Yung, S, Ledran, M, Moreno-Gimeno, I, Conesa, A, Montaner, D, Dopazo, J et al.. Large-scale transcriptional profiling and functional assays reveal important roles for Rho-GTPase signalling and SCL during haematopoietic differentiation of human embryonic stem cells. Hum Mol Genet 20: 4932-4946

Institute of Genetic Medicine, Newcastle University, Newcastle upon Tyne, UK.
Human Molecular Genetics (Impact Factor: 6.39). 09/2011; 20(24):4932-46. DOI: 10.1093/hmg/ddr431
Source: PubMed


Understanding the transcriptional cues that direct differentiation of human embryonic stem cells (hESCs) and human-induced pluripotent stem cells to defined and functional cell types is essential for future clinical applications. In this study, we have compared transcriptional profiles of haematopoietic progenitors derived from hESCs at various developmental stages of a feeder- and serum-free differentiation method and show that the largest transcriptional changes occur during the first 4 days of differentiation. Data mining on the basis of molecular function revealed Rho-GTPase signalling as a key regulator of differentiation. Inhibition of this pathway resulted in a significant reduction in the numbers of emerging haematopoietic progenitors throughout the differentiation window, thereby uncovering a previously unappreciated role for Rho-GTPase signalling during human haematopoietic development. Our analysis indicated that SCL was the 11th most upregulated transcript during the first 4 days of the hESC differentiation process. Overexpression of SCL in hESCs promoted differentiation to meso-endodermal lineages, the emergence of haematopoietic and erythro-megakaryocytic progenitors and accelerated erythroid differentiation. Importantly, intrasplenic transplantation of SCL-overexpressing hESC-derived haematopoietic cells enhanced recovery from induced acute anaemia without significant cell engraftment, suggesting a paracrine-mediated effect.

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Available from: Pedro J Real, Aug 14, 2014
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    • "Endothelial differentiation was induced as previously described [11]. Hematopoietic and neural progenitor cells were generated from H1 hESC as previously described [33, 34]. The cells were harvested at defined time points as indicated and expressed characteristic markers of hESC-derived hematopoiesis (hES-hematopoietic progenitor cell [HPC] CD34 [41%] and CD45 [54%]) as analyzed by flow cytometric analysis. "
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    ABSTRACT: MicroRNAs (miRNAs) are short noncoding RNAs, which post-transcriptionally regulate gene expression. miRNAs are transcribed as precursors and matured to active forms by a series of enzymes, including Dicer. miRNAs are important in governing cell differentiation, development, and disease. We have recently developed a feeder- and serum-free protocol for direct derivation of endothelial cells (ECs) from human embryonic stem cells (hESCs) and provided evidence of increases in angiogenesis-associated miRNAs (miR-126 and -210) during the process. However, the functional role of miRNAs in hESC differentiation to vascular EC remains to be fully interrogated. Here, we show that the reduction of miRNA maturation induced by Dicer knockdown suppressed hES-EC differentiation. A miRNA microarray was performed to quantify hES-EC miRNA profiles during defined stages of endothelial differentiation. miR-99b, -181a, and -181b were identified as increasing in a time- and differentiation-dependent manner to peak in mature hESC-ECs and adult ECs. Augmentation of miR-99b, -181a, and -181b levels by lentiviral-mediated transfer potentiated the mRNA and protein expression of EC-specific markers, Pecam1 and VE Cadherin, increased nitric oxide production, and improved hES-EC-induced therapeutic neovascularization in vivo. Conversely, knockdown did not impact endothelial differentiation. Our results suggest that miR-99b, -181a, and -181b comprise a component of an endothelial-miRNA signature and are capable of potentiating EC differentiation from pluripotent hESCs.
    Stem Cells 04/2012; 30(4):643-54. DOI:10.1002/stem.1026 · 6.52 Impact Factor
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    ABSTRACT: Determining the molecular regulators/pathways responsible for the specification of human embryonic stem cells (hESCs) into hematopoietic precursors has far-reaching implications for potential cell therapies and disease modeling. Mouse models lacking SCL/TAL1 (stem cell leukemia/T-cell acute lymphocytic leukemia 1) do not survive beyond early embryogenesis because of complete absence of hematopoiesis, indicating that SCL is a master early hematopoietic regulator. SCL is commonly found rearranged in human leukemias. However, there is barely information on the role of SCL on human embryonic hematopoietic development. Differentiation and sorting assays show that endogenous SCL expression parallels hematopoietic specification of hESCs and that SCL is specifically expressed in hematoendothelial progenitors (CD45(-)CD31(+)CD34(+)) and, to a lesser extent, on CD45(+) hematopoietic cells. Enforced expression of SCL in hESCs accelerates the emergence of hematoendothelial progenitors and robustly promotes subsequent differentiation into primitive (CD34(+)CD45(+)) and total (CD45(+)) blood cells with higher clonogenic potential. Short-hairpin RNA-based silencing of endogenous SCL abrogates hematopoietic specification of hESCs, confirming the early hematopoiesis-promoting effect of SCL. Unfortunately, SCL expression on its own is not sufficient to confer in vivo engraftment to hESC-derived hematopoietic cells, suggesting that additional yet undefined master regulators are required to orchestrate the stepwise hematopoietic developmental process leading to the generation of definitive in vivo functional hematopoiesis from hESCs.
    Molecular Therapy 04/2012; 20(7):1443-53. DOI:10.1038/mt.2012.49 · 6.23 Impact Factor
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    ABSTRACT: The therapeutic potential of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) is vast, allowing disease modelling, drug discovery and testing and perhaps most importantly regenerative therapies. However, problems abound; techniques for cultivating self-renewing hESCs tend to give a heterogeneous population of self-renewing and partially differentiated cells and general include animal-derived products which can be cost-prohibitive for large scale production and effective lineage specific differentiation protocols also still remain relatively undefined and are inefficient in producing large amounts of cells for therapeutic use. Further, the mechanisms and signalling pathways which mediate pluripotency and differentiation are still to be fully appreciated. However, over the recent years, the development/discovery of a range of effective small molecule inhibitors/activators has had a huge impact in hESC biology. Large scale screening techniques, coupled with greater knowledge of the pathways involved, have generated pharmacological agents which can boost hESC pluripotency/self-renewal and survival and has allowed great increases in the efficiency of various differentiation protocols, while also aiding the delineation of several important signalling pathways. Within this review, we hope to describe the current uses of small molecule inhibitors/activators in hESC biology and their potential uses in the future. © 2012 The Authors. British Journal of Pharmacology © 2012 The British Pharmacological Society.
    British Journal of Pharmacology 04/2012; 169(2). DOI:10.1111/j.1476-5381.2012.01978.x · 4.84 Impact Factor
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