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.

Effects of Different Culture Condition in vitro on Growth of Cashmere Goat Primary Hair Follicles

[Objective] The aim of this study is to lay a foundation for illustrating the biological characteristics and growth regulation mechanism of hair follicles.[Method]Cashmere goat primary hair follicles were separated under aseptic condition and cultured in serum-free DMEM and serum-free Williams E media respectively;subsequently,the growth rate and morphological changes were observed under the inverted microscope.[Result]Hair follicles cultured in serum-free DMEM media showed a growth rate of 0.034 mm/d during the first 3 days,whose structure and morphological characteristics could maintian a stable status for a long time in the growth process.Hair follicles grew much faster in the serum-free Williams E media with a growth rate of 0.077 mm/d during the first 3 days.[Conclusion]There were significant differences(P<0.05)between the growth of cashmere goat hair follicles cultured in the 2 kinds of media.Serum-free Williams E medium was superior to serum-free DMEM medium.

Effects of Estradiol on Cashmere Goat Primary Hair Follicles Cultured in vitro

[Objective] The aim of this study was to preliminarily explore the effects of estradiol on morphology and growth of cashmere goat primary hair follicles. [Method] Cashmere goat primary hair follicles were cultured in serum-free Williams E media supplemented with different doses of 17 β-E2 (0, 0.1, 1.0, 10.0, 100.0 nmol/L), and their growth rates and morphological changes were observed. [Result] The growth rate of 0.1 nmol/L 17 β-E2 group was quite comparable with that of the control group(0 nmol/L), but the 17 β-E2 with concentrations of 1.0, 10.0 and 100.0 nmol/L displayed different degrees of inhibition on the growth of hair follicles. Different morphological changes of hair follicles could also be discovered in different concentration treatments. [Conclusion] The study laid a certain foundation for exploring the regulation mechanism of estrogen on growth of cashmere goat hair follicles.

Comparison on Several Methods for Extracting DNA from Raccoon Dog Hair Follicle

[Objective] This study aimed to investigate a reliable method for DNA ex- traction from Wusuli raccoon dog＇s hair. [Method] Several DNA extraction methods were used to extract DNA from Wusuli raccoon dog hair, including Chelex-100 method, PCR buffer method, organic phenol-chloroform method and centrifugal col- umn type kit method. The extracted DNA was analyzed by using PCR amplification and electrophoresis to compare these four DNA extraction methods. [Result] Accord- ing to the results of spectrophotometer detection and gel electrophoresis, nucleic acid extracted by Chetex-100 method had proteins and other impurities; nucleic acid ex- tracted by PCR buffer method was low in concentration; however, DNA extracted by organic phenol-chloroform method and centrifugal column type kit was high in con- centration with no impurity band. [Conclusion] This study had laid the strong founda- tion of scientific theory to further explore the efficient and simple method for extracting DNA from Wusuli raccoon dog hair follicle.

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.

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.

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.

Hair follicle stem cells: In vitro and in vivo neural differentiation

Hair follicle stem cells(HFSCs) normally give rise to keratinocytes, sebocytes, and transient amplifying progenitor cells. Along with the capacity to proliferate rapidly, HFSCs provide the basis for establishing a putative source of stem cells for cell therapy. HFSCs are multipotent stem cells originating from the bulge area. The importance of these cells arises from two important characteristics, distinguishing them from all other adult stem cells. First, they are accessible and proliferate for long periods. Second, they are multipotent, possessing the ability to differentiate into mesodermal and ectodermal cell types. In addition to a developmental capacity in vitro, HFSCs display an ability to form differentiated cells in vivo. During the last two decades, numerous studies have led to the development of an appropriate culture condition for producing various cell lineages from HFSCs. Therefore, these stem cells are considered as a novel source for cell therapy of a broad spectrum of neurodegenerative disorders. This review presents the current status of human, rat, and mouse HFSCs from both the cellular and molecular biology and cell therapy perspectives. The first section of this review highlights the importance of HFSCs and in vitro differentiation, while the final section emphasizes the significance of cell differentiation in vivo.

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.

Dynamic Wnt5a expression in murine hair follicle cycle and its inhibitory effects on follicular in vivo

Objective:To analyze the dynamic expression of Wnt family member 5A(Wingless-type MMTV integration Wnt site family,member 5a)in murine hair cycle and its inhibitory effects on follicle in vivo.Methods:Situ hybridization in full-thickness skin was used to observe the change of mouse protein expression in different growth stages,and Ad-Wnt5a was injected after defeathering to observe the hair follicle growth in vivo.Results:The Wnt5a mRNA was expressed at birth,and was firstly increased then decreased along with the progress of the hair cycle.It reached the peak in advanced stage of growth cycle(P<0.05).Rhoa andβ-catenin expression levels were significantly decreased in three groups.Rac2 expression was significantly up-regulated,and the expression level of Wnt5a,Shh and Frizzled2 was increased,but less significantly than group 2.Conclusions:The expression of Wnt5a mRNA is consistent with change of murine follicle cycle,and has obvious inhibitory effects on the growth of hair follicle in vivo,indicating that it is antagonistic to Wnts pathway and interferes the growth of follicle together.

NANOG Alleviates the Damage of Human Hair Follicle Mesenchymal Stem Cells Caused by H_2O_2 through Activation of AKT Pathway

Objective To explore the protective effect of NANOG against hydrogen peroxide(H_2O_2)-induced cell damage in the human hair follicle mesenchymal stem cells(hHF-MSCs). Methods NANOG was expressed from a lentiviral vector, pLVX-IRES-ZsGreen. NANOG hHF-MSCs and vector hHF-MSCs were treated with 400 μmol/L hydrogen peroxide(H_2O_2) for 2 h, the cell survival rate, cell morphology, ROS production, apoptosis and expression of AKT, ERK, and p21 were determined and compared. Results Our results showed that NANOG could activate AKT and upregulate the expression of p-AKT, but not p-ERK. When treated with 400 μmol/L H_2O_2, NANOG hHF-MSCs showed higher cell survival rate, lower ROS production and apoptosis, higher expression of p-AKT, higher ratio of p-AKT/AKT. Conclusion Our results suggest that NANOG could protect hHF-MSCs against cell damage caused by H_2O_2 through activating AKT signaling pathway.

Cyclosporine A stimulated hair growth from mouse vibrissae follicles in an organ culture model

Hypertrichosis is one of the most common side effects of systemic cyclosporine A therapy.It has been previously shown that cyclosporine A induces anagen and inhibits catagen development in mice.In the present study,to explore the mechanisms of cyclosporine A,we investigated the effects of cyclosporine A on hair shaft elongation,hair follicle cell proliferation,apoptosis,and mRNA expression of selected growth factors using an organ culture model of mouse vibrissae.In this model,cyclosporine A stimulated hair growth of normal mouse vibrissae follicles by inhibiting catagen-like development and promoting matrix cell proliferation.In addition,cyclosporine A caused an increase in the expression of vascular endothelial growth factor（VEGF）,hepatocyte growth factor（HGF）,and nerve growth factor（NGF）,and inhibited follistatin expression.Our findings provide an explanation for the clinically observed effects of cyclosporine A on hair growth.The mouse vibrissae organ culture offers an attractive model for identifying factors involved in the modulation of hair growth.

Flavonoids in safflower extract reduce cisplatin-induced damage to human follicle dermal papilla cells by inhibiting DNA damage and Rad17/Chk1/Cdc25C signaling

Background:Cisplatin is a chemotherapeutic agent commonly used clinically for the treatment of various human cancers.Patients often reduce the use of cisplatin due to its side effects,which in turn affects its treatment.This study explored the mechanism of action of safflower extract as an adjuvant traditional Chinese medicine for chemotherapy.Methods:Primary human follicle dermal papilla cells(HFDPCs)were used as target cells for cisplatininduced damage to hair cells.Western blotting was used to investigate the molecular targets of cisplatin and safflower extract in causing HFDPCs damage.Cell survival and cell cycle were analyzed by mitochondrial staining reagent WST-1 and propidium iodide.Results:Cisplatin could reduce the viability of HFDPCs without causing cell death.Cisplatin increased the level of phospho-Rad17 in HFDPCs and activated the Chk1/Cdc25C signaling to reduce the expression of Cdc2 protein,thereby arresting the cells in the G2/M phase.The combination of safflower extract and the flavonoids could effectively inhibit the signal transduction of Rad17/Chk1/Cdc25 in cisplatin-treated cells and reduce the cell population in the G2/M phase.Finally,we also confirmed that safflower extract could effectively inhibit the damage to HFDPCs caused by cisplatin,mainly at the level of reducing the DNA damage caused by cisplatin.Conclusions:Safflower extract can be used as an adjuvant Chinese medicine for chemotherapy to reduce the damage caused by chemotherapy to normal hair follicle cells.

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.

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.

Histological Characteristics of Hair Follicle Structure of Hezuo Swine from Gansu Province

The characteristics of skin hair follicle structure of Hezuo swine were in- vestigated using frozen section method and HE staining. Experimental results showed that the hair root, hair tip, outer sheath, inner sheath, hair follicle group, fi- brous sheath, outer epidermis, inner epidermis, sebaceous gland and other organiza- tional structures of hair follicles of Hezuo swine can be clearly observed. The hair follicle pore size varied extremely significantly （P〈0.01） among different parts, showing a downward trend of shoulder 〈 body side 〈 buttocks; the hair follicle density ranged from 5.59 to 7.26 hair follicles/mm^2. This study provides reference for the in-dustrial and medical applications.

Vitamin B12 Activates the Wnt-Pathway in Human Hair Follicle Cells by Induction of <i>beta</i>-Catenin and Inhibition of Glycogensynthase Kinase-3 Transcription

Background and Objectives: Micrograft transplantation is accompanied by a transient induction of telogen in transplanted hair follicles (HF), which might be avoided by supporting the metabolic pathways of the micrograft during the ex vivo period. Vitamin B12 (cobalamin) has been suggested to influence HF growth and cycling in humans, but the mechanisms are unclear. Method: HFs were obtained from patients undergoing routine micrograft transplantation and were cultured for 5 days in Dulbecco’s modified Eagles Medium, supplemented with different amounts of vitamin B12. Hair shaft elongation (HSE) of the isolated HFs as well as quantitative changes of mRNA for beta-catenin, glykogensynthase kinase-3 (GSK-3) and TCF/Lef-1 in HF cells were determined. Results: In vitro HSE demonstrated a dose dependent induction of HSE after stimulation with 2.5 ug/ml and 25 ug/ml vitamin B12 (6.2 +/- 2.1% and 15.4 +/- 3.8% respectively). A dose dependent induction of beta-catenin-mRNA could be demonstrated in cultured HFs after stimulation with 2.5 ug/ml and 25 ug/ml vitamin B12 (fold change compared to DMEM: 9.5 +/- 2.7, p < 0.05 and 23.1 +/- 7.4, p < 0.01;respectively). Concomitantly the amounts of GSK-3 were significantly reduced after stimulation with 25 ug/ml vitamin B12 (fold change compared to DMEM: 0.76 +/- 0.12, p < 0.05). Conclusions: Our data demonstrate a hair growth promoting effect of vitamin B12 in vitro. This effect is accompanied by the modulation of intracellular signal transduction molecules of the wnt-pathway and might promote hair growth after micrograft transplantation.

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.