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Isolation and characterization of hair follicle stem cells from Arbas Cashmere goat.

ABSTRACT: In this study, highly purified hair follicle stem cells from Arbas Cashmere goat (gHFSCs) were isolated using enzyme digestion and adhesion to type IV collagen. The biological characteristics of the gHFSCs were identified by morphological observation, growth curve, markers assay and differentiation in vitro. The gHFSCs were in small cell size with typical cobblestone morphology, good adhesion and high refractive index. Immunocytochemistry staining showed the cells were expressing Krt15, Krt19, CD34, Itg?1 and Krt14. Cell growth curve indicated that cultured gHFSCs had strong proliferation ability. Krt14 and CD34 were high expressed at the mRNA level, respectively, 39.68 and 24.37 times of the Cashmere goat keratinocytes, and krt15 expression was 5.62 times and itg?1 expression was 1.81 times higher (p < 0.01). Western blot detected the expression of all the above markers. After osteogenic induction, the cells were positive for Von Kossa staining and expressed Osteocalcin. Sulfated proteoglycans in cartilaginous matrices were positively stained by Alcian blue after chondrogenic induction and COL2A1 was expressed. In myogenic induction, Hoechst 33342 staining evidenced cytoplasm fusion and positive expression of MyoG was detected by immunocytochemistry.

SUBMITTER: He N

PROVIDER: S-EPMC5101329 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Isolation and characterization of hair follicle stem cells from Arbas Cashmere goat.

He Nimantana N Dong Zhenguo Z Tao Li L Zhao Sirguleng S Bou Shorgan S Liu Dongjun D

Cytotechnology 20160518 6

In this study, highly purified hair follicle stem cells from Arbas Cashmere goat (gHFSCs) were isolated using enzyme digestion and adhesion to type IV collagen. The biological characteristics of the gHFSCs were identified by morphological observation, growth curve, markers assay and differentiation in vitro. The gHFSCs were in small cell size with typical cobblestone morphology, good adhesion and high refractive index. Immunocytochemistry staining showed the cells were expressing Krt15, Krt19, C ...[more]

PMID: 27193423

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Project description:Cashmere goat from Inner Mongolia is an excellent local breed in China, and the related cashmere product is a kind of precious textile raw material with high price. Cashmere is generated from secondary hair follicles, which has obvious annual periodicity and includes three different stages: anagen, catagen, and telogen. Therefore, we investigated skin transcriptome data for 12 months using weighted gene co-expression network analysis (WGCNA) to explore essential modules, pathways, and genes responsible for the periodic growth and development of secondary hair follicles. A total of 17 co-expression modules were discovered by WGCNA, and there is a strong correlation between steelblue module and month (0.65, p = 3E-09), anagen (0.52, p = 1E-05), telogen (-0.6, p = 8E-08). Gene expression was generally high during late anagen to catagen (June to December), while expression was downregulated from telogen to early anagen (January-May), which is similar to the growth rule of hair follicle cycle. KEGG pathway enrichment analyses of the genes of steelblue module indicated that genes are mainly enriched in Cell cycle, Wnt signaling pathway, p53 signaling pathway and other important signal pathways. These genes were also significantly enriched in GO functional annotation of the cell cycle, microtubule movement, microtubule binding, tubulin binding, and so on. Ten genes (WIF1, WNT11, BAMBI, FZD10, NKD1, LEF1, CCND3, E2F3, CDC6, and CDC25A) were selected from these modules, and further identified as candidate biomarkers to regulate periodic development of hair follicles using qRT-PCR. The Wnt signaling pathway and Cell cycle play an important role in the periodic development of hair follicles. Ten genes were identified as essential functional molecules related to periodic development of hair follicle. These findings laid a foundation for understanding molecular mechanisms in biological functions such as hair follicle development and hair growth in cashmere goats.

Project description:Studies in humans and mice have revealed that hair follicle morphogenesis relies on tightly coordinated ectodermal-mesodermal interactions, involving multiple signals and regulatory factors. DNA methylation and long non-coding RNA (lncRNA) play a critical role in early embryonic skin development by controlling gene expression. Acting as an indirect regulator, lncRNA could recruit DNA methyltransferases to specific genomic sites to methylate DNA. However, the molecular regulation mechanisms underlying hair follicle morphogenesis is unclear in cashmere goat. In this study, RNA-seq and whole-genome bisulfite sequencing (WGBS) in embryonic day 65 (E 65) and E 120 skin tissues of cashmere goat were used to reveal this complex regulatory process. The RNA-seq, qRT-PCR, and immunohistochemistry results showed that Wnt signaling played an important role in both hair follicle induction and differentiation stage; transcriptional factors (TFs), including HOXC13, SOX9, SOX21, JUNB, LHX2, VDR, and GATA3, participated in hair follicle differentiation via specific expression at E 120. Subsequently, the combination of WGBS and RNA-seq analysis showed that the expression of some hair follicle differentiation genes and TF genes were negatively correlated with the DNA methylation level generally. A portion of hair follicle differentiation genes were methylated and repressed in the hair follicle induction stage but were subsequently demethylated and expressed during the hair follicle differentiation stage, suggesting that DNA methylation plays an important role in hair morphogenesis by regulating associated gene expression. Furthermore, 45 upregulated and 147 downregulated lncRNAs in E 120 compared with E 65 were identified by lncRNA mapping, and then the potential differentially expressed lncRNAs associated with DNA methylation on the target gene were revealed. In conclusion, critical signals and genes were revealed during hair follicle morphogenesis in the cashmere goat. In this process, DNA methylation was lower in the hair follicle differentiation compared with the hair follicle induction stage and may play an important role in hair morphogenesis by regulating associated gene expression. Furthermore, potential lncRNAs associated with DNA methylation on target genes were delineated. This study enriches the regulatory network and molecular mechanisms on hair morphogenesis.

Dataset Information

Isolation and characterization of hair follicle stem cells from Arbas Cashmere goat.

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Isolation and characterization of hair follicle stem cells from Arbas Cashmere goat.

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