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

DNA double-strand breaks (DSB) are the most cytotoxic lesions induced by topoisomerase II poisons. Nonhomologous end joining (NHEJ) is a major pathway for DSB repair and requires DNA-dependent protein kinase (DNA-PK) activity. DNA-PK catalytic subunit (DNA-PKcs) is structurally similar to PI-3K,which promotes cell survival and proliferation and is upregulated in many cancers. KU-0060648 is a dual inhibitor of DNA-PK and PI-3K in vitro. KU-0060648 was investigated in a panel of human breast and colon cancer cells. The compound inhibited cellular DNA-PK autophosphorylation with IC(50) values of 0.019 $\mu$mol/L (MCF7 cells) and 0.17 $\mu$mol/L (SW620 cells),and PI-3K-mediated AKT phosphorylation with IC(50) values of 0.039 $\mu$mol/L (MCF7 cells) and more than 10 $\mu$mol/L (SW620 cells). Five-day exposure to 1 $\mu$mol/L KU-0060648 inhibited cell proliferation by more than 95{\%} in MCF7 cells but only by 55{\%} in SW620 cells. In clonogenic survival assays,KU-0060648 increased the cytotoxicity of etoposide and doxorubicin across the panel of DNA-PKcs-proficient cells,but not in DNA-PKcs-deficient cells,thus confirming that enhanced cytotoxicity was due to DNA-PK inhibition. In mice bearing SW620 and MCF7 xenografts,concentrations of KU-0060648 that were sufficient for in vitro growth inhibition and chemosensitization were maintained within the tumor for at least 4 hours at nontoxic doses. KU-0060648 alone delayed the growth of MCF7 xenografts and increased etoposide-induced tumor growth delay in both in SW620 and MCF7 xenografts by up to 4.5-fold,without exacerbating etoposide toxicity to unacceptable levels. The proof-of-principle in vitro and in vivo chemosensitization with KU-0060648 justifies further evaluation of dual DNA-PK and PI-3K inhibitors. View Publication

Lymphocyte activation gene-3 (LAG-3) is an inhibitory receptor expressed by CD4+ T cells and tempers their homeostatic expansion. Because CD4+ T cell proliferation is tightly coupled to bioenergetics,we investigate the role of LAG-3 in modulating naive CD4+ T cell metabolism. LAG-3 deficiency enhances the metabolic profile of naive CD4+ T cells by elevating levels of mitochondrial biogenesis. In vivo,LAG-3 blockade partially restores expansion and the metabolic phenotype of wild-type CD4+ T cells to levels of Lag3-/- CD4+ T cells,solidifying that LAG-3 controls these processes. Lag3-/- CD4+ T cells also demonstrate greater signal transducer and activator of transcription 5 (STAT5) activation,enabling resistance to interleukin-7 (IL-7) deprivation. These results implicate this pathway as a target of LAG-3-mediated inhibition. Additionally,enhancement of STAT5 activation,as a result of LAG-3 deficiency,contributes to greater activation potential in these cells. These results identify an additional mode of regulation elicited by LAG-3 in controlling CD4+ T cell responses. View Publication

Early erythroid progenitors are transit-amplifying cells with high proliferative capacity committed to undergoing red cell differentiation. CD71/CD24low progenitors are less mature and have greater proliferative capacity than CD71/CD24high. We present protocols for isolation of CD71/CD24low progenitors from mouse fetal liver using both fluorescence-activated cell sorting (FACS) and immunomagnetic enrichment. CD71/CD24low progenitors isolated with both approaches show similar transcriptomes at single-cell resolution and exhibit characteristic proliferative responses to glucocorticoids. For complete details on the use and execution of this protocol,please refer to Li et al. (2019). View Publication

Adipose tissue dysfunction is strongly linked to the development of chronic inflammation and cardiometabolic disorders in aging. While much attention has been given to the role of resident adipose tissue immune cells in the disruption of homeostasis in obesity,age-specific effects remain understudied. Here,we identified and characterized a population of ?? T cells,which show unique age-dependent accumulation in the visceral adipose tissue (VAT) of both mice and humans. Diet-induced obesity likewise increased ?? T cell numbers; however,the effect was greater in the aged where the increase was independent of fat mass. ?? T cells in VAT express a tissue-resident memory T cell phenotype (CD44hiCD62LlowCD69+) and are predominantly IL-17A-producing cells. Transcriptome analyses of immunomagnetically purified ?? T cells identified significant age-associated differences in expression of genes related to inflammation,immune cell composition,and adipocyte differentiation,suggesting age-dependent qualitative changes in addition to the quantitative increase. Genetic deficiency of ?? T cells in old age improved the metabolic phenotype,characterized by increased respiratory exchange ratio,and lowered levels of IL-6 both systemically and locally in VAT. Decreased IL-6 was predominantly due to reduced production by non-immune stromal cells,primarily preadipocytes,and adipose-derived stem cells. Collectively,these findings suggest that an age-dependent increase of tissue-resident ?? T cells in VAT contributes to local and systemic chronic inflammation and metabolic dysfunction in aging. View Publication

Immune-mediated red blood cell (RBC) destruction due to antibodies is an ongoing problem in transfusion medicine for the selection of the safest blood. Serological testing often revealed incompatibility with donors' RBCs. When this incompatible blood was transfused,destruction was due mostly to extravascular-mediated phagocytosis of the antibody-opsonized RBCs; however,intravascular hemolysis was sometimes observed without explanation. Based on serology,antibodies with potential for clinical sequalae could not be ascertained; thus,antigen-negative blood was usually selected for transfusion to avoid problems. Antibodies to antigens having very high frequency in the general population (>95%),however,made selection of antigen-negative blood difficult and sometimes impossible. Some patients,who were sensitized by previous transfusions or by pregnancy,developed multiple antibodies,again creating a problem for finding compatible blood for transfusion,without the ability to discern which of the antibodies may be clinically irrelevant and ignored. Transfusion medicine scientists began searching for an in vitro means to determine the in vivo outcome of transfusion of blood that was serologically incompatible. Methods such as chemiluminescence,monocyte-macrophage phagocytosis,and antibody-dependent cellular cytotoxicity (ADCC) were described. Over the years,the monocyte monolayer assay (MMA) has emerged as the most reliable in vitro assay for the prediction of the clinical relevance of a given antibody. ADCC has not been fully studied but has the potential to be useful for predicting which antibodies may result in intravascular hemolysis. This article captures the protocols for the implementation and readout of the MMA and ADCC assays for use in predicting the clinical significance of antibodies in a transfusion setting. {\textcopyright} 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Monocyte monolayer assay (MMA) Basic Protocol 2: Antibody-dependent cellular cytotoxicity assay (ADCC). View Publication

Long interspersed element 1 (LINE-1; L1) are a family of transposons that occupy ~17% of the human genome. Though a small number of L1 copies remain capable of autonomous transposition,the overwhelming majority of copies are degenerate and immobile. Nevertheless,both mobile and immobile L1s can exert pleiotropic effects (promoting genome instability,inflammation,or cellular senescence) on their hosts,and L1’s contributions to aging and aging diseases is an area of active research. However,because of the cell type-specific nature of transposon control,the catalogue of L1 regulators remains incomplete. Here,we employ an eQTL approach leveraging transcriptomic and genomic data from the GEUVADIS and 1000Genomes projects to computationally identify new candidate regulators of L1 RNA levels in lymphoblastoid cell lines. To cement the role of candidate genes in L1 regulation,we experimentally modulate the levels of top candidates in vitro,including IL16,STARD5,HSD17B12,and RNF5,and assess changes in TE family expression by Gene Set Enrichment Analysis (GSEA). Remarkably,we observe subtle but widespread upregulation of TE family expression following IL16 and STARD5 overexpression. Moreover,a short-term 24-hour exposure to recombinant human IL16 was sufficient to transiently induce subtle,but widespread,upregulation of L1 subfamilies. Finally,we find that many L1 expression-associated genetic variants are co-associated with aging traits across genome-wide association study databases. Our results expand the catalogue of genes implicated in L1 RNA control and further suggest that L1-derived RNA contributes to aging processes. Given the ever-increasing availability of paired genomic and transcriptomic data,we anticipate this new approach to be a starting point for more comprehensive computational scans for regulators of transposon RNA levels. Author summaryTransposable elements,or jumping genes,are fragments of DNA that have or once had the ability to mobilize to a new location within our genome. In humans,the most abundant transposable element is LINE-1 (L1),accounting for ~17% of our total DNA. Though L1 is generally repressed in healthy human cells,derepression of transposable elements (including L1) has been observed in aging and in aging-associated diseases. Additionally,there is increasing evidence that L1 transcriptional levels may promote features of aging,highlighting the importance of understanding the mechanisms that regulate L1 RNA levels. Here,we computationally identify new candidate regulators of L1 RNA levels,provide experimental evidence that candidate regulators influence L1 RNA levels,and demonstrate that genetic variants associated with differences in L1 RNA levels are co-associated with aging phenotypes. Our approach expands the toolkit that can be used to characterize transposable element regulation and highlights specific genes for further study. Importantly,our results reiterate the notion that L1 levels are linked with aging phenotypes and represent a potential therapeutic target for age-related decline. View Publication

The in vitro oxygen microenvironment profoundly affects the capacity of cell cultures to model physiological and pathophysiological states. Cell culture is often considered to be hyperoxic,but pericellular oxygen levels,which are affected by oxygen diffusivity and consumption,are rarely reported. Here,we provide evidence that several cell types in culture actually experience local hypoxia,with important implications for cell metabolism and function. We focused initially on adipocytes,as adipose tissue hypoxia is frequently observed in obesity and precedes diminished adipocyte function. Under standard conditions,cultured adipocytes are highly glycolytic and exhibit a transcriptional profile indicative of physiological hypoxia. Increasing pericellular oxygen diverted glucose flux toward mitochondria,lowered HIF1α activity,and resulted in widespread transcriptional rewiring. Functionally,adipocytes increased adipokine secretion and sensitivity to insulin and lipolytic stimuli,recapitulating a healthier adipocyte model. The functional benefits of increasing pericellular oxygen were also observed in macrophages,hPSC-derived hepatocytes and cardiac organoids. Our findings demonstrate that oxygen is limiting in many terminally-differentiated cell types,and that considering pericellular oxygen improves the quality,reproducibility and translatability of culture models. View Publication

Solid cancers Myeloid cells are prevalent in solid cancers,but they frequently exhibit an anti-inflammatory pro-tumor phenotype that contribute to the immunosuppressive tumor microenvironment (TME),which hinders the effectiveness of cancer immunotherapies. Myeloid cells’ natural ability of tumor trafficking makes engineered myeloid cell therapy an intriguing approach to tackle the challenges posed by solid cancers,including tumor infiltration,tumor cell heterogenicity and the immunosuppressive TME. One such engineering approach is to target the checkpoint molecule PD-L1,which is often upregulated by solid cancers to evade immune responses. Here we devised an adoptive cell therapy strategy based on myeloid cells expressing a Chimeric Antigen Receptor (CAR)-like immune receptor (CARIR). The extracellular domain of CARIR is derived from the natural inhibitory receptor PD-1,while the intracellular domain(s) are derived from CD40 and/or CD3ζ. To assess the efficacy of CARIR-engineered myeloid cells,we conducted proof-of-principle experiments using co-culture and flow cytometry-based phagocytosis assays in vitro. Additionally,we employed a fully immune-competent syngeneic tumor mouse model to evaluate the strategy’s effectiveness in vivo. Co-culturing CARIR-expressing human monocytic THP-1 cells with PD-L1 expressing target cells lead to upregulation of the costimulatory molecule CD86 along with expression of proinflammatory cytokines TNF-1α and IL-1β. Moreover,CARIR expression significantly enhanced phagocytosis of multiple PD-L1 expressing cancer cell lines in vitro. Similar outcomes were observed with CARIR-expressing human primary macrophages. In experiments conducted in syngeneic BALB/c mice bearing 4T1 mammary tumors,infusing murine myeloid cells that express a murine version of CARIR significantly slowed tumor growth and prolonged survival. Taken together,these results demonstrate that adoptive transfer of PD-1 CARIR-engineered myeloid cells represents a promising strategy for treating PD-L1 positive solid cancers. View Publication

The manufacturing of allogeneic cell therapeutics based on human-induced pluripotent stem cells (hiPSCs) holds considerable potential to revolutionize the accessibility and affordability of modern healthcare. However,achieving the cell yields necessary to ensure robust production hinges on identifying suitable and scalable single-use (SU) bioreactor systems. While specific stirred SU bioreactor types have demonstrated proficiency in supporting hiPSC expansion at L -scale,others,notably instrumented SU multiplate and fixed-bed bioreactors,remain relatively unexplored. By characterizing these bioreactors using both computational fluid dynamics and experimental bioengineering methods,operating ranges were identified for the Xpansion ® 10 and Ascent™ 1 m 2 bioreactors in which satisfactory hiPSC expansion under serum-free conditions was achieved. These operating ranges were shown not only to effectively limit cell exposure to wall shear stress but also facilitated sufficient oxygen transfer and mixing. Through their application,almost 5 × 10 9 viable cells could be produced within 5 days,achieving expansion factors of up to 35 without discernable impact on cell viability,identity,or differentiation potential. Key Points • Bioengineering characterizations allowed the identification of operating ranges that supported satisfactory hiPSC expansion • Both the Xpansion ® 10 multiplate and Ascent™ 1 m 2 fixed-bed reactor accommodated the production of almost 5 × 10 9 viable cells within 5 days • Exposing the hiPSCs to a median wall shear stress of up to 8.2 × 10 −5 N cm −2 did not impair quality The online version contains supplementary material available at 10.1007/s00253-024-13373-2. View Publication

Trogocytosis is an underappreciated phenomenon that shapes the immune microenvironment surrounding many types of solid tumors. The consequences of membrane-bound proteins being deposited from a donor immune cell to a recipient cancer cell via trogocytosis are still unclear. Here,we report that human clear cell renal carcinoma tumors stably express the lymphoid markers CD45,CD56,CD14,and CD16. Flow cytometry performed on fresh kidney tumors revealed consistent CD45 expression on tumor cells,as well as varying levels of the other markers mentioned previously. These results were consistent with our immunofluorescent analysis,which also revealed colocalization of lymphoid markers with carbonic anhydrase 9,a standard kidney tumor marker. RNA analysis showed a significant upregulation of genes typically associated with immune cells by tumor cells. Finally,we show evidence of chromosomal DNA being transferred from immune cells to tumor cells through physical contact. This horizontal gene transfer has transcriptional consequences in the recipient tumor cell,resulting in a fusion phenotype that expresses both immune and cancer specific proteins. This work demonstrates a novel mechanism by which tumor cell protein expression is altered through the acquisition of surface membrane fragments and genomic DNA from infiltrating lymphocytes. These results alter the way in which we understand tumor-immune cell interactions and may reveal new insights into the mechanisms by which tumors develop. Additionally,further studies into trogocytosis and other mechanisms of contact-mediated cellular transfer will help push the field towards the next generation of immunotherapies and biomarkers for treating renal cell carcinoma and other cancers. View Publication

Circulating tumor cells (CTCs) hold significant promise for cancer diagnosis,prognosis,and treatment monitoring. We previously developed a technique for a single‐cell filtering device known as the microcavity array (MCA),specifically designed for the efficient recovery of CTCs from whole blood samples. Efficient enrichment and release of cells from the MCA remains challenging because of cell adhesion that occurs on the MCA surface during the enrichment phase. This study investigated the effects of surface modification with 2‐methacryloyloxyethyl phosphorylcholine (MPC) on the recovery efficiency of cancer cell lines from MCA. Scanning electron microscope (SEM) demonstrated reduced cell‐substrate interactions,leading to improved recovery efficiency. Comparative analyses showed that the MCA method provided superior recovery efficiency and reduced processing time compared to traditional methods such as density gradient centrifugation (DGC),while maintaining cell viability and proliferative capacity. CTCs were successfully detected in patients with gastric cancer,and short‐term cultures were achieved even when fewer than 20 CTCs per milliliter of blood were isolated. These findings emphasize the importance of surface modification for enhancing CTC isolation and the need for optimized culture conditions. The optimized MCA method offers a promising approach for rapid CTC recovery and potential integration with automated systems. Practical application : The Microcavity array (MCA) is a device specifically designed for efficient recovery of CTCs from whole blood. However cell adhesion on the MCA surface can limit release efficiency. This study demonstrated that surface modification with MPC signigicantly reduces cell‐substrate adhesion,improving recovery efficiency while maintaining cell viability and proliferative capacity. Compared to traditional density gradient centrifugation,the MPC‐modified MCA offers shorter processing time and better performance. CTCs were successfully detected in gastric cancer,and short‐term cultures were achieved even when fewer than 20 CTCs per mL of blood were isolated. The method supports downstearm applications such as cancer cell characterization and treatment monitoring. With potential for integration into automated system,the optimized MCA provides a practical,scalable solution for clinical liquid biopsy and personalized oncology. View Publication