| Hair follicle (HF) is a dynamic mini organ, not only having important biological functions such as prevention of skin tissue damage, important sensory and immunologic functions, but also containing diverse stem cells for infinite proliferation, differentiation, hair growth regulation, and skin homeostasis maintenance. Hair follicle stem cells (HFSCs) are required to initiate, maintain and renew the continuously HF cycle. Ever since their identification in mice, the biology of HFSCs has become a promising frontier in the dermatology field, HF field, and also in regenerative medicine.The Inner Mongolia Arbas white Cashmere goat(Capra hircus) is a local cashmere and meat dual-purpose breed, which produces fine, soft, quality cashmere. The Arbas cashmere goats have primary and secondary two different types of HFs with obvious hair cycle and promptly becoming a popular model for hair follicle morphogenesis research.Transcription factor Sox9 has vital functions in the embryonic development and three layer differentiation, stem cell determination and maintenance, and congenital diseases. In the previous years, researchers found Sox9 in the formation of the hair placode and morphogenesis of the hair follicles in the early embryo.However, domestic animal HFSCs remain unclear compared to human and murine HFSCs and there are few studies about Arbas Cashmere goat HFSCs (gHFSCs). Therefore, in this study, we isolated and characterized gHFSCs, analyzed its pluripotency; preliminary verified the function of Sox9 in the gHFSCs and performed chromatin immunoprecipitation (ChIP) and ChIP-seq to explore the mechanism of Sox9 aiming to provide experimental material and basis for the future hair follicle research.1. Isolation and identification of the gHFSCsThe gHFSCs were isolated using tissue adhesion and purified with type IV collagen. The biological characteristics of gHFSCs were identified by morphological observation, FACS, growth curve, markers assay and differentiation in vitro. The results showed that the cells were in small cell size with typical cobblestone-like morphology, good adhesion capacity and high refractivity, and no significant morphological changes were appeared after 20 passages. Immunocytochemistry results showed the cells were positively expressed Krt15,Krt19, CD34, Itgβ1 and Krt14. FASC positive rate of CD34 was 99.8%. It was obvious from the cell growth curve that cultured gHFSCs have strong proliferation ability. Krtl4 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, Itgβ1 expression was lower,1.81 times (p<0.01). Similarly, western blot detected different expression of all of the above markers in the gHFSCs. After osteogenic induction, calcium nodules were positive for Von Kossa staining and Osteocalcin was expressed. Sulfated proteoglycans in cartilaginous matrices were positively stained by Alcian blue and Col2A1 was expressed after chondrogenic differentiation. In myogenic induction, Hoechst33342 staining observed cytoplasm fusion and positive expression of MyoG was detected by immunohistochemistry. After osteogenic and cartilage induction, the cells were positively stained by Von Kossa and alcian blue, respectively. The cells positively expressed MyoG after myogenic differentiation and cell fusion was observed by Hoechst33342 staining.2. Analysis of pluripotency and stemness of gHFSCsWe analyzed the expression of pluripotency and self-renewal associated factors, such as Oct4, Nanog, Sox2, AKP and TERT by Immunocytochemistry, FACS, Q-PCR and Western blot. Immunocytochemistry result showed that the gHFSCs were positive for all five markers; FASC positive rate of Oct4, Nanog and Sox2 was greater than 99.9%. Compared with Arbas Cashmere goat adipose-derived stem cells (gADSCs) at the mRNA expression level, Oct4 was relatively highly expressed in gHFSCs,41.36 times of the gADSCs, and Nanog was 5.61, AKP was 2.74, and TERT was 2.10 times, respectively (p<0.01). Western blot indicated that all markers expressed at the protein level in the gHFSCs. When compared with gADSCs, using a-Tubulin as a reference protein, gray intensity analysis showed that the expression of Oct4, Nanog, AKP, and TERT were respectively 5.94,10.78,1.33 and 1.39 times of gADSCs. Additionally, mRNA and protein expression of Sox2 were detected in the gHFSCs but not in the gADSCs.3. The function of Sox9 in the gHFSCsWe identified Sox9 expression in the gHF and gHFSCs using frozen section histochemistry, immunocytochemistry, Q-PCR and Western blot. Then, Sox9 expression in the gHFSCs was interfered by Sox9-shRNA vector and detected genes related with proliferation, differentiation, gHFSCs maintenance, and pluripotency at the mRNA and protein expression level. We also detected Sox9 and Loricrin expression after calcium induction culture.The results showed that Sox9 expressed in the outer root sheath of the gHF, focus to the bulge region, and also expressed weakly in the matrix. The immunocytochemistry showed that the gHFSCs were positive for Sox9 and Q-PCR and Western blot also detected the expression of Sox9 at the mRNA and protein level. The Sox9-shRNA vector interference efficiency was 53.58%. After Sox9 expression interference:Cell proliferation got slow, the cells cannot go into logarithmic phase and with increasing cell apoptosis; Flow cytometry analysis showed that the cells cannot go into G2/M phase form S phase; Q-PCR analysis showed that the relative mRNA expression of p21 and PCNA was reduced significantly (p<0.01), and p53 expression had no difference (p>0.05), and relative expression of terminal differentiation marker Lor was increased significantly (p<0.01). The gHFSCs not only lost the hair follicle stem cells morphological characteristics but also the mRNA and protein expression of the gHFSCs bibmarkers were reduced significantly (p<0.01). The relative mRNA and protein expression of stem cell pluripotency markers Oct4, Nanog, Sox2, AKP and TERT were decreased significantly (p<0.01). When compared with normal cultured gHFSCs, low calcium cultured cells had no significant morphological changes while cells cultured with high calcium changed into long spindle like morphology. Western blot indicated that Sox9 expression was reduced and Lor expression was increased after high calcium culture, and in the normal and low calcium cultured cells Sox9 and Lor expression had no differences.4. Finding out Sox9 binding genes by ChIP and ChIP-seq assayChIP is the only means to study protein and DNA interaction. We obtained protein-DNA complex by Sox9-ChIP and sequenced by ChIP-seq assay to find out the proteins which interact with Sox9.After the ChIP-seq of purified DNA-protein complex from sonification, we got 10736 MACS-peaks, and 2008 peaks were in exonic region,2827 peaks were in intronic region,5433 peaks were in intergenic region,48 in downstream,61 in upstream,292 in UTR3 region,28 in UTR5 region and 39 in splicing region, respectively; 36 motifs were obtained from de novo finding, and the top motif matches with Sox9 in vitro motif; 96 known motifs including well-defined stem cell pluripotency associated transcription factors Nanog、Oct4、Sox2、Tcf were found using GO database and motif database; We got 278 pathways after KEGG-pathway analysis, and 24 pathways were significantly enriched (p<0.05) including FoxO signaling pathway, MAPK signaling pathway, cAMP signaling pathway, signaling pathway regulating pluripotency of stem cells and actin cytoskeleton regulation etc.9 highly significant (p<0.01) pathways, including Focal adhesion, Adherens junction, Rap1 signaling pathway, Wnt signaling pathway and Hippo signaling pathway. |
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