Purification and Ex Vivo Expansion of Fully Functional Salivary Gland Stem Cells.

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Purification and Ex Vivo Expansion of Fully Functional Salivary Gland Stem Cells.

Stem Cell Reports. 2014 Oct 23;

Authors: Nanduri LS, Baanstra M, Faber H, Rocchi C, Zwart E, de Haan G, van Os R, Coppes RP

Abstract
Hyposalivation often leads to irreversible and untreatable xerostomia. Salivary gland (SG) stem cell therapy is an attractive putative option to salvage these patients but is impeded by the limited availability of adult human tissue. Here, using murine SG cells, we demonstrate single-cell self-renewal, differentiation, enrichment of SG stem cells, and robust in vitro expansion. Dependent on stem cell marker expression, SG sphere-derived single cells could be differentiated in vitro into distinct lobular or ductal/lobular organoids, suggestive of progenitor or stem cell potency. Expanded cells were able to form miniglands/organoids containing multiple SG cell lineages. Expansion of these multipotent cells through serial passaging resulted in selection of a cell population, homogenous for stem cell marker expression (CD24(hi)/CD29(hi)). Cells highly expressing CD24 and CD29 could be prospectively isolated and were able to efficiently restore radiation-damaged SG function. Our approach will facilitate the use of adult SG stem cells for a variety of scientific and therapeutic purposes.

PMID: 25448065 [PubMed – as supplied by publisher]

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Tracing Dynamics and Clonal Heterogeneity of Cbx7-Induced Leukemic Stem Cells by Cellular Barcoding.

Tracing Dynamics and Clonal Heterogeneity of Cbx7-Induced Leukemic Stem Cells by Cellular Barcoding.

Stem Cell Reports. 2014 Nov 25;

Authors: Klauke K, Broekhuis MJ, Weersing E, Dethmers-Ausema A, Ritsema M, González MV, Zwart E, Bystrykh LV, de Haan G

Abstract
Accurate monitoring of tumor dynamics and leukemic stem cell (LSC) heterogeneity is important for the development of personalized cancer therapies. In this study, we experimentally induced distinct types of leukemia in mice by enforced expression of Cbx7. Simultaneous cellular barcoding allowed for thorough analysis of leukemias at the clonal level and revealed high and unpredictable tumor complexity. Multiple LSC clones with distinct leukemic properties coexisted. Some of these clones remained dormant but bore leukemic potential, as they progressed to full-blown leukemia after challenge. LSC clones could retain multilineage differentiation capacities, where one clone induced phenotypically distinct leukemias. Beyond a detailed insight into CBX7-driven leukemic biology, our model is of general relevance for the understanding of tumor dynamics and clonal evolution.

PMID: 25434821 [PubMed – as supplied by publisher]

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Kinase activity profiling reveals active signal transduction pathways in pediatric acute lymphoblastic leukemia; a new approach for target discovery.

Kinase activity profiling reveals active signal transduction pathways in pediatric acute lymphoblastic leukemia; a new approach for target discovery.

Proteomics. 2014 Nov 25;

Authors: van der Sligte NE, Scherpen FJ, Meeuwsen-de Boer TG, Lourens HJ, Ter Elst A, Diks SH, Guryev V, Peppelenbosch MP, van Leeuwen FN, de Bont ES

Abstract
Still about 20% of patients with acute lymphoblastic leukemia (ALL) struggle with relapse, despite intensive chemotherapy. We and others have shown that kinase activity profiling is able to give more insights in active signal transduction pathways and point out interesting signaling hubs as well as new potential druggable targets. With this technique the gap between newly designed drugs and ALL may be bridged. The aim of this study was to perform kinome profiling on 20 pediatric ALL samples (14 BCP-ALL and 6 T-ALL) to identify signaling proteins relevant to ALL. We defined 250 peptides commonly activated in both BCP-ALL and T-ALL representing major signal transduction pathways including MAPK, PI3K/Akt, and regulators of the cell cycle/p53 pathway. For 27 peptides, differentially phosphorylation between BCP-ALL and T-ALL was observed. Among these, 10 peptides were more highly phosphorylated in BCP-ALL while 17 peptides showed increased phosphorylation in T-ALL. Furthermore we selected one lead of the list of commonly activated peptides (HGFR_Y1235) in order to test its efficacy as a potential target and provide proof of principle for this approach. In conclusion kinome profiling is an elegant approach to study active signaling and identify interesting potential druggable targets. This article is protected by copyright. All rights reserved.

PMID: 25422122 [PubMed – as supplied by publisher]

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Genome-wide RNA Tomography in the Zebrafish Embryo.

Genome-wide RNA Tomography in the Zebrafish Embryo.

Cell. 2014 Oct 23;159(3):662-75

Authors: Junker JP, Noël ES, Guryev V, Peterson KA, Shah G, Huisken J, McMahon AP, Berezikov E, Bakkers J, van Oudenaarden A

Abstract
Advancing our understanding of embryonic development is heavily dependent on identification of novel pathways or regulators. Although genome-wide techniques such as RNA sequencing are ideally suited for discovering novel candidate genes, they are unable to yield spatially resolved information in embryos or tissues. Microscopy-based approaches, using in situ hybridization, for example, can provide spatial information about gene expression, but are limited to analyzing one or a few genes at a time. Here, we present a method where we combine traditional histological techniques with low-input RNA sequencing and mathematical image reconstruction to generate a high-resolution genome-wide 3D atlas of gene expression in the zebrafish embryo at three developmental stages. Importantly, our technique enables searching for genes that are expressed in specific spatial patterns without manual image annotation. We envision broad applicability of RNA tomography as an accurate and sensitive approach for spatially resolved transcriptomics in whole embryos and dissected organs.

PMID: 25417113 [PubMed – in process]

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Intrinsically disordered linker and plasma membrane binding motif sorts Ist2 and Ssy1 to junctions.

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Intrinsically disordered linker and plasma membrane binding motif sorts Ist2 and Ssy1 to junctions.

Traffic. 2014 Nov 20;

Authors: Kralt A, Carretta M, Mari M, Reggiori F, Steen A, Poolman B, Veenhoff LM

Abstract
Membrane junctions or contact sites are close associations of lipid bilayers of heterologous organelles. Ist2 is an endoplasmic reticulum (ER) resident transmembrane protein that mediates associations between the plasma membrane (PM) and the cortical ER (cER) in baker’s yeast. We asked the question what structure in Ist2 bridges the up to 30?nm distance between the PM and the cER and we noted that the region spacing the transmembrane domain from the cortical sorting signal interacting with the PM, is predicted to be intrinsically disordered. In Ssy1, a protein that was not previously described to reside at membrane junctions, we recognized a domain organization similar to Ist2. We found that the localization at the cell periphery of both Ist2 and Ssy1 depends on the presence of a PM binding domain, an intrinsically disordered linker region of sufficient length and a transmembrane domain that most likely resides in the ER. We show for the first time that an intrinsically disordered amino acid domain bridges adjacent heterologous membranes. The length and flexibility of intrinsically disordered domains make them uniquely eligible for spanning large distances and we suggest that this domain structure more frequently occurs in proteins that mediate the formation of membrane contact sites.

PMID: 25409870 [PubMed – as supplied by publisher]

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Probing the Disordered Domain of the Nuclear Pore Complex through Coarse-Grained Molecular Dynamics Simulations.

Probing the Disordered Domain of the Nuclear Pore Complex through Coarse-Grained Molecular Dynamics Simulations.

Biophys J. 2014 Sep 16;107(6):1393-1402

Authors: Ghavami A, Veenhoff LM, van der Giessen E, Onck PR

Abstract
The distribution of disordered proteins (FG-nups) that line the transport channel of the nuclear pore complex (NPC) is investigated by means of coarse-grained molecular dynamics simulations. A one-bead-per-amino-acid model is presented that accounts for the hydrophobic/hydrophilic and electrostatic interactions between different amino acids, polarity of the solvent, and screening of free ions. The results indicate that the interaction of the FG-nups forms a high-density, doughnut-like distribution inside the NPC, which is rich in FG-repeats. We show that the obtained distribution is encoded in the amino-acid sequence of the FG-nups and is driven by both electrostatic and hydrophobic interactions. To explore the relation between structure and function, we have systematically removed different combinations of FG-nups from the pore to simulate inviable and viable NPCs that were previously studied experimentally. The obtained density distributions show that the maximum density of the FG-nups inside the pore does not exceed 185 mg/mL in the inviable NPCs, whereas for the wild-type and viable NPCs, this value increases to 300 mg/mL. Interestingly, this maximum density is not correlated to the total mass of the FG-nups, but depends sensitively on the specific combination of essential Nups located in the central plane of the NPC.

PMID: 25229147 [PubMed – as supplied by publisher]

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Chromosome instability induced by Mps1 and p53 mutation generates aggressive lymphomas exhibiting aneuploidy-induced stress.

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Chromosome instability induced by Mps1 and p53 mutation generates aggressive lymphomas exhibiting aneuploidy-induced stress.

Proc Natl Acad Sci U S A. 2014 Sep 2;

Authors: Foijer F, Xie SZ, Simon JE, Bakker PL, Conte N, Davis SH, Kregel E, Jonkers J, Bradley A, Sorger PK

Abstract
Aneuploidy is a hallmark of human solid cancers that arises from errors in mitosis and results in gain and loss of oncogenes and tumor suppressors. Aneuploidy poses a growth disadvantage for cells grown in vitro, suggesting that cancer cells adapt to this burden. To understand better the consequences of aneuploidy in a rapidly proliferating adult tissue, we engineered a mouse in which chromosome instability was selectively induced in T cells. A flanked by Lox mutation was introduced into the monopolar spindle 1 (Mps1) spindle-assembly checkpoint gene so that Cre-mediated recombination would create a truncated protein (Mps1(DK)) that retained the kinase domain but lacked the kinetochore-binding domain and thereby weakened the checkpoint. In a sensitized p53(+/-) background we observed that Mps1(DK/DK) mice suffered from rapid-onset acute lymphoblastic lymphoma. The tumors were highly aneuploid and exhibited a metabolic burden similar to that previously characterized in aneuploid yeast and cultured cells. The tumors nonetheless grew rapidly and were lethal within 3-4 mo after birth.

PMID: 25197064 [PubMed – as supplied by publisher]

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Clonal heterogeneity as a driver of disease variability in the evolution of myeloproliferative neoplasms.

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Clonal heterogeneity as a driver of disease variability in the evolution of myeloproliferative neoplasms.

Exp Hematol. 2014 Sep 5;

Authors: Prick J, de Haan G, Green AR, Kent DG

Abstract
Myeloproliferative neoplasms (MPNs) are clonal hematological diseases in which cells of the myelo-erythroid lineage are overproduced and patients are predisposed to leukemic transformation. Hematopoietic stem cells (HSCs) are the suspected disease initiating cells and these cells must acquire a clonal advantage relative to non-mutant HSCs in order to perpetuate disease. In 2005, several groups identified a single gain-of-function point mutation in JAK2 that associated with the majority of MPNs, and subsequent studies have led to a comprehensive understanding of the mutational landscape in MPNs. However, confusion still exists as to how a single genetic aberration can be associated with multiple distinct disease entities. Many explanations have been proposed, including JAK2V617F homozygosity, individual patient heterogeneity and the differential regulation of downstream JAK2 signaling pathways. Several groups have made knock-in mouse models expressing JAK2V617F and have observed divergent phenotypes, each recapitulating some aspects of disease. Intriguingly, most of these models do not observe a strong HSC self-renewal advantage compared to WT littermate controls, raising the question of how a clonal advantage is established in MPN patients. This review summarizes the current molecular understanding of MPNs, the diversity of disease phenotypes and proposes that the increased proliferation induced by JAK2V617F applies a selection pressure on the mutant clone that results in highly diverse clonal evolution in individuals.

PMID: 25201757 [PubMed – as supplied by publisher]

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microRNAs in hematopoiesis.

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microRNAs in hematopoiesis.

Exp Cell Res. 2014 Sep 2;

Authors: Lazare SS, Wojtowicz EE, Bystrykh LV, de Haan G

Abstract
miRNAs have been implicated in all stages of hematopoiesis including maintenance of self-renewal of hematopoietic stem cells (HSCs) and differentiation into mature blood cells. Regulation bv miRNAs is markedly intertwined with transcription factors. In this review, we highlight miRNAs shown to be important for HSC maintenance and lineage differentiation with focus on their interaction with transcription factors. We also pay attention to the diverse modes of miRNA regulation.

PMID: 25192911 [PubMed – as supplied by publisher]

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The Developmental Potential of iPSCs Is Greatly Influenced by Reprogramming Factor Selection.

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The Developmental Potential of iPSCs Is Greatly Influenced by Reprogramming Factor Selection.

Cell Stem Cell. 2014 Sep 4;15(3):295-309

Authors: Buganim Y, Markoulaki S, van Wietmarschen N, Hoke H, Wu T, Ganz K, Akhtar-Zaidi B, He Y, Abraham BJ, Porubsky D, Kulenkampff E, Faddah DA, Shi L, Gao Q, Sarkar S, Cohen M, Goldmann J, Nery JR, Schultz MD, Ecker JR, Xiao A, Young RA, Lansdorp PM, Jaenisch R

Abstract
Induced pluripotent stem cells (iPSCs) are commonly generated by transduction of Oct4, Sox2, Klf4, and Myc (OSKM) into cells. Although iPSCs are pluripotent, they frequently exhibit high variation in terms of quality, as measured in mice by chimera contribution and tetraploid complementation. Reliably high-quality iPSCs will be needed for future therapeutic applications. Here, we show that one major determinant of iPSC quality is the combination of reprogramming factors used. Based on tetraploid complementation, we found that ectopic expression of Sall4, Nanog, Esrrb, and Lin28 (SNEL) in mouse embryonic fibroblasts (MEFs) generated high-quality iPSCs more efficiently than other combinations of factors including OSKM. Although differentially methylated regions, transcript number of master regulators, establishment of specific superenhancers, and global aneuploidy were comparable between high- and low-quality lines, aberrant gene expression, trisomy of chromosome 8, and abnormal H2A.X deposition were distinguishing features that could potentially also be applicable to human.

PMID: 25192464 [PubMed – in process]

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