Differential and morphogenetic potential of rat dermal papilla cells

Dermal papilla (DP) cells were isolated from rat vibrissae and put into a culture. The homogeneity of the obtained culture was confirmed using immunohistochemical staining with antibodies specific for this type of cell extracellular matrix protein (versican) and staining for alkaline phosphatase. It was demonstrated that the rat vibrissae DP cell culture participates in the development of hair follicles de novo. The ability of the DP culture cells to differentiate in neurons and glia was proved.     

Hair multiplication with dermal papilla like tissue containing human dermal papilla cells

Alopecia is not a critical disease; however it is a disease that can affect the quality of life. Many remedies have been developed to cure alopecia, but only two have been approved by the FDA. Due to the steadily increasing number of young alopecia patients, the need for new therapies for curing alopecia is very high. Recent studies on cell therapy have reported using technique to treat various diseases. We introduce upgraded hair cell therapy which tested hair structure inducing activity with bioartificial dermal papilla tissue. Hair follicles contain two types of stem cells: Outer root sheath cells (ORSCs) derived epithelial cells, and dermal cells (DPCs). In this study, we reconstructed DP-like tissues (DPLTs) using cultured dermal papilla cells (DPCs) from human hair follicles. The DPLTs were produced special media (Dermal Papilla Forming Media: DPFM) conditions in vitro, which can induce epithelial stands from implanted healthy hair without DP. We tested in vivo hair-inducing with a modified hair sandwich model. Two to three weeks DPLT injection into the mouse scalp skin, we observed new hair in the injected site and detected injected human cells from DPLTs and Outer Root Sheath Cells (ORSCs) in the new hair via human Alu-DNA-specific probe. In the future, reconstructed DPLTs may be used in in vitro studies of hair development and the morphogenesis mechanism, as well as in vitro studies of the efficacy and toxicity of drugs for baldness. These tissues will be used as an alternative medicine product for hair transplantation     

Comparative secretome analysis of human follicular dermal papilla cells and fibroblasts using shotgun proteomics

The dermal papilla cells (DPCs) of hair follicles are known to secrete paracrine factors for follicular cells. Shotgun proteomic analysis was performed to compare the expression profiles of the secretomes of human DPCs and dermal fibroblasts (DFs). In this study, the proteins secreted by DPCs and matched DFs were analyzed by 1DE/LTQ FTICR MS/MS, semi-quantitatively determined using emPAI mole percent values and then characterized using protein interaction network analysis. Among the 1,271 and 1,188 proteins identified in DFs and DPCs, respectively, 1,529 were further analyzed using the Ingenuity Pathway Analysis tool. We identified 28 DPC-specific extracellular matrix proteins including transporters (ECM1, A2M), enzymes (LOX, PON2), and peptidases (C3, C1R). The biochemically- validated DPC-specific proteins included thrombospondin 1 (THBS1), an insulin-like growth factor binding protein3 (IGFBP3), and, of particular interest, an integrin beta1 subunit (ITGB1) as a key network core protein. Using the shotgun proteomic technique and network analysis, we selected ITGB1, IGFBP3, and THBS1 as being possible hair-growth modulating protein biomarkers.     

Arctiin blocks hydrogen peroxide-induced senescence and cell death though microRNA expression changes in human dermal papilla cells

Background

Accumulating evidence indicates that reactive oxygen species (ROS) are an important etiological factor for the induction of dermal papilla cell senescence and hair loss, which is also known alopecia. Arctiin is an active lignin isolated from Arctium lappa and has anti-inflammation, anti-microbial, and anti-carcinogenic effects. In the present study, we found that arctiin exerts anti-oxidative effects on human hair dermal papilla cells (HHDPCs).

Results

To better understand the mechanism, we analyzed the level of hydrogen peroxide (H₂O₂)-induced cytotoxicity, cell death, ROS production and senescence after arctiin pretreatment of HHDPCs. The results showed that arctiin pretreatment significantly inhibited the H₂O₂-induced reduction in cell viability. Moreover, H₂O₂-induced sub-G1 phase accumulation and G2 cell cycle arrest were also downregulated by arctiin pretreatment. Interestingly, the increase in intracellular ROS mediated by H₂O₂ was drastically decreased in HHDPCs cultured in the presence of arctiin. This effect was confirmed by senescence associated-beta galactosidase (SA-β-gal) assay results; we found that arctiin pretreatment impaired H₂O₂-induced senescence in HHDPCs. Using microRNA (miRNA) microarray and bioinformatic analysis, we showed that this anti-oxidative effect of arctiin in HHDPCs was related with mitogen-activated protein kinase (MAPK) and Wnt signaling pathways.

Conclusions

Taken together, our data suggest that arctiin has a protective effect on ROS-induced cell dysfunction in HHDPCs and may therefore be useful for alopecia prevention and treatment strategies.     

Impact of taxol on dermal papilla cells--a proteomics and bioinformatics analysis

Dermal papilla (DP) cells play a regulatory role in hair growth, and also play a role in alopecia (hair loss). However, effects of taxol, which is a widely used chemotherapy drug, on DP cells remain unclear, despite that theoretically taxol can impact on DP cells to contribute to taxol-induced alopecia. To better understand pathophysiology of taxol-induced damage in DP cells, morphological and biochemical analyses were performed to check whether taxol can cause apoptosis in cultured DP cells or not. If it can, proteomics and bioinformatics analyses were then performed to investigate the protein networks which are impacted by the taxol treatment. Our data showed that taxol can cause apoptotic damage in DP cells in a concentration-dependant manner, as demonstrated by various apoptotic markers. Proteomic analysis on DP cells treated with the lowest apoptosis-inducible concentration of taxol revealed that taxol can affect expression of proteins involved in Ca2+-regulated biological processes, vesicles transport, protein folding, reductive detoxification, and biomolecules metabolism. Furthermore, bioinformatics analysis indicated that taxol can impact on multiple biological networks. Taken together, this biochemical, proteomics, and bioinformatics data may give an insight into pathophysiology of taxol-induced damage in DP cells and shed light on mechanisms underlying taxol-induced alopecia.     

Different patterns of 5alpha-reductase expression, cellular distribution, and testosterone metabolism in human follicular dermal papilla cells

Androgens regulate hair growth, and 5α-reductase (5αR) plays a pivotal role in the action of androgens on target organs. To clarify the molecular mechanisms responsible for controlling hair growth, the present study presents evidence that the human follicular dermal papilla cells (DPCs) from either beard (bDPCs) or scalp hair (sDPCs) possess endogenous 5αR activity. Real-time RT-PCR revealed that the highest level of 5αR1 mRNA was found in bDPCs, followed by sDPCs, and a low but detectable level of 5αR1 mRNA was observed in fibroblasts. Minimally detectable levels of 5αR2 mRNA were found in all three cell types. A weak band at 26 kDa corresponding to the human 5αR1 protein was detected by Western blot in both DPCs, but not in fibroblasts. Immuonofluorescence analysis confirmed that 5αR1 was localized to the cytoplasm rather than in the nuclei in both DPCs Furthermore, a 5αR assay using [¹⁴C]testosterone labeling in intact cells revealed that testosterone was transformed primarily into androstenedione, and in small amounts, into DHT. Our results demonstrate that the 5αR activities of either bDPCs or sDPCs are stronger than that of dermal fibroblasts, despite the fact that the major steroidogenic activity is attributed to 17β-HSD rather than 5αR among the three cell types. The 5αR1 inhibitor MK386 exhibited a more potent inhibitory effect on 5αR activity than finasteride (5αR2 inhibitor) in bDPCs.     

Gene expression analysis of in vivo fluorescent cells

Background

The analysis of gene expression for tissue homogenates is of limited value because of the considerable cell heterogeneity in tissues. However, several methods are available to isolate a cell type of interest from a complex tissue, the most reliable one being Laser Microdissection (LMD). Cells may be distinguished by their morphology or by specific antigens, but the obligatory staining often results in RNA degradation. Alternatively, particular cell types can be detected in vivo by expression of fluorescent proteins from cell type-specific promoters.

Methodology/Principal Findings

We developed a technique for fixing in vivo fluorescence in brain cells and isolating them by LMD followed by an optimized RNA isolation procedure. RNA isolated from these cells was of equal quality as from unfixed frozen tissue, with clear 28S and 18S rRNA bands of a mass ratio of ∼2∶1. We confirmed the specificity of the amplified RNA from the microdissected fluorescent cells as well as its usefulness and reproducibility for microarray hybridization and quantitative real-time PCR (qRT-PCR).

Conclusions/Significance

Our technique guarantees the isolation of sufficient high quality RNA obtained from specific cell populations of the brain expressing soluble fluorescent marker, which is a critical prerequisite for subsequent gene expression studies by microarray analysis or qRT-PCR.     

Gene expression analysis in post-embryonic pericardial cells of Drosophila

Increasing evidence suggests conservation of cardiovascular molecules between vertebrates and invertebrates. Vertebrate Rudhira, an evolutionary conserved WD40 protein is expressed during primitive erythropoiesis, neoangiogenesis and tumors. We report here the expression profile of the Drosophila ortholog of Rudhira (DRudh) in the fly life cycle. DRudh is expressed specifically in all post-embryonic pericardial cells (PCs) and garland cells (GCs). This is the first report of a cytoplasmic marker highly specific to post-embryonic PCs. Embryonic PCs belong to three distinct genetic classes based on Odd-skipped (Odd), Even-skipped (Eve) and Tinman (Tin) expression. To identify which among these three classes of PCs expresses DRudh in post-embryonic stages, we analyzed expression of embryonic PC markers in the post-embryonic stages. Unlike in the embryo all larval PCs show an identical gene expression profile. While Odd and Eve expression is mutually exclusive in the embryonic PCs, these two markers are co-expressed in larval PCs but show a distinct subcellular localization. Tin is not expressed in any post-embryonic PC. Additionally larval PCs also express the GATA factor, Serpent (Srp) and the extracellular matrix protein, Pericardin (Prc). While PC number is known to decrease post-embryogenesis, which of the Odd or Eve lineage embryonic PCs persists is not known. Co-expression of the two distinct lineage markers only in post-embryonic stages indicates a complex temporal regulation of gene expression in PCs.     

Gene expression analysis following hypoxia-reoxygenation in rat gastric epithelial cells using a high-density oligonucleotide array

Recent investigations have demonstrated that the signaling of hypoxia-re-oxygenation is a major contributing pathway leading to gastric mucosal injury induced by stress, non-steroidal anti-inflammatory drugs, and Helicobacter pylori. The aim of the present study was to perform a gene expression analysis on the gastric mucosal cellular response to hypoxia-reoxygenation using a high-density oligonucleotide array. Cells were subjected to hypoxia with 95% N(2) and 5% CO(2) at 37 degrees C for 2 h. Reoxygenation was initiated by placing the cells in an environment of normoxia for 2 h. Total RNA was extracted, and differences in gene expression profiles between the normoxia and hypoxia-reoxygenation groups were investigated using a GeneChip of Rat Toxicology U34 array (Affymetrix). Hypoxia-reoxygenation up-regulated the stress-related genes (heat shock protein-70 [HSP-70], catalase). The enhanced expression of HSP-70 was confirmed by Western blot analysis. In conclusion, these results suggest that up-regulation of the HSP-70 gene after reoxygenation may play a role in maintaining cell survival and supporting cell function as a molecular chaperone.     

Gene expression microarray analysis of early oxygen-induced retinopathy in the rat

Different inbred strains of rat differ in their susceptibility to oxygen-induced retinopathy (OIR), an animal model of human retinopathy of prematurity. We examined gene expression in Sprague–Dawley (susceptible) and Fischer 344 (resistant) neonatal rats after 3 days exposure to cyclic hyperoxia or room air, using Affymetrix rat Genearrays. False discovery rate analysis was used to identify differentially regulated genes. Such genes were then ranked by fold change and submitted to the online database, DAVID. The Sprague–Dawley list returned the term “response to hypoxia,” absent from the Fischer 344 output. Manual analysis indicated that many genes known to be upregulated by hypoxia-inducible factor-1α were downregulated by cyclic hyperoxia. Quantitative real-time RT-PCR analysis of Egln3, Bnip3, Slc16a3, and Hk2 confirmed the microarray results. We conclude that combined methodologies are required for adequate dissection of the pathophysiology of strain susceptibility to OIR in the rat.     

Gene expression in rat brain

191 randomly selected cDNA clones prepared from rat brain cytoplasmic poly (A)+ RNA were screened by Northern blot hybridization to rat brain, liver and kidney RNA to determine the tissue distribution, abundance and size of the corresponding brain mRNA. 18% hybridized to mRNAs each present equally in the three tissues, 26% to mRNAs differentially expressed in the tissues, and 30% to mRNAs present only in the brain. An additional 26% of the clones failed to detect mRNA in the three tissues at an abundance level of about 0.01%, but did contain rat cDNA as demonstrated by Southern blotting; this class probably represents rare mRNAs expressed in only some brain cells. Therefore, most mRNA expressed in brain is either specific to brain or otherwise displays regulation. Rarer mRNA species tend to be larger than the more abundant species, and tend to be brain specific; the rarest, specific mRNAs average 5000 nucleotides in length. Ten percent of the clones hybridize to multiple mRNAs, some of which are expressed from small multigenic families. From these data we estimate that there are probably at most 30,000 distinct mRNA species expressed in the rat brain, the majority of which are uniquely expressed in the brain.     

Optimization of the reconstruction of dermal papilla like tissues employing umbilical cord mesenchymal stem cells

Alopecia is not life threatening, but patients who undergo alopecia often experience severe mental stress. In addition, the number of individuals afflicted by alopecia has been increasing steadily. The most effective treatment of alopecia developed to date is auto hair transplantation. To overcome the limitations associated with current therapies for the treatment of alopecia, many researchers have attempted to revive hair follicles by in vitro culture of hair follicle cells and subsequent implantation in the treatment area. Previously, we demonstrated that umbilical cord-derived mesenchymal stem cells (UC-MSCs) could be isolated and expanded successfully from the Wharton’s Jelly. Cultureexpanded UC-MSCs formed aggregates similar to native dermal papilla (DP) in special media (DPFM) and reconstructed dermal papilla like tissues (DPLTs) could induce new hair follicles. The purpose of the present study was to optimize the reconstruction of DPLTs. As in the case of MSCs, when compared to differentiated cells, DPLTs require an additional step to induce differentiation into dermal papilla cells. However, it is necessary to use hepatocyte growth factor (HGF) in the differentiation step, which is relatively expensive. To reduce the expenses associated with cell therapy using MSCs, it is necessary to optimize this differentiation step. To accomplish this, we evaluated the effects of cell inoculation density and growth factors during differentiation.     

Gene expression analysis

The response of cells to extracellular signals usually requires altered expression of many genes, possibly including several distinct metabolic pathways. In some cases, only a subset of genes involved in such responses are known, which requires techniques to analyze changes in the expression of multiple genes, both known and unknown. Three techniques, two-dimensional gel electrophoresis, differential display, and gene discovery arrays, provide opportunities for measuring changes in gene expression levels, as well as for identifying novel gene products.     

DigiNorthern,digital expression analysis of query genes based on ESTs

DigiNorthern (DN) is a web-based tool for virtually displaying expression profiles of query genes based on EST sequences. Two utilities are available: DN1 takes one query gene and quantitatively display its expression levels in tissues/organs that express the gene with comparison between normal and neoplastic status of each tissue; DN2 takes two sequences as query genes and compares their expression profiles side by side.