MAB4343 Sigma-AldrichAnti-SOX-2 Antibody

Genome-wide mapping of transcriptional regulatory elements is an essential tool for understanding the molecular events orchestrating self-renewal, commitment and differentiation of stem cells. We combined high-throughput identification of transcription start sites with genome-wide profiling of histones modifications to map active promoters and enhancers in embryonic stem cells (ESCs) induced to neuroepithelial-like stem cells (NESCs). Our analysis showed that most promoters are active in both cell types while approximately half of the enhancers are cell-specific and account for most of the epigenetic changes occurring during neural induction, and most likely for the modulation of the promoters to generate cell-specific gene expression programs. Interestingly, the majority of the promoters activated or up-regulated during neural induction have a "bivalent" histone modification signature in ESCs, suggesting that developmentally-regulated promoters are already poised for transcription in ESCs, which are apparently pre-committed to neuroectodermal differentiation. Overall, our study provides a collection of differentially used enhancers, promoters, transcription starts sites, protein-coding and non-coding RNAs in human ESCs and ESC-derived NESCs, and a broad, genome-wide description of promoter and enhancer usage and of gene expression programs characterizing the transition from a pluripotent to a neural-restricted cell fate.

Recent advances in human embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) biology enable generation of dopaminergic neurons for potential therapy and drug screening. However, our current understanding of molecular and cellular signaling that controls human dopaminergic development and function is limited. Here, we report on a whole genome analysis of gene expression during dopaminergic differentiation of human ESC/iPSC using Illumina bead microarrays. We generated a transcriptome data set containing the expression levels of 28,688 unique transcripts by profiling five lines (three ESC and two iPSC lines) at four stages of differentiation: (1) undifferentiated ESC/iPSC, (2) neural stem cells, (3) dopaminergic precursors, and (4) dopaminergic neurons. This data set provides comprehensive information about genes expressed at each stage of differentiation. Our data indicate that distinct pathways are activated during neural and dopaminergic neuronal differentiation. For example, WNT, sonic hedgehog (SHH), and cAMP signaling pathways were found over-represented in dopaminergic populations by gene enrichment and pathway analysis, and their role was confirmed by perturbation analyses using RNAi (small interfering RNA of SHH and WNT) or small molecule [dibutyryl cyclic AMP (dcAMP)]. In summary, whole genome profiling of dopaminergic differentiation enables systematic analysis of genes/pathways, networks, and cellular/molecular processes that control cell fate decisions. Such analyses will serve as the foundation for better understanding of dopaminergic development, function, and development of future stem cell-based therapies.

Human induced pluripotent stem (iPS) cells have great value for regenerative medicine, but are facing problems of low efficiency. MicroRNAs are a recently discovered class of 19-25 nt small RNAs that negatively target mRNAs. miR302/367 cluster has been demonstrated to reprogram mouse and human somatic cells to iPS cells without exogenous transcription factors, however, the repetition and differentiation potentiality of miR302/367-induced pluripotent stem (mirPS) cells need to be improved. Here, we showed overexpression of miR302/367 cluster reprogrammed human embryonic kidney 293T cells into mirPS cells in serum-free N2B27-based medium. The mirPS cells had similar morphology with embryonic stem cells, and expressed pluripotent markers including Oct4, Sox2, Klf4, and Nanog. In addition, through formation of embryoid bodies, various cells and tissues from three germ layers could be determined. Moreover, we examined the potential of mirPS cells differentiating into germ cells both in vitro and in vivo. Taken together, these data might provide a new source of cells and technique for the investigation of the mechanisms underlying reprogramming and pluripotency.

Little is known about how the immune system impacts human colorectal cancer invasiveness and stemness. Here we detected interleukin-22 (IL-22) in patient colorectal cancer tissues that was produced predominantly by CD4(+) T cells. In a mouse model, migration of these cells into the colon cancer microenvironment required the chemokine receptor CCR6 and its ligand CCL20. IL-22 acted on cancer cells to promote activation of the transcription factor STAT3 and expression of the histone 3 lysine 79 (H3K79) methytransferase DOT1L. The DOT1L complex induced the core stem cell genes NANOG, SOX2, and Pou5F1, resulting in increased cancer stemness and tumorigenic potential. Furthermore, high DOT1L expression and H3K79me2 in colorectal cancer tissues was a predictor of poor patient survival. Thus, IL-22(+) cells promote colon cancer stemness via regulation of stemness genes that negatively affects patient outcome. Efforts to target this network might be a strategy in treating colorectal cancer patients.

Extraneural metastases (ENM) rarely occur in medulloblastoma (MBL) patients and only few cases of subcutaneous localizations have been described. ENM indicate an aggressive disease associated with a worse prognosis. The characterization of metastatic tumours might be useful to understand their pathogenesis and to identify the most appropriate therapeutic strategies.We present the case of a child with Large Cell Anaplastic (LC/A) MBL, who developed multiple subcutaneous metastases in the scalp area after a ventriculo-peritoneal shunting procedure. The disease rapidly progressed and the child died despite chemotherapy and primary tumour surgical debulking.We molecularly classified the tumour as a group 3 MBL; in addition, we derived stem-like cells (SLC) from a metastatic lesion. Primary tumour, metastases and SLC were further analysed, particularly focusing on features linked to the cutaneous dissemination. Indeed, molecules involved in angiogenesis, cell invasion and epidermal growth factor signalling resulted highly expressed.The present report describes a very rare case of subcutaneous metastatic MBL. The tumour, metastases and SLC have been clinically, pathologically and molecularly characterized. Our case is an example of multidisciplinary approach aiming to characterize MBL aggressive behaviour.

The expression pattern of pluripotency markers in adipose tissue-derived stem cells (ADSCs) is a subject of controversy. Moreover, there is no data about the signaling molecules that regulate these markers in ADSCs. In the present study, we studied the roles of leukemia inhibitory factor (LIF) and miR-302 in this regard. Freshly isolated mouse ADSCs expressed hematopoietic, mesenchymal, and pluripotency markers. One day after plating, ADSCs expressed OCT4 and Sox2 proteins. After three passages, the expression of hematopoietic and pluripotency markers decreased, while the expression of mesenchymal stem cell markers exhibited a striking rise. Both supplementation of culture media with LIF and transfection of the ADSCs with miR-302 family upregulated the expression levels of OCT4, Nanog, and Sox2 mRNAs. These findings showed that mouse adipose tissue contains a population of cells with molecular resemblance to embryonic stem cells, and LIF and miR-302 family positively affect the expression of pluripotency markers.

Neuronal activity regulates the phosphorylation states at multiple sites on MeCP2 in postmitotic neurons. The precise control of the phosphorylation status of MeCP2 in neurons is critical for the normal development and function of the mammalian brain. However, it is unknown whether phosphorylation at any of the previously identified sites on MeCP2 can be induced by signals other than neuronal activity in other cell types, and what functions MeCP2 phosphorylation may have in those contexts. Here we show that in neural progenitor cells isolated from the adult mouse hippocampus, cell cycle-linked phosphorylation at serine 421 on MeCP2 is directly regulated by aurora kinase B and modulates the balance between proliferation and neural differentiation through the Notch signalling pathway. Our findings suggest MeCP2 S421 phosphorylation may function as a general epigenetic switch accessible by different extracellular stimuli through different signalling pathways for regulating diverse biological functions in different cell types.

The promise of genetic reprogramming has prompted initiatives to develop banks of induced pluripotent stem cells (iPSCs) from diverse sources. Sentinel assays for pluripotency could maximize available resources for generating iPSCs. Neural rosettes represent a primitive neural tissue that is unique to differentiating PSCs and commonly used to identify derivative neural/stem progenitors. Here, neural rosettes were used as a sentinel assay for pluripotency in selection of candidates to advance to validation assays. Candidate iPSCs were generated from independent populations of amniotic cells with episomal vectors. Phase imaging of living back up cultures showed neural rosettes in 2 of the 5 candidate populations. Rosettes were immunopositive for the Sox1, Sox2, Pax6 and Pax7 transcription factors that govern neural development in the earliest stage of development and for the Isl1/2 and Otx2 transcription factors that are expressed in the dorsal and ventral domains, respectively, of the neural tube in vivo. Dissociation of rosettes produced cultures of differentiation competent neural/stem progenitors that generated immature neurons that were immunopositive for βIII-tubulin and glia that were immunopositive for GFAP. Subsequent validation assays of selected candidates showed induced expression of endogenous pluripotency genes, epigenetic modification of chromatin and formation of teratomas in immunodeficient mice that contained derivatives of the 3 embryonic germ layers. Validated lines were vector-free and maintained a normal karyotype for more than 60 passages. The credibility of rosette assembly as a sentinel assay for PSCs is supported by coordinate loss of nuclear-localized pluripotency factors Oct4 and Nanog in neural rosettes that emerge spontaneously in cultures of self-renewing validated lines. Taken together, these findings demonstrate value in neural rosettes as sentinels for pluripotency and selection of promising candidates for advance to validation assays.

Myeloid-derived suppressor cells (MDSCs) and cancer stem cells (CSCs) are important cellular components in the cancer microenvironment and may affect cancer phenotype and patient outcome. The nature of MDSCs and their interaction with CSCs in ovarian carcinoma are unclear. We examined the interaction between MDSCs and CSCs in patients with ovarian carcinoma and showed that MDSCs inhibited T cell activation and enhanced CSC gene expression, sphere formation, and cancer metastasis. MDSCs triggered miRNA101 expression in cancer cells. miRNA101 subsequently repressesed the corepressor gene C-terminal binding protein-2 (CtBP2), and CtBP2 directly targeted stem cell core genes resulting in increased cancer cell stemness and increasing metastatic and tumorigenic potential. Increased MDSC density and tumor microRNA101 expression predict poor survival, as does decreased tumor CtBP2 expression, independent of each other. Collectively, our work identifies an immune-associated cellular, molecular, and clinical network involving MDSCs-microRNA101-CtBP2-stem cell core genes, which extrinsically controls cancer stemness and impacts patient outcome.

Recent reports have demonstrated that somatic cells can be directly converted to other differentiated cell types through ectopic expression of sets of transcription factors, directly avoiding the transition through a pluripotent state. Our previous experiments generated induced neural progenitor-like cells (iNPCs) by a novel combination of five transcription factors (Sox2, Brn2, TLX, Bmi1 and c-Myc). Here we demonstrated that the iNPCs not only possess NPC-specific marker genes, but also have qualities of primary brain-derived NPCs (WT-NPCs), including tripotent differentiation potential, mature neuron differentiation capability and synapse formation. Importantly, the mature neurons derived from iNPCs exhibit significant physiological properties, such as potassium channel activity and generation of action potential-like spikes. These results suggest that directly reprogrammed iNPCs closely resemble WT-NPCs, which may suggest an alternative strategy to overcome the restricted proliferative and lineage potential of induced neurons (iNCs) and broaden applications of cell therapy in the treatment of neurodegenerative disorders.