参考文献

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

The corneal epithelium is maintained by stem cells located at the periphery of the cornea in a region known as the limbus. Depletion of limbal stem cells (LSCs) results in limbal stem cell deficiency. Treatments for this disease are based on limbal replacement or transplantation of ex vivo expanded LSCs. It is,therefore,crucial to identify cell surface markers for LSCs that can be used for their enrichment and characterization. Aldehyde dehydrogenases (ALDHs) are enzymes which protect cells from the toxic effects of peroxidic aldehydes. In this manuscript,we show for the first time that ALDH1 is absent from the basal cells of the limbal and corneal epithelium. We separated limbal epithelial cells on the basis of ALDH activity and showed that ALDH(dim) cells expressed significantly higher levels of DeltaNp63 and ABCG2 as well as having a greater colony forming efficiency (CFE) when compared to ALDH(bright) cells. Large scale transcriptional analysis of these two populations led to identification of a new cell surface marker,RHAMM/HMMR,which is located in all layers of corneal epithelium and in the suprabasal layers of the limbal epithelium but is completely absent from the basal layer of the limbus. Our studies indicate that absence of RHAMM/HMMR expression is correlated with properties associated with LSCs. RHAMM/HMMR- limbal epithelial cells are smaller in size,express negligible CK3,have higher levels of DeltaNp63 and have a higher CFE compared to RHAMM/HMMR+ cells. Taken together these results suggest a putative role for RHAMM/ HMMR as a negative marker of stem cell containing limbal epithelial cells. Cell selection based on Hoechst exclusion and lack of cell surface RHAMM/HMMR expression resulted in increased colony forming efficiency compared to negative selection using RHAMM/HMMR alone or positive selection using Hoechst on its own. Combination of these two cell selection methods presents a novel method for LSC enrichment and characterization. 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

Chimerism analysis has become an important tool to manage patients in the peri-transplant period of allogenic stem cell transplantation. During this period,cells of donor and host origin can coexist and increasing proportion of cells of host origin is considered as a recurrence of the underlying disease. We currently performed chimerism analysis on separate peripheral blood cell subsets,lymphocytes and granulocytes. To improve our isolation method,a new automated device from Stem Cell Technology Roboseptrade mark was tested and compared to our manual separation technique. The results obtained on T cell purification showed an improvement of the purity (98.42% with Robosep vs. 92.42% with the manual technique Rosettesep) and of the recovery (63.43% with Robosep and 38% with Rosettesep). The results were significantly improved on patient samples with less than 10% CD3 positive cells (purity: 90% vs. 44.44%; recovery: 73.79% vs. 43.98%). Granulocytes separation was based on CD15 expression. The results showed an improvement of the purity with Robosep (96.90% vs. 86.20% with the manual technique Polymorphprep) but the recovery was impaired (35.2% vs. 52.30%). Using a myeloid (CD66/CD33) cocktail,recovery was improved with the Robosep device (64.04% with the myeloid cocktail vs. 22.4% with the CD15 cocktail). Our data demonstrated that Robosep allowed a performant cell purification in the early period post-transplantation even for populations representing less than 10% of the peripheral blood 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

Mef2c is a MADS (MCM1-agamous-deficient serum response factor) transcription factor best known for its role in muscle and cardiovascular development. A causal role of up-regulated MEF2C expression in myelomonocytic acute myeloid leukemia (AML) has recently been demonstrated. Due to the pronounced monocytic component observed in Mef2c-induced AML,this study was designed to assess the importance of Mef2c in normal myeloid differentiation. Analysis of bone marrow (BM) cells manipulated to constitutively express Mef2c demonstrated increased monopoiesis at the expense of granulopoiesis,whereas BM isolated from Mef2c(Delta/-) mice showed reduced levels of monocytic differentiation in response to cytokines. Mechanistic studies showed that loss of Mef2c expression correlated with reduced levels of transcripts encoding c-Jun,but not PU.1,C/EBPalpha,or JunB transcription factors. Inhibiting Jun expression by short-interfering RNA impaired Mef2c-mediated inhibition of granulocyte development. Moreover,retroviral expression of c-Jun in BM cells promoted monocytic differentiation. The ability of Mef2c to modulate cell-fate decisions between monocyte and granulocyte differentiation,coupled with its functional sensitivity to extracellular stimuli,demonstrate an important role in immunity--and,consistent with findings of other myeloid transcription factors,a target of oncogenic lesions in AML. 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

Dyskeratosis congenita (DC) is an inherited bone marrow (BM) failure syndrome associated with mutations in telomerase genes and the acquisition of shortened telomeres in blood cells. To investigate the basis of the compromised hematopoiesis seen in DC,we analyzed cells from granulocyte colony-stimulating factor mobilized peripheral blood (mPB) collections from 5 members of a family with autosomal dominant DC with a hTERC mutation. Premobilization BM samples were hypocellular,and percentages of CD34(+) cells in marrow and mPB collections were significantly below values for age-matched controls in 4 DC subjects. Directly clonogenic cells,although present at normal frequencies within the CD34(+) subset,were therefore absolutely decreased. In contrast,even the frequency of long-term culture-initiating cells within the CD34(+) DC mPB cells was decreased,and the telomere lengths of these cells were also markedly reduced. Nevertheless,the different lineages of mature cells were produced in normal numbers in vitro. These results suggest that marrow failure in DC is caused by a reduction in the ability of hematopoietic stem cells to sustain their numbers due to telomere impairment rather than a qualitative defect in their commitment to specific lineages or in the ability of their lineage-restricted progeny to execute normal differentiation programs. View Publication

Lysophospholipids are signaling molecules that play broad and major roles within the nervous system during both early development and neural injury. We used neural differentiation of human embryonic stem cells (hESC) as an in vitro model to examine the specific effects of lysophosphatidic acid (LPA) at various stages of neural development,from neural induction to mature neurons and glia. We report that LPA inhibits neurosphere formation and the differentiation of neural stem cells (NSC) toward neurons,without modifying NSC proliferation,apoptosis,or astrocytic differentiation. LPA acts through the activation of the Rho/ROCK and the phosphatidylinositol 3-kinase/Akt pathways to inhibit neuronal differentiation. This study is the first demonstration of a role for LPA signaling in neuronal differentiation of hESC. As LPA concentrations increase during inflammation,the inhibition of neuronal differentiation by LPA might contribute to the low level of neurogenesis observed following neurotrauma. View Publication

In response to inflammatory stimuli,monocytes/macrophages secrete greater quantities of the proinflammatory cytokines tumour necrosis factor-alpha (TNF-alpha),interleukin-1beta (IL-1beta) and IL-6. The inflammatory process and the innate immune response are related to the activation of several transcription factors,such as nuclear factor kappaB (NF-kappaB) and activator protein 1 (AP-1). The proteasome is a multimeric protease complex,which plays a vital role in several cellular functions,including the regulation of transcription factors like NF-kappaB. In this study,we used the human monocyte cell line U937 stimulated with lipopolysaccharide (LPS) and phorbol 12-myristate 13-acetate (PMA) as a model to investigate the in vitro effects of MG132,a proteasome inhibitor,on the release of TNF-alpha,IL-1beta and IL-6 and on the expression of their membrane and soluble receptors TNF-R1,IL-1R1 and IL-6R. We also analysed the effects of MG132 on the activation of NF-kappaB and AP-1 and on the IkappaB molecule. MG132 significantly inhibited the secretion of those proinflammatory cytokines. MG132 increased the release of the soluble receptors TNF-R1 and IL-1R1 from U937 cells and decreased their cell-surface expression. MG132 also increased IL-6R cell-surface expression and decreased its release. Proteasome inhibition also led to an increase in LPS+PMA-induced AP-1 activation and the attenuation of LPS+PMA-induced IkappaB degradation,resulting in the abolition of NF-kappaB activation. Our experiments strongly suggest that the proteasome is an important factor in the regulation of proinflammatory cytokines and their receptors. View Publication