Characterization of gene expression profile of dental pulp stem cells from human exfoliated deciduous teeth (SHED) in down syndrome

Single cell gene expression profiling of cortical osteoblast lineage cells

Characterization of gene expression profile in rat Kupffer cells stimulated with IFN-α or IFN-γ

Characterization of Gene Expression Profiles of Plasma Cells in Patients with Multiple Myeloma.

Global Expression Analysis of Prostate Cancer-associated Stroma and Epithelia

Neonatal neuroinflammation, driven by microglial activation and cytokine signaling, contributes to brain injury and adverse neurodevelopment outcomes. Perinatal inflammatory mediators, including interleukin-6, cyclooxygenase-2, and interleukin-17, prime microglia and influence circuit vulnerability. This study investigated whether oxytocin pretreatment attenuates lipopolysaccharide-induced inflammatory priming in BV-2 microglial cells. BV-2 microglia were preincubated with oxytocin (33 ng/mL) for 2 h, followed by lipopolysaccharide (0.5 µg/mL) for 2 h. Expression of ionized calcium-binding adapter molecule 1, a microglia marker, in BV-2 cells was assessed by immunofluorescence. After lipopolysaccharide treatment, the gene expression of BV-2 cells was assayed at 1, 2, and 6 h post stimulation by RT-qPCR and RNA-seq. Functional characterization of gene expression profile was performed. Analyses of gene expression profile of BV-2 cells by RT-qPCR and RNA-seq revealed that oxytocin pretreatment attenuated lipopolysaccharide-induced transcriptional activation, including interleukin-6 and cyclooxygenase-2 upregulation. Pathway enrichment analyses suggested that oxytocin-responsive genes were linked to the interleukin-17 signaling pathway. Gene Ontology enrichment analysis showed enrichment for genes related to cytokine production, membrane raft, and chemokine activity. Oxytocin pretreatment mitigates lipopolysaccharide-induced microglial activation by modulating the interleukin-17–interleukin-6/cyclooxygenase-2 axis, suggesting its potential role for oxytocin as an endogenous modulator of neuroinflammation during early brain development.

Our preclinical experiments indicated that Romidepsin (Depsipeptide FK228; DP) mediates growth arrest and apoptosis in cultured lung cancer cells. A phase II trial was done to examine clinical and molecular responses mediated by this histone deacetylase inhibitor in lung cancer patients. Nineteen patients with neoplasms refractory to standard therapy received 4-h DP infusions (17.8 mg/m(2)) on days 1 and 7 of a 21-day cycle. Each full course of therapy consisted of two identical 21-day cycles. Plasma DP levels were evaluated by liquid chromatography-mass spectrometry techniques. A variety of molecular end points were assessed in tumor biopsies via immunohistochemistry techniques. Long oligo arrays were used to examine gene expression profiles in laser-captured tumor cells before and after DP exposure, relative to lung cancer cells and adjacent normal bronchial epithelia from patients undergoing pulmonary resections. Nineteen patients were evaluable for toxicity assessment; 18 were evaluable for treatment response. Myelosuppression was dose limiting in one individual. No significant cardiac toxicities were observed. Maximum steady-state plasma DP concentrations ranged from 384 to 1,114 ng/mL. No objective responses were observed. Transient stabilization of disease was noted in nine patients. DP enhanced acetylation of histone H4, increased p21 expression in lung cancer cells, and seemed to shift global gene expression profiles in these cells toward those detected in normal bronchial epithelia. Although exhibiting minimal clinical efficacy at this dose and schedule, DP mediates biological effects that may warrant further evaluation of this histone deacetylase inhibitor in combination with novel-targeted agents in lung cancer patients.

536 Characterization of gene expression profile in Kupffer cells stimulated with IFN alpha and gamma

PurposeThe aim of this study is to compare the gene expression profile in mesenchymal stem cells derived from dental tissues and bone marrow for characterization of dental stem cells.MethodsWe employed GeneChip analysis to the expression levels of approximately 32,321 kinds of transcripts in 5 samples of bone-marrow-derived mesenchymal stem cells (BMSCs) (n=1), periodontal ligament stem cells (PDLSCs) (n=2), and dental pulp stem cells (DPSCs) (n=2). Each cell was sorted by a FACS Vantage Sorter using immunocytochemical staining of the early mesenchymal stem cell surface marker STRO-1 before the microarray analysis.ResultsWe identified 379 up-regulated and 133 down-regulated transcripts in BMSCs, 68 up-regulated and 64 down-regulated transcripts in PDLSCs, and 218 up-regulated and 231 down-regulated transcripts in DPSCs. In addition, anatomical structure development and anatomical structure morphogenesis gene ontology (GO) terms were over-represented in all three different mesenchymal stem cells and GO terms related to blood vessels, and neurons were over-represented only in DPSCs.ConclusionsThis study demonstrated the genome-wide gene expression patterns of STRO-1+ mesenchymal stem cells derived from dental tissues and bone marrow. The differences among the expression profiles of BMSCs, PDLSCs, and DPSCs were shown, and 999 candidate genes were found to be definitely up- or down-regulated. In addition, GOstat analyses of regulated gene products provided over-represented GO classes. These data provide a first step for discovering molecules key to the characteristics of dental stem cells.

Objective:Bone marrow (BM) is the most utilized and well-studied source of stem cells. Stem cells from dental tissues have provided an alternate source of mesenchymal stem cells (MSCs). Dental pulp stem cells (DPSCs) have been shown to share a similar pattern of protein expression with BMMSCs in vitro. However, differences have been noted between DPSCs and BMMSCs. This study focuses on variation in expression of stem cell and differentiation markers between DPSCs and BMMSCs.Materials and Methods:The two stem cells were isolated and compared for clonogenic potential, growth characteristics, multipotency, and stem cell marker expression. Specifically, the fatty acid binding protein 4, perilipin, alkaline phosphatase and osteonectic gene expression was analyzed by real-time polymerase chain reaction to confirm the capacity for adipogenic and osteogenic differentiation.Results:MSCs from these cell sources were similar in their morphology and immune phenotype except for the expression of CD105. Growth curves and colony formation assay revealed proliferation rate of DPSCs was significantly faster than BMMSCs (P < 0.05). DPSCs appeared less able to differentiate into adipogenic lineage, although more able to differentiate into osteogenic lineage.Conclusion:Data from the present study indicate how DPSCs are different from BMMSCs though they are a population of MSCs. DPSCs are a novel population of MSCs as observed by their unique expression of differentiation and lineage specific genes. Further microarray analysis could be used to determine, which genes are differentially regulated in BMMSCs and DPSCs to establish uniqueness of each population of MSCs.

Mechanisms under immune response against Candida albicans (C. albicans) remain largely unknown. To better understand the mechanisms of innate immune response against C. albicans, we analyzed the gene expression profile of THP-1 cells stimulated with heat-killed C. albicans. THP-1 cells were stimulated with heat-killed C. albicans for 9 hours at a ratio of 1:1, and gene expression profile of the cells was analyzed using Whole Human Genome Oligo Microarray. Differentially expressed genes were defined as change folds more than 2 and with statistical significance. Gene ontology (GO) and pathway analysis were used to systematically identify biological connections of differentially expressed genes, as well as the pathways associated with the immune response against C. albicans. A total of 355 genes were up-regulated and 715 genes were down-regulated significantly. The up-regulated genes were particularly involved in biological process of RNA processing and pathway of the spliceosome. In case of down-regulated genes, the particularly involved immune-related pathways were G-protein coupled receptor signaling pathway, calcium signaling pathway, MAPK signaling pathway and Ras pathway. We depict the gene expression profile of heat-killed C. albicans stimulated THP-1 cells, and identify the major pathways involved in immune response against C. albicans. These pathways are potential candidate targets for developing anti-C. albicans agent.

Sporamin, a proteinase inhibitor isolated from the sweet potato (Ipomoea batatas), has shown promising anticancer effect against colorectal cancer (CRC) in vitro and in vivo but its mechanisms of action are poorly understood. In the present study, high throughput RNA sequencing (RNA-seq) technology was applied to explore the transcriptomic changes induced by sporamin in the presence of thapsigargin (TG), a non-12-O-tetradecanolphorbol-13-acetate type cancer promoter, in the LoVo human CRC cells. Cellular total RNA was extracted from the cells after they were treated with vehicle (CTL), 1 μM of thapsigargin (TG), or 1 μM of TG plus 30 μM of sporamin (TGSP) for 24 h. The migratory capacity of the cells was determined by wound healing assay. The gene expression profiles of the cells were determined by RNA-seq on an Illumina platform. GO enrichment analysis, KEGG pathway analysis, protein-protein interaction (PPI) network construction, and transcription factors (TF) prediction were all performed based on the differentially expressed genes (DEGs) across groups with a series of bioinformatics tools. Finally, the effect and potential molecular targets of the sporamin at the transcriptome level were evaluated. Sporamin significantly inhibited the migration of cells induced by TG. Among the 17915 genes detected in RNA-seq, 46 DEGs were attributable to the effect of sporamin. RT-PCR experiment validated that the expression of RGPD2, SULT1A3, and BIVM-ERCC5 were up-regulated while NYP4R, FOXN1, PAK6, and CEACAM20 were down-regulated. Sporamin enhanced the mineral absorption pathway, worm longevity regulating pathway, and pyrimidine metabolism pathway. Two TFs (SMIM11A and ATOH8) were down-regulated by sporamin. HMOX1 (up-regulated) and NME1-NME2 (down-regulated) were the main nodes in a PPI network consisting of 16 DEGs that were modulated by sporamin in the presence of TG. Sporamin could favorably alter the gene expression profile of CRC cells, up-regulating the genes that contribute to the homeostasis of intracellular metal ions and the activities of essential enzymes and DNA damage repairment. More studies are warranted to verify its effect on specific genes and delineate the mechanism of action implicated in the process.

Purpose: Neuroblastoma (NB) is one of the most common childhood tumors, accounting for ~15% of all pediatric cancer deaths. Improved neuroblastoma disease modeling is needed to improve our understanding and ultimately lead to the development of more effective therapies. NB is an embryonal tumor of the sympathetic nervous system believed to arise from sympathoadrenal (SA) precursor cells belonging to the primitive neural crest. Because the neural crest is a transient embryologic structure, there have been few studies comparing NB tumors to normal human neural crest stem cells (NCSC). However, recent advances have enabled the in vitro differentiation of neural crest cells from pluripotent stem cells. We compared gene expression profiles of pluripotent stem cells, NCSC, and NB tumors to characterize the gene expression profile of the putative cell of origin of NB as the first step in the development of a human NCSC based model of NB. Methods: Human embryonic and induced pluripotent stem (iPS) cells were differentiated to early NCSC and SA precursors in vitro using modifications of previously published protocols. Specifically, undifferentiated human embryonic stem cell GFP reporter and iPS cell lines were induced to differentiate into SA cells in a sequential, stepwise manner over the course of ~4 weeks. After differentiation, SA precursors were identified by expression of the lineage-specific transcription factors ASCL1 and PHOX2B, as well as other markers including tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). We subsequently compared the gene expression profiles of these cells to early, multi-potent neural crest stem cells, autonomic and enteric neurons, 12 NB cell lines and 39 non-MYCN amplified primary NB tumors (3 stage 1, 5 stage 2, 3 stage 3, 24 stage 4, and 4 stage 4S) using Illumina HT-12 bead arrays. These profiles were also compared to the gene expression profiles of laser-capture microdissected fetal adrenal neuroblasts previously reported by De Preter et. al. (Genome Biology 2006, 7:R84). Results were normalized using quantile normalization and analyzed with principle components analysis (PCA); differential expression analysis was performed using the Linear Models for Microarray Data (Limma) method. Pathway enrichment analysis was performed using DAVID annotation tools and cross-platform normalization was performed using the CONOR package in the R software program. All analyses were corrected for multiple comparisons and results considered significant when False Discovery Rate (FDR) was &amp;lt;0.05. Results: PCA showed that undifferentiated iPS cells and early NCSC cluster relatively closely together and away from primary tumors and fetal adrenal neuroblasts. The NB cell lines and primary tumors do not cluster closely together suggesting there are significant differences between them. Gene expression profiles of in vitro differentiated ASCL1 and PHOX2B(+) SA precursor cells were similar to fetal adrenal neuroblasts and more closely resembled NB tumors than did earlier multi-potent NCSC. Comparison between SA precursor cells, NCSC, and primary tumors identified numerous differentially expressed genes (FDR&amp;lt;0.05) the majority of which are regulators of embryologic and/or neural development (SOX3, DPPA4, HES5, LIN28, IRX2, SOX21, PAX6, FABP7, and OTX2 among others). Most were overexpressed in normal neural crest derivatives relative to NB cell lines and tumors. Conclusions: This study, the first of its kind for this disease, demonstrates the feasibility and potential utility of using human pluripotent stem cells and in vitro derived sympathoadrenal precursors cells to simulate early NB pathogenesis. Gene expression profiling of NB identified down regulation of multiple developmentally important genes of normal neural crest differentiation. Further refinement of this in vitro neuroblastoma tumor model as well as investigations of its disrupted gene pathways is underway. Note: This abstract was not presented at the conference. Citation Format: Stephen S. Roberts, Yudelca Ogando, Irina Ostrovnaya, Faranak Fattahi, Irene Cheung, Nai-Kong V. Cheung, Lorenz Studer, Mark Tomishima. Using directed differentiation of human pluripotent stem cells and gene expression profiling to characterize the cell of origin of neuroblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A08.

Introduction: Dental pulp stem cell (DPSC)-mediated dental pulp regeneration is considered a promising method for the treatment of deep caries with pulpitis. However, mesenchymal stem cell (MSC) senescence is an adverse factor from the perspective of cell-based therapies. In this study, we investigated the characteristics and expression profiles of DPSCs from young and old donors. Methods: DPSCs from young and old donors were cultured in differentiation medium, and their differentiation potentials were assessed. Long noncoding RNA (LncRNA) microarray assays and a bioinformatic analysis were performed to investigate differences in LncRNA and mRNA expression profiles between DPSCs from young and old donors. Results: We found that DPSCs from young donors exhibited more powerful proliferation ability and greater osteogenic and adipogenic differentiation potentials than DPSCs from old donors. In DPSCs from young donors, numerous LncRNAs were significantly up- (n = 389) or down-regulated (n = 172) compared to DPSCs from old donors. Furthermore, 304 mRNAs were differentially expressed, including 247 up-regulated genes and 57 down-regulated genes in DPSCs from young donors. The bioinformatic analysis identified that several pathways may be associated with DPSC characteristics, such as those involved in the cell cycle and RNA transport, and revealed nuclear transcription factor Y subunit β, general transcription factor IIB, and nuclear receptor subfamily 3 group C member 1 as core regulatory factors and FR249114, FR299091, and ENST00000450004 as core LncRNAs. Conclusions: Our results indicated that senescence impaired the proliferation and differentiation potentials of DPSCs and that donor age is an important factor that affects their use for tooth regeneration. We also provide insight into the mechanisms responsible for senescence in DPSCs.

Introduction The etiology of the spinal deformity in idiopathic scoliosis is unclear to date, both with respect to initiation and progression of the disease. While the influence of certain genetic factors has been established, the role of the intervertebral disc (IVD) in the development of idiopathic scoliosis has scarcely been investigated. The aim of this study was to identify molecular differences between disc cells from patients with idiopathic scoliosis in comparison with trauma patients and healthy individuals. To address this aim, cellular gene expression profiles were analyzed by microarray and quantitative RT-PCR. Material and Methods Surgical samples from IVDs of patients with idiopathic scoliosis were obtained after informed consent and approval of the local ethical commission at the time of discectomy during spinal fusion surgery. The disorder and exact curve pattern were documented for further records. Control disc samples were obtained from trauma fusion cases and from organ donors with no known disc disorders according to local and institutional ethical guidelines. Annulus fibrosus (AF) and nucleus pulposus (NP) tissues were separated and cells were isolated by enzymatic digestion within 24 hours. Total RNA was extracted from the cells and subjected to Affymetrix GeneChip® expression profiling. Genes with significant differences between scoliotic and control samples were further analyzed using real time RT-PCR. Results After exclusion of RNA samples with insufficient quality or quantity, the following numbers of samples were used for microarray profiling: 10 AF and 6 NP samples from scoliotic discs; 5 AF and 4 NP samples from traumatic discs; 4 AF and 4 NP samples from healthy discs of organ donors. Microarray data revealed that 52 genes were more highly expressed in scoliotic vs. healthy AF and 26 genes in scoliotic vs. traumatic AF, whereby 21 genes showed higher expression in scoliotic AF compared with both control groups. In addition, 116 genes were more highly expressed in scoliotic vs. healthy NP, 45 genes in scoliotic vs. traumatic NP, and 40 of those in scoliotic NP compared with both control groups. Quantitative gene expression analysis by real time RT-PCR ( n = 6 per group) confirmed significantly increased mRNA levels of S100A8, S100A12, MMP8, MMP13 and Collagen X in annulus fibrosus cells from scoliotic discs ( p &lt; 0.05; Kruskal-Wallis test of log2 transformed data). Conclusion Results of this study reveal significant changes in the gene expression profile of IVD cells from patients with idiopathic scoliosis compared with patients with traumatic disc damage or donors with no known disc disorders. MMP8 and MMP13 are important collagenases involved in disc matrix degradation; while elevated S100 calcium binding proteins may indicate an inflammatory reaction. Interestingly, MMP13 has also been up-regulated by imbalanced loading in an IVD organ culture model. Better knowledge of the dysregulation of structural or regulatory molecules may identify underlying mechanisms of spinal deformities, which will help defining new targets for early therapeutic intervention. Acknowledgment This study is supported by AOSpine International.

This study evaluated the gene expression profile of the human adipose-derived stem cells (hASCs) grown on the Biosilicate® /F18 glass (BioS-2P/F18) scaffolds. hASCs were cultured using the osteogenic medium (control), the scaffolds, and their ionic extract. We observed that ALP activity was higher in hASCs grown on the BioS-2P/F18 scaffolds than in hASCs cultured with the ionic extract or the osteogenic medium on day 14. Moreover, the dissolution product group and the control exhibited deposited calcium, which peaked on day 21. Gene expression profiles of cell cultured using the BioS-2P/F18 scaffolds and their extract were evaluated in vitro using the RT2 Profiler polymerase chain reaction (PCR) microarray on day 21. Mineralizing tissue-associated proteins, differentiation factors, and extracellular matrix enzyme expressions were measured using quantitative PCR. The gene expression of different proteins involved in osteoblast differentiation was significantly up-regulated in hASCs grown on the scaffolds, especially BMP1, BMP2, SPP1, BMPR1B, ITGA1, ITGA2, ITGB1, SMAD1, and SMAD2, showing that both the composition and topographic features of the biomaterial could stimulate osteogenesis. This study demonstrated that gene expression of hASCs grown on the scaffold surface showed significantly increased gene expression related to hASCs cultured with the ionic extract or the osteogenic medium, evidencing that the BioS-2P/F18 scaffolds have a substantial effect on cellular behavior of hASCs.