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

Until recently,culturing human pluripotent stem cells was hampered by three prominent technical problems: a high degree of unwanted cellular stress when the cells are passaged,unacceptably low cloning efficiency and poor recovery of cryopreserved stocks. This review discusses recent developments that address these problems. A major focus of the review is the use of p160 Rho-associated coiled-coil kinase inhibitors for improving both the cloning efficiency and the recovery of cryopreserved human embryonic stem cells and human induced pluripotent stem cells. An underlying theme of this review is that the three problems have a common cause: separation of human pluripotent stem cells from one another increases cellular stress,which greatly decreases their viability unless special steps are taken. View Publication

The generation of induced pluripotent stem cells (iPSCs) often results in aberrant epigenetic silencing of the imprinted Dlk1-Dio3 gene cluster,compromising the ability to generate entirely iPSC-derived adult mice ('all-iPSC mice'). Here,we show that reprogramming in the presence of ascorbic acid attenuates hypermethylation of Dlk1-Dio3 by enabling a chromatin configuration that interferes with binding of the de novo DNA methyltransferase Dnmt3a. This approach allowed us to generate all-iPSC mice from mature B cells,which have until now failed to support the development of exclusively iPSC-derived postnatal animals. Our data show that transcription factor-mediated reprogramming can endow a defined,terminally differentiated cell type with a developmental potential equivalent to that of embryonic stem cells. More generally,these findings indicate that culture conditions during cellular reprogramming can strongly influence the epigenetic and biological properties of the resultant iPSCs. View Publication

Notoginsenoside R1 (NGR1) is the main monomeric component extracted from the dried roots and rhizomes of Panax notoginseng,and exerts pharmacological action against myocardial infarction (MI). Owing to the differences in compound distribution,absorption,and metabolism in vivo,exploring a more effective drug delivery system with a high therapeutic targeting effect is crucial. In the early stages of MI,CD11b-expressing monocytes and neutrophils accumulate at infarct sites. Thus,we designed a mesoporous silica nanoparticle-conjugated CD11b antibody with loaded NGR1 (MSN-NGR1-CD11b antibody),which allowed NGR1 precise targeted delivery to the heart in a noninvasively manner. By increasing targeting to the injured myocardium,intravenous injection of MSN-NGR1-CD11b antibody nanoparticle in MI mice improved cardiac function and angiogenesis,reduced cell apoptosis,and regulate macrophage phenotype and inflammatory factors and chemokines. In order to further explore the mechanism of NGR1 protecting myocardium,cell oxidative stress model and oxygen-glucose deprivation (OGD) model were established. NGR1 protected H9C2 cells and primary cardiomyocytes against oxidative injury induced by H2O2 and OGD treatment. Further network pharmacology and molecular docking analyses suggested that the AKT,MAPK and Hippo signaling pathways were involved in the regulation of NGR1 in myocardial protection. Indeed,NGR1 could elevate the levels of p-Akt and p-ERK,and promote the nuclear translocation of YAP. Furthermore,LY294002 (AKT inhibitor),U0126 (ERK1/2 inhibitor) and Verteporfin (YAP inhibitor) administration in H9C2 cells indicated the involvement of AKT,MAPK and Hippo signaling pathways in NGR1 effects. Meanwhile,MSN-NGR1-CD11b antibody nanoparticles enhanced the activation of AKT and MAPK signaling pathways and the nuclear translocation of YAP at the infarcted site. Our research demonstrated that MSN-NGR1-CD11b antibody nanoparticle injection after MI enhanced the targeting of NGR1 to the infarcted myocardium and improved cardiac function. More importantly,our pioneering research provides a new strategy for targeting drug delivery systems to the ischemic niche. View Publication

Background and aimsMortality rates for hepatocellular carcinoma (HCC) remain high,while multimodal treatment approaches offer new perspectives. Here,we investigated the association of extracellular nicotinamide adenine dinucleotide (eNAD+) on ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (CD203a,ENPP1 or PC-1) on Th17 cells in relation to the likelihood of HCC recurrence following liver resection.MethodThe study compared heparinized blood plasma samples from 95 patients who underwent liver resection,including 25 patients with HCC and 24 control patients without liver disease. Plasma eNAD+ concentrations were determined using a heat-based dichotomous pH extraction method,followed by enzymatic cycling and a colorimetric assay for quantification. Fibrosis was graded histologically using the Desmet score (F0–F4). Surface expression analysis was performed using flow cytometry.ResultsWith increasing grades of liver fibrosis predominant in HCC patients,a significant reduction in plasma eNAD+ concentrations was measured (p < 0.05). Further,a significant correlation was found between HCC patients and CD203a expression on CD4+,CCR4+ as well as CCR6+ T cells (p < 0.05). Patients who exhibited high proportions of CD203a expressing Th17 cells (CD4+,CCR6+ CCR4+) post surgery were found to be at a sixfold increased risk (HR 6.38,95% Cl 1.51–27.00) of HCC recurrence and had a median recurrence-free survival of 233 days (p < 0.05),compared to patients with low CD203a expressing Th17 cells (CD4+ CCR6+ CCR4+). Similarly,patients who had a high proportion of CD203a expressing Th17 cells (CD4+ CCR6+) following surgery had a fivefold increased risk (HR 5.56,95% Cl 1.58–19.59) of HCC recurrence and a median recurrence-free survival of 334 days (p < 0.05) compared to those with low CD203a expressing Th17 cells (CCR6+).ConclusionThe data indicates that eNAD+ levels are decreased in patients with liver fibrosis or cirrhosis. Strikingly,patients with high CD203a expression on Th17 cells had a significantly increased likelihood of recurrence,highlighting its potential as a valuable prognostic marker and a possible therapeutic target.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00432-025-06155-4. View Publication

Circulating tumour cell (CTC) enumeration and profiling has been established as a valuable clinical tool in many solid malignancies. A key challenge in CTC research is the limited number of cells available for study. Ex vivo CTC culture permits expansion of these rare cell populations for detailed characterisation,functional assays including drug sensitivity testing,and investigation of the pathobiology of metastases. We report for the first time the establishment and characterisation of two continuous CTC lines from patients with gastroesophageal cancer. The two cell lines (designated UWG01CTC and UWG02CTC) demonstrated rapid tumorigenic growth in immunodeficient mice and exhibit distinct genotypic and phenotypic profiles which are consistent with the tumours of origin. UWG02CTC exhibits an EpCAM+,cytokeratin+,CD44+ phenotype,while UWG01CTC,which was derived from a patient with metastatic neuroendocrine cancer,displays an EpCAM-,weak cytokeratin phenotype,with strong expression of neuroendocrine markers. Further,the two cell lines show distinct differences in drug and radiation sensitivity which match differential cancer-associated gene expression pathways. This is strong evidence implicating EpCAM negative CTCs in metastasis. These novel,well characterised,long-term CTC cell lines from gastroesophageal cancer will facilitate ongoing research into metastasis and the discovery of therapeutic targets. View Publication

Some cases of pre-B cell acute lymphoblastic leukemia (pre-B-ALL) are caused by the Philadelphia (Ph) chromosome-encoded BCR-ABL oncogene,and these tend to have a poor prognosis. Inhibitors of the PI3K/AKT pathway reduce BCR-ABL-mediated transformation in vitro; however,the specific PI3K isoforms involved are poorly defined. Using a murine model of Ph+ pre-B-ALL,we found that deletion of both Pik3r1 and Pik3r2,genes encoding class IA PI3K regulatory isoforms,severely impaired transformation. BCR-ABL-dependent pre/pro-B cell lines could be established at low frequency from progenitors that lacked these genes,but the cells were smaller,proliferated more slowly,and failed to cause leukemia in vivo. These cell lines displayed nearly undetectable PI3K signaling function and were resistant to the PI3K inhibitor wortmannin. However,they maintained activation of mammalian target of rapamycin (mTOR) and were more sensitive to rapamycin. Treatment with rapamycin caused feedback activation of AKT in WT cell lines but not PI3K-deficient lines. A dual inhibitor of PI3K and mTOR,PI-103,was more effective than rapamycin at suppressing proliferation of mouse pre-B-ALL and human CD19+CD34+)Ph+ ALL leukemia cells treated with the ABL kinase inhibitor imatinib. Our findings provide mechanistic insights into PI3K dependency in oncogenic networks and provide a rationale for targeting class IA PI3K,alone or together with mTOR,in the treatment of Ph+ ALL. 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

Little is known about how neural stem cells are formed initially during development. We investigated whether a default mechanism of neural specification could regulate acquisition of neural stem cell identity directly from embryonic stem (ES) cells. ES cells cultured in defined,low-density conditions readily acquire a neural identity. We characterize a novel primitive neural stem cell as a component of neural lineage specification that is negatively regulated by TGFbeta-related signaling. Primitive neural stem cells have distinct growth factor requirements,express neural precursor markers,generate neurons and glia in vitro,and have neural and non-neural lineage potential in vivo. These results are consistent with a default mechanism for neural fate specification and support a model whereby definitive neural stem cell formation is preceded by a primitive neural stem cell stage during neural lineage commitment. View Publication

Efforts to improve the efficacy of adoptive T-cell therapies and immune checkpoint therapies in myelogenous leukemia are desired. In this study,we evaluated the antileukemia activity of adoptively transferred polyclonal cancer antigen-reactive T cells deficient in the regulator diacylglycerol kinase zeta (DGKζ) with or without PD-1/PD-L1 blockade. In the C1498 mouse model of myeloid leukemia,we showed that leukemia was eradicated more effectively in DGKζ-deficient (DGKζ-/-) mice than wild-type mice. T cells transferred from DGKζ-deficient mice to wild-type tumor-bearing recipients conferred this benefit. Leukemia clearance was similar to mice treated with anti-PD-L1. Strikingly,we found that the activity of adoptively transferred DGKζ-/- T cells relied partly on induction of sustainable host T-cell immunity. Transferring DGKζ-deficient T cells increased the levels of IFNγ and other cytokines in recipient mice,especially with coadministration of anti-PD-L1. Overall,our results offered evidence that targeting DGKζ may leverage the efficacy of adoptive T-cell and immune checkpoint therapies in leukemia treatment. Furthermore,they suggest that DGKζ targeting might decrease risks of antigen escape or resistance to immune checkpoint blockade. Cancer Res; 77(20); 5676-86. textcopyright2017 AACR. View Publication

FOXM1 is an important cell cycle regulator and regulates cell proliferation. In addition,FOXM1 has been reported to contribute to oncogenesis in various cancers. However,it is not clearly understood how FOXM1 contributes to acute myeloid leukemia (AML) cell proliferation. In this study,we investigated the cellular and molecular function of FOXM1 in AML cells. The FOXM1 messenger RNA (mRNA) expressed in AML cell lines was predominantly the FOXM1B isoform,and its levels were significantly higher than in normal high aldehyde dehydrogenase activity (ALDH(hi)) cells. Reduction of FOXM1 expression in AML cells inhibited cell proliferation compared with control cells,through induction of G(2)/M cell cycle arrest,a decrease in the protein expression of Aurora kinase B,Survivin,Cyclin B1,S-phase kinase-associated protein 2 and Cdc25B and an increase in the protein expression of p21(Cip1) and p27(Kip1). FOXM1 messenger RNA (mRNA) was overexpressed in all 127 AML clinical specimens tested (n = 21,56,32 and 18 for M1,M2,M4 and M5 subtypes,respectively). Compared with normal ALDH(hi) cells,FOXM1 gene expression was 1.65- to 2.26-fold higher in AML cells. Moreover,the FOXM1 protein was more strongly expressed in AML-derived ALDH(hi) cells compared with normal ALDH(hi) cells. In addition,depletion of FOXM1 reduced colony formation of AML-derived ALDH(hi) cells due to inhibition of Cdc25B and Cyclin B1 expression. In summary,we found that FOXM1B mRNA is predominantly expressed in AML cells and that aberrant expression of FOXM1 induces AML cell proliferation through modulation of cell cycle progression. Thus,inhibition of FOXM1 expression represents an attractive target for AML therapy. View Publication

Pluripotent stem cells hold great promise for regenerative medicine,due to their unlimited self-renewal potential and the ability to differentiate into all somatic cell types. Differences between the rodent disease models and the situation in humans can be narrowed down with non-human primate models. The common marmoset monkey (Callithrix jacchus) is an interesting model for biomedical research because these animals are easy to breed,get relatively old (≤ 13 years),are small in size,are relatively cost-effective and have a high genetic proximity to the human. In particular,diseases of the liver and pancreas are interesting for cell replacement therapies but the in vitro differentiation of ESCs into the definitive endoderm germ layer is still a demanding task. Membrane-permeable,chemically defined small molecules can possibly replace recombinant growth factors used in most directed differentiation protocols. However,the potent small molecules IDE-1 and IDE-2 were not able to induce definitive endoderm-like cells when ESCs from the common marmoset were treated with these compounds,whereas the recombinant growth factor Activin A could force the differentiation into this lineage. Our results indicate that ESCs from the common marmoset are less sensitive or even insensitive to these small molecules. Thus,differences between the species of human ESCs and ESCs of this non-human primate might be a useful model to further evaluate the exact mode of action of these compounds. View Publication