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.

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.

Coexistence of esophageal blue nevus, hair follicles and basaloid sqamous carcinoma:A case report

We present the case of a 57-year-old man who underwent esophagectomy for esophageal carcinoma found at barium meal and gastroscopic examination. He was diagnosed as esophageal basaloid squamous carcinoma （BSC） and gastric stromal tumor, which were associated with focal proliferation of melanocytes/ pigmentophages and hair follicles in esophageal mucosa. Melanocytic hyperplasia （melanocytosis） has previously been recognized as an occasional reactive lesion, which can accompany esophageal inflammation and invasive squamous carcinoma. The present case is unusual because of its hyperplasia of not only melanocytes but also hair follicles. To our knowledge, this is the first report of esophageal blue nevus and hair follicle coexisting with BSC.

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.

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.

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.

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.

Comparative characterization and osteogenic/adipogenic differentiation of mesenchymal stem cells derived from male rat hair follicles and bone marrow

Clinical applications of cell therapy and tissue regeneration under different conditions need a multiplicity of adult stem cell sources.Up to date,little is available on the comparative isolation,characterization,proliferation,rapid amplification,and osteogenic/adipogenic differentiation of rat mesenchymal stem cells(MSCs)isolated from living bulge cells of the hair follicle(HF)and bone marrow(BM)from the same animal.This work hopes to use HF-MSCs as an additional adult stem cell source for research and application.After reaching 80%confluence,the cell counting,viability%,and yields of HF-MSCs and BM-MSCs were nearly similar.The viability%was 91.41±2.98 and 93.11±3.06 while the cells yield of initial seeding was 33.15±2.76 and 34.22±3.99 and of second passage was 28.76±1.01 and 29.56±3.11 for HF-MSCs and BM-MSCs respectively.Clusters of differentiation(CDs)analysis revealed that HF-MSCs were positively expressed CD34,CD73 and CD200 and negatively expressed CD45.BM-MSCs were positively expressed CD73 and CD200 and negatively expressed of CD34 and CD45.The proliferation of HFMSCs and BM-MSCs was determined by means of incorporation of Brd-U,population doubling time(PDT)assays and the quantity of formazan release.The percentage of Brd-U positive cells and PDT were relatively similar in both types of cells.The proliferation,as expressed by the quantity of formazan assay in confluent cells,revealed that the quantity of release by BM-MSCs was slightly higher than HF-MSCs.Adipogenic differentiated BM-MSCs showed moderate accumulation of oil red-O stained lipid droplets when compared to that of HF-MSCs which exhibited high stain.The total lipid concentration was significantly higher in adipogenic differentiated HF-MSCs than BMMSCs(P<0.05).It was found that activity of bone alkaline phosphatase and calcium concentration were significantly higher(P<0.01 and P<0.05 respectively)in osteogenic differentiated BM-MSCs than that of HF-MSCs.The present findings demonstrate that the HF-MSCs are very similar in most tested characteristics to BM-MSCs with the exception of differentiation.Additionally;no issues have been reported during the collection of HF-MSCs.Therefore,the HF may represent a suitable and accessible source for adult stem cells and can be considered an ideal cell source for adipogenesis research.

Peptide Cy_(RL-QN15)accelerates hair regeneration in diabetic mice by binding to the Frizzled-7 receptor

Individuals with diabetes frequently face serious challenges,including delayed wound healing and increased risk of infection.Notably,the regeneration of hair follicles plays a crucial role in accelerating diabetic skin damage repair,reducing the risk of infection,and enhancing overall skin health.Research has predominantly emphasized the re-epithelialization of diabetic wounds,with a paucity of in-depth studies on hair follicle regeneration.In the current study,we explored the effects of a bioactive amphibian-derived peptide,Cy_(RL-QN15),on promoting hair regeneration in a diabetic skin model.In vivo experiments demonstrated that local treatment with Cy_(RL-QN15)not only accelerated wound healing of scalded skin on the backs of diabetic Kunming(KM)mice but also improved growth of damaged hair follicles.Additionally,back-shaved diabetic C57BL/6 mice showed a significant increase in the growth of newly formed hair after 28 days of continuous Cy_(RL-QN15)treatment.Further analysis indicated that the hairregenerating effects of Cy_(RL-QN15)were closely associated with the proliferation and migration of hair follicle stem cells(HFSCs).Cy_(RL-QN15)enhanced intracellularβ-catenin expression by binding to the Frizzled-7 receptor on the surface of HFSCs.The up-regulation inβ-catenin modulated the levels of downstream proteins,such as cMYC,Cyclin D1,and Lef1,ultimately inducing hair regeneration.This study not only reveals the robust effects of the bioactive peptide Cy_(RL-QN15)in hair follicle regeneration but also provides novel avenues for the development of more targeted and effective therapeutics for diabetic wound healing in the future.

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.

A review of methods for evaluating effectiveness of cosmetic products and ingredients based on mechanisms of the human hair growth cycle

With the increasing incidence of hair loss and especially among younger population in recent years,the demand for diversified products on anti-hair loss solutions is growing and the market of cosmetic products expanding accordingly.Worldwide attention is being paid to the research and development of anti-hair loss actives,while there is no unified standard for the efficacy evaluation and testing methods of anti-hair loss cosmetics.Therefore,it is critical to establish standardized methodology for assessing the effectiveness of anti-hair loss actives and their cosmetic products.In this review,an overview of the human hair follicle structure,the hair follicle cycle,and the biological pathways and signaling mechanisms involved in hair growth are summarized.This provides the fundamental understanding of human hair growth for reviewing anti-hair loss actives and cosmetic hair loss products,and respective methods and techniques applied for evaluating their efficacy.Additionally,changes in the normal hair cycle,and corresponding cosmetic strategies to slow down and reduce hair loss have been provided.This review aims to provide recommendations for evaluating the effectiveness of anti-hair loss actives on the mechanisms involved in the hair follicle growth cycle,and so provide criteria for new technology development and scientific research for cosmetic products.

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.

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.

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.

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.

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.