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FUS/TLS (fused in sarcoma/translocated in liposarcoma) and TDP-43 are integrally involved in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. We found that FUS/TLS binds to RNAs from textgreater5,500 genes in mouse and human brain,primarily through a GUGGU-binding motif. We identified a sawtooth-like binding pattern,consistent with co-transcriptional deposition of FUS/TLS. Depletion of FUS/TLS from the adult nervous system altered the levels or splicing of textgreater950 mRNAs,most of which are distinct from RNAs dependent on TDP-43. Abundance of only 45 RNAs was reduced after depletion of either TDP-43 or FUS/TLS from mouse brain,but among these were mRNAs that were transcribed from genes with exceptionally long introns and that encode proteins that are essential for neuronal integrity. Expression levels of a subset of these were lowered after TDP-43 or FUS/TLS depletion in stem cell-derived human neurons and in View Publication

OBJECTIVE: The S100A9 and S100A8 proteins are highly expressed by neutrophils and monocytes and are part of a group of damage-associated molecular pattern molecules that trigger inflammatory responses. Sera and synovial fluids of patients with rheumatoid arthritis (RA) contain high concentrations of S100A8/A9 that correlate with disease activity.backslashnbackslashnMETHODS: In this study,we investigated the importance of S100A9 in RA by using neutralizing antibodies in a murine lipopolysaccharide-synchronized collagen-induced arthritis model. We also used an in vitro model of stimulation of human immune cells to decipher the role played by S100A9 in leukocyte migration and pro-inflammatory cytokine secretion.backslashnbackslashnRESULTS: Treatment with anti-S100A9 antibodies improved the clinical score by 50%,diminished immune cell infiltration,reduced inflammatory cytokines,both in serum and in the joints,and preserved bone/collagen integrity. Stimulation of neutrophils with S100A9 protein led to the enhancement of neutrophil transendothelial migration. S100A9 protein also induced the secretion by monocytes of proinflammatory cytokines like TNFα,IL-1β and IL-6,and of chemokines like MIP-1α and MCP-1.backslashnbackslashnCONCLUSION: The effects of anti-S100A9 treatment are likely direct consequences of inhibiting the S100A9-mediated promotion of neutrophil transmigration and secretion of pro-inflammatory cytokines from monocytes. Collectively,our results show that treatment with anti-S100A9 may inhibit amplification of the immune response and help preserve tissue integrity. Therefore,S100A9 is a promising potential therapeutic target for inflammatory diseases like rheumatoid arthritis for which alternative therapeutic strategies are needed. View Publication

BACKGROUND: The histone variant H2A.Z has been implicated in nucleosome exchange,transcriptional activation and Polycomb repression. However,the relationships among these seemingly disparate functions remain obscure.backslashnbackslashnRESULTS: We mapped H2A.Z genome-wide in mammalian ES cells and neural progenitors. H2A.Z is deposited promiscuously at promoters and enhancers,and correlates strongly with H3K4 methylation. Accordingly,H2A.Z is present at poised promoters with bivalent chromatin and at active promoters with H3K4 methylation,but is absent from stably repressed promoters that are specifically enriched for H3K27 trimethylation. We also characterized post-translational modification states of H2A.Z,including a novel species dually-modified by ubiquitination and acetylation that is enriched at bivalent chromatin.backslashnbackslashnCONCLUSIONS: Our findings associate H2A.Z with functionally distinct genomic elements,and suggest that post-translational modifications may reconcile its contrasting locations and roles. View Publication

Human embryonic stem cells (hESCs) provide a valuable window into the dissection of the molecular circuitry underlying the early formation of the human forebrain. However,dissection of signaling events in forebrain development using current protocols is complicated by non-neural contamination and fluctuation of extrinsic influences. Here,we show that SMAD7,a cell-intrinsic inhibitor of transforming growth factor-β (TGFβ) signaling,is sufficient to directly convert pluripotent hESCs to an anterior neural fate. Time course gene expression revealed downregulation of MAPK components,and combining MEK1/2 inhibition with SMAD7-mediated TGFβ inhibition promoted telencephalic conversion. Fibroblast growth factor-MEK and TGFβ-SMAD signaling maintain hESCs by promoting pluripotency genes and repressing neural genes. Our findings suggest that in the absence of these cues,pluripotent cells simply revert to a program of neural conversion. Hence,the primed" state of hESCs requires inhibition of the "default" state of neural fate acquisition. This has parallels in amphibians� View Publication

We isolated neural stem cells/neural progenitors (NSC) from 1-day-old rat pups born to mothers fed diets that were deficient or supplemented with n-3 polyunsaturated fatty acids (PUFAs) and compared their proliferation and differentiation in vitro. The cells isolated from the n-3PUFA-deficient pups consistently proliferated more slowly than cells that were isolated from n-3PUFA-supplemented pups,despite the fact that both were cultured under the same conditions. The differences in the proliferation rates were evaluated up until 40 days of culture and were highly significant. When the cells were allowed to differentiate,the deficient cells exhibited a higher degree of neuronal maturation in response to the addition of PUFAs in the medium,as demonstrated by an increase in neurite length,whereas the neurons derived from the supplemented pups showed no change. This result was consistent,regardless of the age of the culture. The properties of the NSC were durably modified throughout the length of the culture,although the membrane phospholipid compositions were similar. We examined the differential expression of selected mRNAs and micro RNAs. We found significant differences in the gene expression of proliferating and differentiating cells,and a group of genes involved in neurogenesis was specifically modified by n-3 PUFA treatment. We conclude that n-3 PUFA levels in the maternal diet can induce persistent modifications of the proliferation and differentiation of NSCs and of their transcriptome. Therefore,the n-3 supply received in utero may condition on a long-term basis cell renewal in the brain. View Publication

De novo DNA methylation is an essential aspect of the epigenetic reprogramming that takes place during early development,yet factors responsible for its instatement at particular genomic loci are poorly defined. Here,we demonstrate that the KRAB-ZFP-mediated recruitment of KAP1 to DNA in embryonic stem cells (ESCs) induces cytosine methylation. This process is preceded by H3K9 trimethylation,and genome-wide analyses reveal that it spreads over short distances from KAP1-binding sites so as to involve nearby CpG islands. In sharp contrast,in differentiated cells,KRAB/KAP1-induced heterochromatin formation does not lead to DNA methylation. Correspondingly,the methylation status of CpG islands in the adult mouse liver correlates with their proximity to KAP1-binding sites in ESCs,not in hepatocytes. Therefore,KRAB-ZFPs and their cofactor KAP1 are in part responsible for the establishment during early embryogenesis of site-specific DNA methylation patterns that are maintained through development View Publication

Recent evidence suggests that Notch signaling may play a role in regulation of cancer stem cell (CSC) self-renewal and differentiation hence presenting a promising target for development of novel therapies for aggressive cancers such as triple negative breast cancer (TNBC). We generated Notch1 monoclonal antibodies (mAbs) that specifically bind to the negative regulatory region of human Notch1. Notch1 inhibition in TNBC Sum149 and patient derived xenograft (PDX) 144580 models led to significant TGI particularly in combination with docetaxel. More interestingly,Notch1 mAbs caused a reduction in mammosphere formation and CD44+/CD24-/lo cell population. It also resulted in decreased tumor incidence upon re-implantation and delay in tumor recurrence. Our data demonstrated a potent antitumor efficacy of Notch1 mAbs,with a remarkable activity against CSCs. These findings suggest that anti-Notch1 mAbs may provide novel therapies to improve the efficacy of conventional therapies by directly targeting the CSC niche. They may also delay tumor recurrence and hence have a major impact on cancer patient survival. View Publication

Neural cells derived from induced pluripotent stem cells (iPSCs) have the potential for autologous cell therapies in treating patients with severe neurological disorders or injury. However,further study of efficacy and safety are needed in large animal preclinical models that have similar neural anatomy and physiology to humans such as the pig. The pig model for pluripotent stem cell therapy has been made possible for the first time with the development of pig iPSCs (piPSCs) capable of in vitro and in vivo differentiation into tissues of all three germ layers. Still,the question remains if piPSCs are capable of undergoing robust neural differentiation using a system similar to those being used with human iPSCs. In this study,we generated a new line of piPSCs from fibroblast cells that expressed pluripotency markers and were capable of embryoid body differentiation into all three germ layers. piPSCs demonstrated robust neural differentiation forming βIII-TUB/MAP2+ neurons,GFAP+ astrocytes,and O4+ oligodendrocytes and demonstrated strong upregulation of neural cell genes representative of all three major neural lineages of the central nervous system. In the presence of motor neuron signaling factors,piPSC-derived neurons showed expression of transcription factors associated with motor neuron differentiation (HB9 and ISLET1). Our findings demonstrate that SSEA4 expression is required for piPSCs to differentiate into neurons,astrocytes,and oligodendrocytes and furthermore develop specific neuronal subtypes. This indicates that the pigs can fill the need for a powerful model to study autologous neural iPSC therapies in a system similar to humans. View Publication

Trisomy 21 is associated with hematopoietic abnormalities in the fetal liver,a preleukemic condition termed transient myeloproliferative disorder,and increased incidence of acute megakaryoblastic leukemia. Human trisomy 21 pluripotent cells of various origins,human embryonic stem (hES),and induced pluripotent stem (iPS) cells,were differentiated in vitro as a model to recapitulate the effects of trisomy on hematopoiesis. To mitigate clonal variation,we isolated disomic and trisomic subclones from the same parental iPS line,thereby generating subclones isogenic except for chromosome 21. Under differentiation conditions favoring development of fetal liver-like,γ-globin expressing,definitive hematopoiesis,we found that trisomic cells of hES,iPS,or isogenic origins exhibited a two- to fivefold increase in a population of CD43(+)(Leukosialin)/CD235(+)(Glycophorin A) hematopoietic cells,accompanied by increased multilineage colony-forming potential in colony-forming assays. These findings establish an intrinsic disturbance of multilineage myeloid hematopoiesis in trisomy 21 at the fetal liver stage. View Publication

We investigated the expression status of periostin in breast cancer stem cells and its clinical implications in order to lay a foundation for managing breast cancer. CD44+/CD24-/line- tumor cells (CSC) from clinical specimens were sorted using flow cytometry. Periostin expression status was detected in CSC cells and 1,086 breast cancer specimens by Western blot and immunohistochemistry staining,with the CSC ratio determined by immunofluorescence double staining. The relationship between the periostin protein and clinico-pathological parameters and prognosis was subsequently determined. As a result,CSC cells are more likely to generate new tumors in mice and cell microspheres that are deficient in NOD/SCID compared to the control group. Periostin protein was expressed higher in CSC cells compared to the control cells and was found to be related to CSC chemotherapy resistance. Moreover,periostin expression was found to be related to the CSC ratio in 1,086 breast cancer specimens (P = 0.001). In total,334 (30.76%) of the 1,086 breast cases showed high periostin expression. After universal and Spearman regression correlation analysis,periostin was observed to be related to histological grade,CSC ratio,lymph node metastasis,tumor size,and triple-negative breast cancer (all Ptextless0.05). Furthermore,periostin was shown to attain a significantly more distant bone metastasis and worse disease-specific survival than those with none or low-expressed periostin protein (P = 0.001). In the Cox regression test,periostin protein was detected as an independent prognostic factor (P = 0.001). In conclusion,periostin was found to be related to the CSC and an independent prognostic factor for breast cancer. It is also perhaps a potential target to breast cancer. View Publication

Multi-parameter flow cytometry analysis of T regulatory (Treg) cells is a widely used approach in basic and translational research studies. This approach has been complicated by a lack of specific markers for Treg cells and lack of uniformity in the quantification of Treg cells. Given the central role of Treg cells in the inception and perpetuation of diverse immune responses as well as its target as a therapeutic,it is imperative to have established methodologies for Treg cell analysis that are robust and usable for studies with multiple subjects as well as multicenter studies. In this study,we describe an optimized multi-parameter flow cytometry protocol for the quantification of human Treg cells from freshly obtained and viably frozen samples and correlations with epigenetic Treg cell analysis (TSDR demethylation). We apply these two methodologies to characterize Treg cell differences between cord blood and adult peripheral blood. In summary,the optimized protocol appears to be robust for Treg cell quantification from freshly isolated or viably frozen cells and the multi-parameter flow cytometry findings are strongly positively correlated with TSDR demethylation thus providing several options for the characterization of Treg cell frequency and function in large translational or clinical studies. View Publication

The pathogenic mechanisms of frontotemporal dementia (FTD) remain poorly understood. Here we generated multiple induced pluripotent stem cell lines from a control subject,a patient with sporadic FTD,and an FTD patient with a novel heterozygous GRN mutation (progranulin [PGRN] S116X). In neurons and microglia differentiated from PGRN S116X induced pluripotent stem cells,the levels of intracellular and secreted PGRN were reduced,establishing patient-specific cellular models of PGRN haploinsufficiency. Through a systematic screen of inducers of cellular stress,we found that PGRN S116X neurons,but not sporadic FTD neurons,exhibited increased sensitivity to staurosporine and other kinase inhibitors. Moreover,the serine/threonine kinase S6K2,a component of the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways,was specifically downregulated in PGRN S116X neurons. Both increased sensitivity to kinase inhibitors and reduced S6K2 were rescued by PGRN expression. Our findings identify cell-autonomous,reversible defects in patient neurons with PGRN deficiency,and provide a compelling model for studying PGRN-dependent pathogenic mechanisms and testing potential therapies View Publication