Human Palatal Mesenchymal Cells Research Articles

Exosomes Derived from Human Palatal Mesenchymal Cells Mediate Intercellular Communication During Palatal Fusion by Promoting Oral Epithelial Cell Migration.

Exosomes are important "messengers" in cell-cell interactions, but their potential effects on palatal fusion are still unknown. This study aimed to explore the role and mechanism of exosomes derived from palatal mesenchymal cells in epithelial-mesenchymal communication during palatogenesis. The expression of exosome marker CD63 and CD81 in palatal cells during palatogenesis was detected by immunofluorescence staining. After being purified from the supernatant of human embryonic palatal mesenchymal (HEPM) cells, exosomes (HEPM-EXO) were characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and Western blot. HEPM-EXO were co-cultured with human immortalized oral epithelial cells (HIOEC). The effects of HEPM-EXO on the cell proliferation, migration, apoptosis and epithelial-mesenchymal transition (EMT) of HIOEC were evaluated. The proteins encapsulated in HEPM-EXO were analyzed by proteomic analysis. The extensive expression of CD63 and CD81 in palatal epithelial and mesenchymal cells were continuously detected during E12.5~E14.5, suggesting that exosomes were involved in the process of palatal fusion. The expression of CD63 was also observed in the acellular basement membrane between the palatal epithelium and the mesenchyme in vivo, and HEPM-EXO could be internalized by HIOEC in vitro, suggesting that exosomes are potent to diffuse through the cellular tissue boundary to mediate palatal cell-cell communication. Exposure of HEPM-EXO to HIOEC substantially inhibited the proliferation and stimulated the migration of HIOEC, but had no significant effect on cell apoptosis and EMT. Proteomic analysis revealed the basic characteristics of the proteins in HEPM-EXO and that exosomal THBS1 may potentially regulate the cell behaviors of HIOEC, which needs further verification. Gene ontology (GO) analysis uncovered that the proteins highly expressed in HEPM-EXO are closely related to wound healing, implying a promising therapeutic opportunity of HEPM-EXO in tissue injury treatment with future studies. HEPM-EXO mediated cell-cell communication by regulating cell proliferation and migration of oral epithelial cells during palatogenesis.

MicroRNA-374a, -4680, and -133b suppress cell proliferation through the regulation of genes associated with human cleft palate in cultured human palate cells

BackgroundCleft palate (CP) is the second most common congenital birth defect; however, the relationship between CP-associated genes and epigenetic regulation remains largely unknown. In this study, we investigated the contribution of microRNAs (miRNAs) to cell proliferation and regulation of genes involved in CP development.MethodsIn order to identify all genes for which mutations or association/linkage have been found in individuals with CP, we conducted a systematic literature search, followed by bioinformatics analyses for these genes. We validated the bioinformatics results experimentally by conducting cell proliferation assays and miRNA-gene regulatory analyses in cultured human palatal mesenchymal cells treated with each miRNA mimic.ResultsWe identified 131 CP-associated genes in the systematic review. The bioinformatics analysis indicated that the CP genes were associated with signaling pathways, microRNAs (miRNAs), metabolic pathways, and cell proliferation. A total 17 miRNAs were recognized as potential modifiers of human CP genes. To validate miRNA function in cell proliferation, a main cause of CP, we conducted cell proliferation/viability assays for the top 11 candidate miRNAs from our bioinformatics analysis. Overexpression of miR-133b, miR-374a-5p, and miR-4680-3p resulted in a more than 30% reduction in cell proliferation activity in human palatal mesenchymal cell cultures. We found that several downstream target CP genes predicted by the bioinformatics analyses were significantly downregulated through induction of these miRNAs (FGFR1, GCH1, PAX7, SMC2, and SUMO1 by miR-133b; ARNT, BMP2, CRISPLD1, FGFR2, JARID2, MSX1, NOG, RHPN2, RUNX2, WNT5A and ZNF236 by miR-374a-5p; and ERBB2, JADE1, MTHFD1 and WNT5A by miR-4680-3p) in cultured cells.ConclusionsOur results indicate that miR-374a-5p, miR-4680-3p, and miR-133b regulate expression of genes that are involved in the etiology of human CP, providing insight into the association between CP-associated genes and potential targets of miRNAs in palate development.

Thirdhand smoke: Chemical dynamics, cytotoxicity, and genotoxicity in outdoor and indoor environments

We tested the toxicity of thirdhand smoke (THS) using two controlled laboratory exposure scenarios and low levels of THS. One exposure modeled THS in a car parked outdoors, while the second modeled THS in a room without sunlight. The fabrics were exposed to cigarette smoke and then extracted in culture medium. Concentrations of nicotine, nicotine related alkaloids, and tobacco specific nitrosamines (TSNAs) were determined in fresh and aged extracts. The concentration of TSNAs increased with aging in the indoor experiment. THS extracts were used for cytotoxicity testing using mouse neural stem cells (mNSC), human dermal fibroblasts (hDF) and human palatal mesenchyme cells (hPM). Extracts from the car experiment inhibited mNSC proliferation in a live cell imaging assay and induced single strand DNA breaks in mNSC and hDF. In the indoor experiment, THS extracts made with medium containing serum proteins were significantly more toxic than extracts made with basal medium, and mNSC and hPM were more sensitive than hDF. These data indicate that: (1) aging of THS chemical differs on different fabrics and differs with and without sunlight; (2) very few cigarettes are sufficient to produce a toxic THS residue; and (3) protein enhances the efficiency of extraction of cytotoxic chemicals.

Biotinylated Biodegradable Nanotemplated Hydrogel Networks for Cell Interactive Applications

We describe the synthesis of a novel biotinylated nanotextured degradable hydrogel that can be rapidly surface engineered with a diverse range of biotinylated moieties. The hydrogel is synthesized by reacting methacrylated biotin-PEG with dimethacrylated P LA-b- PEG-b-P LA (LPLDMA, PEG = poly(ethylene glycol), PLA = poly(lactic acid)),or dimethacrylated PEG-b-P LA-b- PEG (PLPDMA). Methacrylated biotin-PEG is prepared by reacting biotin-PEG-OH with methacrylic anhydride. Biotin-PEG-OH is prepared by reacting alpha-hydroxy-omega-amine PEG with N-hydroxysuccinimide-biotin. Confirmation of the final product is determined using (1)H NMR and Fourier transform infrared spectroscopy (FTIR). The integrity and surface presentation of the biotin units is observed spectrophotometrically using the HABA/avidin assay. To produce nanostructured polymer topography, a self-assembling lyotropic liquid crystalline mesophase is used as a polymerization template, generating biotinylated hydrogels with highly organized lamellar matrix geometry. Traditionally processed isotropic hydrogels are used for comparison. Scanning electron microscopy shows that isotropic hydrogels have a smooth glassy appearance while lamellar templated hydrogels have defined surface topographical features that enhance preosteoblast human palatal mesenchymal cell (HEPM) attachment. Engineering the surfaces of the hydrogels with cell adhesive Arg-Gly-Asp (RGD) peptide sequences using the biotin-avidin interaction significantly enhances cell attachment. Surface engineering of cell adhesive peptides in conjunction with the lamellar template induced surface topography generates additive enhancements in cell attachment.

Spatial control over cell attachment by partial solvent entrapment of poly lysine in microfluidic channels

We demonstrate spatial control over cell attachment on biodegradable surfaces by flowing cell adhesive poly (D-lysine) (PDL) in a trifluoroethanol (TFE)-water mixture through microfluidic channels placed on a biodegradable poly (lactic acid)-poly (ethylene glycol) (PLA-PEG) substrate. The partial solvent mixture swells the PLA-PEG within the confines of the microfluidic channels allowing PDL to diffuse on to the surface gel layer. When excess water is flowed through the channels substituting the TFE-water mixture, the swollen PLA surface collapses, entrapping PDL polymer. Results using preosteoblast human palatal mesenchymal cells (HEPM) indicate that this new procedure can be used for facile attachment of cells in localized regions. The PEG component of the PLA-PEG copolymer prevents cells from binding to the nonpatterned regions.

Expression and characterization of recombinant TGF-beta 2 proteins produced in mammalian cells.

Recombinant DNA plasmids coding for transforming growth factor beta 2 (TGF-beta 2) precursor and a hybrid TGF-beta 1(NH2)/beta 2(COOH) molecule consisting of the amino-terminal precursor portion of transforming growth factor-beta 1 (TGF-beta 1) linked in phase to the carboxyl terminus of mature TGF-beta 2 were constructed and transfected into COS cells. Both plasmids directed the synthesis of active TGF-beta 2 which was secreted into the supernatants of transfected cells. The TGF-beta 2 was secreted in a latent form, as an acidification step was required to demonstrate optimal biological activity. Using site-specific anti-peptide antibodies, we show that precursor and mature forms of TGF-beta 2 are produced. A stable Chinese hamster ovary (CHO) cell line expressing the hybrid TGF-beta 1(NH2)/beta 2(COOH) protein was isolated. This cell line secreted both precursor and mature forms of TGF-beta 1(NH2)/beta 2(COOH); acidification was required to demonstrate biological activity. Protein sequence analysis of recombinant TGF-beta 2 produced by this CHO clone demonstrated that correct proteolytic cleavage had occurred, suggesting that the processing signals contained within the TGF-beta 1 amino portion can function in producing mature TGF-beta 2. Receptor binding studies showed that TGF-beta 2 specifically bound predominantly to type III receptors on the surface of human palatal mesenchymal cells. The availability of active TGF-beta 2 should aid in determining its potential therapeutic use as a growth modulator.

Human foetal palatal corticoid receptors and teratogens for cleft palate.

CLEFT secondary palate can be induced experimentally in various mammalian species by the administration of glucocorticoids to pregnant animals1–4. The well known differences in susceptibility to cortisone-induced cleft palate in inbred strains of mice have been explained in terms of genetic differences in foetal facial mesenchymal glucocorticoid receptor levels5,6. The quantity of one protein in particular, with an isoelectric point (pI) of approximately 7.0, seems to be correlated rather closely with the susceptibility of various strains to cortisone-induced cleft palate6. It has also been shown that much of the difference in cleft palate susceptibility between C57BL/10 (B10) and A/J mice can be accounted for by H–2-linked genes7,8, and that there is H–2-linked variation in the level of cortisol-binding proteins obtained from the cytosol fraction of foetal palatal tissue6. As corticoids form a specific cytoplasmic cortisol receptor complex in target tissues which interacts with the genome resulting in activation or depression of transcription9,10, a product of a gene in or near the H–2 locus seems to be the glucocorticoid receptor, the level of which has to be raised for cleft palate to occur. As a first step to determine whether such a mechanism may operate in humans, we have examined whether human palatal mesenchymal cells, obtained in cultures from foetuses shortly after the critical period of secondary palatal closure, contain glucocorticoid receptors. We show here that these cells have specific glucocorticoid receptor proteins with high affinity for dexamethasone and triamcinolone acetonide, and that dexamethasone is displaced from its binding sites only by drugs which produce cleft palate in sensitive mouse strains. Thus, the human foetus contains an active glucocorticoid receptor mechanism immediately after the critical period of palatal organogenesis, which may be involved in palatal clefting.