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Pluripotent cells can be derived from fibroblasts by ectopic expression of defined transcription factors. A fundamental unresolved question is whether terminally differentiated cells can be reprogrammed to pluripotency. We utilized transgenic and inducible expression of four transcription factors (Oct4,Sox2,Klf4,and c-Myc) to reprogram mouse B lymphocytes. These factors were sufficient to convert nonterminally differentiated B cells to a pluripotent state. However,reprogramming of mature B cells required additional interruption with the transcriptional state maintaining B cell identity by either ectopic expression of the myeloid transcription factor CCAAT/enhancer-binding-protein-alpha (C/EBPalpha) or specific knockdown of the B cell transcription factor Pax5. Multiple iPS lines were clonally derived from both nonfully and fully differentiated B lymphocytes,which gave rise to adult chimeras with germline contribution,and to late-term embryos when injected into tetraploid blastocysts. Our study provides definite proof for the direct nuclear reprogramming of terminally differentiated adult cells to pluripotency. View Publication

We identify the tumor necrosis factor receptor superfamily 25 (TNFRSF25)/TNFSF15 pair as critical trigger for allergic lung inflammation,which is a cardinal feature of asthma. TNFRSF25 (TNFR25) signals are required to exert T helper cell 2 (Th2) effector function in Th2-polarized CD4 cells and co-stimulate interleukin (IL)-13 production by glycosphingolipid-activated NKT cells. In vivo,antibody blockade of TNFSF15 (TL1A),which is the ligand for TNFR25,inhibits lung inflammation and production of Th2 cytokines such as IL-13,even when administered days after airway antigen exposure. Similarly,blockade of TNFR25 by a dominant-negative (DN) transgene,DN TNFR25,confers resistance to lung inflammation in mice. Allergic lung inflammation-resistant,NKT-deficient mice become susceptible upon adoptive transfer of wild-type NKT cells,but not after transfer of DN TNFR25 transgenic NKT cells. The TNFR25/TL1A pair appears to provide an early signal for Th2 cytokine production in the lung,and therefore may be a drug target in attempts to attenuate lung inflammation in asthmatics. View Publication

Recent data suggest that the practice of using frozen allogeneic grafts is becoming increasingly common among transplant centres. Therefore,we retrospectively analysed 31 frozen allogeneic PBSC and 8 BM grafts by flow cytometry with regard to their CD34+ content,membrane integrity (7-AAD) and stem cell-specific enzyme activity (aldehyde dehydrogenase,ALDH) in relation to individual transplantation results. Membrane integrity of CD34+ cells was significantly impaired in cryopreserved PBSC but not in BM compared to unfrozen allografts. In 9 out of 31 frozen PBSC (but none of the BM) grafts numbers of SSC(lo)ALDH(br) cells per kg body weight (BW) were significantly reduced while in the same grafts the numbers of CD34+ cells per kg BW were close to normal. Overall,9 out of 33 patients (27%) who received unrelated PBSC allografts cryopreserved after transportation did not achieve engraftment. For comparison,primary graft failure was observed in our centre in only 7 out of 493 recipients (1.4%) of fresh allogeneic PBSC grafts. Moreover,we did not see any graft failure in patients receiving frozen/thawed BM or autologous PBSC transplants. We,therefore,conclude that PBSC grafts become much more sensitive to cryopreservation after transport and/or storage. Importantly,the engraftment potential of frozen HSC grafts may reliably be predicted by measuring ALDH activity. View Publication

The generation of amyloid-beta peptide (Abeta) and its accumulation in amyloid plaques are generally recognized as key characteristics of Alzheimer's disease. A number of reports have indicated that Abeta can regulate the proliferation of neural precursor cells and adult neurogenesis,suggesting that this may underpin the cognitive decline and compromised olfaction also associated with the condition. Here we report that Abeta(1-42) treatment both in vitro and in vivo,as well as endogenous generation of Abeta in C100 and APP/PS1 transgenic models of Alzheimer's disease,stimulate neurogenesis of young adult subventricular zone precursors. The neurogenic effect of Abeta(1-42) was found to require expression of the p75 neurotrophin receptor (p75(NTR)) by the precursor cells,and activation of p75(NTR) by metalloprotease cleavage. However,precursors from 12-month-old APP/PS1 mice failed to respond to Abeta(1-42). Our results suggest that overstimulation of p75(NTR)-positive progenitors during early life might result in depletion of the stem cell pool and thus a more rapid decline in basal neurogenesis. This,in turn,could lead to impaired neurogenic function in later life. View Publication

Ectopic expression of the transcription factors Oct4,Sox2,c-Myc,and Klf4 in fibroblasts generates induced pluripotent stem (iPS) cells. Little is known about the nature and sequence of molecular events accompanying nuclear reprogramming. Using doxycycline-inducible vectors,we have shown that exogenous factors are required for about 10 days,after which cells enter a self-sustaining pluripotent state. We have identified markers that define cell populations prior to and during this transition period. While downregulation of Thy1 and subsequent upregulation of SSEA-1 occur at early time points,reactivation of endogenous Oct4,Sox2,telomerase,and the silent X chromosome mark late events in the reprogramming process. Cell sorting with these markers allows for a significant enrichment of cells with the potential to become iPS cells. Our results suggest that factor-induced reprogramming is a gradual process with defined intermediate cell populations that contain the majority of cells poised to become iPS cells. View Publication

Pluripotency can be induced in differentiated murine and human cells by retroviral transduction of Oct4,Sox2,Klf4,and c-Myc. We have devised a reprogramming strategy in which these four transcription factors are expressed from doxycycline (dox)-inducible lentiviral vectors. Using these inducible constructs,we derived induced pluripotent stem (iPS) cells from mouse embryonic fibroblasts (MEFs) and found that transgene silencing is a prerequisite for normal cell differentiation. We have analyzed the timing of known pluripotency marker activation during mouse iPS cell derivation and observed that alkaline phosphatase (AP) was activated first,followed by stage-specific embryonic antigen 1 (SSEA1). Expression of Nanog and the endogenous Oct4 gene,marking fully reprogrammed cells,was only observed late in the process. Importantly,the virally transduced cDNAs needed to be expressed for at least 12 days in order to generate iPS cells. Our results are a step toward understanding some of the molecular events governing epigenetic reprogramming. View Publication

Uniparental zygotes with two paternal (androgenetic [AG]) or two maternal (gynogenetic [GG]; parthenogenetic [PG]) genomes are not able to develop into viable offspring but can form blastocysts from which embryonic stem cells (ESCs) can be derived. Although some aspects of the in vitro and in vivo differentiation potential of PG and GG ESCs of several species have been studied,the developmental capacity of AG ESCs is much less clear. Here,we investigate the potential of murine AG ESCs to undergo neural differentiation. We observed that AG ESCs differentiate in vitro into pan-neural progenitor cells (pnPCs) that further give rise to cells that express neuronal- and astroglial-specific markers. Neural progeny of in vitro-differentiated AG ESCs exhibited fidelity of expression of six imprinted genes analyzed,with the exception of Ube3a. Bisulfite sequencing for two imprinting control regions suggested that pnPCs predominantly maintained their methylation pattern. Following blastocyst injection of AG and biparental (normal fertilized [N]) ESCs,we found widespread and evenly distributed contribution of ESC-derived cells in both AG and N chimeric early fetal brains. AG and N ESC-derived cells isolated from chimeric fetal brains by fluorescence-activated cell sorting exhibited similar neurosphere-initiating cell frequencies and neural multilineage differentiation potential. Our results indicate that AG ESC-derived neural progenitor/stem cells do not differ from N neural progenitor/stem cells in their self-renewal and neural multilineage differentiation potential. Disclosure of potential conflicts of interest is found at the end of this article. View Publication

Most tumors grow in immunocompetent hosts despite expressing NKG2D ligands (NKG2DLs) such as the MHC class I chain-related genes A and B (MICA/B). However,their participation in tumor cell evasion is still not completely understood. Here we demonstrate that several human melanomas (cell lines and freshly isolated metastases) do not express MICA on the cell surface but have intracellular deposits of this NKG2DL. Susceptibility to NK cell-mediated cytotoxicity correlated with the ratio of NKG2DLs to HLA class I molecules but not with the amounts of MICA on the cell surface of tumor cells. Transfection-mediated overexpression of MICA restored cell surface expression and resulted in an increased in vitro cytotoxicity and IFN-gamma secretion by human NK cells. In xenografted nude mice,these melanomas exhibited a delayed growth and extensive in vivo apoptosis. Retardation of tumor growth was due to NK cell-mediated antitumor activity against MICA-transfected tumors,given that this effect was not observed in NK cell-depleted mice. Also,mouse NK cells killed MICA-overexpressing melanomas in vitro. A mechanistic analysis revealed the retention of MICA in the endoplasmic reticulum,an effect that was associated with accumulation of endoH-sensitive (immature) forms of MICA,retrograde transport to the cytoplasm,and degradation by the proteasome. Our study identifies a novel strategy developed by melanoma cells to evade NK cell-mediated immune surveillance based on the intracellular sequestration of immature forms of MICA in the endoplasmic reticulum. Furthermore,this tumor immune escape strategy can be overcome by gene therapy approaches aimed at overexpressing MICA on tumor cells. View Publication

The JAK2(V617F) mutation was found in most patients with myeloproliferative disorders (MPDs),including polycythemia vera,essential thrombocythemia,and primary myelofibrosis. We have generated transgenic mice expressing the mutated enzyme in the hematopoietic system driven by a vav gene promoter. The mice are viable and fertile. One line of the transgenic mice,which expressed a lower level of JAK2(V617F),showed moderate elevations of blood cell counts,whereas another line with a higher level of JAK2(V617F) expression displayed marked increases in blood counts and developed phenotypes that closely resembled human essential thrombocythemia and polycythemia vera. The latter line of mice also developed primary myelofibrosis-like symptoms as they aged. The transgenic mice showed erythroid,megakaryocytic,and granulocytic hyperplasia in the bone marrow and spleen,displayed splenomegaly,and had reduced levels of plasma erythropoietin and thrombopoietin. They possessed an increased number of hematopoietic progenitor cells in peripheral blood,spleen,and bone marrow,and these cells formed autonomous colonies in the absence of growth factors and cytokines. The data show that JAK2(V617F) can cause MPDs in mice. Our study thus provides a mouse model to study the pathologic role of JAK2(V617F) and to develop treatment for MPDs. View Publication

Abelson kinase (c-abl) and platelet-derived growth factor (PDGF) are key players in the pathogenesis of systemic sclerosis (SSc). The aim of the present study was to evaluate the antifibrotic potential of dasatinib and nilotinib,2 novel inhibitors of c-abl and PDGF,which are well tolerated and have recently been approved. Dasatinib and nilotinib dose-dependently reduced the mRNA and protein levels of extracellular matrix proteins in human stimulated dermal fibroblasts from SSc patients (IC(50) of 0.5-2.0 nM for dasatinib and 0.8-2.5 nM for nilotinib). In a mouse model of bleomycin-induced dermal fibrosis,dasatinib and nilotinib potently reduced the dermal thickness,the number of myofibroblasts,and the collagen content of the skin in a dose-dependent manner at well-tolerated doses. These data indicate that dasatinib and nilotinib potently inhibit the synthesis of extracellular matrix in vitro and in vivo at biologically relevant concentrations. Thus,we provide the first evidence that dasatinib and nilotinib might be promising drugs for the treatment of patients with SSc. View Publication

Epigenetic regulation through chromatin is thought to play a critical role in the establishment and maintenance of pluripotency. Traditionally,antibody-based technologies were used to probe for specific posttranslational modifications (PTMs) present on histone tails,but these methods do not generally reveal the presence of multiple modifications on a single-histone tail (combinatorial codes). Here,we describe technology for the discovery and quantification of histone combinatorial codes that is based on chromatography and mass spectrometry. We applied this methodology to decipher 74 discrete combinatorial codes on the tail of histone H4 from human embryonic stem (ES) cells. Finally,we quantified the abundances of these codes as human ES cells undergo differentiation to reveal striking changes in methylation and acetylation patterns. For example,H4R3 methylation was observed only in the presence of H4K20 dimethylation; such context-specific patterning exemplifies the power of this technique. View Publication

Natural killer (NK) cells have been originally defined by their naturally occurring" effector function. However View Publication