Reprogramming of Human Peripheral Blood Cells to Induced Pluripotent Stem Cells

Embryonic stem cells are pluripotent cells derived from the inner cell mass of the developing embryo that have the capacity to differentiate into every cell type of the adult (Evans and Kaufman, 1981, Martin, 1981, Martin and Evans, 1975 and Thomson et al., 1998). The generation of patient-specific pluripotent cells is therefore an important goal of regenerative medicine. A major step to achieve this was the recent discovery that ectopic expression of defined transcription factors induces pluripotency in somatic cells (Lowry et al., 2008, Park et al., 2008b, Takahashi et al., 2007 and Yu et al., 2007). Until now, the most common source from which to derive human iPSCs has been skin fibroblasts (Lowry et al., 2008, Park et al., 2008a, Park et al., 2008b, Takahashi et al., 2007 and Yu et al., 2009). However, the requirement for skin biopsies and the need to expand fibroblast cells for several passages in vitro represent a hurdle that must be overcome to make iPSC technology broadly applicable. Peripheral blood can be utilized as an easily accessible source of patient tissue for reprogramming. Here we derived iPSCs from frozen human peripheral blood samples. Some of the iPSCs had rearrangements of the T cell receptor (TCR), indicating that T cells can be reprogrammed to pluripotency.National Institutes of Health (U.S.) (Grant 5-RO1-HDO45022)National Institutes of Health (U.S.) (Grant 5-R37-CA084198)National Institutes of Health (U.S.). (Grant 5-RO1-CA087869)National Center for Research Resources (U.S.) (Grant UL1 RR025758

Rapid genome editing by CRISPR-Cas9-POLD3 fusion

Precision CRISPR gene editing relies on the cellular homology-directed DNA repair (HDR) to introduce custom DNA sequences to target sites. The HDR editing efficiency varies between cell types and genomic sites, and the sources of this variation are incompletely understood. Here, we have studied the effect of 450 DNA repair protein-Cas9 fusions on CRISPR genome editing outcomes. We find the majority of fusions to improve precision genome editing only modestly in a locus- and cell-type specific manner. We identify Cas9-POLD3 fusion that enhances editing by speeding up the initiation of DNA repair. We conclude that while DNA repair protein fusions to Cas9 can improve HDR CRISPR editing, most need to be optimized to the cell type and genomic site, highlighting the diversity of factors contributing to locus-specific genome editing outcomes.Peer reviewe

The ubiquitin-mediated degradation of Jak1 modulates osteoclastogenesis by limiting interferon-beta-induced inhibitory signaling.

Interferons (IFNs) have been shown to negatively regulate osteoclastogenesis. In a proteomic study to assess protein expression during osteoclastogenesis, we discovered that the expression level of Jak1 was significantly decreased during the early stage of osteoclast differentiation from mouse bone marrow macrophages (BMMs) upon stimulation with receptor activator of nuclear factor kappaB ligand (RANKL). RANKL induced Jak1 ubiquitination, and a proteasome inhibitor MG132 efficiently blocked the RANKL-induced degradation of Jak1. The expression level of Jak1 correlated with the susceptibility of osteoclast precursors to the negative regulatory effects of IFN-beta on osteoclastogenesis, since preosteoclasts (pOCs) in which Jak1 expression is significantly reduced could proceed with osteoclastogenesis in the presence of IFN-beta. Forced down-regulation of Jak1 by small interfering RNA (siRNA) resulted in the efficient osteoclast differentiation of BMMs in the presence of inhibitory IFN-beta, while overexpression of Jak1 in pOCs elicited IFN-beta-dependent inhibition of osteoclastogenesis. Furthermore, we found that the IFN-beta-induced inhibition of osteoclastogenesis required STAT3 downstream of Jak1. These data suggest that the regulation of Jak1 expression during osteoclast differentiation might serve as an intrinsic mechanism that determines osteoclast lineage commitment by modulating the negative regulation by IFN-beta

Metastable Pluripotent States in NOD Mouse Derived ES Cells

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.National Institutes of Health (U.S.) (Grant RO1-HDO45022)National Institutes of Health (U.S.) (Grant R37-CA084198)National Institutes of Health (U.S.) (Grant RO1-CA087869

The JAK-STAT pathway and hematopoietic stem cells from the JAK2 V617F perspective

Janus kinases (JAKs) are non-receptor tyrosine kinases essential for activation of signaling mediated by cytokine receptors that lack catalytic activity, including receptors for erythropoietin, thrombopoietin, most interleukins and interferon. Upon hormone binding, JAKs phosphorylate tyrosine residues in the receptor cytoplasmic domains and in JAKs themselves leading to recruitment and activation of downstream signaling proteins such as signal transducer and activator of transcription (STAT). The JAK-STAT pathway is important for functional hematopoiesis and several activating mutations in JAK proteins have recently been described as underlying cause of blood disorders. One of the best studied examples is the JAK2 V617F mutant which is found in 95% of polycythemia vera patients and 50% of patients suffering from essential thrombocythemia and primary myelofibrosis. Much effort has been made to understand how the JAK2 V617F affects hematopoietic stem cell (HSC) renewal and lineage differentiation, since convincing evidence has been provided to support the notion that the mutation is acquired at the HSC level. We discuss several in vivo models that support contrary conclusions with respect to the advantage given to HSCs by JAK2 V617F. Moreover, we provide the current knowledge about STAT5 activation and its link to HSC expansion as well as amplification of the erythroid compartment. Evidence for both JAK2 V617F mutated HSCs exhibiting skewed differentiation potential and for amplification occurring after erythroid commitment has been provided, and we will discuss whether this evidence is relevant for the „disease

Janus kinases affect thrombopoietin receptor cell surface localization and stability.

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

JAK1 and Tyk2 activation by the homologous polycythemia vera JAK2 V617F mutation - Cross-talk with IGF1 receptor

The majority of polycythemia vera (PV) patients harbor a unique somatic mutation (V617F) in the pseudokinase domain of JAK2, which leads to constitutive signaling. Here we show that the homologous mutations in JAK1 (V658F) and in Tyk2 (V678F) lead to constitutive activation of these kinases. Their expression induces autonomous growth of cytokine-dependent cells and constitutive activation of STAT5, STAT3, mitogen-activated protein kinase, and Akt signaling in Ba/F3 cells. The mutant JAKs exhibit constitutive signaling also when expressed in fibrosarcoma cells deficient in JAK proteins. Expression of the JAK2 V617F mutant renders Ba/F3 cells hypersensitive to insulin-like growth factor 1 (IGF1), which is a hallmark of PV erythroid progenitors. Upon selection of Ba/F3 cells for autonomous growth induced by the JAK2 V617F mutant, cells respond to IGF1 by activating STAT5, STAT3, Erk1/2, and Akt on top of the constitutive activation characteristic of autonomous cells. The synergic effect on proliferation and STAT activation appears specific to the JAK2 V617F mutant. Our results show that the homologous V617F mutation induces activation of JAK1 and Tyk2, suggesting a common mechanism of activation for the JAK1, JAK2, and Tyk2 mutants. JAK3 is not activated by the homologous mutation M592F, despite the presence of the conserved GVC preceding sequence. We suggest that mutations in the JAK1 and Tyk2 genes may be identified as initial molecular defects in human cancers and autoimmune diseases

DNMT3B deficiency alters mitochondrial biogenesis and α‐ketoglutarate levels in human embryonic stem cells

Embryonic stem cell renewal and differentiation is regulated by metabolites that serve as cofactors for epigenetic enzymes. An increase of α‐ketoglutarate (α‐KG), a cofactor for histone and DNA demethylases, triggers multilineage differentiation in human embryonic stem cells (hESCs). To gain further insight into how the metabolic fluxes in pluripotent stem cells can be influenced by inactivating mutations in epigenetic enzymes, we generated hESCs deficient for de novo DNA methyltransferases (DNMTs) 3A and 3B. Our data reveal a bidirectional dependence between DNMT3B and α‐KG levels: a‐KG is significantly upregulated in cells deficient for DNMT3B, while DNMT3B expression is downregulated in hESCs treated with α‐KG. In addition, DNMT3B null hESCs exhibit a disturbed mitochondrial fission and fusion balance and a switch from glycolysis to oxidative phosphorylation. Taken together, our data reveal a novel link between DNMT3B and the metabolic flux of hESCs

An amphipathic motif at the transmembrane-cytoplasmic junction prevents autonomous activation of the thrombopoietin receptor

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 (Δ5TpoR), where this sequence was deleted, is constitutively active. In the absence of ligand, Δ5TpoR activates Jak2, Tyk2, STAT5, and mitogen-activated protein (MAP) kinase, but does not appear to induce STAT3 phosphorylation. Δ5TpoR induces hematopoietic myeloid differentiation in the absence of Tpo. In the presence of Tpo, the Δ5TpoR 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

Cytokinesis arrest and multiple centrosomes in B cell chronic lymphocytic leukaemia

Cytokinesis failure leads to the emergence of tetraploid cells and multiple centrosomes. Chronic lymphocytic leukaemia (CLL) is the most common haematological malignancy in adults and is characterized by clonal B cell expansion. Here, we show that a significant number of peripheral blood CLL cells are arrested in cytokinesis and that this event occurred after nuclear envelope reformation and before cytoplasmic abscission. mRNA expression data showed that several genes known to be crucial for cell cycle regulation, checkpoint and centromere function, such as ING4, ING5, CDKN1A and CDK4, were significantly dysregulated in CLL samples. Our results demonstrate that CLL cells exhibit difficulties in completing mitosis, which is different from but may, at least in part, explain the previously reported accumulation of CLL cells in G0/1