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Abstract
Dermal papilla cells (DPCs) are specialized fibroblasts found in the hair follicle papilla, which are associated with the development and cycle regulation of hair follicles (HFs). DPCs exhibit a multilayer aggregative growth character, which is closely related to induction of HF formation. Versican, a large chondroitin sulphate proteoglycan and one of the major components of the extracellular matrix, is involved in the formation of HF.
To confirm the relationship between versican and the aggregative growth of DPCs, we first induced and established an aggregative cell model in DPCs in vitro, with cells taken to passage 8. Simultaneously, aggregative passage 2 DPCs and nonaggregative passage 8 DPCs were selected as parallel controls. RNA interference (RNAi) targeted to versican was used in passage 2 DPCs using a lentiviral vector. Reverse transcriptase (RT)-PCR and western blotting were used to assay the expression of versican in DPCs.
RNAi targeted to versican efficiently suppressed the aggregative growth of passage 2 DPCs, and the inhibitory effect was significant 3 days after RNAi treatment. The mRNA and protein levels of versican were also downregulated in passage 2 DPCs, and were lower than levels in nonaggregative passage 8 DPCs. Notably, the aggregative growth of nonaggregative passage 8 DPCs was restored after induction in a 1 : 1 v/v mixture of fresh DMEM and medium recycled from a previous passage.
Versican is a key gene for the aggregative growth of DPCs, and might be significant in the regeneration of HF.
... Characterization of protein and gene expression of signature genes in DP spheroids RT-PCR showed that the expression of signature genes, which is associated with HF inductivity of DP cells, 6,7,10,17,29,30 was preserved in DP spheroids (Fig. 4). Compared with adherent growth P8 DP cells (negative control), NCAM, Versican, and a-SMA were significantly upregulated during cultivation in sphere-forming conditions for P8 DP cells (Fig. 4). ...
... inductive capabilities. 6,7 Studies have been presented that detail the maintenance of Versican expression in vitro, 29,30 thought to be a way of maintaining the antigen characteristics and inductive capabilities of DP cells. 30 As in previous studies, 38,39 we also showed that P8 DP cells can restore expression of DP markers after sphere formation. ...
... 6,7 Studies have been presented that detail the maintenance of Versican expression in vitro, 29,30 thought to be a way of maintaining the antigen characteristics and inductive capabilities of DP cells. 30 As in previous studies, 38,39 we also showed that P8 DP cells can restore expression of DP markers after sphere formation. Although the expression of these markers was regarded as an indicator of DP cells in vitro, the hair-inducing ability of DP cells was usually verified by an in vivo experiment. ...
We have succeeded in culturing human dermal papilla (DP) cells spheroids and developed a three-dimensional Matrigel (basement membrane matrix) culture technique that can enhance and restores DP cells unique characteristics in vitro. When 10000 DP cells were cultured on the 96 well plates pre-coated with Matrigel for 5 days, both passage 2 and passage 8 DP cells formed spheroidal microtissues with a diameter of 150-250 μm in an aggregative and proliferative manner. We transferred and re-cultured these DP spheroids onto commercial plates. Cells within DP spheres could disaggregate and migrate out, which was similar to primary DP. Moreover, we examined the expression of several genes and proteins associated with hair follicle inductivity of DP cells, such as NCAM, Versican and α-SMA, and confirmed that their expression level was elevated in the spheres compared with the dissociated DP cells. To examine hair-inducing ability of DP spheres, hair germinal matrix cells and DP spheres were mixed and cultured on Matrigel. Unlike the dissociated DP cells and hair germinal matrix cells co-cultured in two dimensions, hair germinal matrix cells can differentiate into hair-like fibers under the induction of the DP spheres made from the high passage cells (passage 8) in vitro. We are the first to show that passage 3 human hair germinal matrix cells differentiate into hair-like fiber in the presence of human DP spheroids. These results suggest that three-dimensional Matrigel culture technique is an ideal culture model for forming DP spheroids and that sphere formation partially models the intact DP, resulting in hair induction, even by high passage DP cells.
... The agglutinative growth of DPCs in this manuscript is defined as DPCs gather in clusters when growing. A number of reports have shown that following a fifth passage, hDPCs in culture lose both their ability to coagulate as well as their hair inductivity (11,12). Furthermore, the expression of marker proteins including alkaline phosphatase (ALP) (13), and SOX-2 (14) are also decreased in hDPCs. ...
... that VCAN (chondroitin sulfate proteoglycan matrix core protein) is essential component in the processes of mesenchymal condensation and induction of hair growth (12,15). ...
The dermal papilla (DP) cells in hair follicles function as critical regulators of hair growth. In particular, Alopecia Areata (AA) is closely related to the malfunctioning of the human dermal papilla cells(hDPCs). Thus, identifying the regulatory mechanism of hDPCs is important in inducing hair follicle (HFs) regeneration in AA patients. Recently, growing evidence indicated that 3’ untranslated regions (3’UTR) of key genes may participate in the regulatory circuitry underlying cell differentiation and diseases though a so-called competing endogenous mechanism, but none has been reported in HFs regeneration. Here, we demonstrate that the 3’UTR of JAM-A could act as an essential competing endogenous RNA to maintain hDPCs function and promote HFs regeneration in AA. We showed that the 3’ UTR of junctional adhesion molecule A(JAM-A) shares many microRNAs(miRNAs) response elements, especially miR-221–3p, with versican(VCAN) mRNA, and JAM-A 3’UTR could directly modulate the miRNA-mediated suppression of VCAN in self-renewing hDPCs. Furthermore, upregulated VCAN can in turn promote the expression level of JAM-A. Overall, we propose that JAM-A 3’UTR forms a feedback loop with VCAN and miR-221–3p to regulate hDPCs maintenance, proliferation, and differentiation, which may lead to developing new therapies for hair loss.
... (3) Certain types of proteoglycans act directly as potent anagen inducers to the extent that their local injection leads to hair growth induction and prolongation [13,24]. (4) Gene silencing of a specific proteoglycan, versican, results in the loss of aggregative growth in DP cells and a significant decline in their proliferative ability [34], whereas its forced expression restores the hair inducibility of DP cells [14]. ...
... A more specific study has revealed that either enhanced endogenous expression or treatment with exogenous recombinant versican significantly stimulates fibroblast cell proliferation through the EGF-like motif of its G3 domain [120]. Later, this finding has been confirmed by showing that the versican level increases in aggregative DP cells, and its suppression by RNA interference can lead to a considerable loss of aggregative growth and proliferative ability of DP cells [34]. Further research has revealed that versican is the specific mediator of Wnt/ β-catenin pathway in regulating the aggregative growth of DP cells [38]. ...
Follicular proteoglycans are key players with structural, functional, and regulatory roles in the growth and cycling behaviour of the hair follicles. The expression pattern of specific proteoglycans is strongly correlated with follicular phase transitions, which further affirms their functional involvement. Research shows that bioactive proteoglycans, e.g., versican and decorin, can actively trigger follicular phase shift by their anagen-inducing, anagen-maintaining, and immunoregulatory properties. This emerging insight has led to the recognition of “dysregulated proteoglycan metabolism” as a plausible causal or mediating pathology in hair growth disorders in both men and women. In support of this, declined expression of proteoglycans has been reported in cases of anagen shortening and follicular miniaturisation. To facilitate scientific communication, we propose designating this pathology “follicular hypoglycania (FHG),” which results from an impaired ability of follicular cells to replenish and maintain a minimum relative concentration of key proteoglycans during anagen. Lasting FHG may advance to structural decay, called proteoglycan follicular atrophy (PFA). This process is suggested to be an integral pathogenetic factor in pattern hair loss (PHL) and telogen effluvium (TE). To address FHG and PFA, a proteoglycan replacement therapy (PRT) program using oral administration of a marine-derived extract (Nourkrin® with Marilex®, produced by Pharma Medico Aps, Aarhus, Denmark) containing specific proteoglycans has been developed. In clinical studies, this treatment significantly reduced hair fall, promoted hair growth, and improved quality of life in patients with male- and female-pattern hair loss. Accordingly, PRT (using Nourkrin® with Marilex®) can be recommended as an add-on treatment or monotherapy in patients with PHL and TE.
... 64,65 Dermal papillae cells isolated from Vcan promoter driven-GFP mice were applied to a spheroid culture, and a half-diluted combination of adipogenic and osteogenic medium together with FGF2 and PDGF-AA promoted expression of dermal papillae-specific gene expression. 66 Although several researchers used Vcan expression as a dermal papillae cell marker, 67 68 HA is unlikely to be involved, as it is not colocalized in dermal papillae where Vcan is present. 63 Whereas Vcan is used as a marker for dermal papilla cells, the role of Vcan in the growth and maintenance of hair follicles remains to be understood. ...
Versican is a large chondroitin sulfate/dermatan sulfate proteoglycan in the extracellular matrix and one of the aggrecan/lectican family. Whereas versican is constitutively expressed and serves as a structural macromolecule in some tissues, it is transiently expressed at high levels when the extracellular matrix dynamically changes. There, versican plays an important role in forming the provisional matrix, which is replaced with the “authentic” extracellular matrix, that is, the matrix as it should be. ADAMTS‐1, 4, 5, 9, 15, and 20 cleave versican core protein and are therefore named versicanases. These proteinases have been believed to play a critical role in versican turnover. A cleaved N‐terminal fragment harbors biological functions, and it is termed “versikine.” This review discusses recent advances in the research on the in vivo function of versican and versikine generated by versicanases.
... CD133 þ DPCs are able to secret Wnt ligands and regulate the interaction between the mesenchyme and the epithelial to further promote HF growth and regeneration [54]. The HF regeneration is also highly dependent on DPC aggregation growth, which was mainly composed of versican þ DPCs [55]. Other functional molecules that involved in the positive regulation of DPC HF-inducive capacity also include but not limited to Wnt/b-catenin, SHH/platelet-derived growth factor-A (PDGF-A), fibroblast growth factor (FGF), BMP, TGF-b, and Edar/nuclear factor-kappa B (NF-kB) signaling pathways [56e58]. ...
Alopecia is a common and distressing medical condition that has affected a majority of people worldwide, which leads to great effects on the quality of life and self-esteem. Numerous treatments had been used to cure alopecia, including hair growth stimulants, herbal products, and hair transplantation. However, these treatments have their side effects, such as hypertrichosis, edema, and even cardiovascular adverse effects, which lead to the urgent requirement to explore a new hair-follicle (HF) regeneration approach. Tissue engineering could be the potential way for HF regeneration by simulating the epithelial-mesenchymal interaction and cell-extracellular matrix interactions. This review summarized the potential cells that are used in tissue engineering, commonly used tissue engineering techniques, and most importantly, the biomaterials that have been applied for in vitro three-dimensional cell culture or in vivo co-transplantation in HF regeneration. The literature shows that advances in this field toward functional HF development have progressively increased. Although the clinical application of biomaterial co-transplantation for HF regeneration still faces various challenges, numerous studies have proved that this is a promising direction that could be achieved in the future.
... CD133 + DPCs have been shown to be a specific subpopulation of cells in DPCs, and they can produce Wnt ligands and mediate signalling crosstalk between the mesenchyme and the epithelial compartment, further promoting adult HF growth and regeneration. 21 In addition, Versican + DPCs exhibit the typical characteristics of aggregation growth, 22 on which HF formation is highly dependent. 23 In addition, many functional molecules are involved in the positive regulation of DPC HF-inducive capacity (Fig. 4), such as endothelin-1 and stem cell growth factor, 24 insulin-like growth factor-1 (IGF-1) 25 and histidine decarboxylase, 26 but matricellular protein connective tissue growth factor (CCN2) negatively regulates HF regeneration, physiologically curbing HF formation by the destabilization of β-catenin. ...
The hair follicle (HF) is a highly conserved sensory organ associated with the immune response against pathogens, thermoregulation, sebum production, angiogenesis, neurogenesis and wound healing. Although recent advances in lineage-tracing techniques and the ability to profile gene expression in small populations of cells have increased the understanding of how stem cells operate during hair growth and regeneration, the construction of functional follicles with cycling activity is still a great challenge for the hair research field and for translational and clinical applications. Given that hair formation and cycling rely on tightly coordinated epithelial-mesenchymal interactions, we thus review potential cell sources with HF-inducive capacities and summarize current bioengineering strategies for HF regeneration with functional restoration.
... Compared to the negative control, versican was expressed more in the OSAtreated DPCs and, particularly, in the RBM-treated DPCs. Versican is a hair follicle formation related proteoglycan [35] found in the ECM [36,37]. According to Diana et al, versican is a widespread ECM component of rodent and human skin, with a unique pattern of distribution in murine hair follicle morphogenesis and cycling. ...
The objective of this study is to evaluate of rice bran mineral extract (RBM) increases the expression of anagen-related molecules in human dermal papilla (DOCs). Four treatment groups were established to evaluate the efficacy of RBM, including a negative control, positive control (ascorbic acid), RBM and ortho-silicic acid (Si(OH)4) (OSA) group. Three days after the DPCs were administered the various treatments, western blot analysis showed that type I collagen expression was increased 2.5-fold in the OSA group and 4-fold in the RBM group, and ALP expression was increased 1.5-fold in the OSA and RBM group while the expression of fibronectin was increased ~3-fold in the OSA group and 2.5-fold in the RBM group. Also, the expression of Wnt-3α and β-catenin protein was increased in OSA and RBM group compared to control group. Furthermore, the expression of IL-1a was decreased by more than 50% in the OSA and RBM groups compared to the negative control. Analysis of mRNA expression by RT-qPCR showed that type I collagen increased 1.2-fold in the OSA- and RBM-treated DPCs, whereas type IV collagen increased 2.7-fold in the OSA group and 3.5-fold in the RBM group. However, TGF-β2 mRNA decreased about 80% in the OSA and RBM groups, respectively. Immunohistochemical staining of the DPCs for versican protein showed a significant increase in the OSA- and RBM-treated groups compared to the negative control. Thus, RBM have a potential to recover of DPCs activity and decreased inflammatory-related markers. It can be expected that hair loss prevention and hair growth enhancement can be expected when RBM is applied as a cosmetic product.
... Besides hair-inducing abilities, the key feature of DP cells is a tendency to aggregate in culture reproducing the initial steps of HF formation. This process depends on ECM components, especially DP-specific protein versican [15]. Human HF development has not been well investigated, but basing on evidences obtained from rodent research, soluble factors mentioned above and other molecules are involved in the process of DP cell condensation. ...
Hair follicle (HF) reconstruction in vitro is a promising field in alopecia treatment and human HF development research. Here, we combined postnatal human dermal papilla (DP) cells and skin epidermal keratinocytes (KCs) in a hanging drop culture to develop an artificial HF germ. The method is based on DP cell hair-inducing properties and KC self-organization. We evaluated two protocols of aggregate assembling. Mixed HF germ-like structures demonstrated the initiation of epithelial-mesenchymal interaction, including WNT pathway activation and expression of follicular markers. We analyzed the influence of possible DP cell niche components including soluble factors and extracellular matrix (ECM) molecules in the process of the organoid assembling and growth. Our results demonstrated that soluble factors had little impact on HF germ generation and Ki67 ⁺ cell score inside the organoids although BMP6 and VD3 maintained effectively the DP identity in the monolayer culture. Aggrecan, biglycan, fibronectin, and hyaluronic acid (HA) significantly stimulated cell proliferation in DP cell monolayer culture without any effect on DP cell identity. Most of ECM compounds prevented the formation of cell aggregates while HA promoted the formation of larger organoids. In conclusion, our model could be suitable to study cell-cell and cell-niche interactions during HF reconstruction in vitro .
... Many lines of evidence show that the aggregation growth of DPC is associated with versican, an extracellular matrix proteoglycan. Feng et al. reported that both mRNA and protein levels of versican decline along with a decrease in aggregative growth of high-passage DPCs, and RNA interference targeting versican suppressed DPC aggregation 34 . Furthermore, versican is found to be specifically expressed in DPCs during hair anagen, but downregulated during catagen and absent during telogen 35 . ...
Dermal papilla cells (DPCs) are important components of hair follicles and play a critical role in hair follicle development. However, the mechanisms by which DPCs induce hair follicle development remain unclear. In the present study, we identified the mitotic arrest deficient protein MAD2B as a modifier of DPCs. Overexpression of MAD2B inhibited DPC aggregative growth and proliferation induced by the Wnt signaling activator T cell factor 4 (TCF4), and decreased TCF4-induced expression and the release of hair growth-related cytokines, including hepatocyte growth factor, insulin-like growth factor-1, and vascular endothelial growth factor in DPCs. In contrast, knockdown of MAD2B promoted TCF4-induced DPC proliferation, but did not affect the expression and secretion of cytokines by TCF4-induced DPCs. These results suggest a functional antagonism between MAD2B and TCF4 in DPC-induced hair follicle development. Mechanistically, MAD2B physically interacted with TCF4 to repress TCF4 transcriptional activity via β-catenin mediation, leading to reduced β-catenin/TCF4-dependent transactivation and Wnt signaling activity. These results demonstrate, for the first time, that MAD2B plays a negative role in TCF4-induced DPC growth and proliferation.
... The Vcan Δ3/Δ3 dermal papilla showed decrease in versican deposition ( Figure 1F) as well as the number of hair follicles (data not shown). Similar results were observed in a previous in vitro study, wherein RNAi targeted to versican efficiently suppressed the aggregative growth of dermal papilla cells in the hair follicle (34). Versican was detected in WT dermal papilla at a high level as well as in Vcan Δ3/Δ3 dermal papilla that lacks HA deposition ( Figure 3C and D). ...
Versican, a large chondroitin sulfate proteoglycan, serves as a structural macromolecule of the extracellular matrix and regulates cell behavior. We determined the function of versican in dermal development using VcanΔ3/Δ3 mutant mice expressing versican with deleted A-subdomain of the N-terminal G1 domain. The mutant versican showed a decreased hyaluronan-binding ability and failed to accumulate in the extracellular matrix. In the early developmental stage, VcanΔ3/Δ3 dermis showed a decrease in versican expression as compared with WT. As development proceeded, versican expression further decreased to a barely detectable level, and VcanΔ3/Δ3 mice died at the neonatal period (P0). At P0, VcanΔ3/Δ3 dermis exhibited impaired the extracellular matrix structure structure and decreased cell density. While the level of collagen deposition was similar in both genotypes, collagen biosynthesis significantly decreased in VcanΔ3/Δ3 fibroblasts as compared with that in wild type. Transforming growth factor β (TGFβ) signaling mediated through the Smad2/3-dependent pathway was down-regulated in VcanΔ3/Δ3 fibroblasts and a reduced TGFβ storage in the extracellular matrix was observed. Microarray analysis revealed a decrease in the expression levels of transcription factors, early growth response (Egr) 2 and 4, which act downstream of TGFβ signaling. Thus, our results suggest that A-subdomain is necessary for adequate versican expression in dermis and that versican is involved in the formation of the extracellular matrix and regulation of TGFβ signaling.
... [7] Decreased versican expression is correlated with increased passage number of cultured human DP cells and the reduction of both aggregative ability in vitro and hair induction in vivo. [26] In three strains of ovine DP cells, this study confirmed that versican expression is specific for aggregates, and it is not expressed in the cells which were still in a monolayer state [4] [ Figure 2]. ...
Context:
The dermal papilla (DP) is a condensation of mesenchymal cells at the proximal end of the hair follicle, which determines hair shaft size and regulates matrix cell proliferation and differentiation. DP cells have the ability to regenerate new hair follicles. These cells tend to aggregate both in vitro and in vivo. This tendency is associated with the ability of papilla cells to induce hair growth. However, human papilla cells lose their hair-inducing activity in later passage number. Ovine DP cells are different from human DP cells since they do not lose their aggregative behavior or hair-inducing activity in culture. Nonetheless, our understanding of ovine DP cells is still limited.
Aim:
The aim of this study was to observe the expression of established DP markers in ovine cells and their association with aggregation.
Subjects and methods:
Ovine DP cells from three different sheep were compared. Histochemistry, immunoflourescence, and polymerase chain reaction experiments were done to analyze the DP markers.
Results:
We found that ovine DP aggregates expressed all the 16 markers evaluated, including alkaline phosphatase and versican. Expression of the versican V0 and V3 isoforms, neural cell adhesion molecule, and corin was increased significantly with aggregation, while hey-1 expression was significantly decreased.
Conclusions:
Overall, the stable expression of numerous markers suggests that aggregating ovine DP cells have a similar phenotype to papillae in vivo. The stability of their molecular phenotype is consistent with their robust aggregative behavior and retained follicle-inducing activity after prolonged culture. Their phenotypic stability in culture contrasts with DP cells from other species, and suggests that a better understanding of ovine DP cells might provide opportunities to improve the hair-inducing activity and therapeutic potential of human cells.
... Interestingly, targeted RNAi against versican resulted in less aggregative behavior in dermal papilla fibroblasts associated with hair follicles. 124 Although these data are of unclear importance for wound healing, they point toward a further investigation into the effect of versican on fibrosis. ...
Significance: Fibroblasts play a critical role in normal wound healing. Various extracellular matrix (ECM) components, including collagens, fibrin, fibronectin, proteoglycans, glycosaminoglycans, and matricellular proteins, can be considered potent protagonists of fibroblast survival, migration, and metabolism. Recent Advances: Advances in tissue culture, tissue engineering, and ex vivo models have made the examination and precise measurements of ECM components in wound healing possible. Likewise, the development of specific transgenic animal models has created the opportunity to characterize the role of various ECM molecules in healing wounds. In addition, the recent characterization of new ECM molecules, including matricellular proteins, dermatopontin, and FACIT collagens (Fibril-Associated Collagens with Interrupted Triple helices), further demonstrates our cursory knowledge of the ECM in coordinated wound healing. Critical Issues: The manipulation and augmentation of ECM components in the healing wound is emerging in patient care, as demonstrated by the use of acellular dermal matrices, tissue scaffolds, and wound dressings or topical products bearing ECM proteins such as collagen, hyaluronan (HA), or elastin. Once thought of as neutral structural proteins, these molecules are now known to directly influence many aspects of cellular wound healing. Future Directions: The role that ECM molecules, such as CCN2, osteopontin, and secreted protein, acidic and rich in cysteine, play in signaling homing of fibroblast progenitor cells to sites of injury invites future research as we continue investigating the heterotopic origin of certain populations of fibroblasts in a healing wound. Likewise, research into differently sized fragments of the same polymeric ECM molecule is warranted as we learn that fragments of molecules such as HA and tenascin-C can have opposing effects on dermal fibroblasts.
... The size and shape of hair shafts were not noticeably different in vehicle-treated versus propolistreated HFs. Versican, which is a marker protein for functional dermal papilla cells, 36 was also distributed normally in propolistreated HFs. These results indicate that propolis facilitates normal hair growth. ...
Propolis is a natural honeybee hive product with the potential for use in the treatment of dermatological conditions, such as cutaneous abrasions, burns, and acne. In this study, we investigated whether propolis stimulates hair growth in mice. Ethanol-extracted propolis, which contains various physiologically active substances such as caffeic acid and kaempferol, stimulated anagen induction in the shaved back skin. Anagen induction occurred without any detectable abnormalities in the shape of the hair follicles (HFs), hair stem cells in the bulge, proliferating hair matrix keratinocytes in the hair bulb, or in the localization of versican in the dermal papilla. Propolis treatment also stimulated migration of hair matrix keratinocytes into the hair shaft in HFs during late anagen in the depilated back skin. Organotypic culture of skin containing anagen stage HFs revealed significant stimulation of hair matrix keratinocyte proliferation by propolis. Furthermore, propolis facilitated the proliferation of epidermal keratinocytes. These results indicate that propolis stimulates hair growth by inducing hair keratinocyte proliferation.
... Another property of DP cells is the co-expression of SCF (stem cell factor), ET-1 (endothelin-1) and bFGF (basic fibroblast growth factor) (Lu et al., 2006). Versican secretion by DP is implicated in the induction of hair morphogenesis and the initiation of hair regeneration (Kishimoto et al., 1999;Feng et al., 2011) also play important roles in the maintenance of hair growth in mouse species (Soma et al., 2005). A previous report has demonstrated that CD133 is a novel surface marker useful for collecting DP cells (Ito et al., 2007). ...
DP (dermal papilla) is a mesenchyme-derived structure situated at the base of the HF (hair follicle) that plays an important role in embryonic hair morphogenesis and maintenance of the hair growth cycle. hMSCs (human mesenchymal stem cells) have gained widespread attention in the field of tissue engineering, but not much is known about the differentiation of hMSCs into DP cells. hMSCs involved in HF formation were examined in our previous study. Here, we have explored the differentiation potential of hMSCs into DP cells by co-culturing hMSCs with DP cells, which proved to be the case. During the differentiation process, the expression of versican, CD133, SCF (stem cell factor), ET-1 (endothelin-1) and bFGF (basic fibroblast growth factor) increased. Compared with hMSCs alone, the aggregate number clearly increased when co-cultured with DP cells. The expression in vivo of HLA-I (human leucocyte antigen class I) was confined to DP of the newly formed HF. The data suggest that hMSCs possess the potential to differentiate into DP cells in vivo and in vitro.
Pathological hair loss (also known as alopecia) and shortage of hair follicle (HF) donors have posed an urgent requirement for HF regeneration. With the revelation of mechanisms in tissue engineering, the proliferation of HFs in vitro has achieved more promising trust for the treatments of alopecia and other skin impairments. Theoretically, HF organoids have great potential to develop into native HFs and attachments such as sweat glands after transplantation. However, since the rich extracellular matrix (ECM) deficiency, the induction characteristics of skin-derived cells gradually fade away along with their trichogenic capacity after continuous cell passaging in vitro. Therefore, ECM-mimicking support is an essential prelude before HF transplantation is implemented. This review summarizes the status of providing various epidermal and dermal cells with a three-dimensional (3D) scaffold to support the cell homeostasis and better mimic in vivo environments for the sake of HF regeneration. HF-relevant cells including dermal papilla cells (DPCs), hair follicle stem cells (HFSCs), and mesenchymal stem cells (MSCs) are able to be induced to form HF organoids in the vitro culture system. The niche microenvironment simulated by different forms of biomaterial scaffold can offer the cells a network of ordered growth environment to alleviate inductivity loss and promote the expression of functional proteins. The scaffolds often play the role of ECM substrates and bring about epithelial-mesenchymal interaction (EMI) through coculture to ensure the functional preservation of HF cells during in vitro passage. Functional HF organoids can be formed either before or after transplantation into the dermis layer. Here, we review and emphasize the importance of 3D culture in HF regeneration in vitro. Finally, the latest progress in treatment trials and critical analysis of the properties and benefits of different emerging biomaterials for HF regeneration along with the main challenges and prospects of HF regenerative approaches are discussed.
The lncRNA-599547 (619-nt in length) is identified in secondary hair follicle (SHF) of cashmere goat, but its functional roles in regulating the inductive property of dermal papilla cells (DPCs) remains unknown. We found that lncRNA-599547 had significantly higher expression in dermal papilla of cashmere goat SHF at anagen than its counterpart at telogen. The overexpression of lncRNA-599547 led to a significant increase of ALP and LEF1 expression in DPCs (p < 0.05), whereas, the siLncRNA-1 mediated silencing of lncRNA-599547 significantly down-regulated the expression of ALP and LEF1 in DPCs (p < 0.05). Based on biotin-labeled RNA pull-down assay, we found that lncRNA-599547 directly interacted with chi-miR-15b-5p in DPCs. Based on both overexpression and silencing analysis of lncRNA-599547, our results indicate that lncRNA-599547 promotes the expression of Wnt10b in DPCs but without modulating its promoter methylation level. Using the mRNA-3′UTR fragments of goat Wnt10b containing the predicted binding sites of chi-miR-15b-5p in Dual-luciferase Reporter Assays, we show that lncRNA-599547 modulates the expression of Wnt10b at the chi-miR-15b-5p mediated post-transcriptional level. Taken together, our results indicate that lncRNA-599547 sponges miR-15b-5p to positively regulate the expression of Wnt10 gene, and thereby contributes the inductive property of DPCs in cashmere goat.
Long noncoding RNAs (lncRNAs), a class of non-protein conding RNAs > 200 nt in length, were thought to play critical roles in regulating the expression of protein-coding genes. Here, we identified and characterized a novel lncRNA-000133 from the secondary hair follicle (SHF) of cashmere goat with its ceRNA network analysis, as well as, its potential effects on inductive property of dermal papilla cells were evaluated through overexpression analysis. Expression analysis indicated that lncRNA-000133 had a significantly higher expression at anagen than that at telogen in SHF of Cashmere goat, suggesting that lncRNA-000133 might be involved in the reconstruction of SHF with the formation and growth of cashmere fiber. Taken together with methylation analysis, we showed that 5’ regulatory region methylation of the lncRNA-000133 gene might be involved in its expression suppression in SHF of Cashmere goat. The ceRNA regulatory network showed that a rich and complex regulatory relationship between lncRNA-000133 and related miRNAs with their target genes. The overexpression of lncRNA-000133 led to a significant increasing in the relative expression of ET-1, SCF, ALP and LEF1 in dermal papilla cells suggesting that lncRNA-000133 appears to contribute the inductive property of dermal papilla cells.
Extracellular matrix (ECM) is a dynamic network of macromolecules, playing a regulatory role in cell functions, tissue regeneration and remodeling. Wound healing is a tissue repair process necessary for the maintenance of the functionality of tissues and organs. This highly orchestrated process is divided into four temporally overlapping phases, including hemostasis, inflammation, proliferation and tissue remodeling. The dynamic interplay between ECM and resident cells exerts its critical role in many aspects of wound healing, including cell proliferation, migration, differentiation, survival, matrix degradation and biosynthesis. Several epigenetic regulatory factors, such as the endogenous non-coding microRNAs (miRNAs), are the drivers of the wound healing response. microRNAs have pivotal roles in regulating ECM composition during wound healing and dermal regeneration.Their expression is associated with the distinct phases of wound healing, and they serve as target biomarkers and targets for systematic regulation of wound repair. In this article we critically present the importance of epigenetics with particular emphasis on miRNAs regulating ECM components (i.e. glycoproteins, proteoglycans and matrix proteases) that are key players in wound healing. The clinical relevance of miRNA targeting as well as the delivery strategies designed for clinical applications are also presented and discussed.
The skin is a complex stratified organ which acts not only as a permeability barrier and defense against external agents, but also has essential thermoregulatory, sensory and metabolic functions. Due to its high versatility and activity, the skin undergoes continuous self-renewal to repair damaged tissue and replace old cells. Consequently, the skin is a reservoir for adult stem cells of different embryonic origins. Skin stem cell populations reside in the adult hair follicle, sebaceous gland, dermis and epidermis. However, the origin of most of the stem cell populations found in the adult epidermis is still unknown. Far more unknown is the embryonic origin of other stem cells that populate the other layers of this tissue. In this review we attempt to clarify the emergence, structure, markers and embryonic development of diverse populations of stem cells from the epidermis, dermis and related appendages such as the sebaceous gland and hair follicle.
The skin is a complex stratified organ which acts not only as a permeability barrier and defense against external agents, but also has essential thermoregulatory, sensory and metabolic functions. Due to its high versatility and activity, the skin undergoes continuous self-renewal to repair damaged tissue and replace old cells. Consequently, the skin is a reservoir for adult stem cells of different embryonic origins. Skin stem cell populations reside in the adult hair follicle, sebaceous gland, dermis and epidermis. However, the origin of most of the stem cell populations found in the adult epidermis is still unknown. Far more unknown is the embryonic origin of other stem cells that populate the other layers of this tissue. In this review we attempt to clarify the emergence, structure, markers and embryonic development of diverse populations of stem cells from the epidermis, dermis and related appendages such as the sebaceous gland and hair follicle.
The determination of animal form depends on the coordination of events that lead to the morphological patterning of cells. This epigenetic view of development suggests that embryonic structures arise as a consequence of environmental influences acting on the properties of cells, rather than an unfolding of a completely genetically specified and preexisting invisible pattern. Specialized cells of developing multicellular organisms are surrounded by a complex extracellular matrix (ECM), comprised largely of different collagens, proteoglycans, and glycoproteins. This ECM is a substrate for tissue morphogenesis, lends support and flexibility to mature tissues, and acts as an epigenetic informational entity in the sense that it transduces and integrates intracellular signals via distinct cell surface receptors. Consequently, ECM-receptor interactions have a profound influence on major cellular programs including growth, differentiation, migration, and survival. In contrast to many other ECM proteins, the tenascin (TN) family of glycoproteins (TN-C, TN-R, TN-W, TN-X, and TN-Y) display highly restricted and dynamic patterns of expression in the embryo, particularly during neural development, skeletogenesis, and vasculogenesis. These molecules are reexpressed in the adult during normal processes such as wound healing, nerve regeneration, and tissue involution, and in pathological states including vascular disease, tumorigenesis, and metastasis. In concert with a multitude of associated ECM proteins and cell surface receptors that include members of the integrin family, TN proteins impart contrary cellular functions, depending on their mode of presentation (i.e., soluble or substrate-bound) and the cell types and differentiation states of the target tissues. Expression of tenascins is regulated by a variety of growth factors, cytokines, vasoactive peptides, ECM proteins, and biomechanical factors. The signals generated by these factors converge on particular combinations of cis-regulatory elements within the recently identified TN gene promoters via specific transcriptional activators or repressors. Additional complexity in regulating TN gene expression is achieved through alternative splicing, resulting in variants of TN polypeptides that exhibit different combinations of functional protein domains. In this review, we discuss some of the recent advances in TN biology that provide insights into the complex way in which the ECM is regulated and how it functions to regulate tissue morphogenesis and gene expression. Dev Dyn;218:235–259. © 2000 Wiley-Liss, Inc.
We have demonstrated previously that chick embryo fibroblasts synthesize and secrete a large chondroitin sulfate proteoglycan (designated PG-M) that binds to fibronectin. We now report the possibility that PG-M interactions with cell surfaces can modulate cell-substrate adhesion. When PG-M was added to the medium, various types of trypsinized cells failed to adhere not only to fibronectin-coated substrates but also to collagen- or vitronectin-coated substrates. Adhesion of the cells to laminin or glycyl-arginyl-glycyl-aspartyl-serine derivatized serum albumin (arginyl-glycyl-aspartic acid-containing molecules with no capacity to bind PG-M) was also inhibited by PG-M. Treatment of the proteoglycan with either proteolytic enzymes or chondroitinase abolished its inhibitory effects on the cell adhesion. These results suggest that direct binding between PG-M and fibronectin, if any, is not a cause of the inhibition by PG-M and that only the proteoglycan form is responsible for the activity. When the immobilization of added PG-M to available plastic surfaces of coated dishes was blocked by pretreating the dishes with serum albumin, the inhibitory effect of PG-M was abolished, suggesting that the immobilized fraction of PG-M can act as a cell adhesion inhibitor. In immobilized form, both cartilage chondroitin sulfate proteoglycan (designated PG-H) and chondroitin sulfate-derivatized serum albumin also inhibited cell adhesion. In contrast, heparan sulfate proteoglycan form LD and heparan sulfate-derivatized serum albumin had far lower inhibitory activities, indicating that the active site for the interaction between cells and PG-M is on the chondroitin sulfate chains.
The hairless mutation of mice was caused by insertion of a murine leukemia virus. Starting with sequences flanking the provirus, a series of overlapping clones surrounding the viral integration site were obtained. By using a combination of sequencing, PCR, and exon-trapping techniques, the hairless gene was identified. It encodes a predicted protein of 1182 amino acids, including a potential zinc-finger domain. The expression patterns of the gene closely reflect the phenotype of animals carrying the hairless mutation.
Ligands for a leukocyte adhesion molecule, L-selectin, are expressed not only in the specific vascular endothelium in lymph nodes and Peyer's patches but also in the extravascular tissues such as the brain white matter, choroid plexus and the kidney distal straight tubuli. However, the biological significance of these extravascular ligands is currently unknown. We now report the purification and characterization of a novel extravascular ligand for L-selectin in the kidney using a tubule-derived cell line, ACHN. Binding of L-selectin-IgG chimera (LEC-IgG) to the isolated ligand was specifically blocked with either (i) anti-L-selectin mAb, (ii) EDTA, (iii) fucoidan, (iv) chondroitin sulfate (CS) B or CS E, or (v) treatment with chondroitinases. Partial amino acid sequencing, Western blotting and immunoprecipitation analyses showed that a major ligand for L-selectin in ACHN cells is versican of 1600 kDa. Histochemical as well as biochemical analyses verified that a versican subspecies in the kidney was indeed reactive with L-selectin. Studies with cell lines including those derived from the kidney indicated that a certain glycoform and/or splice form of versican is reactive with L-selectin. Under pathological conditions such as those induced by unilateral ureteral obstruction, versican was shed from the distal straight tubuli and became localized in the adjacent vascular bundles around which a substantial leukocyte infiltration was concomitantly observed. Possible involvement of versican in leukocyte trafficking into the kidney under diseased conditions is discussed.
Strategies to generate highly concentrated HIV-1 vector pseudotypes involving different envelope (Env) proteins including the vesicular stomatitis virus (VSV) G glycoprotein, the Moloney murine leukemia virus (MLV) 4070A amphotropic Env and the rabies G glycoprotein were established. Virus stocks were prepared by transient transfection using standard cell culture media or serum-free media. Such stocks were concentrated 50- to 300-fold by ultracentrifugation or by ultrafiltration using Centricon Plus-80 units yielding titers of up to 109transducing units per milliliter. There was no loss in titer with any of the pseudotypes tested. Thus, like lentiviral vectors pseudotyped with VSV-G, HIV-1-based vectors pseudotyped with the MLV 4070A amphotropic Env and the rabies G glycoprotein resist inactivation during concentration. This opens up the possibility to generate highly concentrated HIV-1 vector stocks carrying alternative Env proteins on a large scale.
Here we show that a large chondroitin sulfate proteoglycan, versican, derived from a renal adenocarcinoma cell line ACHN, binds L-selectin, P-selectin, and CD44. The binding was mediated by the interaction of the chondroitin sulfate (CS) chain of versican with the carbohydrate-binding domain of L- and P-selectin and CD44. The binding of versican to L- and P-selectin was inhibited by CS B, CS E, and heparan sulfate (HS) but not by any other glycosaminoglycans tested. On the other hand, the binding to CD44 was inhibited by hyaluronic acid, chondroitin (CH), CS A, CS B, CS C, CS D, and CS E but not by HS or keratan sulfate. A cross-blocking study indicated that L- and P-selectin recognize close or overlapping sites on versican, whereas CD44 recognizes separate sites. We also show that soluble L- and P-selectin directly bind to immobilized CS B, CS E, and HS and that soluble CD44 directly binds to immobilized hyaluronic acid, CH, and all the CS chains examined. Consistent with these results, structural analysis showed that versican is modified with at least CS B and CS C. Thus, proteoglycans sufficiently modified with the appropriate glycosaminoglycans should be able to bind L-selectin, P-selectin, and/or CD44.
De novo hair follicle formation in embryonic skin and new hair growth in adult skin are initiated when specialized mesenchymal dermal papilla (DP) cells send cues to multipotent epithelial stem cells. Subsequently, DP cells are enveloped by epithelial stem cell progeny and other cell types to form a niche orchestrating hair growth. Understanding the general biological principles that govern the mesenchymal-epithelial interactions within the DP niche, however, has been hampered so far by the lack of systematic approaches to dissect the complete molecular make-up of this complex tissue. Here, we take a novel multicolor labeling approach, using cell type-specific transgenic expression of red and green fluorescent proteins in combination with immunolabeling of specific antigens, to isolate pure populations of DP and four of its surrounding cell types: dermal fibroblasts, melanocytes, and two different populations of epithelial progenitors (matrix and outer root sheath cells). By defining their transcriptional profiles, we develop molecular signatures characteristic for the DP and its niche. Validating the functional importance of these signatures is a group of genes linked to hair disorders that have been largely unexplored. Additionally, the DP signature reveals novel signaling and transcription regulators that distinguish them from other cell types. The mesenchymal-epithelial signatures include key factors previously implicated in ectodermal-neural fate determination, as well as a myriad of regulators of bone morphogenetic protein signaling. These findings establish a foundation for future functional analyses of the roles of these genes in hair development. Overall, our strategy illustrates how knowledge of the genes uniquely expressed by each cell type residing in a complex niche can reveal important new insights into the biology of the tissue and its associated disease states.
Versican, a chondroitin sulfate proteoglycan, is one of the main components of the extracellular matrix, which provides a loose and hydrated matrix during key events in development and disease. Versican participates in cell adhesion, proliferation, migration, and angiogenesis, and hence plays a central role in tissue morphogenesis and maintenance. In addition, versican contributes to the development of a number of pathologic processes including atherosclerotic vascular diseases, cancer, tendon remodeling, hair follicle cycling, central nervous system injury, and neurite outgrowth. Versican is a complex molecule consisting of modular core protein domains and glycosaminoglycan side chains, and there are various steps of synthesis and processes regulating them. Also, there is differential temporal and spatial expression of versican by multiple cell types and in different developmental and pathological time frames. To fully appreciate the functional roles of versican as it relates to changing patterns of expression in development and disease, an in depth knowledge of versicans biosynthetic processing is necessary. The goal of this review is to evaluate the current status of our knowledge regarding the transcriptional control of versican gene regulation. We will be focusing on the signal transduction pathways, promoter regions, cis-acting elements, and trans-factors that have been characterized.
Lentiviral vectors offer unique versatility and robustness as vehicles for gene delivery. They can transduce a wide range of cell types and integrate into the host genome in both dividing and post-mitotic cells, resulting in long-term expression of the transgene both in vitro and in vivo. This protocol describes how lentiviral vectors can be produced, purified and titrated. High titer suspensions can be routinely prepared with relative ease: a low-titer (10(6) viral particles/ml) unpurified preparation can be obtained 3 d after transfecting cells with lentiviral vector and packaging plasmids; a high-titer (10(9) viral particles/ml) purified preparation requires 2 more days.
Ligands for a leukocyte adhesion molecule, L-selectin, are expressed not only in the specific vascular endothelium in lymph nodes and Peyer’s patches but also in the extravascular tissues such as the brain white matter, choroid plexus and the kidney distal straight tubuli. However, the biological significance of these extravascular ligands is currently unknown. We now report the purification and characterization of a novel extravascular ligand for L-selectin in the kidney using a tubule-derived cell line, ACHN. Binding of L-selectin‐IgG chimera (LEC‐IgG) to the isolated ligand was specifically blocked with either (i) anti-L-selectin mAb, (ii) EDTA, (iii) fucoidan, (iv) chondroitin sulfate (CS) B or CS E, or (v) treatment with chondroitinases. Partial amino acid sequencing, Western blotting and immunoprecipitation analyses showed that a major ligand for L-selectin in ACHN cells is versican of 1600 kDa. Histochemical as well as biochemical analyses verified that a versican subspecies in the kidney was indeed reactive with L-selectin. Studies with cell lines including those derived from the kidney indicated that a certain glycoform and/or splice form of versican is reactive with L-selectin. Under pathological conditions such as those induced by unilateral ureteral obstruction, versican was shed from the distal straight tubuli and became localized in the adjacent vascular bundles around which a substantial leukocyte infiltration was concomitantly observed. Possible involvement of versican in leukocyte trafficking into the kidney under diseased conditions is discussed.
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-DeltaDeltaCr) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-DeltaDeltaCr) method. In addition, we present the derivation and applications of two variations of the 2(-DeltaDeltaCr) method that may be useful in the analysis of real-time, quantitative PCR data. (C) 2001 Elsevier science.
Versican is a member of the group of aggregating proteoglycans involved in matrix assembly and structure and in cell adhesion. We examined changes in the distribution of versican in mammalian skin, with emphasis on hair follicle development and cycling. In adult human skin, immunostaining for versican appeared predominantly in the dermis, with intense staining of the reticular dermis. Weak staining was observed at the dermoepidermal junction and the connective tissue sheath of hair follicles. Versican expression was also noted in the reticular dermis of rat skin, within dermal papillae, and possibly associated with follicle basement membranes. During mouse hair follicle development, versican was not expressed until the hair follicles were beginning to produce fibers. With follicle maturation, versican expression intensified in the dermal papillae, reaching a maximum at the height of the growth phase (anagen), after which it diminished as the end of this phase approached. Versican immunoreactivity in the papillae decreased further during catagen and was absent from these structures during telogen. However, intense staining for versican was then observed in the neck regions of telogen follicles. As the follicles entered the next hair cycle, versican disappeared from the necks and was again seen in the dermal papillae when follicles began producing fibers. This type of expression continued throughout subsequent hair cycles and is unlike any other dermal papilla component. The results of this study are consistent with a distinct supportive role for versican in the follicle matrices during hair follicle morphogenesis and cycling.
Combinations of cultured and uncultured epidermal and dermal cell preparations from newborn and perinatal mice were grafted onto the backs of athymic nude mouse hosts to elucidate the cellular requirements for skin appendage formation. All epidermal populations studied, including a total epidermal keratinocyte preparation from trypsin-split skin, developing hair follicle buds isolated from epidermis, and preformed hair follicles isolated from dermis, make haired skin when grafted with fresh dermal cells. Only pre-formed hair follicles produce haired skin on grafts without an additional dermal component. Hair follicle buds grafted alone or with cultured dermal cells will reconstitute skin but without appendage formation. Thus, cells or factors present in fresh, but not cultured, dermal cells are essential for supporting hair growth from budding follicles, whereas more developed (pre-formed) follicles appear to contain all the necessary components for hair formation. Dissociation of isolated hair follicles by trypsin/ethylenediaminetetraacetic acid prior to grafting is permissive for hair growth, suggesting that follicle cells can be re-induced or reassociate in vivo. Dermal papilla cells, microdissected from rat vibrissal follicles and cultured for up to 14 passages, stimulate hair growth from follicle buds and influence the quality of hair growth from pre-formed hair follicles. Thus, dermal papilla cells maintain inductive capacity in culture and contribute to the reconstituted skin. This reconstitution model should be useful for identifying cell populations within the hair follicle compartment necessary for hair growth and for examining the effects of specific gene products on hair follicle growth and development in vivo.
Low passage cultured dermal papilla cells from adult rats stimulate complete hair follicle neogenesis when re-implanted into heterotypic skin. In contrast, cultured sheath cells are non-inductive despite sharing other behavioural characteristics (a common lineage and in situ proximity) with papilla cells. However, since sheath cells can behave inductively in amputated follicles after regenerating the papilla, this poses the question of what influences the sheath to papilla cell transition? During reciprocal tissue interactions specific epidermal cues are crucial to skin appendage development, and while in vivo assays to date have focussed on dermal interactive influence, our aim was to investigate epidermal potential. We have previously observed that hair follicle epidermal cells display exceptional interactive behaviour when combined with follicle dermal cells in vitro. Thus in the present study, hair follicle germinative, outer root sheath or skin basal epidermal cells were separately combined with each of three non-inductive dermal cell types (high passage papilla, low passage sheath or fibroblast) and then implanted into small ear skin wounds. The sheath/germinative and papilla/germinative cell implants repeatedly induced giant vibrissa-type follicles and fibres. In complete contrast, any single cell type and all other forms of recombination were consistently non-inductive. Hence, the adult germinative epidermal cells enable non-inductive adult dermal cells to stimulate hair follicle neogenesis, effectively, by altering their 'status', causing the sheath cells to 'specialise' and the 'aged' papilla cells to 'rejuvenate'.
Hair dermal papilla cells are specialized mesenchymal cells that exist in the dermal papilla located at the bottom of hair follicles. These cells play pivotal roles in hair formation, growth, and cycling. Hair follicle formation is usually directed by an aggregation of dermal mesenchymal cells, the origin of dermal papilla cells, in the embryonic skin. We noticed that cultured dermal papilla cells also have hair-forming activity and do not lose the activity even after long-term cultivation, if they are cultured with conditioned medium from keratinocytes obtained from the sole or with a medium containing fibroblast growth factor. The secreted factors from keratinocytes and fibroblast growth factor are, therefore, important for maintaining the cellular properties of dermal papilla cells. Even if the hair bulb, including the hair matrix and the dermal papilla, has been removed from vibrissal follicles in vivo, the new hair matrix and papilla can regenerate from the rest of the follicle, and eventually a hair shaft regrows. It has been reported that hair bulb regeneration does not occur when the lower half of a hair follicle is removed. However, new hair bulbs were formed in the remaining upper halves of vibrissal follicles if the amputated follicles had been implanted under the kidney capsule. The formed bulbs were small and pelage-type, not large vibrissa-type. Histological studies showed that the new dermal papillae were derived from dermal sheath cells surrounding upper follicular epidermis, and the new hair matrices were produced from the follicular epidermis. Moreover, the upper halves of vibrissal follicles reformed large vibrissa-type bulbs when they were associated with dermal papillae or cultured papilla cells and implanted in the kidney. Thus, dermal papilla cells and probably dermal sheath cells have the ability to induce and form hair bulbs under preferred environmental conditions. Attempts to identify the genes and proteins associated with hair-forming activity of dermal papilla cells have been carried out. We and other groups successfully isolated the molecules that were specifically expressed in dermal papilla cells. The nature of the hair-producing factors could be understood through the studies of these molecules.
Human follicle cells can be induced to grow in an incompatible host of
the other sex.
Heparan sulphate proteoglycans are abundant cell-surface molecules that
consist of a protein core to which heparan sulphate glycosaminoglycan chains
are attached. The functions of these molecules have remained mostly underappreciated
by developmental biologists; however, the actions of important signalling
molecules, for example Wnt and Hedgehog, depend on them. To understand both
the mechanisms by which ligands involved in development interact with their
receptors and how morphogens pattern tissues, biologists need to consider
the functions of heparan sulphate proteoglycans in signalling and developmental
patterning.
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.
Chondroitin sulphate, fibronectin, laminin and the hyaluronan receptor, CD44, were localized in ovine skin during follicle morphogenesis. Prior to initiation, chondroitin sulphate was detected in the mesenchyme adjacent to the dermal-epidermal junction and showed an approximately regular periodicity in staining intensity. With the appearance of follicle primordia, the more strongly stained regions of the matrix were associated with mesenchymal condensations. During later development and in the mature follicle, staining was localized to the matrices of cells of the dermal sheath and papilla. CD44 was also localized in the mesenchymal condensations at follicle initiation and, subsequently, in the dermal sheath. Fibronectin staining was confined to the mesenchyme prior to follicle formation and became associated with presumptive papilla and dermal sheath cells during follicle formation and maturation. Fibronectin antisera detected an approximately 220 kDa protein in western blots of adult and fetal skin. An additional band of 150 kDa was also observed prior to follicle initiation. In contrast, laminin was predominantly restricted to the basal laminae of developing and mature follicles. The aggregative behaviour of ovine papilla cells was examined in vitro. The number and size of aggregates were not affected by inclusion of chondroitin sulphate or fibronectin in the culture medium, but both increased in the presence of hyaluronidase. Chondroitinase had the opposite effect and beta-D-xyloside completely abolished aggregative behaviour. In conclusion, the appearance of certain matrix molecules may presage morphogenetic movements of cells at follicle initiation and regulate patterns of follicle distribution in skin during fetal life.
This study examines histologically the degeneration and subsequent regeneration processes of human hair follicles whose bulb is severely damaged. Human scalp hair follicles were isolated and grafted onto immunodeficient mice after their bulb was amputated. On day 14, thickening and corrugation of the vitreous membrane, apoptosis of follicular keratinocytes, and regression of the lower portion of the follicles were observed. By day 20, mesenchymal cells had accumulated around the lower end of the follicles. From day 14 through 50, the follicular regression and apoptosis continued, and between days 30 and 40 the follicles became maximally shortened, and the vitreous membrane disappeared. By day 50 the lower end of the follicles had become cup-shaped, and the cup surrounded an aggregate of mesenchymal cells that corresponded to the dermal papilla. By day 60, all the grafted follicles had developed into anagen VI follicles, and the apoptosis had ceased. These results indicate that human scalp hair follicles whose bulb is completely destroyed enter into dystrophic telogen after restoration of the dermal papilla, then into anagen, and that the duration of the dystrophic telogen is shorter than that of the normal hair cycle.
Clinical conditions causing hair loss, such as androgenetic alopecia, alopecia areata, and scarring alopecia, can be psychologically devastating to individuals and are the target of a multimillion dollar pharmaceutical industry. The importance of the hair follicle in skin biology, however, does not rest solely with its ability to produce hair. Hair follicles are self-renewing and contain reservoirs of multipotent stem cells that are capable of regenerating the epidermis and are thought to be utilized in wound healing. Hair follicles are also the sites of origin of many neoplasias, including some basal cell carcinomas and pilomatricoma. These diseases result from inappropriate activation of signaling pathways that regulate hair follicle morphogenesis. Identification of the signaling molecules and pathways operating in developing and postnatal, cycling, hair follicles is therefore vital to our understanding of pathogenic states in the skin and may ultimately permit the development of novel therapies for skin tumors as well as for hair loss disease. The purpose of this review is to summarize recent progress in our understanding of the molecular mechanisms regulating hair follicle formation, and to discuss ways in which this information may eventually be utilized in the clinic.
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.
Androgenetic alopecia (AGA) is hereditary and androgen-dependent, progressive thinning of the scalp hair that follows a defined pattern. While the genetic involvement is pronounced but poorly understood, major advances have been achieved in understanding principal elements of the androgen metabolism involved: androgen-dependent processes are predominantly due to the binding of dihydrotestosterone (DHT) to the androgen receptor (AR). DHT-dependent cell functions depend on the availability of weak androgens, their conversion to more potent androgens via the action of 5 alpha-reductase, low enzymatic activity of androgen inactivating enzymes, and functionally active AR present in high numbers. The predisposed scalp exhibits high levels of DHT, and increased expression of the AR. Conversion of testosterone to DHT within the dermal papilla plays a central role, while androgen-regulated factors deriving from dermal papilla cells are believed to influence growth of other components of the hair follicle. Current available treatment modalities with proven efficacy are oral finasteride, a competitive inhibitor of type 2 5 alpha-reductase, and topical minoxidil, an adenosine-triphosphate-sensitive potassium channel opener which has been reported to stimulate the production of vascular endothelial growth factor in cultured dermal papilla cells. Since the clinical success rate of treatment of AGA with modulators of androgen metabolism or hair growth promoters is limited, sustained microscopic follicular inflammation with connective tissue remodeling, eventually resulting in permanent hair loss, is considered a possible cofactor in the complex etiology of AGA.
Epithelial-mesenchymal interactions play pivotal roles in the morphogenesis of many organs and various types of appendages. During hair follicle development, extensive interactions between two embryologically different hair follicle compartments (epidermal keratinocytes and dermal papilla fibroblasts) lead to the formation of the hair shaft-producing mini-organ that shows cyclic activity during postnatal life with periods of active growth, involution and resting. During the hair cycle, the epithelium and the mesenchyme are regulated by a distinct set of molecular signals that are unique for every distinct phase of the hair cycle. In telogen hair follicles, epithelial-mesenchymal interactions are characterized by a predominance of inhibitory signals that retain the hair follicle in a quiescent state. During anagen, a large variety of growth stimulatory pathways are activated in the epithelium and in the mesenchyme, the coordination of which are essential for proper hair fiber formation. During catagen, the termination of anagen-specific signaling interactions between the epithelium and the mesenchyme leads to apoptosis in the hair follicle epithelium, while activation of selected signaling pathways promotes the transition of the dermal papilla into a quiescent state. The signaling exchange between the follicular epithelium and the mesenchyme is modulated by proteoglycans, such as versican, which may significantly enhance or reduce the biological activities of secreted growth stimulators. However, additional research will be required to bridge the gap between our current understanding of mechanisms underlying epithelial-mesenchymal interactions in hair follicles and the potential clinical application of growth modulators involved in those interactions. Further progress in this area of research will hopefully lead to the development of new drugs for the treatment of hair growth disorders.
To master tissue and organ morphogenesis necessitates a thorough understanding of the cellular and molecular events involved in development, renewal, repair and regeneration. Skin reconstruction is the paradigm of tissue engineering. The transplantation of autologous adult epidermal stem cells is a life-saving procedure as it regenerates the indispensable barrier function of the skin, but the reconstruction of fully functional skin has been hampered by the complexity of the process. The recent identification of multipotent epithelial stem cells in adult hair follicles and of multipotent stem cells in dermis raises new hopes.
Adult epidermal stem cells renew the epithelial compartment of the skin throughout life and are the most accessible of all adult stem cells. Most importantly, epidermal stem cells can be efficiently cultivated and transplanted, a significant advantage for cell and gene therapy. Recent work has pointed to the hair follicle as the main repository of multipotent stem cells in skin. Hair follicles, which are often affected in the mouse by spontaneous or man-made mutations, have become superb model systems to study the cellular and molecular factors that regulate the proliferation, migration and fate of adult stem cells.
Green fluorescent protein (GFP)-expressing wild-type, and nontransgenic mouse vibrissa follicle cells were cultured and implanted to mouse ears and footpads. Dermal papiller (DP)-derived cells and cells from the peribulbar dermal sheath "cup" (DSC) induced new hair follicles in both implanted ears and footpads, while nonbulbar dermal sheath cells did not. Confocal microscopy revealed that GFP-expressing DP and DSC cells induced hair growth associated with the formation of DP exclusively comprised of fluorescent cells. In mouse ears, but not footpads, fluorescent DP and DSC cells could also be identified in DP along with nonfluorescent cells. DSC cells were characterized in vivo and in vitro by low alkaline phosphatase activity in contrast to high alkaline phosphatase in DP cells. The results indicate transplanted DP and DSC cells were equally capable of DP formation and hair follicle induction. This suggests the DP and peribulbar DSC may be functionally similar. In addition to observing papillae exclusively composed of GFP-expressing cells, DP and DSC cells may also have combined with resident cells to form papillae composed of implanted GFP-expressing cells and host-derived non-GFP-expressing cells. Alkaline phosphatase expression may be utilized as a simple marker to identify hair follicle mesenchyme derived cells with hair follicle inductive abilities.
The epithelial-mesenchymal interactions between keratinocyte stem cells and dermal papilla (DP) cells are crucial for normal development of the hair follicle as well as during hair cycling. During the cyclical regrowth of a new lower follicle, the multipotent hair follicle stem cells are stimulated to proliferate and differentiate through interactions with the underlying mesenchymal DP cells. To characterize the events occurring during the process of epithelial stem cell fate determination, we utilized a coculture system by incubating human hair follicle keratinocyte stem cells with DP cells. Using GeneChip microarrays, we analyzed changes in gene expression within the stem cells upon coculture with the DP over a 5-day time course. A number of important signaling pathways and growth factors were regulated. The hair-specific keratin 6hf (K6hf) gene proved a particularly good marker of hair differentiation, with a 7.9-fold increase in mRNA and resulting increased protein levels. The high expression of K6hf was unique to DP-induced keratinocyte differentiation, since expression of K6hf was not induced by high calcium. Since the beta-catenin signaling pathway has been implicated in hair follicle development, we examined the role of beta-catenin in our system and demonstrated that beta-catenin/lef-1 signaling is required for DP-induced hair differentiation.
The goal of the present study was to develop an efficient transient transfection method for large-scale production of high titer lentivirus vector stocks of eight different pseudotypes. The envelope genes used for this purpose were those from VSV-G, Mokola, Rabies, MLV-Ampho, MLV-10A1, LCMV-WE, and LCMV-Arm53b. All envelopes were cloned into phCMV, which yielded lentivirus vector titers one, two, or three orders of magnitude higher than the original plasmids for the Rabies, MLV-10A1, and MLV-Ampho envelopes, respectively. When these newly constructed envelope expression plasmids were used for packaging, treatment with sodium butyrate resulted in almost five-fold increase in titers for some of the pseudotypes, had no effect for others (VSV-G and Rabies), and negatively impacted titers for the LCMV-derived pseudotypes. Production of vectors in serum-free media yielded titers only slightly lower than those obtained in the presence of serum. The efficiency of concentrating vector supernatants by ultracentrifugation or ultrafiltration was compared, with higher recovery efficiencies for the latter method, but the highest titers for most pseudotypes were obtained by ultracentrifugation. The best conditions for each individual pseudotype yielded lentivirus vector stocks with titers above 1 x 10(9) tu/mL for most pseudotypes, and higher than 1 x 10(10) tu/mL for VSV-G.
Dermal papilla cells of the hair follicle can be maintained in an active, hair-inducing state in vitro when cocultured with cells secreting Wnt3a. By inducing cultured dermal papilla cells to secrete Wnt themselves, we demonstrate that this activity is a direct effect of Wnt signaling to dermal papilla cells. We further demonstrate that the effects of Wnt3a are exerted through activation of the beta-catenin signal transduction pathway and do not require alternative Wnt transduction cascades. Once dermal papilla cells have lost hair-inducing properties in vitro, neither treatment with Wnt nor expression of a truncated and activating form of beta-catenin is sufficient to restore these properties to the cultured cells.
Versican, a large chondroitin sulfate proteoglycan molecule, is implicated in the induction of hair morphogenesis, the initiation of hair regeneration, and the maintenance of hair growth in mouse species. In contrast, in human hair follicles, the distribution and the roles of versican remains obscure.
To elucidate the implication of versican in normal human hair growth.
Versican expression was examined by in situ hybridization (mRNA) and immunohistochemistry (protein).
The results clearly showed specific versican gene expression in the dermal papilla of anagen, which apparently decreased in the dermal papilla of catagen hair follicles. No specific signal was detectable in telogen hair follicles. Consistent with ISH results, versican immunoreactivity was extended over the dermal papilla of anagen hair follicles, and again, this staining diminished in the catagen phase of human hair follicles. Interestingly, versican proteins were deposited outside K15-positive epithelial cells in the bulge throughout the hair cycle. Versican immunoreactivity in the dermal papilla was almost lost in vellus-like hair follicles affected by male pattern baldness.
Specific expression of versican in the anagen hair follicles suggests its importance to maintain the normal growing phase of human as well as mouse.
In this study, we show a more efficient method for isolation and cultivation of dermal papilla cells from hair follicles of human scalp skin. The dermal partments of low hair follicles were pulled out from cutaneous fat and the bulb epithelium was teased out from the fibrous sheath with attached dermal papilla by applying gentle pressure with the tip of an occal forceps. When these fibrous sheaths were entirely digested into isolated cells by collagenase D but the dermal papillae were justly to be digested, collagenase D was discarded and the dermal papillae were isolated completely out from the resuspension solution by repeated low-speed centrifugation and transferred to another dish for free-floating culture. This procedure markedly simplifies the steps of isolated dermal papilla operation and relieves the laborious tension. Furthermore, dermal papillae could be isolated on a large-scale and remained intact. After collagenase digestion, the dermal papillae showed very high adherent rate and quicker growth than that of microdissection, which suggests that the definition factor of dermal papilla cell migration was relaxed and some structure had been activated or exposed. The cells exhibited a multi-layer forming property and spread-out growth style. They showed positive with alcian blue, with toluidine blue O for different gradient pH and PAS, which was similar to the staining results of in situ dermal papilla. It suggests that the culture papilla cells still synthesize and excrete neutral and acid mucopolysaccharides. Our results demonstrate that the papilla cells in culture condition still remain the ability to synthesize the specific extracellular matrix components of in situ dermal papilla, which supports the concept that the dermal papilla cell, a highly specialized fibroblast, especially is involved in hair growth regulation.
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.
Versican is an abundant proteoglycan in the blood vessel wall that is increased after vascular injury and accumulates in advanced atherosclerotic plaques. Versican is a large molecule with domains that mediate binding to cytokines, enzymes, lipoproteins, other extracellular matrix molecules, and signaling receptors. There is evidence that versican exists in the normal, as well as the diseased, vessel wall as discrete fragments, which represent these functional domains. We review the literature on versican degradation in vascular tissue and the function of versican domains, all of which suggest that proteolytic modification of versican may have physiologic as well as pathologic implications for the vascular system.
We have succeeded in culturing dermal papilla (DP) cells long term and developed new techniques that enhance their hair follicle-inducing efficiency in a patch assay. The outgrowing DP cells from mouse vibrissae were markedly stimulated by 10% fetal bovine serum-Dulbecco's modified essential medium that included fibroblast growth factor-2 (FGF-2). Moreover, the potency of proliferation was maintained during serial cultivations (more than 30 passages). We combined these established DP cells with epidermal cells and implanted them subcutaneously into athymic mice to examine their hair follicle-inducing ability. New hair follicles were induced by dissociated DP cells at earlier passages (under passage 4), but the cells from later passages could not induce follicles. We next aggregated the DP cells to form spheres and then injected them with epidermal cells. Unlike the dissociated DP cells, the spheres made from the later passaged cells (more than 10 passages) did induce new hair follicles. We examined several genes specific for DP of anagen follicles and confirmed that their expression level was elevated in the spheres compared with their expression level in adherent DP cells. These results suggest that FGF-2 is essential for dermal papilla cell culture and that sphere formation partially models the intact DP, resulting in hair follicle induction, even by later passaged cells.
The Author(s) 84 CED Ó 2010 British Association of Dermatologists @BULLET Clinical and Experimental Dermatology RNAi versican-targeting suppresses aggregative growth of dermal papilla cells @BULLET M
- Feng
Ó The Author(s) 84 CED Ó 2010 British Association of Dermatologists @BULLET Clinical and Experimental Dermatology, 36, 77–84 RNAi versican-targeting suppresses aggregative growth of dermal papilla cells @BULLET M. Feng et al.
The multifaceted adult epi-24 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2 )DDCT method
- L Gambardella
- Barrandon
Gambardella L, Barrandon Y. The multifaceted adult epi-24 Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2 )DDCT method. Methods 2001; 25: 402–8.
Selective activation of the versican promoter by epithelial-mesenchymal interactions during hair follicle development
- J Kishimoto
- R Ehama
- L Wu
- Etal
Production and purification of lentiviral vectors
- G Tiscornia
- O Singer
- Im Verma
Optimized large-scale production of high titer lentivirus vector pseudotypes
- M Sena-Esteves
- Jc Tebbets
- S Steffens
- Etal
Selective activation of the versican promoter by epithelial-mesenchymal interactions during hair follicle development
- Kishimoto
Production and purification of lentiviral vectors
- Tiscornia
Optimized large-scale production of high titer lentivirus vector pseudotypes
- Sena-Esteves