-inducible mesenchymal cells in the rat embryo

Rat vibrissa follicle morphogenesis starts around 13 days of gestation. By day 14 mesenchymal cells have already aggregated as 'condensations' beneath the initial hair bud. Some of the mesenchymal cells will form a dermal papilla, having profound effects on hair follicle formation. The appearance of follicle-inducing mesenchymal cells in the process of vibrissa follicle development was examined. Mesenchymal cells were isolated from the developing site of vibrissa follicles at 13 days or at later stages and amplified in mass culture, harvested and transplanted in association with the epithelium. It was demonstrated that 13-day mesenchymal cells did not induce any hair bulbs but those from 14 days or later stages could induce hair-producing new bulbs or new follicles depending on the association with the follicle epithelium or with the glabrous sole epidermis of the adult rats, respectively. Further, clones having hair bulb-inducing ability were obtained from 14- and 15-day mass-cultured mesenchymal cells. Based on these and other results, it was concluded that mesenchymal cells having follicle-inducing ability are present at least by 14 days in the future whisker pad region. This suggests that the differentiation of the dermal papilla cells must start before the initial hair bud stage.     

The multipotency of adult vibrissa follicle stem cells

Several studies focused on the characterization of bulge keratinocytes have proved that they are multipotent stem cells, being recruited not only to regenerate the hair follicle itself, but also the sebaceous gland and the epidermis. However, due to the difficulty in preparing transplantable cell sheets harvested with conventional enzymatic digestion, there is still no direct evidence of the bulge stem cells’ multipotency. Whether they can respond to adult dermal papilla (DP) signals in recombination experiments also remains unclear. In this study, we addressed this problem by culturing and detaching intact bulge keratinocyte sheets from thermo-responsive culture dishes, only by reducing its temperature. When sheets of mass cultured bulge keratinocytes isolated from rat vibrissa follicles were recombined with fresh adult DPs and sole skin dermis in vivo, regeneration of epidermis and sebaceous gland-like structures, and formation of hair bulb with differentiating inner root sheath and hair cuticle were observed within 3 weeks. However, regardless the expression of stem cells markers like CD34, SA1004 and SA1006, no structures were observed when cloned bulge keratinocytes were used to prepare cell sheets and recombinants, revealing the possible existence of monoclonal stem cells within the bulge region. This report is the first to succeed in harvesting adult bulge keratinocyte sheets. Using these sheets it is demonstrated that bulge stem cells directly respond to adult DP signals to induce hair bulb formation in vivo.     

Cultured dermal papilla cells induce follicle formation and hair growth by transdifferentiation of an adult epidermis.

Adult rat pelage follicle dermal papilla cells induced follicle neogenesis and external hair growth when associated with adult footpad skin epidermis. They thus demonstrated a capacity to completely change the structural arrangement and gene expression of adult epidermis--an ability previously undocumented for cultured adult cells. Isolation chambers ensured that de novo follicle formation must have occurred by eliminating the possibility of cellular contributions, and/or inductive influences, from local skin follicles. These findings argue against previous suggestions of vibrissa follicle specificity, and imply that the potential for hair follicle induction may be common to all adult papilla cells.     

Induction of follicle formation and hair growth by vibrissa dermal papillae implanted into rat ear wounds: vibrissa-type fibres are specified.

Adult vibrissa follicle dermal papillae have the capacity to induce hair growth and follicle formation when associated with epidermis from various sources. However, the range of conditions under which hair follicle induction will take place has not been established. The question of whether or not the adult papilla carries information to impose fibre-type specificity has also not been fully answered. This study describes how the implantation of isolated papillae into small incisional cuts on the rat ear pinna resulted in the subsequent emergence of abnormally large hair fibres from the wound sites. Many of these hairs were found to display vibrissa-type characteristics. Histological observations indicated that the papillae had interacted with the edges of the wound epidermis to produce new, and particularly large follicles, while immunohistochemical staining revealed that early follicle construction was accompanied by a profusion of the basement membrane constituents laminin and type IV collagen in the subjacent dermis. These findings show that adult rat papillae retain the capacity, as displayed by embryonic dermis, to determine vibrissa specificity in induced follicles.     

Dynamic regulation of retinoic acid-binding proteins in developing, adult and neoplastic skin reveals roles for beta-catenin and Notch signalling

Retinoic acid (RA) signalling is essential for epidermal differentiation; however, the mechanisms by which it acts are largely unexplored. Partitioning of RA between different nuclear receptors is regulated by RA-binding proteins. We show that cellular RA-binding proteins CRABP1 and CRABP2 and the fatty acid-binding protein FABP5 are dynamically expressed during skin development and in adult tissue. CRABP1 is expressed in embryonic dermis and in the stroma of skin tumours, but confined to the hair follicle dermal papilla in normal postnatal skin. CRABP2 and FABP5 are expressed in the differentiating cells of sebaceous gland, interfollicular epidermis and hair follicles, with FABP5 being a prominent marker of sebaceous glands and anagen follicle bulbs. All three proteins are upregulated in response to RA treatment or Notch activation and are negatively regulated by Wnt/β-catenin signalling. Ectopic follicles induced by β-catenin arise from areas of the sebaceous gland that have lost CRABP2 and FABP5; conversely, inhibition of hair follicle formation by N-terminally truncated Lef1 results in upregulation of CRABP2 and FABP5. Our findings demonstrate that there is dynamic regulation of RA signalling in different regions of the skin and provide evidence for interactions between the RA, β-catenin and Notch pathways.     

Reprogramming adult dermis to a neonatal state through epidermal activation of β-catenin

Hair follicle formation depends on reciprocal epidermal-dermal interactions and occurs during skin development, but not in adult life. This suggests that the properties of dermal fibroblasts change during postnatal development. To examine this, we used a PdgfraEGFP mouse line to isolate GFP-positive fibroblasts from neonatal skin, adult telogen and anagen skin and adult skin in which ectopic hair follicles had been induced by transgenic epidermal activation of β-catenin (EF skin). We also isolated epidermal cells from each mouse. The gene expression profile of EF epidermis was most similar to that of anagen epidermis, consistent with activation of β-catenin signalling. By contrast, adult dermis with ectopic hair follicles more closely resembled neonatal dermis than adult telogen or anagen dermis. In particular, genes associated with mitosis were upregulated and extracellular matrix-associated genes were downregulated in neonatal and EF fibroblasts. We confirmed that sustained epidermal β-catenin activation stimulated fibroblasts to proliferate to reach the high cell density of neonatal skin. In addition, the extracellular matrix was comprehensively remodelled, with mature collagen being replaced by collagen subtypes normally present only in developing skin. The changes in proliferation and extracellular matrix composition originated from a specific subpopulation of fibroblasts located beneath the sebaceous gland. Our results show that adult dermis is an unexpectedly plastic tissue that can be reprogrammed to acquire the molecular, cellular and structural characteristics of neonatal dermis in response to cues from the overlying epidermis.     

小鼠皮肤及其毛囊早期发育的组织学观察

目的探讨小鼠皮肤及其毛囊的早期发育规律。方法采用常规石蜡切片和H-E染色技术,观察昆明系小鼠出生前后皮肤及其毛囊的形态发育。结果(1)孕龄16 d胎鼠的皮肤表面形成凹凸不平的深褶皱,但在生后3 d~5 d不仅皱褶的数量减少,而且凹陷变浅;(2)胎鼠孕龄16 d至19 d,其皮肤的表皮、真皮及皮肤总厚度呈现平稳增厚。但是,出生后,其表皮、真皮和皮肤总厚度急剧降低;在生后第1天至第9天,表皮呈现平稳增厚,而真皮则在生后快速厚度,第7天达到最高值(1861.50μm);(3)孕龄16 d的胎鼠皮肤中可观察到初级毛囊,至生后第7天其密度呈现平稳增长;与其相比,次级毛囊从18 d胎鼠开始出现,其密度增长非常迅速,出生后第7天达到1257.14/mm;毛囊的总密度与次级毛囊呈现相似的变化趋势。出生第7天后,由于毛囊的数量急剧增加,无法观察初级毛囊和次级毛囊的变化规律;(4)初级毛囊和次级毛囊的长度与深度变化在出生前后的相对缓慢,与其相比在第3天以后至第7天呈现迅速变化趋势。结论小鼠皮肤及其毛囊的生长性发育发生在胎儿晚期和生后的早期,而其周期性变化可能从出生后的第9天以后开始出现;在孕期16 d至生后第7天可能是检测毛囊特异性基因表达的最佳期。     

Transformation of amnion epithelium into skin and hair follicles

There is increasing interest into the extent to which epithelial differentiation can be altered by mesenchymal influence, and the molecular basis for these changes. In this study, we investigated whether amnion epithelium could be transformed into skin and hair follicles by associating E12.5 to E14.5 mouse amnion from the ROSA 26 strain, with mouse embryonic hair-forming dermis from a wild-type strain. These associations were able to produce fully formed hair follicles with associated sebaceous glands, and skin epidermis. Using beta-galactosidase staining we were able to demonstrate that the follicular epithelium and skin epidermis, but not the associated dermal cells, originated from the amnion. As Noggin and Sonic hedgehog (Shh) were recently shown to be required for early chick ventral skin formation, and able to trigger skin and feather formation from chick amnion, we associated cells engineered to produce those two factors with mouse amnion. In a few cases, we obtained hair buds connected to a pluristratified epithelium; however, the transformation of the amnion was impeded by uncontrolled fibroblastic proliferation. In contrast to an earlier report, none of our control amnion specimens autonomously transformed into skin and hair follicles, indicating that specific influences are necessary to elicit follicle formation from the mouse amnion. The ability to turn amnion into skin and its appendages has practical potential for the tissue engineering of replacement skin, and related biotechnological approaches.     

Essential roles of BMPR-IA signaling in differentiation and growth of hair follicles and in skin tumorigenesis

Hair differentiation and growth are controlled by complex reciprocal signaling between epithelial and mesenchymal cells. To better understand the requirement and molecular mechanism of BMP signaling in hair follicle development, we performed genetic analyses of bone morphogenetic protein receptor 1A (BMPR-IA) function during hair follicle development by using a conditional knockout approach. The conditional mutation of Bmpr1a in ventral limb ectoderm and its derivatives (epidermis and hair follicles) resulted in a lack of hair outgrowth from the affected skin regions. Mutant hair follicles exhibited abnormal morphology and lacked hair formation and pigment deposition during anagen. The timing of the hair cycle and the proliferation of hair matrix cells were also affected in the mutant follicles. We demonstrate that signaling via epithelial BMPR-IA is required for differentiation of both hair shaft and inner root sheath from hair matrix precursor cells in anagen hair follicles but is dispensable for embryonic hair follicle induction. Surprisingly, aberrant de novo hair follicle morphogenesis together with hair matrix cell hyperplasia was observed in the absence of BMPR-IA signaling within the affected skin of adult mutants. They developed hair follicle tumors from 3 months of age, indicating that inactivation of epidermal BMPR-IA signaling can lead to hair tumor formation. Taken together, our data provide genetic evidence that BMPR-IA signaling plays critical and multiple roles in controlling cell fate decisions or maintenance, proliferation, and differentiation during hair morphogenesis and growth, and implicate Bmpr1a as a tumor suppressor in skin tumorigenesis.     

Derivation of Hair-Inducing Cell from Human Pluripotent Stem Cells

Dermal Papillae (DP) is a unique population of mesenchymal cells that was shown to regulate hair follicle formation and growth cycle. During development most DP cells are derived from mesoderm, however, functionally equivalent DP cells of cephalic hairs originate from Neural Crest (NC). Here we directed human embryonic stem cells (hESCs) to generate first NC cells and then hair-inducing DP-like cells in culture. We showed that hESC-derived DP-like cells (hESC-DPs) express markers typically found in adult human DP cells (e.g. p-75, nestin, versican, SMA, alkaline phosphatase) and are able to induce hair follicle formation when transplanted under the skin of immunodeficient NUDE mice. Engineered to express GFP, hESC-derived DP-like cells incorporate into DP of newly formed hair follicles and express appropriate markers. We demonstrated that BMP signaling is critical for hESC-DP derivation since BMP inhibitor dorsomorphin completely eliminated hair-inducing activity from hESC-DP cultures. DP cells were proposed as the cell-based treatment for hair loss diseases. Unfortunately human DP cells are not suitable for this purpose because they cannot be obtained in necessary amounts and rapidly loose their ability to induce hair follicle formation when cultured. In this context derivation of functional hESC-DP cells capable of inducing a robust hair growth for the first time shown here can become an important finding for the biomedical science.     

Enrichment of epidermal stem cells by rapid adherence and analysis of the reciprocal interaction of epidermal stem cells with neighboring cells using an organotypic system

Keratinocytes have the ability to adhere to extracellular matrix rapidly. With this in mind, in this study we isolated keratinocytes known as rapidly adhering (RA) cells. To compare epidermal regenerative abilities, skin substitutes were reconstructed by adding keratinocytes or RA cells to two groups of bioengineered dermis made by fibroblasts and hair follicle dermal cells respectively. After transplantation, the results illustrated that the skin substitutes including RA cells were integrated into the host tissue. Furthermore, with hair follicle dermal cells' influences, the RA cells could form structures very similar to normal hair follicles. These results indicate that RA cells are predominately comprised of epidermal stem cells. The results also demonstrated that besides the reciprocal interaction of epidermal stem cells with dermal cells, the interaction of epidermal stem cells with keratinocytes were critical in epidermis morphogenesis and self-renewal, and application of RA cells could optimize engineering of skin substitutes.     

Role of Sonic hedgehog signaling in epithelial and mesenchymal development of hair follicles in an organ culture of embryonic mouse skin

Studies with gene knockout mice have shown that Sonic hedgehog (Shh) is required for early development of hair follicles, but the role of this gene in the late stages of follicle development is not clear. By using an organ culture system of embryonic mouse skin, the role of Shh signaling in the early and late stages of follicle development was investigated. In the early stage of follicle development, the downward growth of the follicular epithelium was suppressed by cyclopamine, an inhibitor of Shh signaling, and accelerated by recombinant Shh. In addition, cyclopamine impaired dermal papilla formation, accompanied by the rearrangement of papilla cells, but not the elongation of the follicular epithelium at the later stage. These results suggest that Shh signaling is required for the proliferation of epithelial cells in the early development of hair follicles and for the morphogenetic movement of mesenchymal cells at the later stage of follicle development.