Myeloproliferative disorders are clonal haematopoietic stem cell malignancies characterized by independency or hypersensitivity of haematopoietic progenitors to numerous cytokines. The molecular basis of most myeloproliferative disorders is unknown. On the basis of the model of chronic myeloid leukaemia, it is expected that a constitutive tyrosine kinase activity could be at the origin of these diseases. Polycythaemia vera is an acquired myeloproliferative disorder, characterized by the presence of polycythaemia diversely associated with thrombocytosis, leukocytosis and splenomegaly. Polycythaemia vera progenitors are hypersensitive to erythropoietin and other cytokines. Here, we describe a clonal and recurrent mutation in the JH2 pseudo-kinase domain of the Janus kinase 2 (JAK2) gene in most (> 80%) polycythaemia vera patients. The mutation, a valine-to-phenylalanine substitution at amino acid position 617, leads to constitutive tyrosine phosphorylation activity that promotes cytokine hypersensitivity and induces erythrocytosis in a mouse model. As this mutation is also found in other myeloproliferative disorders, this unique mutation will permit a new molecular classification of these disorders and novel therapeutical approaches.

The study of induced pluripotency is complicated by the need for infection with high-titer retroviral vectors, which results in genetically heterogeneous cell populations. We generated genetically homogeneous ‘secondary’ somatic cells that carry the reprogramming factors as defined doxycycline (dox)-inducible transgenes. These cells were produced by infecting fibroblasts with dox-inducible lentiviruses, reprogramming by dox addition, selecting induced pluripotent stem cells and producing chimeric mice. Cells derived from these chimeras reprogram upon dox exposure without the need for viral infection with efficiencies 25- to 50-fold greater than those observed using direct infection and drug selection for pluripotency marker reactivation. We demonstrate that (i) various induction levels of the reprogramming factors can induce pluripotency, (ii) the duration of transgene activity directly correlates with reprogramming efficiency, (iii) cells from many somatic tissues can be reprogrammed and (iv) different cell types require different induction levels. This system facilitates the characterization of reprogramming and provides a tool for genetic or chemical screens to enhance reprogramming.

Direct reprogramming of human fibroblasts to induced pluripotent stem cells (iPS) has been achieved by ectopic expression of defined transcription factors. Derivation of human fibroblasts however is a time consuming process and requires punch biopsies or isolation of patient foreskin. Here we use a polycistronic vector encoding Oct4, Klf4, Sox2 and c-Myc to generate iPS cells from from frozen peripheral blood of several donors. Genomic DNA analyses indicated that iPS cells were derived from mature T cells as well as myeloid donor cells. Inducing pluripotency in peripheral blood would allow utilization of easy to get samples from the adult and, more importantly, provide convenient access to numerous patient samples stored in blood banks. The latter is of major interest as frozen blood samples, when reprogrammed to iPS cells, would allow the retrospective molecular analyses of rare diseases.

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Binding of erythropoietin to the erythropoietin receptor (EpoR) extracellular domain orients the transmembrane (TM) and cytosolic regions of the receptor dimer into an unknown activated conformation. By replacing the EpoR extracellular domain with a dimeric coiled coil, we engineered TM EpoR fusion proteins where the helical TM domains were constrained into seven possible relative orientations. We identify one dimeric TM conformation that imparts full activity to the cytosolic domain of the receptor and signals via JAK2, STAT proteins, and MAP kinase, one partially active orientation that preferentially activates MAP kinase, and one conformation corresponding to the inactive receptor. The active and inactive conformations were independently identified by computational searches for low-energy TM dimeric structures. We propose a specific EpoR-activated interface and suggest its use for structural and signaling studies.

The thrombopoietin receptor (TpoR) regulates hematopoietic stem cell renewal, megakaryocyte differentiation, and platelet formation. TpoR signals by activating Janus kinases JAK2 and Tyk2. Here we show that, in addition to signaling downstream from the activated TpoR, JAK2 and Tyk2 strongly promote cell surface localization and enhance total protein levels of the TpoR. This effect is caused by stabilization of the mature endoglycosidase H-resistant form of the receptor. Confocal microscopy indicates that TpoR colocalizes partially with recycling transferrin in Ba/F3 cells. The interaction with JAK2 or Tyk2 appears to protect the receptor from proteasome degradation. Sequences encompassing Box1 and Box2 regions of the receptor cytosolic domain and an intact JAK2 or Tyk2 FERM domain are required for these effects. We discuss the relevance of our results to the reported defects of TpoR processing in myeloproliferative diseases and to the mechanisms of Tpo signaling and clearance via the TpoR.

Embryonic stem (ES) cells are isolated from the inner cell mass (ICM) of blastocysts, whereas epiblast stem cells (EpiSCs) are derived from the post-implantation epiblast and display a restricted developmental potential. Here we characterize pluripotent states in the non-obese diabetic (NOD) mouse strain, which prior to this study was considered “non-permissive” for ES cell derivation. We find that NOD stem cells can be stabilized by providing constitutive expression of Klf4 or c-Myc or small molecules that can replace these factors during in vitro reprogramming. The NOD ES and iPS cells appear “metastable”, as they acquire an alternative EpiSC-like identity after removal of the exogenous factors, while their reintroduction converts the cells back to ICM-like pluripotency. Our findings suggest that stem cells from different genetic backgrounds can assume distinct states of pluripotency in vitro, the stability of which is regulated by endogenous genetic determinants and can be modified by exogenous factors.

Ligand binding to the thrombopoietin receptor (TpoR) is thought to impose a dimeric receptor conformation(s) leading to hematopoietic stem cell renewal, megakaryocyte differentiation, and platelet formation. Unlike other cytokine receptors, such as the erythropoietin receptor, TpoR contains an amphipathic KWQFP motif at the junction between the transmembrane (TM) and cytoplasmic domains. We show here that a mutant TpoR (⌬5TpoR), where this sequence was deleted, is constitutively active. In the absence of ligand, ⌬5TpoR activates Jak2, Tyk2, STAT5, and mitogen-activated protein (MAP) kinase, but does not appear to induce STAT3 phosphorylation. ⌬5TpoR induces hematopoietic myeloid differentiation in the absence of Tpo. In the presence of Tpo, the ⌬5TpoR mutant appears to enhance erythroid differentiation when compared with the Tpo-activated wild-type TpoR. Strikingly, individual substitution of K507 or W508 to alanine also induces constitutive TpoR activation, indicating that the K and W residues within the amphipathic KWQFP motif are crucial for maintaining the unliganded receptor inactive. These residues may be targets for activating mutations in humans. Such a motif may exist in other receptors to prevent ligand-independent activation and to allow signaling via multiple flexible interfaces. IntroductionThe thrombopoietin receptor (c-mpl or TpoR) and its ligand thrombopoietin (Tpo) regulate the proliferation of megakaryocytic progenitors, their differentiation into mature megakaryocytes, and formation of platelets. 1,2 Tpo or TpoR knockout mice exhibit a significant reduction of megakaryocytes and circulating platelets, and they also show reduced number of hematopoietic stem cells (HSCs) in the bone marrow, indicating a role for the TpoR and its ligand in HSC self-renewal. 3 Like the erythropoietin receptor (EpoR), the TpoR is thought to function as a homodimer after binding Tpo. Downstream signaling pathways activated by the TpoR are triggered by 2 cytoplasmic tyrosine kinases, the Janus kinase (Jak) 2, and Tyk2. [4][5][6] Upon receptor activation, phosphorylated tyrosine residues in the cytoplasmic domain of the TpoR and in Jaks provide docking sites for the SH2 domains of many signaling proteins, such as STAT3 and STAT5 (the signal transducers and activators of transcription 3 and 5), shc, SHIP, Grb2, and PI3K. 4,5,[7][8][9] Several activating mutations in the TpoR have been identified. In one, the envelope protein of the myeloproliferative virus replaces the extracellular domain of the receptor and activates it by oligomerization. 10 Introduction of a cysteine residue in the extracellular domain of the TpoR at a position equivalent to that of the constitutively active R129C EpoR mutant 11,12 leads to active disulfide-bonded TpoR dimers. 13 Replacement of S498 (or S505) in the transmembrane (TM) domain by asparagine results in constitutively active receptors, 9,14 most likely by ligand-independent dimerization. 9,10,15 Deletion of the membrane distal extracellular cytokine receptor module of the TpoR results...