Establishment of an immortalized mouse dermal papilla cell strain with optimized culture strategy.
Ontology highlight
ABSTRACT:
SUBMITTER: Guo H
PROVIDER: S-EPMC5788059 | biostudies-other | 2018
REPOSITORIES: biostudies-other
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Project description:Alopecia is an exceedingly prevalent problem and lacks effective therapy. Recently, research has focused on early-passage dermal papilla cells (DPCs), which have hair inducing activity both in vivo and in vitro. Our previous study indicated that factors secreted from early-passage DPCs contribute to hair follicle (HF) regeneration. To identify which factors are responsible for HF regeneration and why late-passage DPCs lose this potential，we collected 48-h-culture medium (CM) from both of passage 3 and 9 DPCs and subcutaneously injected the DPC-CM into NU/NU mice. Passage 3 DPC-CM induced HF regeneration, based on the emergence of a white hair coat, but passage 9 DPC-CM not. In order to identify the key factors responsible for hair inductive capacity, CM from passage 3 and 9 DPCs was analyzed by iTRAQ-based quantitative proteomic technology. We identified 1360 proteins, of which 213 proteins were differentially expressed between CM from early-passage vs. late-passage DPCs, including SDF1, MMP3, biglycan and LTBP1.Further analysis indicated that the differentially-expressed proteins regulated the Wnt, TGF-β and BMP signaling pathways, which directly and indirectly participate in HF morphogenesis and regeneration. Subsequently, we selected 19 proteins for further verification by multiple reaction monitoring (MRM) between the two types of CM. These results would be used to identify the key factors for inducing HF regeneration and reveal the molecular mechanisms of losing inductive ability of DPCs. Furthermore, it is possible to explore some drugs for alopecia in the clinic according to this secretome date analysis.
2018-10-26 | PXD004201 | Pride
Project description:Androgenetic alopecia (AGA) is the most common type of hair loss, and is mainly caused by the biological effects of testosterone on dermal papilla cells (DPCs). In vitro culturing of DPCs might be a useful tool for the screening of target molecule of AGA. However, primary DPCs cannot continuously proliferate owing to cellular senescence and cell culture stress. In this study, we introduced mutant cyclin-dependent kinase 4 (CDK4), Cyclin D1, and telomerase reverse transcriptase (TERT) into DPCs. We confirmed protein expression of CDK4 and Cyclin D1, and enzymatic activity of TERT. Furthermore, we found the established cell line was free from cellular senescence. We also introduced the androgen receptor gene using a recombinant retrovirus, to compensate the transcriptional suppressed endogenous androgen receptor in the process of cell proliferation. Furthermore, we detected the efficient nuclear translocation of androgen receptor into the nucleus after the treatment of dihydrotestosterone, indicating the functionality of our introduced receptor. Our established cell line is a useful tool to identify the downstream signaling pathway, which activated by the testosterone.
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Project description:CONTEXT:The dermal papilla (DP) is a condensation of mesenchymal cells at the proximal end of the hair follicle, which determines hair shaft size and regulates matrix cell proliferation and differentiation. DP cells have the ability to regenerate new hair follicles. These cells tend to aggregate both in vitro and in vivo. This tendency is associated with the ability of papilla cells to induce hair growth. However, human papilla cells lose their hair-inducing activity in later passage number. Ovine DP cells are different from human DP cells since they do not lose their aggregative behavior or hair-inducing activity in culture. Nonetheless, our understanding of ovine DP cells is still limited. AIM:The aim of this study was to observe the expression of established DP markers in ovine cells and their association with aggregation. SUBJECTS AND METHODS:Ovine DP cells from three different sheep were compared. Histochemistry, immunoflourescence, and polymerase chain reaction experiments were done to analyze the DP markers. RESULTS:We found that ovine DP aggregates expressed all the 16 markers evaluated, including alkaline phosphatase and versican. Expression of the versican V0 and V3 isoforms, neural cell adhesion molecule, and corin was increased significantly with aggregation, while hey-1 expression was significantly decreased. CONCLUSIONS:Overall, the stable expression of numerous markers suggests that aggregating ovine DP cells have a similar phenotype to papillae in vivo. The stability of their molecular phenotype is consistent with their robust aggregative behavior and retained follicle-inducing activity after prolonged culture. Their phenotypic stability in culture contrasts with DP cells from other species, and suggests that a better understanding of ovine DP cells might provide opportunities to improve the hair-inducing activity and therapeutic potential of human cells.
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Project description:Bone morphogenetic protein 2 (Bmp2) is essential for odontogensis and dentin mineralization. Generation of floxed Bmp2 dental mesenchymal cell lines is a valuable application for studying the effects of Bmp2 on dental mesenchymal cell differentiation and its signaling pathways during dentinogenesis. Limitation of the primary culture of dental mesenchymal cells has led to the development of cell lines that serve as good surrogate models for the study of dental mesenchymal cell differentiation into odontoblasts and mineralization. In this study, we established and characterized immortalized mouse floxed Bmp2 dental papilla mesenchymal cell lines, which were isolated from 1st mouse mandibular molars at postnatal day 1 and immortalized with pSV40 and clonally selected. These transfected cell lines were characterized by RT-PCR, immunohistochemistry, and analyzed for alkaline phosphatase activity and mineralization nodule formation. One of these immortalized cell lines, iBmp2-dp, displayed a higher proliferation rate, but retained the genotypic and phenotypic characteristics similar to primary cells as determined by expression of tooth-specific markers as well as demonstrated the ability to differentiate and form mineralized nodules. In addition, iBmp2-dp cells were inducible and responded to BMP2 stimulation. Thus, we for the first time described the establishment of an immortalized mouse floxed Bmp2 dental papilla mesenchyma cell line that might be used for studying the mechanisms of dental cell differentiation and dentin mineralization mediated by Bmp2 and other growth factor signaling pathways.
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Project description:Transcriptome analysis of DP signature gene expression in hTERT-immortalized balding (BAB) and non-balding (BAN) dermal papilla cells derived from frontal and occipital scalp of male patients with androgenetic alopecia Hamilton grade IV.
2016-04-20 | GSE66663 | GEO
Project description:To search hair growth-promoting herbal extract, a screening platform of having HEK293T fibroblast being transfected with pTOPFLASH DNA construct was developed over a thousand of herbal extracts and phytochemicals were screened. One of the hits was ethanolic extract of Rhizoma Belamcandae, the rhizome of Belamcanda chinensis (L.) DC. Tectoridin, an isoflavone from Rhizoma Belamcandae, was shown to be responsible for this activation of promoter construct, inducing the transcription of pTOPFLASH in the transfected fibroblasts in a dose-dependent manner. The blockage by DKK-1 suggested the action of tectoridin could be mediated by the Wnt receptor. The hair growth-promoting effects of tectoridin were illustrated in human follicular dermal papilla cells and mouse vibrissae organ cultures. In tectoridin-treated dermal papilla cultures, an activation of Wnt signaling was demonstrated by various indicative markers, including TCF/LEF1 transcriptional activity, nuclear translocation of β-catenin, expressions level of mRNAs encoding axin-related protein, (AXIN2), β-catenin, lymphoid enhancer-binding factor-1 (LEF-1), insulin-like growth factor 1 (IGF-1) and alkaline phosphatase (ALP). In addition, an increase of hair shaft elongation was observed in cultured mouse vibrissae upon the treatment of tectoridin. Tectoridin, as well as the herbal extract of Rhizoma Belamcandae, possesses hair promoting activity, which deserves further development.
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Project description:The reprogramming of somatic fibroblasts into alternative cell linages could provide a promising source of cells for regenerative medicine and cell therapy. However, the direct conversion of fibroblasts into other functional cell types is still challenging. In this study, we show that dermal-papilla-cell-like cells (DPC-LCs) can be generated by treating fibroblasts, including L929 mouse fibroblast cell lines and somatic mouse fibroblasts, with small molecules. Based on alkaline phosphatase activity and other molecular markers, different compounds or their combinations are needed for converting the two different fibroblasts into DPC-LCs. Notably, we found that TTNPB alone can efficiently convert primary adult mouse fibroblasts into DPC-LCs. DPC-LCs generated from mouse fibroblasts showed a stronger hair-inducing capacity. Transcriptome analysis reveals that expression of genes associated with a hair-inducing capacity are increased in DPC-LCs. This pharmacological approach to generating functional dermal papilla cells may have many important implications for hair follicle regeneration and hair loss therapy.
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Project description:The dermal papilla is a cluster of mesenchymal cells located at the base of the hair follicle which have a number of important roles in the regulation of hair growth. As a consequence, in vitro models of these cells are widely used to study the molecular mechanisms which underlie hair follicle induction, growth and maintenance. While dermal papilla from rodent hair follicles can be digested prior to cell isolation, the unique extracellular matrix composition found in human dermal papilla renders enzymes such as trypsin and collagenase insufficient for digestion of the dermal papilla into a single cell suspension. As such, to grow human dermal papilla cells in vitro, the papilla has to first be isolated via a micro-dissection approach from the follicle. In this article we describe the micro-dissection and culture methods, which we use within our laboratory, for the study of human dermal papilla cells.
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Project description:Recent development of hepatitis B virus (HBV) culture systems has made it possible to analyze the almost all steps of the viral life cycle. However, the reproducibility of interaction between HBV and host cells seemed inaccurate in those systems because of utilization of cancer cell lines with a difference from hepatocytes in the majority of cases. In this study, in order to resolve this point, a novel HBV culture system using non-cancer-derived immortalized human hepatocytes derived cell lines, producing exogenous human sodium taurocholate cotransporting polypeptide, was developed. One of the cell clones, E/NtG8 cells, was permissive to both blood-borne HBV (HBVbb) and culture-derived recombinant HBV when cultured in the three-dimensional condition. Furthermore, the production of infectious HBV particles, which showed the similar physicochemical properties to HBVbb, was observed for about a month after HBVbb infection in this system, suggesting that it may reproduce whole steps of the HBV lifecycle under the condition analogous to human liver cells infected with HBV. This system seemed to contribute not only to find novel interactions between HBV and host cells but also to understand mechanism of HBV pathogenesis.
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