Project description:Interstitial cells of Cajal (ICC) are electrical pacemakers and mediators of neuromuscular neurotransmission in the gastrointestinal tract. Studies in organotypic cultures of intact gastric muscular wall tissues identified Kit(low)Cd44(+)Cd34(+)Insr(+)Igf1r(+) cells as local ICC progenitors (Lorincz et al., Gastroenterology 2008;134:1083-1093). Subsequently, conditionally immortalized Kit(low)Cd34(+) cells were isolated by immunomagnetic and fluorescent cell sorting and serial cloning from the gastric muscular wall of homozygous, 14-day-old H-2Kb-tsA58 mice and shown to share other phenotypic characteristics with in-situ ICC precursors (2xSCS70 cells; Bardsley et al., Gastroenterology 2010;139:942-952). 2xSCS70 cells were also found to express the ICC marker Ano1, to be able to extensively self-renew in the absence of the SV40 tsA58-TAg when cultured in NeuroCultÂ® Neural Stem Cell Proliferation Medium (NSCPM; STEMCELL Technologies) and to differentiate into Kit(+) ICC both in vitro and in vivo. Wild-type cells with Kit(low)Ano1(+)Cd44(+)Cd34(+)Insr(+)Igf1r(+) phenotype (2xSCS2F10 cells) were prospectively isolated from primary cultures of dissociated gastric muscular wall tissues from 7-day-old C57BL/6J mice by serial cloning following 150-day expansion in NSCPM containing 5% cell-free chick embryo extract (CEE; Accurate Chemical). Long-term culturing of both cell lines was associated with further decline in Kit expression while expression of Ano1 and other markers of ICC precursors was maintained. Following spontaneous transformation, both 2xSCS70 and 2xSCS2F10 cells grafted into nude mice gave rise to tumors containing Kit(+)cells and otherwise resembling gastrointestinal stromal tumors (GIST), which originate from the ICC lineage (Bardsley et al., Gastroenterology 2010;139:942-952). This study utilized RNA-sequencing to complement the companion microarray study in non-transformed 2xSCS70 and 2xSCS2F10 cells. Libraries were made from total RNA isolated from ICC progenitor cell lines using the Illumina TruSeq mRNA Sample Preparation Kit v2 and sequenced on the Illumina HiSeq 2000 platform.

Project description:Interstitial cells of Cajal (ICC) are electrical pacemakers and mediators of neuromuscular neurotransmission in the gastrointestinal tract. Studies in organotypic cultures of intact gastric muscular wall tissues identified Kit(low)Cd44(+)Cd34(+)Insr(+)Igf1r(+) cells as local ICC progenitors (Lorincz et al., Gastroenterology 2008;134:1083-1093). Subsequently, conditionally immortalized Kit(low)Cd34(+) cells were isolated by immunomagnetic and fluorescent cell sorting and serial cloning from the gastric muscular wall of homozygous, 14-day-old H-2Kb-tsA58 mice and shown to share other phenotypic characteristics with in-situ ICC precursors (2xSCS70 cells; Bardsley et al., Gastroenterology 2010;139:942-952). 2xSCS70 cells were also found to express the ICC marker Ano1, be able to extensively self-renew in the absence of the SV40 tsA58-TAg when cultured in NeuroCultÂ® Neural Stem Cell Proliferation Medium (NSCPM; STEMCELL Technologies) and differentiate into Kit(+) ICC both in vitro and in vivo. Wild-type cells with Kit(low)Ano1(+)Cd44(+)Cd34(+)Insr(+)Igf1r(+) phenotype (2xSCS2F10 cells) were prospectively isolated from primary cultures of dissociated gastric muscular wall tissues from 7-day-old C57BL/6J mice by serial cloning following 150-day expansion in NSCPM containing 5% cell-free chick embryo extract (CEE; Accurate Chemical). Long-term culturing of both cell lines was associated with further decline in Kit expression while expression of Ano1 and other markers of ICC precursors was maintained. Following spontaneous transformation, both 2xSCS70 and 2xSCS2F10 cells grafted into nude mice gave rise to tumors containing Kit(+)cells and otherwise resembling gastrointestinal stromal tumors (GIST), which originate from the ICC lineage (Bardsley et al., Gastroenterology 2010;139:942-952). This study utilized oligonucleotide microarray analysis to charaterize the transcriptome of non-transformed 2xSCS70 and 2xSCS2F10 cells relative to their source tissues. Total RNA from ICC progenitor cell lines and their source tissue (gastric tunica muscularis from C57BL/6J mice (source of 2xSCS2F10 cells) and H-2Kb-tsA58 mice (source of 2xSCS70 cells) was isolated. Oligonucleotide micorarray studies were performed using 2-3 Affymetrix Mouse Genome 430.2 GeneChips per sample.

Project description:Several transcription factors are known to be expressed in discrete regions of the otic vesicle and Dlx5 is one of those that is expressed highly in the presumptive dorsal vestibular region. Mice lacking Dlx5 have vestibular defects. Specifically, they fail to form the endolymphatic duct (a defect visible as early as E10) as well as the anterior and posterior semi-circular canals. The lateral canal does form but is smaller, whereas the saccule, the utricle and the cochlea appear relatively normal. The goal of this study was to use microarrays to identify differentially expressed genes between wild-type and Dlx5-null otic vesicles microdissected from E10 and 10.5 and identify downstream targets of Dlx5 by searching the immediate 3kb promoter regions of the differentially expressed genes for homeodomain binding sites followed by chromatin immunoprecipitation in an otic vesicle-derived cell line over-expressing Dlx5. Normal vestibular morphogenesis is compromised in mice lacking Dlx5, a member of the Distal-less family of homeobox transcription factors. We identified its direct downstream targets in the developing mouse inner ear by gene expression profiling wild-type and Dlx5 null otic vesicles from embryonic stages E10 and E10.5. Four hundred genes were differentially expressed in mutants when compared to wild-type in at least one of the two stages. To further constrain the list of likely direct targets of Dlx5, we examined the genomic DNA sequences in the 3kb promoter regions immediately proximal to the transcriptional start sites of these genes. We searched for (i) one or more previously described binding site for Dlx5, (ii) one or more novel 12bp-long motifs with a canonical homeodomain response element (HDRE) shared by promoters of two or more genes, and (iii) 100% conservation of the 12bp-long HDRE-containing motifs in promoter regions of human orthologs. Forty genes passed one or more of these filters, 12 of which are known to be expressed in the developing otic vesicle in domains that at least partially overlap with that of Dlx5 in one or both stages that we examined. Chromatin immunoprecipitation using a Dlx5 antibody confirmed direct binding of Dlx5 to promoter regions of seven of these genes (Atbf1, Bmper, Large, Lrrtm1, and Msx1, all of which were down-regulated in mutants, and Ebf1 and Lhx1, both of which were up-regulated in mutants) in an otic vesicle-derived cell line over-expressing Dlx5. Gene expression profiling of this cell line showed that Bmper and Lrrtm1 transcripts were up-regulated, further supporting their identification as direct targets of Dlx5 activity. Otic vesicles from wild-type and Dlx5-null embryos were microdissected from mouse developmental stages E10 and E10.5 and expression profiled in duplicate for each stage and genotype. Dlx5-transfected and empty vector-transfected 2B1 cell line samples were also profiled in duplicate (independent cell cultures).

Project description:Background Primitive brain tumors are the first cause of cancer-related death in children. Tumor cells with stem-like properties (TSCs), thought to account for tumorigenesis and therapeutic resistance, have been isolated from high-grade gliomas in adults. Whether TSCs are a common component of pediatric brain tumors and are of clinical relevance remains to be determined. Methodology/Principal findings Tumor cells with self-renewal properties were isolated with cell biology techniques from a majority of 55 pediatric brain tumors samples, regardless of their histopathologies and grades of malignancy (57% of embryonal tumors, 57% of low-grade gliomas and neuro-glial tumors, 70% of ependymomas, 91% of high-grade gliomas). The vast majority (10/12) of high-grade glioma-derived oncospheres sustained long-term self-renewal numbers akin to neural stem cells (>7 self-renewals), whereas cells with limited renewing abilities akin to neural progenitors dominated in all other tumor types. Regardless of tumor entities, the young age group was associated with self-renewal properties akin to neural stem cells (P=0.05, chi-square test). TSCs shared a complex molecular profile combining embryonic stem cell markers with elements controlling neural stem cell properties and epithelio-mesenchymal transitions. They were radio- and chemoresistant and formed aggressive tumors after intracerebral grafting. Survival analysis of the cohort showed an association between isolation of TSCs with long-term self-renewal abilities and a patientM-^Rs higher mortality rate (P = 0.022, log-rank test). Patients bearing cells with limited self-renewal properties constituted an intermediate group of survival but which did not reach statistical significance. Conclusions/Significance In brain tumors affecting adult patients, TSC have been isolated only from high-grade gliomas. In contrast, our data show that tumor cells with stem cell-like or progenitor-like properties can be isolated from a wide range of histological sub-types and grades of pediatric brain tumors. They suggest that cellular mechanisms fueling tumor development differ between adult and pediatric brain tumors.

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Transcriptome analysis of two clonally-derived ICC progenitor cell lines

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