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

Experimental autoimmune uveoretinitis (EAU) is a mouse model of human autoimmune uveitis marked by ocular autoantigen-specific regulatory immunity in the spleen. The melanocortin 5 receptor (MC5r) and adenosine 2 A receptor (A2Ar) are required for induction of post-EAU regulatory T cells (Tregs) which provide resistance to EAU. We show that blocking the PD-1/PD-L1 pathway prevented suppression of EAU by post-EAU Tregs. A2Ar induction of PD-1+FoxP3+ Tregs in uveitis patients was similar compared to healthy controls,but was significantly reduced with melanocortin stimulation. Further,lower body mass index correlated with responsiveness to stimulation of this pathway. These observations indicate an importance of the PD-1/PD-L1 pathway to provide resistance to relapsing uveitis and shows a reduced capacity of uveitis patients to induce Tregs when stimulated through melanocortin receptors,but that it is possible to bypass this part of the pathway through direct stimulation of A2Ar. View Publication

Mucopolysaccharidosis (MPS) IVA is a bone-affecting lysosomal storage disease (LSD) caused by impaired degradation of the glycosaminoglycans (GAGs) keratan sulfate (KS) and chondroitin 6-sulfate (C6S) due to deficient N-acetylgalactosamine-6-sulfatase (GALNS) enzyme activity. Previously,we successfully developed and validated a CRISPR/nCas9-based gene therapy (GT) to insert an expression cassette at the AAVS1 and ROSA26 loci in human MPS IVA fibroblasts and MPS IVA mice,respectively. In this study,we have extended our approach to evaluate the effectiveness of our CRISPR/nCas9-based GT in editing human CD34+ cells to mediate cross-correction of MPS IVA fibroblasts. CD34+ cells were electroporated with the CRISPR/nCas9 system,targeting the AAVS1 locus. The nCas9-mediated on-target donor template insertion,and the stemness of the CRISPR/nCas-edited CD34+ cells was evaluated. Additionally,MPS IVA fibroblasts were co-cultured with CRISPR/nCas-edited CD34+ cells to assess cross-correction. CRISPR/nCas9-based gene editing did not affect the stemness of CD34+ cells but did lead to supraphysiological levels of the GALNS enzyme. Upon co-culture,MPS IVA fibroblasts displayed a significant increase in the GALNS enzyme activity along with lysosomal mass reduction,pro-oxidant profile amelioration,mitochondrial mass recovery,and pro-apoptotic and pro-inflammatory profile improvement. These results show the potential of our CRISPR/nCas9-based GT to edit CD34+ cells to mediate cross-correction. View Publication

Re-activation and clonal expansion of tumor-specific antigen (TSA)-reactive T cells are critical to the success of checkpoint blockade and adoptive transfer of tumor-infiltrating lymphocyte (TIL)-based therapies. There are no reliable markers to specifically identify the repertoire of TSA-reactive T cells due to their heterogeneous composition. We introduce FucoID as a general platform to detect endogenous antigen-specific T cells for studying their biology. Through this interaction-dependent labeling approach,intratumoral TSA-reactive CD4+,CD8+ T cells,and TSA-suppressive CD4+ T cells can be detected and separated from bystander T cells based on their cell-surface enzymatic fucosyl-biotinylation. Compared to bystander TILs,TSA-reactive TILs possess a distinct T cell receptor (TCR) repertoire and unique gene features. Although exhibiting a dysfunctional phenotype,TSA-reactive CD8+ TILs possess substantial capabilities of proliferation and tumor-specific killing. Featuring genetic manipulation-free procedures and a quick turnover cycle,FucoID should have the potential of accelerating the pace of personalized cancer treatment. View Publication

The breakthrough in derivation of human-induced pluripotent stem cells (hiPSCs) provides an approach that may help overcome ethical and allergenic challenges posed in numerous medical applications involving human cells,including neural stem/progenitor cells (NSCs). Considering the great potential of NSCs in targeted cancer gene therapy,we investigated in this study the tumor tropism of hiPSC-derived NSCs and attempted to enhance the tropism by manipulation of biological activities of proteins that are involved in regulating the migration of NSCs toward cancer cells. We first demonstrated that hiPSC-NSCs displayed tropism for both glioblastoma cells and breast cancer cells in vitro and in vivo. We then compared gene expression profiles between migratory and non-migratory hiPSC-NSCs toward these cancer cells and observed that the gene encoding neuronal nitric oxide synthase (nNOS) was down-regulated in migratory hiPSC-NSCs. Using nNOS inhibitors and nNOS siRNAs,we demonstrated that this protein is a relevant regulator in controlling migration of hiPSC-NSCs toward cancer cells,and that inhibition of its activity or down-regulation of its expression can sensitize poorly migratory NSCs and be used to improve their tumor tropism. These findings suggest a novel application of nNOS inhibitors in neural stem cell-mediated cancer therapy. View Publication

Recent genomic approaches have revealed that the repertoire of RNA Pol III-transcribed genes varies in different human cell types,and that this variation is likely determined by a combination of the chromatin landscape,cell-specific DNA-binding transcription factors,and collaboration with RNA Pol II. Although much is known about this regulation in differentiated human cells,there is presently little understanding of this aspect of the Pol III system in human ES cells. Here,we determine the occupancy profiles of Pol III components in human H1 ES cells,and also induced pluripotent cells,and compare to known profiles of chromatin,transcription factors,and RNA expression. We find a relatively large fraction of the Pol III repertoire occupied in human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). In ES cells we find clear correlations between Pol III occupancy and active chromatin. Interestingly,we find a highly significant fraction of Pol III-occupied genes with adjacent binding events by pluripotency factors in ES cells,especially NANOG. Notably,in human ES cells we find H3K27me3 adjacent to but not overlapping many active Pol III loci. We observe in all such cases,a peak of H3K4me3 and/or RNA Pol II,between the H3K27me3 and Pol III binding peaks,suggesting that H3K4me3 and Pol II activity may “insulate�? Pol III from neighboring repressive H3K27me3. Further,we find iPSCs have a larger Pol III repertoire than their precursors. Finally,the active Pol III genome in iPSCs is not completely reprogrammed to a hESC like state and partially retains the transcriptional repertoire of the precursor. Together,our correlative results are consistent with Pol III binding and activity in human ES cells being enabled by active/permissive chromatin that is shaped in part by the pluripotency network of transcription factors and RNA Pol II activity. View Publication

The blood-brain barrier (BBB) maintains brain homeostasis but also presents a major obstacle to brain drug delivery. Brain microvascular endothelial cells (BMECs) form the principal barrier and therefore represent the major cellular component of in vitro BBB models. Such models are often used for mechanistic studies of the BBB in health and disease and for drug screening. Recently,human induced pluripotent stem cells (iPSCs) have emerged as a new source for generating BMEC-like cells for use in in vitro human BBB studies. However,the inability to cryopreserve iPSC-BMECs has impeded implementation of this model by requiring a fresh differentiation to generate cells for each experiment. Cryopreservation of differentiated iPSC-BMECs would have a number of distinct advantages,including enabling production of larger scale lots,decreasing lead time to generate purified iPSC-BMEC cultures,and facilitating use of iPSC-BMECs in large-scale screening. In this study,we demonstrate that iPSC-BMECs can be successfully cryopreserved at multiple differentiation stages. Cryopreserved iPSC-BMECs retain high viability,express standard endothelial and BBB markers,and reach a high transendothelial electrical resistance (TEER) of ∼3000 Ωtextperiodcenteredcm(2),equivalent to nonfrozen controls. Rho-associated coiled coil-containing kinase (ROCK) inhibitor Y-27632 substantially increased survival and attachment of cryopreserved iPSC-BMECs,as well as stabilized TEER above 800 Ωtextperiodcenteredcm(2) out to 7 days post-thaw. Overall,cryopreservation will ease handling and storage of high-quality iPSC-BMECs,reducing a key barrier to greater implementation of these cells in modeling the human BBB. View Publication

Induced pluripotent stem (iPS) cells are being used increasingly to complement their embryonic counterparts to understand and develop the therapeutic potential of pluripotent cells. Our objectives were to identify an efficient cardiac differentiation protocol for human iPS cells as monolayers,and demonstrate that the resulting cardiac progenitors could provide a therapeutic benefit in a rodent model of myocardial infarction. Herein,we describe a 14-day protocol for efficient cardiac differentiation of human iPS cells as a monolayer,which routinely yielded a mixed population in which over 50% were cardiomyocytes,endothelium,or smooth muscle cells. When differentiating,cardiac progenitors from day 6 of this protocol were injected into the peri-infarct region of the rat heart; after coronary artery ligation and reperfusion,we were able to show that human iPS cell-derived cardiac progenitor cells engrafted,differentiated into cardiomyocytes and smooth muscle,and persisted for at least 10 weeks postinfarct. Hearts injected with iPS-derived cells showed a nonsignificant trend toward protection from decline in function after myocardial infarction,as assessed by magnetic resonance imaging at 10 weeks,such that the ejection fraction at 10 weeks in iPS treated hearts was 62%±4%,compared to that of control infarcted hearts at 45%±9% (Ptextless0.2). In conclusion,we demonstrated efficient cardiac differentiation of human iPS cells that gave rise to progenitors that were retained within the infarcted rat heart,and reduced remodeling of the heart after ischemic damage. View Publication

Graphene quantum dots (GQDs) have gained significant attention in various biomedical applications. The physicochemical properties of these nanoparticles,including toxic effects,are largely determined by their surface modifications. Previous studies have demonstrated high in vitro cytotoxicity of the hydroxylated GQDs (OH-GQDs). The focus of this study was on the intestinal toxicity of OH-GQDs. Briefly,C57BL/6J mice were given daily oral gavage of 0.05,0.5 or 5 mg/kg OH-GQD for 7 days,and the indices of intestinal damage were evaluated. Higher doses of the OH-GQDs caused significant intestinal injuries,such as enhanced intestinal permeability,shortened villi and crypt loss. The number of Lgr5+ intestinal stem cells also decreased dramatically upon OH-GQDs exposure,which also inhibited the Ki67+ proliferative progenitor cells. In addition,an increased number of crypt cells harboring the oxidized DNA base 8-OHdG and $\gamma$H2AX foci were also detected in the intestines of OH-GQD-treated mice. Mechanistically,the OH-GQDs up-regulated both total and phosphorylated p53. Consistent with this,the average number of TUNEL+ and cleaved caspase-3+ apoptotic intestinal epithelial cells were significantly increased after OH-GQDs treatment. Finally,a 3-dimensional organoid culture was established using isolated crypts,and OH-GQDs treatment significantly reduced the size of the surviving intestinal organoids. Taken together,the intestinal toxicity of the OH-GQDs should be taken into account during biomedical applications. View Publication

Natural killer (NK) cells play a crucial role in immune surveillance by recognizing and eliminating tumor cells. However,tumors employ various mechanisms to evade NK cell-mediated immunity. NKp30 is a potent activating receptor on NK cells,but its function can be inhibited by specific ligands secreted by cancer cells. Here,we identified dipeptidase 1 (DPEP1) as a novel ligand for NKp30 in KM12C colon cancer cells,using co-immunoprecipitation,confocal microscopy,and flow cytometry. We examined how the DPEP1–NKp30 interaction affects NK cell activity and found that NK cytotoxicity increased in KM12C cells with DPEP1 knockdown but was significantly reduced in HCT116 cells overexpressing DPEP1. We further demonstrated that DPEP1 is secreted via extracellular vesicles and that its interaction with NKp30 suppressed the expression and secretion of perforin 1,granzyme B,CD107a,and interferon-γ in NK92 cells. In a xenograft mouse model treated with NK92 cells,tumors derived from HCT116/DPEP1 cells were significantly larger than those from HCT116/mock cells. Using peripheral blood-derived human NK cells,we confirmed that DPEP1 inhibited both cytotoxicity and granzyme B secretion. These findings suggest that disrupting the DPEP1–NKp30 interaction may enhance NK cell-mediated cytotoxicity and represent a novel therapeutic strategy for cancer immunotherapy. The online version contains supplementary material available at 10.1038/s41598-025-18475-z. View Publication

It has long been known that small changes to the structure of the R(2) side chain of nitrogen-containing bisphosphonates can dramatically affect their potency for inhibiting bone resorption in vitro and in vivo,although the reason for these differences in antiresorptive potency have not been explained at the level of a pharmacological target. Recently,several nitrogen-containing bisphosphonates were found to inhibit osteoclast-mediated bone resorption in vitro by inhibiting farnesyl diphosphate synthase,thereby preventing protein prenylation in osteoclasts. In this study,we examined the potency of a wider range of nitrogen-containing bisphosphonates,including the highly potent,heterocycle-containing zoledronic acid and minodronate (YM-529). We found a clear correlation between the ability to inhibit farnesyl diphosphate synthase in vitro,to inhibit protein prenylation in cell-free extracts and in purified osteoclasts in vitro,and to inhibit bone resorption in vivo. The activity of recombinant human farnesyl diphosphate synthase was inhibited at concentrations textgreater or = 1 nM zoledronic acid or minodronate,the order of potency (zoledronic acid approximately equal to minodronate textgreater risedronate textgreater ibandronate textgreater incadronate textgreater alendronate textgreater pamidronate) closely matching the order of antiresorptive potency. Furthermore,minor changes to the structure of the R(2) side chain of heterocycle-containing bisphosphonates,giving rise to less potent inhibitors of bone resorption in vivo,also caused a reduction in potency up to approximately 300-fold for inhibition of farnesyl diphosphate synthase in vitro. These data indicate that farnesyl diphosphate synthase is the major pharmacological target of these drugs in vivo,and that small changes to the structure of the R(2) side chain alter antiresorptive potency by affecting the ability to inhibit farnesyl diphosphate synthase. View Publication

Human noroviruses (HuNoVs) are the leading cause of nonbacterial gastroenteritis worldwide. Histo-blood group antigen (HBGA) expression is an important susceptibility factor for HuNoV infection based on controlled human infection models and epidemiologic studies that show an association of secretor status with infection caused by several genotypes. The fucosyltransferase 2 gene (FUT2) affects HBGA expression in intestinal epithelial cells; secretors express a functional FUT2 enzyme,while nonsecretors lack this enzyme and are highly resistant to infection and gastroenteritis caused by many HuNoV strains. These epidemiologic associations are confirmed by infections in stem cell-derived human intestinal enteroid (HIE) cultures. GII.4 HuNoV does not replicate in HIE cultures derived from nonsecretor individuals,while HIEs from secretors are permissive to infection. However,whether FUT2 expression alone is critical for infection remains unproven,since routinely used secretor-positive transformed cell lines are resistant to HuNoV replication. To evaluate the role of FUT2 in HuNoV replication,we used CRISPR or overexpression to genetically manipulate FUT2 gene function to produce isogenic HIE lines with or without FUT2 expression. We show that FUT2 expression alone affects both HuNoV binding to the HIE cell surface and susceptibility to HuNoV infection. These findings indicate that initial binding to a molecule(s) glycosylated by FUT2 is critical for HuNoV infection and that the HuNoV receptor is present in nonsecretor HIEs. In addition to HuNoV studies,these isogenic HIE lines will be useful tools to study other enteric microbes where infection and/or disease outcome is associated with secretor status.IMPORTANCE Several studies have demonstrated that secretor status is associated with susceptibility to human norovirus (HuNoV) infection; however,previous reports found that FUT2 expression is not sufficient to allow infection with HuNoV in a variety of continuous laboratory cell lines. Which cellular factor(s) regulates susceptibility to HuNoV infection remains unknown. We used genetic manipulation of HIE cultures to show that secretor status determined by FUT2 gene expression is necessary and sufficient to support HuNoV replication based on analyses of isogenic lines that lack or express FUT2. Fucosylation of HBGAs is critical for initial binding and for modification of another putative receptor(s) in HIEs needed for virus uptake or uncoating and necessary for successful infection by GI.1 and several GII HuNoV strains. View Publication