Lin28 acts as a repressor of microRNA processing and as a post-transcriptional regulatory factor for a subset of mRNAs. Here we report that in human embryonic stem cells Lin28 facilitates the expression of the pivotal pluripotency factor Oct4 at the post-transcriptional level. We provide evidence that Lin28 binds Oct4 mRNA directly through high affinity sites within its coding region and that an interaction between Lin28 and RNA helicase A (RHA) may play a part in the observed regulation. We further demonstrate that decreasing RHA levels impairs Lin28-dependent stimulation of translation in a reporter system. Taken together with previous studies showing that RHA is required for efficient translation of a specific class of mRNAs, these findings suggest a novel mechanism by which Lin28 may affect target mRNA expression and represent the first evidence of post-transcriptional regulation of Oct4 expression by Lin28 in human embryonic stem cells.

To examine the fundamental mechanisms governing neural differentiation, we analyzed the transcriptome changes that occur during the differentiation of hESCs into the neural lineage. Undifferentiated hESCs as well as cells at three stages of early neural differentiation-N1 (early initiation), N2 (neural progenitor), and N3 (early glial-like)-were analyzed using a combination of single read, paired-end read, and long read RNA sequencing. The results revealed enormous complexity in gene transcription and splicing dynamics during neural cell differentiation. We found previously unannotated transcripts and spliced isoforms specific for each stage of differentiation. Interestingly, splicing isoform diversity is highest in undifferentiated hESCs and decreases upon differentiation, a phenomenon we call isoform specialization. During neural differentiation, we observed differential expression of many types of genes, including those involved in key signaling pathways, and a large number of extracellular receptors exhibit stage-specific regulation. These results provide a valuable resource for studying neural differentiation and reveal insights into the mechanisms underlying in vitro neural differentiation of hESCs, such as neural fate specification, neural progenitor cell identity maintenance, and the transition from a predominantly neuronal state into one with increased gliogenic potential.RNA-Seq | splicing isoforms | unannotated transcripts | neuron | glial N eural commitment and subsequent differentiation is a complex process. Although the complexity of RNAs expressed in neural tissues is very high (1, 2), a comprehensive analysis of the genes and RNA isoforms that are expressed during the different stages of neural cell differentiation is largely lacking. Such information is expected to be important for understanding mechanisms of neural cell differentiation and ultimately providing therapeutic solutions for neural degenerative diseases, such as Parkinson's and Alzheimer's disease.Our current knowledge of the mechanisms involved in neural cell formation is derived mostly from studying neurogenesis in the developing embryos of animal models (3, 4). However, neurogenesis in animals is a complex process involving many different cell types that differentiate asynchronously. This heterogeneity, along with the relatively small number of cells that can be readily obtained, makes the analysis of the temporal differentiation of individual cell types extremely difficult. One solution is to analyze hESCs during in vitro differentiation to different stages of neural development, which can be performed using a relatively large numbers of cells (5-9). Analysis of the transcriptome in these cells is expected to provide insights into the mechanisms and pathways involved in early cell fate specification, such as the acquisition of neurogenic potential and the transition to gliogenic potential, which may ultimately be extremely useful for pharmacologic screening and neurodegenerative disease therapies.Many high-throughput methods have...

We have previously shown that coculture of human embryonic stem cells (hESCs) for 14 days with immortalized fetal hepatocytes yields CD34 ؉ cells that can be expanded in serum-free liquid culture into large numbers of megaloblastic nucleated erythroblasts resembling yolk sacderived cells. We show here that these primitive erythroblasts undergo a switch in hemoglobin (Hb) composition during late terminal erythroid maturation with the basophilic erythroblasts expressing predominantly Hb Gower I ( 2 ⑀ 2 ) and the orthochromatic erythroblasts hemoglobin Gower II (␣ 2 ⑀ 2 ). This suggests that the switch from Hb Gower I to Hb Gower II, the first hemoglobin switch in humans is a maturation switch not a lineage switch. We also show that extending the coculture of the hESCs with immortalized fetal hepatocytes to 35 days yields CD34 ؉ cells that differentiate into more developmentally mature, fetal liver-like erythroblasts, that are smaller, express mostly fetal hemoglobin, and can enucleate. We conclude that hESC-derived erythropoiesis closely mimics early human development because the first 2 human hemoglobin switches are recapitulated, and because yolk sac-like and fetal liver-like cells are sequentially produced. Development of a method that yields erythroid cells with an adult phenotype remains necessary, because the most mature cells that can be produced with current systems express less than 2% adult ␤-globin mRNA. IntroductionIn humans, primitive erythropoiesis originates from the extraembryonic mesoderm, is first detectable in the yolk sac 14 to 19 days after conception, and persists in this organ until the ninth week of gestation. It has long been known that yolk sac-derived primitive erythrocytes undergo a partial hemoglobin (Hb) switch: At week 5, yolk sac erythroblasts synthesize primarily Hb Gower I ( 2 ⑀ 2 ), but at weeks 6 to 8, they also synthesize large amounts of Hb Gower II (␣ 2 ⑀ 2 ). 1 Definitive erythropoiesis, which originates from the aortagonado-mesonephros region of the embryo proper, 2 is first detectable in the fetal liver during the sixth week of development. Erythroblasts produced in this organ express , ⑀, ␣, ␥, and small amounts of ␤-globin but the ⑀ and -globin genes are rapidly silenced while the ␣ and ␥-globin genes remain expressed at high level until around birth. At that point, bone marrow erythropoiesis, which is first detectable around the eleventh week of gestation, becomes the major site of erythropoiesis and expression of the ␤-globin gene, which had slowly risen during gestation almost completely replaces ␥-globin expression. In addition to these differences in globin-expression patterns, yolk sac, fetal liver, and bone marrow erythrocytes differ in morphology because yolk sac erythroblasts are nucleated and megaloblastic, while both fetal liver and bone marrow erythrocytes are enucleated. Fetal and adult erythrocytes differ by size, with the fetal cells bigger than the adult ones.Human embryonic stem cells (hESCs) can self-renew indefinitely in culture while retaining the ca...

No abstract

No abstract

Steady state cellular microRNA (miRNA) levels represent the balance between miRNA biogenesis and turnover. The kinetics and sequence determinants of mammalian miRNA turnover during and after miRNA maturation are not fully understood. Through a large-scale study on mammalian miRNA turnover, we report the co-existence of multiple cellular miRNA pools with distinct turnover kinetics and biogenesis properties and reveal previously unrecognized sequence features for fast turnover miRNAs. We measured miRNA turnover rates in eight mammalian cell types with a combination of expression profiling and deep sequencing. While most miRNAs are stable, a subset of miRNAs, mostly miRNA*s, turnovers quickly, many of which display a two-step turnover kinetics. Moreover, different sequence isoforms of the same miRNA can possess vastly different turnover rates. Fast turnover miRNA isoforms are enriched for 5′ nucleotide bias against Argonaute-(AGO)-loading, but also additional 3′ and central sequence features. Modeling based on two fast turnover miRNA*s miR-222-5p and miR-125b-1-3p, we unexpectedly found that while both miRNA*s are associated with AGO, they strongly differ in HSP90 association and sensitivity to HSP90 inhibition. Our data characterize the landscape of genome-wide miRNA turnover in cultured mammalian cells and reveal differential HSP90 requirements for different miRNA*s. Our findings also implicate rules for designing stable small RNAs, such as siRNAs.

Megakaryoblastic leukemia 1 (MKL1), identified as part of the t(1;22) translocation specific to acute megakaryoblastic leukemia, is highly expressed in differentiated muscle cells and promotes muscle differentiation by activating serum response factor (SRF). Here we show that Mkl1 expression is up-regulated during murine megakaryocytic differentiation and that enforced overexpression of MKL1 enhances megakaryocytic differentiation.When the human erythroleukemia (HEL) cell line is induced to differentiate with 12-O-tetradecanoylphorbol 13-acetate, overexpression of MKL1 results in an increased number of megakaryocytes with a concurrent increase in ploidy. MKL1 overexpression also promotes megakaryocytic differentiation of primary human CD34 ؉ cells cultured in the presence of thrombopoietin. The effect of MKL1 is abrogated when SRF is knocked down, suggesting that MKL1 acts through SRF. Consistent with these findings in human cells, knockout of Mkl1 in mice leads to reduced platelet counts in peripheral blood, and reduced ploidy in bone marrow megakaryocytes. In conclusion, MKL1 promotes physiologic maturation of human and murine megakaryocytes.

SummaryThere is an urgent need for an efficient approach to obtain a large-scale and renewable source of functional human vascular smooth muscle cells (VSMCs) to establish robust, patient-specific tissue model systems for studying the pathogenesis of vascular disease, and for developing novel therapeutic interventions. Here, we have derived a large quantity of highly enriched functional VSMCs from human induced pluripotent stem cells (hiPSC-VSMCs). Furthermore, we have engineered 3D tissue rings from hiPSC-VSMCs using a facile one-step cellular self-assembly approach. The tissue rings are mechanically robust and can be used for vascular tissue engineering and disease modeling of supravalvular aortic stenosis syndrome. Our method may serve as a model system, extendable to study other vascular proliferative diseases for drug screening. Thus, this report describes an exciting platform technology with broad utility for manufacturing cell-based tissues and materials for various biomedical applications.