搜索结果: 'methocult media formulations for mouse hematopoietic cells serum containing'

Ectopic C/EBPalpha expression in p210(BCR/ABL)-expressing hematopoietic cells induces granulocytic differentiation,inhibits proliferation,and suppresses leukemogenesis. To assess the underlying mechanisms,C/EBPalpha targets were identified by microarray analyses. Upon C/EBPalpha activation,expression of c-Myb and GATA-2 was repressed in 32D-BCR/ABL,K562,and chronic myelogenous leukemia (CML) blast crisis (BC) primary cells but only c-Myb levels decreased slightly in CD34(+) normal progenitors. The role of these 2 genes for the effects of C/EBPalpha was assessed by perturbing their expression in K562 cells. Ectopic c-Myb expression blocked the proliferation inhibition- and differentiation-inducing effects of C/EBPalpha,whereas c-Myb siRNA treatment enhanced C/EBPalpha-mediated proliferation inhibition and induced changes in gene expression indicative of monocytic differentiation. Ectopic GATA-2 expression suppressed the proliferation inhibitory effect of C/EBPalpha but blocked in part the effect on differentiation; GATA-2 siRNA treatment had no effects on C/EBPalpha induction of differentiation but inhibited proliferation of K562 cells,alone or upon C/EBPalpha activation. In summary,the effects of C/EBPalpha in p210(BCR/ABL)-expressing cells depend,in part,on transcriptional repression of c-Myb and GATA-2. Since perturbation of c-Myb and GATA-2 expression has nonidentical consequences for proliferation and differentiation of K562 cells,the effects of C/EBPalpha appear to involve dif-ferent transcription-regulated targets. View Publication

The viral infection of higher vertebrates elicits potent innate and adaptive host immunity. However,an excessive or inappropriate immune response also may lead to host pathology that often is more severe than the direct effects of viral replication. Therefore,several mechanisms exist that regulate the magnitude and class of the immune response. Here,we have examined the potential involvement of regulatory T (Treg) cells in limiting pathology induced by influenza A virus (IAV) infection. Using lymphocyte-deficient mice as hosts,we showed that Treg cell reconstitution resulted in a significant delay in weight loss and prolonged survival following infection. The adoptively transferred Treg cells did not affect the high rate of IAV replication in the lungs of lymphocyte-deficient hosts,and therefore their disease-ameliorating effect was mediated through the suppression of innate immune pathology. Mechanistically,Treg cells reduced the accumulation and altered the distribution of monocytes/macrophages in the lungs of IAV-infected hosts. This reduction in lung monocytosis was associated with a specific delay in monocyte chemotactic protein-2 (MCP-2) induction in the infected lungs. Nevertheless,Treg cells failed to prevent the eventual development of severe disease in lymphocyte-deficient hosts,which likely was caused by the ongoing IAV replication. Indeed,using T-cell-deficient mice,which mounted a T-cell-independent B cell response to IAV,we further showed that the combination of virus-neutralizing antibodies and transferred Treg cells led to the complete prevention of clinical disease following IAV infection. Taken together,these results suggested that innate immune pathology and virus-induced pathology are the two main contributors to pathogenesis during IAV infection. View Publication

UNLABELLED The current protocols for generation of induced pluripotent stem (iPS) cells involve genome integrating viral vectors which may induce tumorgenesis. The aim of this study was to develop and optimize a non-viral method without genetic manipulation for reprogramming of skeletal myoblasts (SMs) using small molecules. METHODS AND RESULTS SMs from young male Oct3/4-GFP(+) transgenic mouse were treated with DNA methyltransferase (DNMT) inhibitor,RG108. Two weeks later,GFP(+) colonies of SM derived iPS cells (SiPS) expressing GFP and with morphological similarity of mouse embryonic stem (ESCs) were formed and propagated in vitro. SiPS were positive for alkaline phosphatase activity,expressed SSEA1,displayed ES cell specific pluripotency markers and formed teratoma in nude mice. Optimization of culture conditions for embryoid body (EBs) formation yielded spontaneously contracting EBs having morphological,molecular,and ultra-structural similarities with cardiomyocytes and expressed early and late cardiac markers. miR profiling showed abrogation of let-7 family and upregulation of ESCs specific miR-290-295 cluster thus indicating that SiPS were similar to ESCs in miR profile. Four weeks after transplantation into the immunocompetent mice model of acute myocardial infarction (n = 12 per group),extensive myogenesis was observed in SiPS transplanted hearts as compared to DMEM controls (n = 6 per group). A significant reduction in fibrosis and improvement in global heart function in the hearts transplanted with SiPS derived cardiac progenitor cells were observed. CONCLUSIONS Reprogramming of SMs by DNMT inhibitor is a simple,reproducible and efficient technique more likely to generate transgene integration-free iPS cells. Cardiac progenitors derived from iPS cells propagated extensively in the infarcted myocardium without tumorgenesis and improved cardiac function. View Publication

Human pluripotent stem cells (hPSCs) provide unprecedented opportunities to study the earliest stages of human development in vitro and have the potential to provide unlimited new sources of cells for regenerative medicine. Although previous studies have reported cytokeratin 14+/p63+ keratinocyte generation from hPSCs,the multipotent progenitors of epithelial lineages have not been described and the developmental pathways regulating epithelial commitment remain largely unknown. Here we report membrane localization of β-catenin during retinoic acid (RA)--induced epithelial differentiation. In addition hPSC treatment with the Src family kinase inhibitor SU6656 modulated β-catenin localization and produced an enriched population of simple epithelial cells under defined culture conditions. SU6656 strongly upregulated expression of cytokeratins 18 and 8 (K18/K8),which are expressed in simple epithelial cells,while repressing expression of the pluripotency gene Oct4. This homogeneous population of K18+K8+Oct4- simple epithelial precursor cells can further differentiate into cells expressing keratinocyte or corneal-specific markers. These enriched hPSC-derived simple epithelial cells may provide a ready source for development and toxicology cell models and may serve as a progenitor for epithelial cell transplantation applications. View Publication

Zoledronic acid,a third-generation bisphosphonate,has been shown to reduce cell migration,invasion,and metastasis. However,the effects of zoledronic acid on the epithelial-mesenchymal transition (EMT),a cellular process essential to the metastatic cascade,remain unclear. Therefore,the effects of zoledronic acid on EMT,using triple-negative breast cancer (TNBC) cells as a model system,were examined in more detail. Zoledronic acid treatment decreased the expression of mesenchymal markers,N-cadherin,Twist,and Snail,and subsequently upregulated expression of E-cadherin. Zoledronic acid also inhibited cell viability,induced cell-cycle arrest,and decreased the proliferative capacity of TNBC,suggesting that zoledronic acid inhibits viability through reduction of cell proliferation. As EMT has been linked to acquisition of a self-renewal phenotype,the effects of zoledronic acid on self-renewal in TNBC were also studied. Treatment with zoledronic acid decreased expression of self-renewal proteins,BMI-1 and Oct-4,and both prevented and eliminated mammosphere formation. To understand the mechanism of these results,the effect of zoledronic acid on established EMT regulator NF-$$B was investigated. Zoledronic acid inhibited phosphorylation of RelA,the active subunit of NF-$$B,at serine 536 and modulated RelA subcellular localization. Treatment with zoledronic acid reduced RelA binding to the Twist promoter,providing a direct link between inactivation of NF-$$B signaling and loss of EMT transcription factor gene expression. Binding of Twist to the BMI-1 promoter was also decreased,correlating modulation of EMT to decreased self-renewal. On the basis of these results,it is proposed that through inactivation of NF-$$B,zoledronic acid reverses EMT,which leads to a decrease in self-renewal. View Publication

BACKGROUND Pluripotent human embryonic stem cells (hESC) are a promising source of repopulating cardiomyocytes. We hypothesized that we could improve maturation of cardiomyocytes and facilitate electrical interconnections by creating a model that more closely resembles heart tissue; that is,containing both endothelial cells (ECs) and cardiomyocytes. METHODS We induced cardiomyocyte differentiation in the coculture of an hESC line expressing the cardiac reporter NKX2.5-green fluorescent protein (GFP),and an Akt-activated EC line (E4(+)ECs). We quantified spontaneous beating rates,synchrony,and coordination between different cardiomyocyte clusters using confocal imaging of Fura Red-detected calcium transients and computer-assisted image analysis. RESULTS After 8 days in culture,94% ± 6% of the NKX2-5GFP(+) cells were beating when hESCs embryonic bodies were plated on E4(+)ECs compared with 34% ± 12.9% for controls consisting of hESCs cultured on BD Matrigel (BD Biosciences) without ECs at Day 11 in culture. The spatial organization of beating areas in cocultures was different. The GFP(+) cardiomyocytes were close to the E4(+)ECs. The average beats/min of the cardiomyocytes in coculture was faster and closer to physiologic heart rates compared with controls (50 ± 14 [n = 13] vs 25 ± 9 [n = 8]; p < 0.05). The coculture with ECs led to synchronized beating relying on the endothelial network,as illustrated by the loss of synchronization upon the disruption of endothelial bridges. CONCLUSIONS The coculturing of differentiating cardiomyocytes with Akt-activated ECs but not EC-conditioned media results in (1) improved efficiency of the cardiomyocyte differentiation protocol and (2) increased maturity leading to better intercellular coupling with improved chronotropy and synchrony. View Publication

Glioblastoma (GBM) is associated with poor prognosis despite aggressive surgical resection,chemotherapy,and radiation therapy. Unfortunately,this standard therapy does not target glioma cancer stem cells (GCSCs),a subpopulation of GBM cells that can give rise to recurrent tumors. GBMs express human cytomegalovirus (HCMV) proteins,and previously we found that the level of expression of HCMV immediate-early (IE) protein in GBMs is a prognostic factor for poor patient survival. In this study,we investigated the relation between HCMV infection of GBM cells and the presence of GCSCs. Primary GBMs were characterized by their expression of HCMV-IE and GCSCs marker CD133 and by patient survival. The extent to which HCMV infection of primary GBM cells induced a GCSC phenotype was evaluated in vitro. In primary GBMs,a large fraction of CD133-positive cells expressed HCMV-IE,and higher co-expression of these two proteins predicted poor patient survival. Infection of GBM cells with HCMV led to upregulation of CD133 and other GSCS markers (Notch1,Sox2,Oct4,Nestin). HCMV infection also promoted the growth of GBM cells as neurospheres,a behavior typically displayed by GCSCs,and this phenotype was prevented by either chemical inhibition of the Notch1 pathway or by treatment with the anti-viral drug ganciclovir. GBM cells that maintained expression of HCMV-IE failed to differentiate into neuronal or astrocytic phenotypes. Our findings imply that HCMV infection induces phenotypic plasticity of GBM cells to promote GCSC features and may thereby increase the aggressiveness of this tumor. View Publication

The airway epithelium provides a first line of defense against pathogens by release of antimicrobial factors and neutrophil-attracting chemokines. Pseudomonas (P.) aeruginosa,a Gram-negative bacterium that expresses flagellin as an important virulence factor,is a common cause of injurious airway inflammation. The aim of our study was to determine the contribution of flagellin to the inflammatory,antimicrobial,and metabolic responses of the airway epithelium to P. aeruginosa . Furthermore,as we previously showed that targeting mTOR limited the glycolytic and inflammatory response induced by flagellin,we assessed the effect of rapamycin on human bronchial epithelial (HBE) cells stimulated with flagellated and non-flagellated P. aeruginosa. Primary pseudostratified HBE cells,cultured on an air-liquid-interface,were treated on the basolateral side with medium,vehicle or rapamycin,exposed on the apical side with flagellated or flagellin-deficient P. aeruginosa,and analyzed for their inflammatory,antimicrobial,and glycolytic responses. Flagellin augmented the P. aeruginosa -induced expression of antimicrobial factors and secretion of chemokines by HBE cells but did not further increase the glycolytic response. Treatment of HBE cells with rapamycin inhibited mTOR activation in general and flagellin-augmented mTOR activation in particular,but did not affect the glycolytic response. Rapamycin,however,diminished the flagellin-augmented inflammatory and antimicrobial response induced by Pseudomonas . These results demonstrate that flagellin is a significant factor that augments the inflammatory and antimicrobial response of human airway epithelial cells upon exposure to P. aeruginosa and suggest that mTOR inhibition by rapamycin in the airway epithelium diminishes these exaggerated responses. View Publication

Prime editing is a promising approach for correcting pathogenic variants,but its efficiency remains variable across genomic contexts. Here,we systematically evaluated 12 modifications of the PEmax system for correcting the CFTR F508del pathogenic variant that caused cystic fibrosis in patient-derived airway basal cells. We chose EXO1 and FEN1 nucleases to improve the original system. While all tested variants showed comparatively low efficiency in this AT-rich genomic region,4-FEN modification demonstrated significantly improved editing rates (up to 2.13 fold) compared to standard PEmax. Our results highlight two key findings: first,the persistent challenge of AT-rich target sequence correction even with optimized editors,and second,the performance of 4-FEN suggests its potential value for other genomic targets. View Publication

Targeting of cancer stem cells (CSC) is expected to be a paradigm-shifting approach for the treatment of cancers. Cell surface proteoglycans bearing sulfated glycosaminoglycan (GAG) chains are known to play a critical role in the regulation of stem cell fate. Here,we show for the first time that G2.2,a sulfated nonsaccharide GAG mimetic (NSGM) of heparin hexasaccharide,selectively inhibits colonic CSCs in vivo G2.2-reduced CSCs (CD133+/CXCR4+,Dual hi) induced HT-29 and HCT 116 colon xenografts' growth in a dose-dependent fashion. G2.2 also significantly delayed the growth of colon xenograft further enriched in CSCs following oxaliplatin and 5-fluorouracil treatment compared with vehicle-treated xenograft controls. In fact,G2.2 robustly inhibited CSCs' abundance (measured by levels of CSC markers,e.g.,CD133,DCMLK1,LGR5,and LRIG1) and self-renewal (quaternary spheroids) in colon cancer xenografts. Intriguingly,G2.2 selectively induced apoptosis in the Dual hi CSCs in vivo eluding to its CSC targeting effects. More importantly,G2.2 displayed none to minimal toxicity as observed through morphologic and biochemical studies of vital organ functions,blood coagulation profile,and ex vivo analyses of normal intestinal (and bone marrow) progenitor cell growth. Through extensive in vitro,in vivo,and ex vivo mechanistic studies,we showed that G2.2's inhibition of CSC self-renewal was mediated through activation of p38$\alpha$,uncovering important signaling that can be targeted to deplete CSCs selectively while minimizing host toxicity. Hence,G2.2 represents a first-in-class (NSGM) anticancer agent to reduce colorectal CSCs. View Publication

Osteoarthritis (OA) is characterized by cartilage degradation and chronic joint inflammation. Mesenchymal stem cells (MSCs) have shown promising results in OA,but their mechanism of action is not fully understood. We hypothesize that MSCs polarize macrophages,which are strongly associated with joint inflammation to more homeostatic sub-types. We tracked ferumoxytol (Feraheme™,iron oxide nanoparticle)-labeled murine MSCs (Fe-MSCs) in murine OA joints,and quantified changes to joint inflammation and fibrosis. 10-week-old C57BL/6 male mice (n = 5/group) were induced to undergo osteoarthritis by destabilization of medical meniscus (DMM) or sham surgery. 3 weeks post-surgery,mice were injected intra-articularly with either fluorescent dye-(DiR) labeled or DiR-Fe-MSC or saline to yield 4 groups (n = 5 per group for each timepoint [1,2 and 4weeks]). 4 weeks after injection,mice were imaged by MRI,and scored for i) OARSI (Osteoarthritis Research Society International) to determine cartilage damage; ii) immunohistochemical changes in iNOS,CD206,F4/80 and Prussian Blue/Sca-1 to detect pro-inflammatory,homeostatic and total macrophages and ferumoxytol -labeled MSCs respectively,and iii) Masson's Trichrome to detect changes in fibrosis. Ferumoxytol-labeled MSCs persisted at greater levels in DMM vs. SHAM-knee joints. We observed no difference in OARSI scores between MSC and vehicle groups. Sca-1 and Prussian Blue co-staining confirmed the ferumoxytol label resides in MSCs,although some ferumoxytol label was detected in proximity to MSCs in macrophages,likely due to phagocytosis of apoptotic MSCs,increasing functionality of these macrophages through MSC efferocytosis. MRI hypertintensity scores related to fluid edema decreased in MSC-treated vs. control animals. For the first time,we show that MSC-treated mice had increased ratios of {\%}CD206+: {\%}F4/80+ (homeostatic macrophages) (p{\textless}0.05),and decreased ratios of {\%}iNOS+: {\%}F4/80+ macrophages (p{\textless}0.01),supporting our hypothesis that MSCs may modulate synovial inflammation. View Publication

ABSTRACTRAD50?interacting protein1 (RINT1) deficiency has been implicated in recurrent acute liver failure (RALF) triggered by fever or infections. RINT1,together with neuroblastoma amplified sequence and Zeste White 10 (forming the NRZ complex),localizes at the interface between the endoplasmic reticulum and Golgi apparatus,where it plays a key role in vesicular trafficking. However,the mechanisms by which RINT1 deficiency leads to RALF remain unclear. This study aimed to describe a woman with RALF harboring a homozygous missense mutation in RINT1. Induced pluripotent stem cells (iPSCs) were generated from the patient's mononuclear cells and differentiated into hepatocyte?like cells (HLCs). Upon exposure to high temperature (40°C),RINT1?deficient HLCs exhibited cellular damage characteristic of RALF. Furthermore,these cells also demonstrated heightened sensitivity to cytokines and viral mimetics while showing comparatively lower responsiveness to bacterial infection?related stimuli. Transcriptome sequencing revealed dysregulated gene expression associated with the extracellular matrix (ECM). Additionally,glycosaminoglycan disaccharide analysis revealed abnormal levels of chondroitin sulfate,heparan sulfate,and hyaluronan in RINT1?deficient HLCs. In conclusion,HLCs derived from RINT1?deficient iPSCs serve as a valuable model for investigating RINT1?related liver pathogenesis. The results suggest that cytokine responses,particularly those triggered by viral infections,play a central role in the development of RALF. Furthermore,ECM alterations provided novel insights into the potential role of RINT1 defects in RALF. RAD50?interacting protein1 (RINT1) deficiency causes recurrent acute liver failure (RALF) during fever or infections. To investigate its underlying mechanism,induced pluripotent stem cells were generated from a patient with RINT1 deficiency and differentiated into hepatocyte?like cells (HLCs). RINT1?deficient HLCs exhibited damage resembling RALF when exposed to high temperatures and were more susceptible to cytokines and viral mimetics than to bacterial infection?related factors. Furthermore,RNA?seq and disaccharide analyses revealed dysregulation of extracellular matrix?related genes and abnormalities in extracellular matrix levels. View Publication