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

BACKGROUND: Previously,we enriched a subpopulation of head and neck cancer-derived tumor initiating cells (HNC-TICs) presented high tumorigenic,chemo-radioresistant,and coupled with epithelial-mesenchymal transition (EMT) properties. The purpose of this study was to investigate the therapeutic effect and molecular mechanisms of quercetin on HNC-TICs. METHOD: ALDH1 activity of head and neck cancer cells with quercetin treatment was assessed by the Aldefluor assay flow cytometry analysis. Self-renewal,invasiveness,and EMT capability of HNC-TICs with different doses of quercetin was presented. RESULTS: We first observed that the treatment of quercetin significantly downregulated the ALDH1 activity of head and neck cancer cells in a dose-dependent manner (p textless .05). Moreover,quercetin reduced self-renewal property and stemness signatures expression in head and neck cancer-derived sphere cells. The migration ability of head and neck cancer-derived sphere cells was lessened under quercetin treatment partially due to the decreased productions of Twist,N-cadherin,and vimentin. CONCLUSION: Quercetin suppressing HNC-TICs characteristics may therefore be valuable therapeutics clinically in combination with standard treatment modalities. View Publication

VIDEO ABSTRACT: Although endogenous recruitment of adult neural stem cells has been proposed as a therapeutic strategy,clinical approaches for achieving this are lacking. Here,we show that metformin,a widely used drug,promotes neurogenesis and enhances spatial memory formation. Specifically,we show that an atypical PKC-CBP pathway is essential for the normal genesis of neurons from neural precursors and that metformin activates this pathway to promote rodent and human neurogenesis in culture. Metformin also enhances neurogenesis in the adult mouse brain in a CBP-dependent fashion,and in so doing enhances spatial reversal learning in the water maze. Thus,metformin,by activating an aPKC-CBP pathway,recruits neural stem cells and enhances neural function,thereby providing a candidate pharmacological approach for nervous system therapy. View Publication

Down syndrome cell adhesion molecule,or DSCAM,has been implicated in many neurodevelopmental processes including axon guidance,dendrite arborization,and synapse formation. Here we show that DSCAM plays an important role in regulating the morphogenesis of cortical pyramidal neurons in the mouse. We report that DSCAM expression is developmentally regulated and localizes to synaptic plasma membranes during a time of robust cortical dendrite arborization and spine formation. Analysis of mice that carry a spontaneous mutation in DSCAM (DSCAM(del17)) revealed gross morphological changes in brain size and shape in addition to subtle changes in cortical organization,volume,and lamination. Early postnatal mutant mice displayed a transient decrease in cortical thickness,but these reductions could not be attributed to changes in neuron production or cell death. DSCAM(del17) mutants showed temporary impairments in the branching of layer V pyramidal neuron dendrites at P10 and P17 that recovered to normal by adulthood. Defects in DSCAM(del17) dendrite branching correlated with a temporal increase in apical branch spine density and lasting changes in spine morphology. At P15 and P42,mutant mice displayed a decrease in the percentage of large,stable spines and an increase in the percentage of small,immature spines. Together,our findings suggest that DSCAM contributes to pyramidal neuron morphogenesis by regulating dendrite arborization and spine formation during cortical circuit development. View Publication

The unique ability of Sox2 to cooperate with Oct4 at selective binding sites in the genome is critical for reprogramming somatic cells into induced pluripotent stem cells (iPSCs). We have recently demonstrated that Sox17 can be converted into a reprogramming factor by alteration of a single amino acid (Sox17EK) within its DNA binding HMG domain. Here we expanded this study by introducing analogous mutations to 10 other Sox proteins and interrogated the role of N-and C-termini on the reprogramming efficiency. We found that point-mutated Sox7 and Sox17 can convert human and mouse fibroblasts into iPSCs,but Sox4,Sox5,Sox6,Sox8,Sox9,Sox11,Sox12,Sox13,and Sox18 cannot. Next we studied regions outside the HMG domain and found that the C-terminal transactivation domain of Sox17 and Sox7 enhances the potency of Sox2 in iPSC assays and confers weak reprogramming potential to the otherwise inactive Sox4EK and Sox18EK proteins. These results suggest that the glutamate (E) to lysine (K) mutation in the HMG domain is necessary but insufficient to swap the function of Sox factors. Moreover,the HMG domain alone fused to the VP16 transactivation domain is able to induce reprogramming,albeit at low efficiency. By molecular dissection of the C-terminus of Sox17,we found that the β-catenin interaction region contributes to the enhanced reprogramming efficiency of Sox17EK. To mechanistically understand the enhanced reprogramming potential of Sox17EK,we analyzed ChIP-sequencing and expression data and identified a subset of candidate genes specifically regulated by Sox17EK and not by Sox2. View Publication

Pathogenic mutations in Bcl2-associated athanogene 3 (BAG3) cause genetic dilated cardiomyopathy (DCM),a disease characterized by ventricular dilation,systolic dysfunction,and fibrosis. Previous studies have demonstrated that BAG3 mediates sarcomeric protein turnover through chaperone-assisted selective autophagy to maintain sarcomere integrity in the human heart. Although mouse models provide valuable insights into whole-organism effects of BAG3 knockout or pathogenic variants,their utility is limited by species-specific differences in pathophysiology,which often do not translate to humans and contribute to the failure of clinical trials. As a result,the development of induced pluripotent stem cell-derived cardiomyocytes (iCM) and engineered heart tissues presents a promising alternative for studying adult-onset cardiac diseases. Here,we used genome engineering to generate an isogenic pseudo-wild-type control cell line from a patient-derived iPSC line carrying a pathogenic BAG3 variant,clinically presenting with DCM. While monolayer iCMs recapitulated some features of BAG3-mediated DCM,such as reduced autophagy,mitochondrial membrane potential,and decreased HSPB8 stability,they failed to develop the age-associated impairment in sarcomere integrity. Therefore,we developed a mature,patient-specific,human engineered heart tissue model of BAG3-mediated DCM and compared it to its isogenic healthy control. We demonstrated successful recapitulation of adult-like features of the clinically observed disorganized sarcomeres and impaired tissue contractility,thereby providing a platform to investigate BAG3-related pathophysiology and therapeutic interventions. Graphical abstract View Publication

RNA constitutes a large fraction of chromatin. Spatial distribution and functional relevance of most of RNA-chromatin interactions remain unknown. We established a landscape analysis of RNA-chromatin interactions in human acute myeloid leukemia (AML). In total more than 50 million interactions were captured in an AML cell line. Protein-coding mRNAs and long non-coding RNAs exhibited a substantial number of interactions with chromatin in cis suggesting transcriptional activity. In contrast,small nucleolar RNAs (snoRNAs) and small nuclear RNAs (snRNAs) associated with chromatin predominantly in trans suggesting chromatin specific functions. Of note,snoRNA-chromatin interaction was associated with chromatin modifications and occurred independently of the classical snoRNA-RNP complex. Two C/D box snoRNAs,namely SNORD118 and SNORD3A,displayed high frequency of trans -association with chromatin. The transcription of SNORD118 and SNORD3A was increased upon leukemia transformation and enriched in leukemia stem cells,but decreased during myeloid differentiation. Suppression of SNORD118 and SNORD3A impaired leukemia cell proliferation and colony forming capacity in AML cell lines and primary patient samples. Notably,this effect was leukemia specific with less impact on healthy CD34+ hematopoietic stem and progenitor cells. These findings highlight the functional importance of chromatin-associated RNAs overall and in particular of SNORD118 and SNORD3A in maintaining leukemia propagation. Subject terms: Acute myeloid leukaemia,Cancer epigenetics View Publication

Expansion of the glutamine tract (poly-Q) in the protein huntingtin (HTT) causes the neurodegenerative disorder Huntington’s disease (HD). Emerging evidence suggests that mutant HTT (mHTT) disrupts brain development. To gain mechanistic insights into the neurodevelopmental impact of human mHTT,we engineered male induced pluripotent stem cells to introduce a biallelic or monoallelic mutant 70Q expansion or to remove the poly-Q tract of HTT. The introduction of a 70Q mutation caused aberrant development of cerebral organoids with loss of neural progenitor organization. The early neurodevelopmental signature of mHTT highlighted the dysregulation of the protein coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2),a transcription factor involved in mitochondrial integrated stress response. CHCHD2 repression was associated with abnormal mitochondrial morpho-dynamics that was reverted upon overexpression of CHCHD2. Removing the poly-Q tract from HTT normalized CHCHD2 levels and corrected key mitochondrial defects. Hence,mHTT-mediated disruption of human neurodevelopment is paralleled by aberrant neurometabolic programming mediated by dysregulation of CHCHD2,which could then serve as an early interventional target for HD. Subject terms: Huntington's disease,Mechanisms of disease,Stem cells View Publication

During embryogenesis,human hematopoietic stem cells (HSCs) first emerge in the aorta-gonad-mesonephros (AGM) region via transformation of specialized hemogenic endothelial (HE) cells into premature HSC precursors. This process is termed endothelial-to-hematopoietic transition (EHT),in which the HE cells undergo drastic functional and morphological changes from flat,anchorage-dependent endothelial cells to free-floating round hematopoietic cells. Despite its essential role in human HSC development,molecular mechanisms underlying the EHT are largely unknown. This is due to lack of methods to visualize the emergence of human HSC precursors in real time in contrast to mouse and other model organisms. In this study,by inducing HE from human pluripotent stem cells in feeder-free monolayer cultures,we achieved real-time observation of the human EHT in vitro . By continuous observation and single-cell tracking in the culture,it was possible to visualize a process that a single endothelial cell gives rise to a hematopoietic cell and subsequently form a hematopoietic-cell cluster. The EHT was also confirmed by a drastic HE-to-HSC switching in molecular marker expressions. Notably,HSC precursor emergence was not linked to asymmetric cell division,whereas the hematopoietic cell cluster was formed through proliferation and assembling of the floating cells after the EHT. These results reveal unappreciated dynamics in the human EHT,and we anticipate that our human EHT model in vitro will provide an opportunity to improve our understanding of the human HSC development. View Publication

Clear cell renal cell carcinoma (ccRCC) demonstrates enhanced glycolysis,critically contributing to tumor development. Programmed death-ligand 1 (PD-L1) aids tumor cells in evading T-cell-mediated immune surveillance. Yet,the specific mechanism by which glycolysis influences PD-L1 expression in ccRCC is not fully understood. Our research identified that the glycolysis-related gene (GRG) HK3 has a unique correlation with PD-L1 expression. HK3 has been identified as a key regulator of O-GlcNAcylation in ccRCC. O-GlcNAcylation exists on the serine 900 (Ser900) site of EP300 and can enhance its stability and oncogenic activity by preventing ubiquitination. Stably expressed EP300 works together with TFAP2A as a co-transcription factor to promote PD-L1 transcription and as an acetyltransferase to stabilize PD-L1 protein. Furthermore,ccRCC exhibits interactive dynamics with tumor-associated macrophages (TAMs). The uridine 5′-diphospho-N-acetylglucosamine (UDP-GlcNAc),which serves as a critical substrate for the O-GlcNAcylation process,facilitates TAMs polarization. In ccRCC cells,HK3 expression is influenced by IL-10 secreted by M2 TAMs. Our study elucidates that HK3-mediated O-GlcNAcylation of EP300 is involved in tumor immune evasion. This finding suggests potential strategies to enhance the efficacy of immune checkpoint blockade therapy. Subject terms: Cancer metabolism,Renal cell carcinoma View Publication

Hereditary spastic paraplegias are rare genetic disorders characterized by corticospinal tract impairment. Spastic paraplegia 83 (SPG83) is associated with biallelic mutations in the HPDL gene,leading to varied severities from neonatal to juvenile onset. The function of HPDL is unclear,though it is speculated to play a role in alternative coenzyme Q10 biosynthesis. Here,we report the generation of hiPS lines from primary skin fibroblasts derived from three SPG83 patients with different HPDL mutations,using episomal reprogramming. The patients’ clinical characteristics are carefully listed. The hiPS lines were meticulously characterized,demonstrating typical pluripotent characteristics through immunofluorescence assays for stemness markers (OCT4,TRA1-60,NANOG,and SSEA4) and RT-PCR for endogenous gene expression. Genetic integrity and identity were confirmed via Sanger sequencing and short tandem repeat analysis. These hiPS cells displayed typical pluripotent characteristics and were able to differentiate into neocortical neurons via a dual SMAD inhibition protocol. In addition,HPDL mutant neurons assessed via long-term culturing were able to achieve effective maturation,similarly to their wild-type counterparts. The HPDL hiPS lines we generated will provide a valuable model for studying SPG83,offering insights into its molecular mechanisms and potential for developing targeted therapies. View Publication

Breast cancer is a heterogeneous disease comprising multiple molecularly distinct subtypes with varied prevalence,prognostics,and treatment strategies. Among them,triple-negative breast cancer,though the least prevalent,is the most aggressive subtype,with limited therapeutic options. Recent emergence of competing endogenous RNA (ceRNA) networks has highlighted how long noncoding RNAs (lncRNAs),microRNAs (miRs),and mRNA orchestrate a complex interplay meticulously modulating mRNA functionality. Focusing on TNBC,this study aimed to construct a ceRNA network using differentially expressed lncRNAs,miRs,and mRNAs. We queried the differentially expressed lncRNAs (DElncRNAs) between TNBC and luminal samples and found 389 upregulated and 386 downregulated lncRNAs,including novel transcripts in TNBC. DElncRNAs were further evaluated for their clinical,functional,and mechanistic relevance to TNBCs using the lnc2cancer 3.0 database,which presented LUCAT1 (lung cancer-associated transcript 1) as a putative node. Next,the ceRNA network (lncRNA–miRNA–mRNA) of LUCAT1 was established. Several miRNA–mRNA connections of LUCAT1 implicated in regulating stemness (LUCAT1-miR-375-Yap1,LUCAT1-miR181-5p-Wnt,LUCAT1-miR-199a-5p-ZEB1),apoptosis (LUCAT1-miR-181c-5p-Bcl2),drug efflux (LUCAT1-miR-200c-ABCB1,LRP1,MRP5,MDR1),and sheddase activities (LUCAT1-miR-493-5p-ADAM10) were identified,indicating an intricate regulatory mechanism of LUCAT1 in TNBC. Indeed,LUCAT1 silencing led to mitigated cell growth,migration,and stem-like features in TNBC. This work sheds light on the LUCAT1 ceRNA network in TNBC and implies its involvement in TNBC growth and progression. View Publication

Natural killer (NK) cells are exploited in cellular therapies for cancer. While NK cell therapies are efficient against haematological cancers,it has been difficult to target solid tumours due to low tumour infiltration and a hostile tumour microenvironment. NK‐cell derived extracellular vesicles (NK‐EVs) target and kill cancer cells in vitro and represent an alternative treatment strategy for solid tumours. To exploit their potential,it is necessary to standardize NK‐EV production protocols. Here,we have performed a comparative analysis of EVs from the human NK‐92 cell line cultured in five serum‐free commercial media optimized for growth of human NK cells and one serum‐free medium for growth of lymphocytes. The effect of growing the NK‐92 cells in static cell cultures versus shaking flasks was compared. EVs were purified via ultracentrifugation followed by size‐exclusion chromatography. We found that there were no significant differences in EV yield from NK‐92 cells grown under static or dynamic conditions. However,we found clear differences between the different culture media in terms of EV purity as assessed by the enrichment of the CD63 and CD81 markers in the isolates that translated into their capacity to induce apoptosis of the colon cancer cell line HCT 116. These findings will be instructive for the design of future production protocols for therapeutic NK‐cell derived EVs. View Publication