Andrew Hinton

Activin A Maintains Pluripotency of Human Embryonic Stem Cells in the Absence of Feeder Layers

To date, all human embryonic stem cells (hESCs) available for research require unidentified soluble factors secreted from feeder layers to maintain the undifferentiated state and pluripotency. Activation of STAT3 by leukemia inhibitory factor is required to maintain “stemness” in mouse embryonic stem cells, but not in hESCs, suggesting the existence of alternate signaling pathways for self‐renewal and pluripotency in human cells. Here we show that activin A is secreted by mouse embryonic feeder layers (mEFs) and that culture medium enriched with activin A is capable of maintaining hESCs in the undifferentiated state for >20 passages without the need for feeder layers, conditioned medium from mEFs, or STAT3 activation. hESCs retained both normal karyotype and markers of undifferentiated cells, including Oct‐4, nanog, and TRA‐1‐60 and remained pluripotent, as shown by the in vivo formation of teratomas.

A Distinct MicroRNA Signature for Definitive Endoderm Derived From Human Embryonic Stem Cells

Human embryonic stem cells (hESCs) have the potential to differentiate into many adult cell types, and they are being explored as a resource for cell replacement therapies for multiple diseases. In order to optimize in vitro differentiation protocols, it will be necessary to elucidate regulatory mechanisms that contribute to lineage specification. MicroRNAs (miRNAs) are emerging as key regulators of hESC differentiation and embryonic development. In this study, we compare miRNA expression profiles between pluripotent hESCs and definitive endoderm (DE), an early step in the pathway toward the pancreatic lineage. Results from microarray analysis showed that DE can be distinguished by its unique miRNA profile, which consists of 37 significantly down-regulated and 17 up-regulated miRNAs in 2 different cell lines and in the presence/absence of feeder layers. Comparison to other hESC-derived lineages showed that most of the highly up-regulated miRNAs are specific to endoderm in early development. Notably, miR-375, which was previously implicated in regulating development and function of later stages of pancreatic development, is highly and specifically up-regulated during DE formation, suggesting that it may have a distinct role very early in development. Examination of potential mRNA targets showed that TIMM8A is repressed by ectopic miR-375 expression in pluripotent hESCs.

The stretch responsive microRNA miR-148a-3p is a novel repressor of IKBKB, NF-κB signaling, and inflammatory gene expression in human aortic valve cells

Bicuspid aortic valves calcify at a significantly higher rate than normal aortic valves, a process that involves increased inflammation. Because we have previously found that bicuspid aortic valve experience greater stretch, we investigated the potential connection between stretch and inflammation in human aortic valve interstitial cells (AVICs). Microarray, quantitative PCR (qPCR), and protein assays performed on AVICs exposed to cyclic stretch showed that stretch was sufficient to increase expression of interleukin and metalloproteinase family members by more than 1.5‐fold. Conditioned medium from stretched AVICs was sufficient to activate leukocytes. microRNA sequencing and qPCR experiments demonstrated that miR‐148a‐3p was repressed in both stretched AVICs (43% repression) and, as a clinical correlate, human bicuspid aortic valves (63% reduction). miR‐148a‐3p was found to be a novel repressor of IKBKB based on data from qPCR, luciferase, and Western blot experiments. Furthermore, increasing miR‐148a‐3p levels in AVICs was sufficient to decrease NF‐κB (nuclear factor kappa‐light‐chain‐enhancer of activated B cells) signaling and NF‐κB target gene expression. Our data demonstrate that stretch‐mediated activation of inflammatory pathways is at least partly the result of stretch‐repression of miR‐148a‐3p and a consequent failure to repress IKBKB. To our knowledge, we are the first to report that cyclic stretch of human AVICs activates inflammatory genes in a tissue‐autonomous manner via a microRNA that regulates a central inflammatory pathway.—Patel, V., Carrion, K., Hollands, A., Hinton, A., Gallegos, T., Dyo, J., Sasik, R., Leire, E., Hardiman, G., Mohamed, S. A., Nigam, S., King, C. C., Nizet, V., Nigam V. The stretch responsive microRNA miR‐148a‐3p is a novel repressor of IKBKB, NF‐κB signaling, and inflammatory gene expression in human aortic valve cells. FASEB J. 29, 1859‐1868 (2015). www.fasebj.org

Transcriptome sequencing uncovers novel long noncoding and small nucleolar RNAs dysregulated in head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma persists as one of the most common and deadly malignancies, with early detection and effective treatment still posing formidable challenges. To expand our currently sparse knowledge of the noncoding alterations involved in the disease and identify potential biomarkers and therapeutic targets, we globally profiled the dysregulation of small nucleolar and long noncoding RNAs in head and neck tumors. Using next-generation RNA-sequencing data from 40 pairs of tumor and matched normal tissues, we found 2808 long noncoding RNA (lncRNA) transcripts significantly differentially expressed by a fold change magnitude ≥2. Meanwhile, RNA-sequencing analysis of 31 tumor-normal pairs yielded 33 significantly dysregulated small nucleolar RNAs (snoRNA). In particular, we identified two dramatically down-regulated lncRNAs and one down-regulated snoRNA whose expression levels correlated significantly with overall patient survival, suggesting their functional significance and clinical relevance in head and neck cancer pathogenesis. We confirmed the dysregulation of these noncoding RNAs in head and neck cancer cell lines derived from different anatomic sites, and determined that ectopic expression of the two lncRNAs inhibited key EMT and stem cell genes and reduced cellular proliferation and migration. As a whole, noncoding RNAs are pervasively dysregulated in head and squamous cell carcinoma. The precise molecular roles of the three transcripts identified warrants further characterization, but our data suggest that they are likely to play substantial roles in head and neck cancer pathogenesis and are significantly associated with patient survival.

sRNA-seq Analysis of Human Embryonic Stem Cells and Definitive Endoderm Reveals Differentially Expressed MicroRNAs and Novel IsomiRs with Distinct Targets

MicroRNAs (miRNAs) are noncoding, regulatory RNAs expressed dynamically during differentiation of human embryonic stem cells (hESCs) into defined lineages. Mapping developmental expression of miRNAs during transition from pluripotency to definitive endoderm (DE) should help to elucidate the mechanisms underlying lineage specification and ultimately enhance differentiation protocols. In this report, next generation sequencing was used to build upon our previous analysis of miRNA expression in human hESCs and DE. From millions of sequencing reads, 747 and 734 annotated miRNAs were identified in pluripotent and DE cells, respectively, including 77 differentially expressed miRNAs. Among these, four of the top five upregulated miRNAs were previously undetected in DE. Furthermore, the stem‐loop for miR‐302a, an important miRNA for both hESCs self‐renewal and endoderm specification, produced several highly expressed miRNA species (isomiRs). Overall, isomiRs represented >10% of sequencing reads in >40% of all detected stem‐loop arms, suggesting that the impact of these abundant miRNA species may have been overlooked in previous studies. Because of their relative abundance, the role of differential isomiR targeting was studied using the miR‐302 cluster as a model system. A miRNA mimetic for miR‐302a‐5p, but not miR‐302a‐5p(+3), decreased expression of orthodenticle homeobox 2 (OTX2). Conversely, isomiR 302a‐5p(+3) selectively decreased expression of tuberous sclerosis protein 1, but not OTX2, indicating nonoverlapping specificity of miRNA processing variants. Taken together, our characterization of miRNA expression, which includes novel miRNAs and isomiRs, helps establish a foundation for understanding the role of miRNAs in DE formation and selective targeting by isomiRs. Stem Cells 2014;32:2360–2372

From Pluripotency to Islets: miRNAs as Critical Regulators of Human Cellular Differentiation

MicroRNAs (miRNAs) actively regulate differentiation as pluripotent cells become cells of pancreatic endocrine lineage, including insulin-producing β cells. The process is dynamic; some miRNAs help maintain pluripotency, while others drive cell fate decisions. Here, we survey the current literature and describe the biological role of selected miRNAs in maintenance of both mouse and human embryonic stem cell (ESC) pluripotency. Subsequently, we review the increasing evidence that miRNAs act at selected points in differentiation to regulate decisions about early cell fate (definitive endoderm and mesoderm), formation of pancreatic precursor cells, endocrine cell function, as well as epithelial to mesenchymal transition.

The non-coding landscape of head and neck squamous cell carcinoma

Head and neck squamous cell carcinoma (HNSCC) is an aggressive disease marked by frequent recurrence and metastasis and stagnant survival rates. To enhance molecular knowledge of HNSCC and define a non-coding RNA (ncRNA) landscape of the disease, we profiled the transcriptome-wide dysregulation of long non-coding RNA (lncRNA), microRNA (miRNA), and PIWI-interacting RNA (piRNA) using RNA-sequencing data from 422 HNSCC patients in The Cancer Genome Atlas (TCGA). 307 non-coding transcripts differentially expressed in HNSCC were significantly correlated with patient survival, and associated with mutations in TP53, CDKN2A, CASP8, PRDM9, and FBXW7 and copy number variations in chromosomes 3, 5, 7, and 18. We also observed widespread ncRNA correlation to concurrent TP53 and chromosome 3p loss, a compelling predictor of poor prognosis in HNSCCs. Three selected ncRNAs were additionally associated with tumor stage, HPV status, and other clinical characteristics, and modulation of their expression in vitro reveals differential regulation of genes involved in epithelial-mesenchymal transition and apoptotic response. This comprehensive characterization of the HNSCC non-coding transcriptome introduces new layers of understanding for the disease, and nominates a novel panel of transcripts with potential utility as prognostic markers or therapeutic targets.

From Pluripotency to Islets

MicroRNAs (miRNAs) actively regulate differentiation as pluripotent cells become cells of pancreatic endocrine lineage, including insulin-producing β cells. The process is dynamic; some miRNAs help maintain pluripotency, while others drive cell fate decisions. Here, we survey the current literature and describe the biological role of selected miRNAs in maintenance of both mouse and human embryonic stem cell (ESC) pluripotency. Subsequently, we review the increasing evidence that miRNAs act at selected points in differentiation to regulate decisions about early cell fate (definitive endoderm and mesoderm), formation of pancreatic precursor cells, endocrine cell function, as well as epithelial to mesenchymal transition.

The SDF-1α/CXCR4 Axis is Required for Proliferation and Maturation of Human Fetal Pancreatic Endocrine Progenitor Cells

The chemokine receptor CXCR4 and ligand SDF-1α are expressed in fetal and adult mouse islets. Neutralization of CXCR4 has previously been shown to diminish ductal cell proliferation and increase apoptosis in the IFNγ transgenic mouse model in which the adult mouse pancreas displays islet regeneration. Here, we demonstrate that CXCR4 and SDF-1α are expressed in the human fetal pancreas and that during early gestation, CXCR4 colocalizes with neurogenin 3 (ngn3), a key transcription factor for endocrine specification in the pancreas. Treatment of islet like clusters (ICCs) derived from human fetal pancreas with SDF-1α resulted in increased proliferation of epithelial cells in ICCs without a concomitant increase in total insulin expression. Exposure of ICCs in vitro to AMD3100, a pharmacological inhibitor of CXCR4, did not alter expression of endocrine hormones insulin and glucagon, or the pancreatic endocrine transcription factors PDX1, Nkx6.1, Ngn3 and PAX4. However, a strong inhibition of β cell genesis was observed when in vitro AMD3100 treatment of ICCs was followed by two weeks of in vivo treatment with AMD3100 after ICC transplantation into mice. Analysis of the grafts for human C-peptide found that inhibition of CXCR4 activity profoundly inhibits islet development. Subsequently, a model pancreatic epithelial cell system (CFPAC-1) was employed to study the signals that regulate proliferation and apoptosis by the SDF-1α/CXCR4 axis. From a selected panel of inhibitors tested, both the PI 3-kinase and MAPK pathways were identified as critical regulators of CFPAC-1 proliferation. SDF-1α stimulated Akt phosphorylation, but failed to increase phosphorylation of Erk above the high basal levels observed. Taken together, these results indicate that SDF-1α/CXCR4 axis plays a critical regulatory role in the genesis of human islets.

MicroRNA dynamics during human embryonic stem cell differentiation to pancreatic endoderm.

MicroRNAs (miRNAs) are small non-coding RNAs that have emerged as critical regulators of human embryonic stem cell (hESC) pluripotency and differentiation. Despite the wealth of information about the role individual that miRNAs play in these two processes, there has yet to be a large-scale temporal analysis of the dynamics of miRNA expression as hESCs move from pluripotency into defined lineages. In this report, we used Next Generation Sequencing (NGS) to map temporal expression of miRNAs over ten 24-hour intervals as pluripotent cells were differentiated into pancreatic endoderm. Of the 2042 known human miRNAs, 694 had non-zero expression on all 11 days. Of these 694 miRNAs, 494 showed statistically significant changes in expression during differentiation. Clusters of miRNAs were identified, each displaying unique expression profiles distributed over multiple days. Selected miRNAs associated with pluripotency/differentiation (miR-302/367 and miR-371/372/373) and development/growth (miR-21, miR-25, miR-103, miR-9, and miR-92a) were found to have distinct expression profiles correlated with changes in media used to drive the differentiation process. Taken together, the clustering of miRNAs to identify expression dynamics that occur over longer periods of time (days vs. hours) provides unique insight into specific stages of differentiation. Major shifts in defined stages of hESC differentiation appear to be heavily dependent upon changes in external environmental factors, rather than intrinsic conditions in the cells.