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     

Myogenic differentiation of dermal papilla cells from bovine skin

Cells from the dermal papilla and dermal sheath of hair follicles exhibit pronounced plasticity in vitro, being capable of adopting fat, bone, hematopoietic, and nerve cell phenotypes. In this study, we show that bovine dermal papilla cells (DPC) are also capable of undergoing skeletal muscle differentiation. DiI labeled DPC incorporated into myotubes when co-cultured with differentiating C(2)C(12) myoblasts. Bovine-specific PCR assays showed that the muscle markers MyoD and myogenin were up-regulated, confirming that the DPC had adopted a myogenic gene expression program. Nine clonal lines of DPC underwent both adipogenic and myogenic differentiation, demonstrating the multipotency of individual cells. Primary populations of both DPC and extra-follicular dermal fibroblasts were also capable of both adipogenic and myogenic differentiation. However, on myogenic differentiation, cells derived from dermal papillae expressed higher levels of myogenin than primary fibroblasts derived from extra-follicular dermis, suggesting that papilla cells undergo myogenesis more efficiently. This result shows that populations of fibroblastic cells derived from different anatomical sites within the skin are not equivalent with respect to their plasticity. Cultured DPC and dermal fibroblasts both expressed Pax3, a marker for the dermomyotome which represents a common embryological origin of muscle and dermis. Quantitative PCR showed that Pax3 expression levels before myogenic induction correlated with myogenin expression levels after myogenesis. These results suggest that a degree of dedifferentiation may underlie the plasticity of dermal cells in vitro, and that this plasticity may be predicted, at least in part, by levels of Pax3 expression.     

Cloning and expression of human GTDC1 gene (glycosyltransferase-like domain containing 1) from human fetal library

The glycosyltransferases (GTs) catalyze the synthesis of the carbohydrate portions of glycoproteins, glycolipids, and proteoglycans. Here we report the cloning and characterization of a novel human GTDC1 (glycosyltransferase-like domain containing 1) gene, which locates on human chromosome 2q22. The GTDC1 cDNA is 2954 bp in length, encoding a putative protein of 458 amino acids. At protein level human GTDC1 has 75 and 37% identity with its homologous counterparts in the mouse and fruitfly, respectively. RT-PCR analysis revealed its relatively high expression level in the adult lung, spleen, testis, and peripheral blood leukocyte.     

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.     

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.     

Differentiation capacity of stromal fibroblast-like cells from human bone marrow, adipose tissue, hair follicle dermal papilla and derma

We compared the morphology and differentiation capacity of human stromal cells derived from bone marrow (BMSC), adipose tissue (ATSC), hair follicle dermal papilla (DPC) and dermal fibroblasts (DFb). All cells have fibroblast-like morphology. ATSC and DPC cells expressed stem cell the surface markers CD105, CD49d, and STRO-1, which were revealed immunocytochemically. CD49d was not found on BMSC. The low expression of CD49d and STRO-1 was registered in the DFb population. ATSC, BMSC, and DPC have similar capacities for adipo- and osteogenic differentiation. These cells, cultured in appropriate induction media, alter the phenotype and synthesize specific proteins. However, the expression of differentiation in the DPC population is lower than in ATSC and BMSC cultures. We propose that these cell populations have primitive progenitor cells with properties of mesenchymal stem cells.     

Preservation of a specialized phenotype of dermal papilla cells of a human hair follicle under cultivation conditions

Possible ways to extend cultivation of dermal papilla cells without the loss of expression of their specific markers were studied. The effect of extracellular matrix components, as well as valproic acid, on the maintenance of the phenotype of dermal papilla cells was studied for the first time. Two ways of cultivation (in a monolayer culture and in spheroids) were used. It was established that a short-term positive effect is reached during the addition of the BMP6 growth factor and vitamin D₃ in the monolayer culture, while cultivation in spheroids or in the presence of valproic acid allows us to preserve most efficiently the initial phenotype of these cells in vitro. The significance of the results obtained for tissue engineering and for the study of regeneration processes is discussed.     

Cloning and pharmacological characterization of a novel gene encoding human nucleoside transporter 1 (hNT1) from a human breast cancer cDNA library

AimsWe isolated a novel gene encoding human nucleoside transporter 1 (hNT1), from a human breast cancer cDNA library.Main methodsA nondirectional cDNA library was screened by an EST clone (GenBank?/EMBL/DDBJ: BU944345). A Xenopus laevis oocyte expression system was used for functional characterization. Membrane localization in the human breast was determined by immunohistochemistry.Key findingsIsolated hNT1 cDNA consisted of 246 base pairs that encoded an 82-amino acid protein. By RT-PCR analysis, hNT1 mRNA was strongly detected in the breast cancer tissues. When expressed in X. oocytes, hNT1 mediated the high affinity transport of [³H]5-fluorouracil (5-FU) with a K_m value of 69.2 ± 24.5 nM in time- and pH-dependent, and Na⁺-independent manners. A cis-inhibition experiment revealed that hNT1 mediated transport of [³H]5-FU is strongly inhibited by various nucleosides such as pyrimidine, uracil, uridine, guanosine, inosine, thymidine, adenosine, cytidine and purine suggesting that hNT1 may be involved in the trans epithelial transport of these endogenous substrates. Immunohistochemical analysis revealed that the hNT1 protein is localized in the lactiferous duct epithelium.SignificanceOur present results indicate that a newly isolated cDNA clone, hNT1, is a key molecule for the breast handling of 5-FU in humans.     

Cloning and characterization of a novel human leptin receptor overlapping transcript-like 1 gene (LEPROTL1)

A new full-length cDNA encoding a novel protein was isolated from our human fetal brain cDNA library. The cDNA consists of 2701 bp and has a putative open reading frame encoding 131 amino acids which possesses a JAK binding site (Pro(46)-Ile-Pro(48) which is preceded by a cluster of hydrophobic residues) and is highly homologous to the leptin receptor gene-related protein (OB-RGRP). Northern blot analysis showed that this new gene is widely expressed in human tissues and radiation hybrid mapping placed the gene to human chromosome 8p21.1-8p21.2.     

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.     

Gene expression in rat dermal papilla cells: analysis of 2529 ESTs

Dermal papilla (DEPA) cells are resident at the base of hair follicles and are fundamental to hair growth and development. Cultured DEPA cells, in contrast to normal fibroblast cells, are capable of inducing de novo hair follicle growth in vivo. By differential screening of a DEPA cDNA library, we have demonstrated that dermal papilla cells are different from fibroblasts at the molecular level. We further studied these cells by random sequencing of 5130 clones from the DEPA cDNA library. Fifty percent had a BLASTX E value < or =1 x 10(-25). Twenty-one percent had similarity to proteins involved in cell structure/motility with 4 of the top 10 most abundant clones encoding extracellular matrix proteins. Clones encoding growth factor molecules were also abundant. The remaining 50.7% of clones had low similarity scores, demonstrating many novel molecules. For example, we identified a new CTGF family member, the rat homologue of Elm1.     

Hydroxytyrosol prevents dermal papilla cells inflammation under oxidative stress by inducing autophagy

Hydroxytyrosol (HT), a primary phenolic antioxidant in olive oil, can afford protection from oxidative stress (OS) in different cells, including skin cells. In particular, it regulates several inflammation‐associated processes as well as in improving the antioxidant defense system. However, there is no information about HT used in the treatment of hair loss. This work aimed at exploring the potential protective actions of HT against OS in rat dermal papilla cells. After treatment, the related expression of protein and messenger RNA were detected using morphological and molecular analyses. The results showed that HT significantly reduced intracellular reactive oxygen species level, apoptotic markers and inflammation induced by OS and enhanced cell survival by regulating autophagy. Furthermore, HT enhanced the secretion of hair growth factors in the anti‐inflammation process. These results suggest that HT has a significant protective ability against OS and encourage the use of this biological ingredient as a possible tool to prevent alopecia.     

Valproic acid induces hair regeneration in murine model and activates alkaline phosphatase activity in human dermal papilla cells

BACKGROUND: Alopecia is the common hair loss problem that can affect many people. However, current therapies for treatment of alopecia are limited by low efficacy and potentially undesirable side effects. We have identified a new function for valproic acid (VPA), a GSK3β inhibitor that activates the Wnt/β-catenin pathway, to promote hair re-growth in vitro and in vivo. METHODOLOGY/ PRINCIPAL FINDINGS: Topical application of VPA to male C3H mice critically stimulated hair re-growth and induced terminally differentiated epidermal markers such as filaggrin and loricrin, and the dermal papilla marker alkaline phosphatase (ALP). VPA induced ALP in human dermal papilla cells by up-regulating the Wnt/β-catenin pathway, whereas minoxidil (MNX), a drug commonly used to treat alopecia, did not significantly affect the Wnt/β-catenin pathway. VPA analogs and other GSK3β inhibitors that activate the Wnt/β-catenin pathway such as 4-phenyl butyric acid, LiCl, and BeCl(2) also exhibited hair growth-promoting activities in vivo. Importantly, VPA, but not MNX, successfully stimulate hair growth in the wounds of C3H mice. CONCLUSIONS/ SIGNIFICANCE: Our findings indicate that small molecules that activate the Wnt/β-catenin pathway, such as VPA, can potentially be developed as drugs to stimulate hair re-growth.     

Oscillation of human period 1 (hPER1) reporter gene activity in human neuroblastoma cells in vivo

The mammalian period (Per) genes, which are components of the circadian clock, are mainly regulated via an autoregulatory feedback loop. Here we provide evidence that human Per1 (hPER1) reporter gene activity shows circadian rhythmicity in a human neuroblastoma, but not in a astrocytoma or a hepatoma cell line. Medium change and various pharmacological stimuli differentially induce this behavior. This circadian oscillation was strongly dampened and could be followed over maximally three cycles. It was even possible to phase-shift the course of this oscillation by repeated application of stimuli.