Recent progress in hair follicle stem cell markers and their regulatory roles

Hair follicle stem cells(HFSCs)in the bulge are a multipotent adult stem cell population.They can periodically give rise to new HFs and even regenerate the epidermis and sebaceous glands during wound healing.An increasing number of biomarkers have been used to isolate,label,and trace HFSCs in recent years.Considering more detailed data from single-cell transcriptomics technology,we mainly focus on the important HFSC molecular markers and their regulatory roles in this review.

Melatonin promotes the development of the secondary hair follicles by regulating circMPP5

Background The quality and yield of cashmere fibre are closely related to the differentiation and development of secondary hair follicles in the skin of cashmere goats.The higher the density of secondary hair follicles,the higher the quality and yield of cashmere from the fleece.Development of secondary hair follicles commences in the embryonic stage of life and is completed 6 months after birth.Preliminary experimental results from our laboratory showed that melatonin(MT)treatment of goat kids after their birth could increase the density of secondary hair follicles and,thus,improve the subsequent yield and quality of cashmere.These changes in the secondary hair follicles resulted from increases in levels of antioxidant and expression of anti-apoptotic protein,and from a reduction in apoptosis.The present study was conducted to explore the molecular mechanism of MT-induced secondary hair follicle differentiation and development by using whole-genome analysis.Results MT had no adverse effect on the growth performance of cashmere kids but significantly improved the character of the secondary hair follicles and the quality of cashmere,and this dominant effect continued to the second year.Melatonin promotes the proliferation of secondary hair follicle cells at an early age.The formation of secondary hair follicles in the MT group was earlier than that in the control group in the second year.The genome-wide data results involved KEGG analysis of 1044 DEmRNAs,91 DElncRNAs,1054 DEcircRNAs,and 61 DEmiRNAs which revealed that the mitogen-activated protein kinase(MAPK)signaling pathway is involved in the development of secondary hair follicles,with key genes(FGF2,FGF21,FGFR3,MAPK3(ERK1))being up-regulated and expressed.We also found that the circMPP5 could sponged miR-211 and regulate the expression of MAPK3.Conclusions We conclude that MT achieves its effects by regulating the MAPK pathway through the circMPP5 sponged the miR-211,regulating the expression of MAPK3,to induce the differentiation and proliferation of secondary hair follicle cells.In addition there is up-regulation of expression of the anti-apoptotic protein causing reduced apoptosis of hair follicle cells.Collectively,these events increase the numbers of secondary hair follicles,thus improving the production of cashmere from these goats.

miR-21 promotes the differentiation of hair folliclederived neural crest stem cells into Schwann cells

Hair follicle-derived neural crest stem cells can be induced to differentiate into Schwann cells in vivo and in vitro. However, the underlying regulatory mechanism during cell differentiation remains poorly understood. This study isolated neural crest stem cells from human hair folli-cles and induced them to differentiate into Schwann cells. Quantitative RT-PCR showed that microRNA （miR）-21 expression was gradually increased during the differentiation of neural crest stem cells into Schwann cells. After transfection with the miR-21 agonist （agomir-21）, the differentiation capacity of neural crest stem cells was enhanced. By contrast, after transfection with the miR-21 antagonist （antagomir-21）, the differentiation capacity was attenuated. Further study results showed that SOX-2 was an effective target of miR-21. Without compromising SOX2 mRNA expression, miR-21 can down-regulate SOX protein expression by binding to the 3′-UTR of miR-21 mRNA. Knocking out the SOX2 gene from the neural crest stem cells significantly reversed the antagomir-21 inhibition of neural crest stem cells differentiating into Schwann cells. The results suggest that miR-21 expression was increased during the differentiation of neural crest stem cells into Schwann cells and miR-21 promoted the differentiation through down-regu-lating SOX protein expression by binding to the 3′-UTR of SOX2 mRNA.

Hoxc13/β-catenin Correlation with Hair Follicle Activity in Cashmere Goat

Seasonal hair follicle activity and fibre growth in some Cashmere-bearing goats （Caprus hircus） is a cyclic process that is well characterized morphologically but understood incompletely at the molecular level. As an initial step in discovering regulators in hair-follicle activity and cycling, we used qPCR to investigate 19 genes expression in Cashmere goat side skin from 12 mon. Many of these genes may be associated with the hair follicle development-relevant genes （HFDRGs） in the literature. Here we show that Hoxc13/β-catenin gene associated with the follicle activity. In addition, Hoxc13 was found to be expressed with an drastic increase between July and November for melatonin treatments. To further investigate the role of Hoxcl3 on HFDRGs, fibroblasts and keratinocytes from Cashmere goat skin were transfected with p-ECFP- Hoxc13. The result suggested that overexpression ofHoxcl3 gene decreased HFDRGs with negative role for hair follicle development and increase HFDRGs with positive role for hair follicle development in vitro. These findings provide data on the Hoxc13 expression profile of normal Cashmere goat skin and Cashmere goat skin with melatonin treatment, and demonstrate hair-follicle-activity dependent regulation of Hoxc13 expression.

Hoxc13 Expression Pattern in Cashmere Goat Skin During Hair Follicle Development

Hoxc13 has an important role in controlling hair formation. In this study, we examine the Hoxc13 RNA expression pattern of skin during embryo development. The result indicated that changes of the Hoxe13 gene expression and thickness of skin have a similar trend during hair follicle morphogenesis. In interpreting these results, we investigated whether the regulation motifs is in Hoxc13 intron, which is a 5.4 kb fragment. To blast with other mammals, we found a very conservative region in all mammal animals and two regions in livestock, such as cow, sheep, horse, dog, and so on, which are not in other Hox genes. We have examined putative pre-miRNA in this region, providing an entry point for elucidating currently unknown mechanisms that are required for regulating quantitative levels of Hoxc13 gene expression.

Rat hair follicle stem cells differentiate and promote recovery following spinal cord injury

Emerging studies of treating spinal cord injury （SCI） with adult stem cells led us to evaluate the effects of transplantation of hair follicle stem cells in rats with a compression-induced spinal cord lesion. Here, we proposed a hypothesis that rat hair follicle stem cell transplantation can promote the recovery of injured spinal cord. Compression-induced spinal cord injury was induced in Wistar rats in this study. The bulge area of the rat vibdssa follicles was isolated, cultivated and characterized with nestin as a stem cell marker. 5-Bromo-2＇-deoxyuridine （BrdU） labeled bulge stem cells were transplanted into rats with spinal cord injury. Immunohistochemical staining results showed that some of the grafted cells could survive and differentiate into oligodendrocytes （receptor-interacting protein positive cells） and neuronal-like cells （~lll-tubulin positive cells） at 3 weeks after transplantation. In addition, recovery of hind limb locomotor function in spinal cord injury rats at 8 weeks following cell transplantation was assessed using the Basso, Beattie and Bresnahan （BBB） locomotor rating scale. The results demon- strate that the grafted hair follicle stem cells can survive for a long time period in vivo and differentiate into neuronal- and glial-like cells. These results suggest that hair follicle stem cells can promote the recovery of spinal cord injury.

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

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

Methionine Levels in the Diet for Pregnant Female Rabbits Affects Hair Follicles Development of Baby Rabbits

[ Objective] This experiment was conducted to study methionine level in diet for prepnant famale rabbits on the development of hair follicles of baby rabbits. [ Method] Fifty-four Angora female rabbits with similar body weight, parities and historicaly hair yield were randomly assigned to three groups with eighteen replicates in each group and one rabbit per replicate. Rabbits during pregnancy were fed diets with 0.5!%, 0.71% and 0.91% methionine, respectively. Hair follicle density and diameter in the back skin were determined at 18-day fetus. 26-day fetus, newborn and 2- month-old,and hair fiber diameter at 2-month-old was also determined. [ Result] The results showed as follows： （1） hair follicle density and diameter at each stage,coarse hair fiber diameter at 2-month. old showed a tendency of increasing with dietary methionine level（ P 〉0.05）, fine hair diameter in 0.91% group was significantly larger than that in 0.51% group at 2-month-old （ P 〈 0.05）. （2） With the development of baby rabbit,the ratio of secondary follicle and primary follicle increased gradually, at newborn of rabbits in group 0.51%, 0.71% and 0.91%were 5.84, 5.56 and 5.81, respectively, and at 2-month-old were 8.47, 7.97 and 8.03, respectively. （3） Baby rabbits in group 0.51%, 0.71% and 0.91% gained increases of primary follicle diameter by 0.58, 0.57 and 0.61pm/d from 26-day fetus to birth, and 0.11,0.11 and 0.12 prn/d from birth to 2-month-old, re- spectively; they also gained increases on secondary follicle diameter by 0.0018,0. 012 and 0. 011prn/d from birth to 2-months-old, respectively. [ Conclusion] These results indicate that increase of methionine level in diet for female rabbits during pregnancy is a stimulation factor for the development of hair follicle of baby rabbits, and can improve their hair fiber diameter.

Detailed Histological Structure of Human Hair Follicle Bulge Region at Different Ages: A Visible Niche for Nesting Adult Stem Cells

In the bulge region of the hair follicle, a densely and concentrically packed cell mass is encircled by the arrector pili muscle (APM), which offers a specilized microenvironment (niche) for housing heterogeneous adult stem cells. However, the detailed histological architecture and the cellular composition of the bulge region warrants intensive study and may have implications for the regulation of hair follicle growth regulation. This study was designed to define the gene-expression pro-files of putative stem cells and lineage-specific precursors in the mid-portions of plucked hair follicles prepared according to the presence of detectable autofluorescence. The structure was also characterized by using a consecutive sectioning technique. The bulge region of the hair follicle with autofluorescence was precisely excised by employing a micro-dissection procedure. Semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was performed to identify the gene expression profiles specific for epithelial, melanocyte and stromal stem cells in the bulge region of the hair follicle visualized by autofluorescence. The morphology and its age-dependent changes of bulge region of the hair follicles with autofluorescence segment were also examined in 9 scalp skin specimens collected from patients aged 30 weeks to 75 years, by serial sectioning and immuno-staining. Gene expression profile analysis revealed that there were cells with mRNA transcripts of DctHiTyraseLo-Tyrp1LoMC1RLoMITFLo/K15Hi/NPNTHi in the bulge region of the hair follicle with autofluorescence segments, which differed from the patterns in hair bulbs. Small cell-protrusions that sprouted from the outer root sheath (ORS) were clearly observed at the APM inserting level in serial sections of hair follicles by immunohistological staining, which were characteristically replete with K15+/K19+expressing cells. Likewise, the muscle bundles of APM positive for smooth muscle actin intimately encircled these cell-protrusions, and the occurrence frequency of the cell-protrusions was increased in fetal scalp skin compared with adult scalp skin. This study provided the evidence that the cell-protrusions occurring at the ORS relative to the APM insertion are more likely to be characteristic of the visible niches that are filled with abundant stem cells. The occurrence frequency of these cell-protrusions was significantly increased in fetal scalp skin samples (128%) as compared with the scalp skins of younger (49.4%) and older (25.4%) adults (P<0.01), but difference in the frequency between the two adult groups were not significant. These results indicated that these cell-protrusions function as a niche house for the myriad stem cells and/or precursors to meet the needs of the development of hair follicles in an embryo. The micro-dissection used in this study was simple and reliable in excising the bulge region of the hair follicle with autofluorescence segments dependent on their autofluorescence is of value for the study of stem cell culture.

Dynamic expression of Wnt signaling molecules and stem-cell marker CK15 in the growth cycle of rat whisker hair follicles

Objective To investigate the distribution and dynamic changes of both Wnt signaling molecules and CK15 throughoutthe three phases of the follicular cycle,and to explore the relationship between Wnt/β-catenin signaling and CK15 in rat whisker hair follicle(HF)growth cycles.Methods Hematoxylin-Eosin(HE)and immunofluorescence stains were used to characterize the expression patterns,including sites and levels of some representative proteins of both canonical and non-canonical Wnt signaling molecules,as well as HF epithelial stem cell marker CK15.Results The expression patterns of bothβ-catenin and Wnt5a were correlated with that of CK15.CK15 was only expressed in anagen.In catagen,β-catenin showed a massive depletion while Wnt5a noticeably increased.In telogen,high level expression ofβ-catenin and low level of Wnt5a were detected.Wnt10b and TCF3 were detected during the entire HF growth cycle.Conclusion These results suggest that Wnt5a is associated with the transition of anagen-catagen phase,accompanied by broad deletion ofβ-catenin and loss of CK15.WntlOb is important for the maintenance of HF activity and is related to the telogenanagen transition.

Isolation, Cultivation, and Morphological Characteristics of Hair Follicle Adult Stem Cells in the Bulge Region in Mouse and Human

Skin contains various populations of stem cells (SCs). Among these are hair follicle stem cells (HFSCs) in the bulge region. The behavior of HFSCs deserves to be widely studied due to the benefits to be derived from their identification, isolation, and amplification. Skin samples of newborn mice (n = 32) and human adults (n = 10) were used, and the bulge region was isolated and cultured. The isolation and characterization of cells were conducted through immunocytochemistry and immunofluorescence, using mainly CD34 and CD200 monoclonal antibodies. Initially, cells grew slowly from the explant around the bulge region, accruing cells with different morphology in both mouse and human, latter being mostly polygonal;the mouse cells reaching confluence faster (5 to 7 days) than the human (12 to 15 days). It was possible to isolate into subcultures cells with small size (10 - 13 μm diameter), round-shape, scant cytoplasm, central prominent nucleus and with nucleolus, which formed colonies, maintaining their phenotype in a high proportion (77% - 83% and 91% in mouse and human, respectively), without showing changes in their morphology during almost 7 months in the mouse cells, and a month and a half in the human. These results demonstrate that the selection, the isolation, and the conditioned mediums allowed population increases of bulge cells and indicate that cultured cells may retain their sternness in that they maintained their phenotypic characteristics, expressed specific markers for SCs, and showed a high proliferative capacity for long periods. Hair follicles, in mice and humans, are important repositories of multipotent stem cells, due to their tendency to differentiate into keratinocytes. Human HFSCs, obtained by depilation, preserve their potential for proliferation and prove to be easily accessible. This suggests that the bulge cells may present an alternative source of autologous stem cells for tissue engineering and regenerative medicine.

Constructing skin-equivalents using hair follicle stem cells

Objective： To establish the method of constructing skin-equivalents （SE） using hair follicle stem cells （HFSC）. Methods： K19 positive cells derived from hair were cultivated using serum-free medium KGM and seeded on dermal equivalents （DE）. After the culture between the air-liquid interface for 14 days, SE were harvested and used for evaluation. Results： K19 positive cells chosen as HFSC were located in bulge of out root sheet in hair follicle. Cultivated HFSC could build a fully developed, multi-layered epidermis on the basis of DE, resembling the skin structure. Conclusion： HFSC located in out root sheet can differentiate into kerafinocyte in vitro and be used for SE construction.

Transcriptome sequencing of black and white hair follicles in the giant panda

With the completion of the draft assembly of the giant panda genome sequence,RNA sequencing technology has been widely used in genetic research on giant pandas.We used RNA-seq to examine black and white hair follicle samples from adult pandas.By comparison with the giant panda genome,75963 SNP loci were labeled,2426 differentially expressed genes(DEGs)were identified,and 2029 new genes were discovered,among which 631 were functionally annotated.A cluster analysis of the DEGs showed that they were mainly related to the Wnt signaling pathway,ECM–receptor interaction,the p53 signaling pathway,and ribosome processing.The enrichment results showed that there were significant differences in the regulatory networks of hair follicles with different colors during the transitional stage of hair follicle resting growth,which may play a regulatory role in melanin synthesis during growth.In conclusion,our results provide new insights and more data support for research on the color formation in giant pandas.

Future application of hair follicle stem cells： capable in differentiation into sweat gland cells

Background Sweat glands （SGs） can not regenerate after complete destruction in the severe skin injury, so it is important to find a ideal stem cell source in order to regenerate functional SGs. Hair follicle stem cells （HFSCs） possess the obvious properties of the adult stem cells, which are multipotent and easily accessible. In this research, we attempted to direct the HFSCs suffered from the sweat gland cells （SGCs） special differentiation by a cooperative co- culture system in vitro. Methods The designed co-culture microenvironment in the transwell was consist of two critial factors： heat shocked SGCs and dermis-like mesenchymal tissue, which appeared independently in the two control groups; after induction, the purified induced SGC-like cells were transplanted into the full-thickness scalded wounds of the nude mice, after 4 weeks, the reconstructed SG-like structures were identified by immunohistochemical and immunofluorescence analysis. Results A part of HFSCs in experimental group finally expressed SGCs phenotypes, by contrast, the control group 1 which just containing dermis-like mesenchymal tissue failed and the control group 2 consisted of heat shocked SGCs was in a poor efficiency; by immunofluorescence staining and flow cytometry analysis, the expression of HFSCs special biomarkers was down regulated, instead of the positive efficiency of SGCs special antigens increased; besides, the induced SGCs displayed a high expression of ectodysplasin A （EDA） and ectodysplasin A receptor （EDAR） genes and proteins; after cell transplantation, the youngest SG-like structures formed and be positive in SGCs special antigens, which never happened in untreated wounds （P 〈0.05）. Conclusion The HFSCs are multipotential and capable in differentiating into SGCs which promise a potential stem cells reservoir for future use; our special co-culture microenvironment is promising for HFSCs differentiating; the induced SGCs are functional and could work well in the regeneration of SGs.

Biological characterization of cultured dermal papilla cells and hair follicle regeneration in vitro and in vivo

Background Dermal papilla cells （DPC） are a group of mesenchyme-derived cells at the base of the hair follicle, where they regulate and control hair follicle growth through the expression and secretion of cytokines. Nevertheless, the role of DPC derived chemokines and other cytokines in the hair follicle biology remain speculative. In this study, we investigated the expression of basic fibroblast growth factor （bFGF）, endothelin-1 （ET-1） and stem cell factor （SCF） in different passages of cultured DPC and their effects on the biological behaviour of DPC. Methods The expression of bFGF, ET-1 and SCF in different passages of cultured DPC and their possible effects on the biological behavior of DPC are investigated using in sire hybridization and immunochemistry. In addition, we performed transplantation of hair follicle cells into nude mice. The cultured DPC, dermal sheath cells and fibroblast of human scalp, respectively, were mixed with cells of the hair follicle epithelium in different ratios, and then were cultured in hair follicle organotypic cultures or implanted into the subcutis of nude mice. Results The expression of ET-1 and SCF in early passages of cultured DPC became stronger, but turned weaker and even negative in late passages （〉6 passages）. Hair follicle-like structures were formed after DPC combined with the cells of hair follicle epithelium cells in hair follicle organotypic cultures. When hair follicle organotypic cultures were implanted into the subcutis of nude mice, the relative intact hair follicles were formed. After the transplantation of hair follicle cells into the nude mice, the hair follicle-like structure was formed in the group that contained DPC mixed with hair follicle epithelium cells. However, no hair follicles were formed in the other two groups. It was found that the higher the expression of ET-1 and SCF in DPC, the stronger the ability of DPC to induce hair follicle regeneration. Conclusions The cultured DPC can induce hair follicle regeneration and sustain hair growth in vivo and in vitro. Moreover, the expression of ET-1 and SCF is correlated with the ability of DPC inducing hair follicle regeneration.

Regulation of signaling pathways in hair follicle stem cells

Hair follicle stem cells(HFSCs)reside in the bulge region of the outer root sheath of the hair follicle.They are considered slow-cycling cells that are endowed with multilineage differentiation potential and superior proliferative capacity.The normal morphology and periodic growth of HFSCs play a significant role in normal skin functions,wound repair and skin regeneration.The HFSCs involved in these pathophysiological processes are regulated by a series of cell signal transduction pathways,such as lymphoid enhancer factor/T-cell factor,Wnt/β-catenin,transforming growth factor-β/bone morphogenetic protein,Notch and Hedgehog.The mechanisms of the interactions among these signaling pathways and their regulatory effects on HFSCs have been previously studied,but many mechanisms are still unclear.This article reviews the regulation of hair follicles,HFSCs and related signaling pathways,with the aims of summarizing previous research results,revealing the regulatory mechanisms of HFSC proliferation and differentiation and providing important references and new ideas for treating clinical diseases.

A Single-cell Transcriptome Atlas of Cashmere Goat Hair Follicle Morphogenesis

Cashmere,also known as soft gold,is produced from the secondary hair follicles(SHFs)of cashmere goats.The number of SHFs determines the yield and quality of cashmere;therefore,it is of interest to investigate the transcriptional profiles present during cashmere goat hair follicle development.However,mechanisms underlying this development process remain largely unexplored,and studies regarding hair follicle development mostly use a murine research model.In this study,to provide a comprehensive understanding of cellular heterogeneity and cell fate decisions,single-cell RNA sequencing was performed on 19,705 single cells of the dorsal skin from cashmere goat fetuses at induction(embryonic day 60;E60),organogenesis(E90),and cytodifferentiation(E120)stages.For the first time,unsupervised clustering analysis identified 16 cell clusters,and their corresponding cell types were also characterized.Based on lineage inference,a detailed molecular landscape was revealed along the dermal and epidermal cell lineage developmental pathways.Notably,our current data also confirmed the heterogeneity of dermal papillae from different hair follicle types,which was further validated by immunofluorescence analysis.The current study identifies different biomarkers during cashmere goat hair follicle development and has implications for cashmere goat breeding in the future.

Design of a biofluid-absorbing bioactive sandwich-structured Zn-Si bioceramic composite wound dressing for hair follicle regeneration and skin burn wound healing

The deep burn skin injures usually severely damage the dermis with the loss of hair follicle loss,which are difficult to regenerate.Furthermore,severe burns often accompanied with large amount of wound exudates making the wound moist,easily infected,and difficult to heal.Therefore,it is of great clinical significance to develop wound dressings to remove wound exudates and promote hair follicle regeneration.In this study,a sandwich-structured wound dressing(SWD)with Janus membrane property was fabricated by hot compression molding using hydrophilic zinc silicate bioceramics(Hardystonite,ZnCS)and hydrophobic polylactic acid(PLA).This unique organic/inorganic Janus membrane structure revealed excellent exudate absorption property and effectively created a dry wound environment.Meanwhile,the incorporation of ZnCS bioceramic particles endowed the dressing with the bioactivity to promote hair follicle regeneration and wound healing through the release of Zn^(2+)and SiO^(2-)_(3)ions,and this bioactivity of the wound dressing is mainly attributed to the synergistic effect of Zn^(2+)and SiO^(2-)_(3)to promote the recruitment,viability,and differentiation of hair follicle cells.Our study demonstrates that the utilization of the Janus membrane and synergistic effect of different type bioactive ions are effective approaches for the design of wound dressings for burn wound healing.

Using bioprinting and spheroid culture to create a skin model with sweat glands and hair follicles

Background:Sweat glands(SGs)and hair follicles(HFs)are two important cutaneous appendages that play crucial roles in homeostatic maintenance and thermoregulation,and their interaction is involved in wound healing.SGs can be regenerated from mesenchymal stem cell-laden 3D bioprinted scaffolds,based on our previous studies,whereas regeneration of HFs could not be achieved in the same model.Due to the lack of an in vitro model,the underlying molecular mechanism of the interaction between SGs and HFs in regeneration could not be fully understood.The purpose of the present study was to establish an in vitro model of skin constructs with SGs and HFs and explore the interaction between these two appendages in regeneration.Methods:To investigate the interaction effects between SGs and HFs during their regeneration processes,a combined model was created by seeding HF spheroids on 3D printed SG scaffolds.The interaction between SG scaffolds and HF spheroids was detected using RNA expression and immunofluorescence staining.The effects of microenvironmental cues on SG and HF regeneration were analysed by altering seed cell types and plantar dermis homogenate in the scaffold.Results:According to this model,we overcame the difficulties in simultaneously inducing SG and HF regeneration and explored the interaction effects between SG scaffolds and HF spheroids.Surprisingly,HF spheroids promoted both SG and HF differentiation in SG scaffolds,while SG scaffolds promoted SG differentiation but had little effect on HF potency in HF spheroids.Specifically,microenvironmental factors(plantar dermis homogenate)in SG scaffolds effectively promoted SG and HF genesis in HF spheroids,no matter what the seed cell type in SG scaffolds was,and the promotion effects were persistent.Conclusions:Our approach elucidated a new model for SG and HF formation in vitro and provided an applicable platform to investigate the interaction between SGs and HFs in vitro.This platform might facilitate 3D skin constructs with multiple appendages and unveil the spatiotemporal molecular program of multiple appendage regeneration.

Pyridoxine regulates hair follicle development via the PI3K/Akt, Wnt and Notch signalling pathways in rex rabbits

This study was conducted to evaluate the effect of pyridoxine on the development of hair follicles in Rex rabbits and the underlying molecular mechanism.Two hundred 3-month-old Rex rabbits were randomly divided into 5 groups and fed diets supplemented with 0,5,10,20,or 40 mg/kg pyridoxine.The hair follicle density on the dorsal skin and the gene and protein expression levels of components of the phosphoinositide 3-kinase(PI3 K)/protein kinase B(PKB or Akt),Wnt,Notch and bone morphogenetic protein(BMP)signalling pathways were measured.In addition,free hair follicles were isolated from Rex rabbits and cultured with pyridoxine in vitro to measure hair shaft growth.Furthermore,dermal papilla cells(DPC)were isolated from the skin of Rex rabbits and cultured with pyridoxine in vitro to measure the gene and protein expression levels of components of the PI3 K/Akt,Wnt,Notch and BMP signalling pathways.The results showed that the addition of dietary pyridoxine significantly increased the total follicle density,secondary follicle density,and secondary-to-primary ratio(S/P,P<0.05),that the growth ratio of hair stems was promoted by pyridoxine in basic culture medium,and that the growth length of tentacle hair follicles cultured in the pyridoxine group was longer than that in the control group(P<0.05).In addition,pyridoxine changed the DPC cycle progression and promoted cell proliferation,and appropriate concentrations of pyridoxine(10 and 20μmol/L)significantly inhibited cell apoptosis(P<0.05).Pyridoxine significantly affected the gene expression of components of the PI3 K/Akt,Wnt and Notch signalling pathways in the skin and DPC of Rex rabbits(P<0.05),increased the levels of phosphorylated catenin beta 1(CTNNB1)and Akt,and decreased the level of phosphorylated glycogen synthase kinase 3 beta(GSK-3β)(P<0.05).Therefore,the molecular mechanism by which pyridoxine promotes hair follicle density in Rex rabbits probably occurs through activation of the PI3 K/Akt,Wnt and Notch signalling pathways,prolonging hair follicle growth and delaying the onset of telogen.