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

Human embryonic stem cells (hESCs) hold great value for future clinical applications. However,standard culture conditions maintain hESCs in a primed state,which bears heterogeneity in pluripotency and a tendency for spontaneous differentiation. To counter these drawbacks,primed hESCs have been converted to a naive state,but this has restricted the efficiency of existing directed differentiation protocols. In mouse,WNT inhibition by inhibitor of WNT production-2,together with a higher dose of fibroblast growth factor 2 (12 ng/mL) in DMEM/F12 basal medium (DhiFI),markedly improved derivation and maintenance of primed mouse epiblast stem cells. In this study,we show that DhiFI conditions similarly improved primed hESC traits,such as conferring a primed transcriptional signature with high levels of pluripotency markers and reduced levels of differentiation markers. When triggered to differentiate to neuronal and cardiac lineages,DhiFI hESCs and isogenic primed hESCs progressed similarly. Moreover,DhiFI conditions supported the derivation of hESC lines from a post-inner cell mass intermediate (PICMI). DhiFI-derived hESCs showed less spontaneous differentiation and expressed significantly lower levels of lineage-specific markers,compared to primed-derived lines from the same PICMI. Overall,DhiFI hESCs retained advantages of both primed and naive pluripotency and may ultimately represent a more favorable starting point for differentiation toward clinically desired cell types. View Publication

The study introduces a new immunotherapy for treating melanoma and other cancers by developing a PROTAC that degrades NR4A1,an intracellular nuclear factor that plays a crucial role in immune suppression. An effective cancer therapy requires killing cancer cells and targeting the tumor microenvironment (TME). Searching for molecules critical for multiple cell types in the TME,we identified NR4A1 as one such molecule that can maintain the immune suppressive TME. Here,we establish NR4A1 as a valid target for cancer immunotherapy and describe a first-of-its-kind proteolysis-targeting chimera (PROTAC,named NR-V04) against NR4A1. NR-V04 degrades NR4A1 within hours in vitro and exhibits long-lasting NR4A1 degradation in tumors with an excellent safety profile. NR-V04 inhibits and frequently eradicates established tumors. At the mechanistic level,NR-V04 induces the tumor-infiltrating (TI) B cells and effector memory CD8+ T (Tem) cells and reduces monocytic myeloid-derived suppressor cells (m-MDSC),all of which are known to be clinically relevant immune cell populations in human melanomas. Overall,NR-V04–mediated NR4A1 degradation holds promise for enhancing anticancer immune responses and offers a new avenue for treating various types of cancers such as melanoma. Graphical Abstract View Publication

The CD31(+) subset of human naive CD4(+) T cells is thought to contain the population of cells that have recently emigrated from the thymus,while their CD31(-) counterparts have been proposed to originate from CD31(+) cells after homeostatic cell division. Naive T-cell maintenance is known to involve homeostatic cytokines such as interleukin-7 (IL-7). It remains to be investigated what role this cytokine has in the homeostasis of naive CD4(+) T-cell subsets defined by CD31 expression. We provide evidence that IL-7 exerts a preferential proliferative effect on CD31(+) naive CD4(+) T cells from adult peripheral blood compared with the CD31(-) subset. IL-7-driven proliferation did not result in loss of CD31 expression,suggesting that CD31(+) naive CD4(+) T cells can undergo cytokine-driven homeostatic proliferation while preserving CD31. Furthermore,IL-7 sustained or increased CD31 expression even in nonproliferating cells. Both proliferation and CD31 maintenance were dependent on the activation of phosphoinositide 3-kinase (PI3K) signaling. Taken together,our data suggest that during adulthood CD31(+) naive CD4(+) T cells are maintained by IL-7 and that IL-7-based therapies may exert a preferential effect on this population. View Publication

Dosage compensation of the X chromosomes in mammals is performed via the formation of facultative heterochromatin on extra X chromosomes in female somatic cells. Facultative heterochromatin of the inactivated X (Xi),as well as constitutive heterochromatin,replicates late during the S-phase. It is generally accepted that Xi is always more compact in the interphase nucleus. The dense chromosomal folding has been proposed to define the late replication of Xi. In contrast to mouse pluripotent stem cells (PSCs),the status of X chromosome inactivation in human PSCs may vary significantly. Fluorescence in situ hybridization with a whole X-chromosome- specific DNA probe revealed that late-replicating Xi may occupy either compact or dispersed territory in human PSCs. Thus,the late replication of the Xi does not depend on the compactness of chromosome territory in human PSCs. However,the Xi reactivation and the synchronization in the replication timing of X chromosomes upon reprogramming are necessarily accompanied by the expansion of X chromosome territory. View Publication

Basic fibroblast growth factor (bFGF) is a crucial factor sustaining human pluripotent stem cells (hPSCs). We designed this study to search the substitutive factors other than bFGF for the maintenance of hPSCs by using human placenta-derived conditioned medium without exogenous bFGF (hPCCM-),containing chemokine (C-X-C motif) receptor 2 (CXCR2) ligands,including interleukin (IL)-8 and growth-related oncogene $\$(GRO$\$),which were developed on the basis of our previous studies. First,we confirmed that IL-8 and/or GRO$\$ independent roles to preserve the phenotype of hPSCs. Then,we tried CXCR2 blockage of hPSCs in hPCCM- and verified the significant decrease of pluripotency-associated genes expression and the proliferation of hPSCs. Interestingly,CXCR2 suppression of hPSCs in mTeSR™1 containing exogenous bFGF decreased the proliferation of hPSCs while maintaining pluripotency characteristics. Lastly,we found that hPSCs proliferated robustly for more than 35 passages in hPCCM- on a gelatin substratum. Higher CXCR2 expression of hPSCs cultured in hPCCM- than those in mTeSR™1 was observable. Our findings suggest that CXCR2 and its related ligands might be novel factors comparable to bFGF supporting the characteristics of hPSCs and hPCCM- might be useful for the maintenance of hPSCs as well as for the accurate evaluation of CXCR2 role in hPSCs without the confounding influence of exogenous bFGF. View Publication

Human induced pluripotent stem cells (hiPSCs) provide a platform for studying human disease in vitro,increase our understanding of human embryonic development,and provide clinically relevant cell types for transplantation,drug testing,and toxicology studies. Since their discovery,numerous advances have been made in order to eliminate issues such as vector integration into the host genome,low reprogramming efficiency,incomplete reprogramming and acquisition of genomic instabilities. One of the ways to achieve integration-free reprogramming is by using RNA-based Sendai virus. Here we describe a method to generate hiPSCs with Sendai virus in both feeder-free and feeder-dependent culture systems. Additionally,we illustrate methods by which to validate pluripotency of the resulting stem cell population. View Publication

Freshly isolated,uncultured,autologous adipose derived regenerative cells (ADRCs) have emerged as a promising tool for regenerative cell therapy. The Transpose RT system (InGeneron,Inc.,Houston,TX,USA) is a system for isolating ADRCs from adipose tissue,commercially available in Europe as a CE-marked medical device and under clinical evaluation in the United States. This system makes use of the proprietary,enzymatic Matrase Reagent for isolating cells. The present study addressed the question whether the use of Matrase Reagent influences cell yield,cell viability,live cell yield,biological characteristics,physiological functions or structural properties of the ADRCs in final cell suspension. Identical samples of subcutaneous adipose tissue from 12 subjects undergoing elective lipoplasty were processed either with or without the use of Matrase Reagent. Then,characteristics of the ADRCs in the respective final cell suspensions were evaluated. Compared to non-enzymatic isolation,enzymatic isolation resulted in approximately twelve times higher mean cell yield (i.e.,numbers of viable cells/ml lipoaspirate) and approximately 16 times more colony forming units. Despite these differences,cells isolated from lipoaspirate both with and without the use of Matrase Reagent were independently able to differentiate into cells of all three germ layers. This indicates that biological characteristics,physiological functions or structural properties relevant for the intended use were not altered or induced using Matrase Reagent. A comprehensive literature review demonstrated that isolation of ADRCs from lipoaspirate using the Transpose RT system and the Matrase Reagent results in the highest viable cell yield among published data regarding isolation of ADRCs from lipoaspirate. View Publication

Different innate immune cell types are known to release extracellular traps (ETs) in response to invasive pathogens,including parasites. These ETs function to trap,immobilize,and eventually kill pathogens. In line with this,monocytes and macrophages have been shown to release ETs,known as monocyte/macrophage extracellular traps (METs). Toxoplasma gondii (T. gondii) is an apicomplexan zoonotic parasite that infects humans and homeothermic animals. While most studies have focused on prolonged exposure of immune cells to T. gondii,this study characterized the early innate immune reaction of mononuclear phagocytes to vital T. gondii tachyzoites. Methods: Primary human and bovine monocytes,monocytic THP-1 cells,and THP-1 cell-derived macrophages (M0-,M1-,and M2-like) were exposed to T. gondii tachyzoites for 4 h. Scanning electron microscopy (SEM),transmission electron microscopy (TEM),immunofluorescencemicroscopy,and confocal microscopy were used to visualize cell activation and the presence of METs. Additionally,the release of pro-inflammatory cytokines interleukin (IL)-1β and IL-6,and expression of Toll-like receptor (TLR) 2 and TLR4 were analyzed. Results and discussion: Microscopic analysis illustrated the activation of all cell types tested within 4 h of exposure to T. gondii tachyzoites. Numerous tachyzoites were found intracellularly in THP-1 cell-derived M1-like macrophages. Furthermore,the co-localization of extracellular DNA (extDNA) and histones in extracellular web-like fibers proved classical characteristics of extruded T. gondii-induced METs,although this was a rare event. In primary human monocytes,an increased release of IL-1β and IL-6 was observed following exposure to T. gondii tachyzoites. When co-stimulated with lipopolysaccharide (LPS),primary human monocytes showed an enhanced release of IL-1β and IL-6 in response to T. gondii. In contrast to monocytic THP-1 cells,THP-1 cell-derived M1-like macrophages released IL-1β in response to T. gondii tachyzoite exposure. When additionally stimulated by LPS,all THP-1 cell-derived macrophages showed an enhanced release of IL-1β,and monocytic THP-1 cells an increased release of IL-6 in response to T. gondii tachyzoites. This study provides insights into the early innate immune response of human and bovine mononuclear phagocytes to T. gondii. While cytokine secretion was prominent,MET formation was rare in the early response (i.e. < 4 h of exposure) to T. gondii tachyzoites. View Publication

SARS-CoV-2 Omicron variant has evolved into sublineages. Here,we compared the neutralization susceptibility and viral fitness of EG.5.1 and XBB.1.9.1. Serum neutralization antibody titer against EG.5.1 was 1.71-fold lower than that for XBB.1.9.1. However,there was no significant difference in virus replication between EG.5.1 and XBB.1.9.1 in human nasal organoids and TMPRSS2/ACE2 over-expressing A549 cells. No significant difference was observed in competitive fitness and cytokine/chemokine response between EG.5.1 and XBB.1.9.1. Both EG.5.1 and XBB.1.9.1 replicated more robustly in the nasal organoid from a younger adult than that from an older adult. Our findings suggest that enhanced immune escape contributes to the dominance of EG.5.1 over earlier sublineages. The combination of population serum susceptibility testing and viral fitness evaluation with nasal organoids may hold promise in risk assessment of upcoming variants. Utilization of serum specimens and nasal organoid derived from older adults provides a targeted risk assessment for this vulnerable population. Subject areas: Immunology,Immune response,Virology View Publication

Treatment of acute liver failure by cell transplantation is hindered by a shortage of human hepatocytes. Current protocols for hepatic differentiation of human induced pluripotent stem cells (hiPSCs) result in low yields,cellular heterogeneity,and limited scalability. In the present study,we have developed a novel multicellular spheroid-based hepatic differentiation protocol starting from embryoid bodies of hiPSCs (hiPSC-EBs) for robust mass production of human hepatocyte-like cells (HLCs) using two novel inhibitors of the Wnt pathway. The resultant hiPSC-EB-HLCs expressed liver-specific genes,secreted hepatic proteins such as Albumin,Alpha Fetoprotein,and Fibrinogen,metabolized ammonia,and displayed cytochrome P450 activities and functional activities typical of mature primary hepatocytes,such as LDL storage and uptake,ICG uptake and release,and glycogen storage. Cell transplantation of hiPSC-EB-HLC in a rat model of acute liver failure significantly prolonged the mean survival time and resolved the liver injury when compared to the no-transplantation control animals. The transplanted hiPSC-EB-HLCs secreted human albumin into the host plasma throughout the examination period (2 weeks). Transplantation successfully bridged the animals through the critical period for survival after acute liver failure,providing promising clues of integration and full in vivo functionality of these cells after treatment with WIF-1 and DKK-1. View Publication

Tamoxifen is used widely in the treatment of endocrine-responsive breast cancers in humans. Studies were undertaken to examine the biological character (estrogenic-antiestrogenic properties) and estrogen receptor (ER) interaction of the cis- and trans-isomers of tamoxifen and hydroxytamoxifen in MCF-7 human breast cancer cells. For each compound,the following parameters were monitored: affinity for ER and effects on cellular ER levels; stimulation-inhibition of cell growth,plasminogen activator activity,and cellular progesterone receptor levels; and isomer interconversion and metabolism in vitro. The relative binding affinities of the compounds cis-tamoxifen,trans-tamoxifen,cis-hydroxytamoxifen,and trans-hydroxytamoxifen for cytosol ER were 0.3,2.5,1.8,and 310%,respectively,in which the affinity of estradiol is considered 100%. cis-Tamoxifen behaved as a weak estrogen agonist in all assays,while trans-tamoxifen was an effective estrogen antagonist. cis-Tamoxifen behaved like estradiol in stimulating MCF-7 cell growth and increasing plasminogen activator activity and cellular progesterone receptor content,although very much higher concentrations of cis-tamoxifen (10(-6) M) were needed to achieve the levels of stimulation observed with 10(-10) M estradiol. trans-Tamoxifen and trans-hydroxytamoxifen suppressed cell growth,inhibited plasminogen activator activity of control cells,and suppressed estradiol-stimulation of plasminogen activator activity,and they evoked minimal increases in cellular progesterone receptor levels. trans-Hydroxytamoxifen had a 100-fold increased affinity for ER and was approximately 100-times more potent than was trans-tamoxifen in suppressing cell growth and plasminogen activator activity. cis-Hydroxytamoxifen behaved as an estrogen antagonist,suppressing cell growth and plasminogen activator activity,and it elicited submaximal increases in progesterone receptor levels. This apparently paradoxical behavior of cis-hydroxytamoxifen was shown to be due to the fact that the cis- and trans-hydroxytamoxifens readily undergo isomeric interconversion upon exposure to our cell culture conditions,resulting in substantial accumulation of the higher-affinity trans-hydroxytamoxifen in the nuclear ER fraction of cells. In contrast to the facile interconversion of the hydroxytamoxifen isomers,there is no metabolism or interconversion of the parent compounds cis- and trans-tamoxifen in vitro. Hence,by the criteria we have used,the biological characters of trans-tamoxifen and trans-hydroxytamoxifen are similar,the major difference being the approximately 100-fold enhanced potency of the hydroxylated form. In contrast,cis-t View Publication