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

Here we demonstrate,both in vivo and in vitro,that growth hormone (GH) mediates precursor cell activation in the subventricular zone (SVZ) of the aged (12-month-old) brain following exercise,and that GH signaling stimulates precursor activation to a similar extent to exercise. Our results reveal that both addition of GH in culture and direct intracerebroventricular infusion of GH stimulate neural precursor cells in the aged brain. In contrast,no increase in neurosphere numbers was observed in GH receptor null animals following exercise. Continuous infusion of a GH antagonist into the lateral ventricle of wild-type animals completely abolished the exercise-induced increase in neural precursor cell number. Given that the aged brain does not recover well after injury,we investigated the direct effect of exercise and GH on neural precursor cell activation following irradiation. This revealed that physical exercise as well as infusion of GH promoted repopulation of neural precursor cells in irradiated aged animals. Conversely,infusion of a GH antagonist during exercise prevented recovery of precursor cells in the SVZ following irradiation. View Publication

Many long noncoding RNA (lncRNA) species have been identified in mammalian cells,but the genomic origin and regulation of these molecules in individual cell types is poorly understood. We have generated catalogs of lncRNA species expressed in human and murine embryonic stem cells and mapped their genomic origin. A surprisingly large fraction of these transcripts (textgreater60%) originate from divergent transcription at promoters of active protein-coding genes. The divergently transcribed lncRNA/mRNA gene pairs exhibit coordinated changes in transcription when embryonic stem cells are differentiated into endoderm. Our results reveal that transcription of most lncRNA genes is coordinated with transcription of protein-coding genes. View Publication

BACKGROUND: Airway remodeling might explain lung function decline among asthmatic children. Extracellular matrix (ECM) deposition by human lung fibroblasts (HLFs) is implicated in airway remodeling. Airway epithelial cell (AEC) signaling might regulate HLF ECM expression. OBJECTIVES: We sought to determine whether AECs from asthmatic children differentially regulate HLF expression of ECM constituents. METHODS: Primary AECs were obtained from well-characterized atopic asthmatic (n = 10) and healthy (n = 10) children intubated during anesthesia for an elective surgical procedure. AECs were differentiated at an air-liquid interface for 3 weeks and then cocultured with HLFs from a healthy child for 96 hours. Collagen I (COL1A1),collagen III (COL3A1),hyaluronan synthase (HAS) 2,and fibronectin expression by HLFs and prostaglandin E2 synthase (PGE2S) expression by AECs were assessed by using RT-PCR. TGF-$$1 and TGF-$$2 concentrations in media were measured by using ELISA. RESULTS: COL1A1 and COL3A1 expression by HLFs cocultured with AECs from asthmatic patients was greater than that by HLFs cocultured with AECs from healthy subjects (2.2-fold,P textless .02; 10.8-fold,P textless .02). HAS2 expression by HLFs cocultured with AECs from asthmatic patients was 2.5-fold higher than that by HLFs cocultured with AECs from healthy subjects (P textless .002). Fibronectin expression by HLFs cocultured with AECs from asthmatic patients was significantly greater than that by HLFs alone. TGF-$$2 activity was increased in cocultures of HLFs with AECs from asthmatic patients (P textless .05),whereas PGES2 was downregulated in AEC-HLF cocultures (2.2-fold,P textless .006). CONCLUSIONS: HLFs cocultured with AECs from asthmatic patients showed differential expression of the ECM constituents COL1A1 and COL3A1 and HAS2 compared with HLFs cocultured with AECs from healthy subjects. These findings support a role for altered ECM production in asthmatic airway remodeling,possibly regulated by unbalanced AEC signaling. View Publication

The microbes in the gastrointestinal tract are separated from the host by a single layer of intestinal epithelial cells (IECs) that plays pivotal roles in maintaining homeostasis by absorbing nutrients and providing a physical and immunological barrier to potential pathogens. Preservation of homeostasis requires the crosstalk between the epithelium and the microbial environment. One epithelial-driven innate immune mechanism that participates in host-microbe communication involves the release of reactive oxygen species (ROS),such as hydrogen peroxide (H2O2),toward the lumen. Phagocytes produce high amounts of ROS which is critical for microbicidal functions; the functional contribution of epithelial ROS,however,has been hindered by the lack of methodologies to reliably quantify extracellular release of ROS. Here,we used a modified Amplex Red assay to investigate the inflammatory and microbial regulation of IEC-generated H2O2 and the potential role of Duox2,a NADPH oxidase that is an important source of H2O2. We found that colonoids respond to interferon-$\gamma$ and flagellin by enhancing production of H2O2 in a Duox2-mediated fashion. To extend these findings,we analyzed ex vivo production of H2O2 by IECs after acute and chronic inflammation,as well as after exposure to dysbiotic microbiota. While acute inflammation did not induce a significant increase in epithelial-driven H2O2,chronic inflammation caused IECs to release higher levels of H2O2. Furthermore,colonization of germ-free mice with dysbiotic microbiota from mice or patients with IBD resulted in increased H2O2 production compared with healthy controls. Collectively,these data suggest that IECs are capable of H2O2 production during chronic inflammation and dysbiotic states. Our results provide insight into luminal production of H2O2 by IECs as a read-out of innate defense by the mucosa. View Publication

Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant neurodegenerative disease caused by repeat expansion of the CAG trinucleotide within exon 10 of the ATXN3 gene. This mutation results in the production of an abnormal ataxin-3 protein containing an extended polyglutamine tract,referred to as mutant ataxin-3. In this study,we investigated the therapeutic potential of CRISPR/Cas9-mediated genome editing for SCA3. First,we designed a specific single-guide RNA targeting the ATXN3 gene and constructed the corresponding targeting vector. Induced pluripotent stem cells (iPSCs) derived from a SCA3 patient were then electroporated with the CRISPR/Cas9 components. Positive clones were screened and validated by PCR and Sanger sequencing to obtain genome-editing iPSCs (GE-iPSCs). Subsequently,the pluripotency of GE-iPSCs was confirmed,and the effects of genome editing on mutant ataxin-3 protein expression and Golgi apparatus morphology were assessed using Western blotting and immunofluorescence analyses. Our results demonstrated that targeted insertion of polyadenylation signals (PAS) upstream of the abnormal CAG repeats effectively suppressed the production of mutant ataxin-3. This intervention also reduced the formation of neuronal nuclear inclusions in differentiated neurons,restored the structural integrity of the Golgi apparatus (which exhibited a loose and enlarged morphology in SCA3 cells),and increased the expression levels of Golgi structural proteins (GM130 and GORASP2). In conclusion,our findings indicate that the targeted insertion of PAS upstream of the abnormal CAG repeats in the ATXN3 gene represents a promising therapeutic strategy for SCA3 through genome editing.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-93369-8. View Publication

SummaryMotivated by the cellular heterogeneity in complex tissues,particularly in brain and induced pluripotent stem cell (iPSC)-derived brain models,we developed a complete workflow to reproducibly characterize cell types in complex tissues. Our approach combines a flow cytometry (FC) antibody panel with our computational pipeline CelltypeR,enabling dataset aligning,unsupervised clustering optimization,cell type annotating,and statistical comparisons. Applied to human iPSC derived midbrain organoids,it successfully identified the major brain cell types. We performed fluorescence-activated cell sorting of CelltypeR-defined astrocytes,radial glia,and neurons,exploring transcriptional states by single-cell RNA sequencing. Among the sorted neurons,we identified subgroups of dopamine neurons: one reminiscent of substantia nigra cells most vulnerable in Parkinson’s disease. Finally,we used our workflow to track cell types across a time course of organoid differentiation. Overall,our adaptable analysis framework provides a generalizable method for reproducibly identifying cell types across FC datasets in complex tissues. Graphical abstract Highlights•CelltypeR is a flow cytometry and computational pipeline for cell type quantification•Identified brain cell types in midbrain organoids and measured changes in proportions•Enriched selected populations using FACS and characterized by single-cell RNA sequencing•Identified substantia nigra–like dopaminergic neurons sensitive in Parkinson’s disease Neuroscience; Cell biology; Omics View Publication

AbstractObjectiveUnique metabolic requirements accompany the development and functional fates of immune cells. How cellular metabolism is important in natural killer (NK) cells and their memory‐like differentiation in bacterial infections remains elusive.MethodsHere,we utilise our established NK cell memory assay to investigate the metabolic requirement for memory‐like NK cell formation and function in response to the Gram‐negative intracellular bacteria Burkholderia pseudomallei (BP),the causative agent of melioidosis.ResultsWe demonstrate that CD160+ memory‐like NK cells upon BP stimulation upregulate glucose and amino acid transporters in a cohort of recovered melioidosis patients which is maintained at least 3‐month post‐hospital admission. Using an in vitro assay,human BP‐specific CD160+ memory‐like NK cells show metabolic priming including increased expression of glucose and amino acid transporters with elevated glucose uptake,increased mTOR activation and mitochondrial membrane potential upon BP re‐stimulation. Antigen‐specific and cytokine‐induced IFN‐γ production of this memory‐like NK cell subset are highly dependent on oxidative phosphorylation (OXPHOS) with some dependency on glycolysis,whereas the formation of CD160+ memory‐like NK cells in vitro is dependent on fatty acid oxidation and OXPHOS and further increased by metformin.ConclusionThis study reveals the link between metabolism and cellular function of memory‐like NK cells,which can be exploited for vaccine design and for monitoring protection against Gram‐negative bacterial infection. This study reveals the link between metabolism and cellular function of memory‐like NK cells in melioidosis. We demonstrate that CD160+ memory‐like NK cells upon Burkholderia pseudomallei (BP) stimulation upregulate glucose and amino acid transporters in a cohort of recovered melioidosis patients. Using an in vitro assay,human BP‐specific CD160+ memory‐like NK cells show metabolic priming including increased expression of glucose and amino acid transporters with elevated glucose uptake,increased mTOR activation and mitochondrial membrane potential upon BP re‐stimulation. View Publication

In humans,recent clinical and experimental data from hematopoietic stem cell transplantation revealed that donor-derived alloreactive NK cells exert a beneficial graft versus leukemia effect. The existence of donor-derived alloreactive NK cells can be predicted on the basis of donor killer cell Ig-like receptor (KIR) gene profile and HLA class I typing of both donor and recipient. Moreover,the size of the alloreactive NK cell population can be directly assessed by the combined use of anti-KIR-specific mAb. In this study,in an attempt to improve the definition of alloreactive NK cell subsets,we assessed the KIR genotype and phenotype in a cohort of 44 donors. This approach allowed the identification of two different KIR2DL3 alleles (KIR2DL3*005 and the novel allele KIR2DL3*015) that did not react with the anti-KIR2DL3-specific ECM41 mAb. In contrast,both alleles were recognized at the cell surface by several mAb reacting with KIR2DL2/L3/S2. Notably,KIR2DL3*005 was also stained by the anti-KIR2DL1/S1-specific EB6B and 11PB6 mAb. Functional analysis revealed that,despite its particular mAb reactivity,the specificity of KIR2DL3*005 for HLA-C molecules did not differ from that of other KIR2DL2/L3 alleles. Finally,site-directed mutagenesis demonstrated that glutamine at position 35 is required for ECM41 staining,whereas glutamic acid 35 and arginine 50 are relevant for staining with EB6B or 11PB6 mAb. Our present data represent a substantial progress in the characterization of the NK cell repertoire and an improved phenotypic/functional definition of given KIR(+) subsets. View Publication

We hypothesized that the neonatal rat heart would bring transplanted human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) to maturity as it grows to adult size. In neonatal rat heart,engrafted hiPSC derivatives developed partially matured myofibrils after 3 months,with increasing cell size and sarcomere length. There was no difference between grafts from hiPSC-CMs or hiPSC-derived cardiac progenitors (hiPSC-CPs) at 3 months,nor was maturation influenced by infarction. Interestingly,the infarcted adult heart induced greater human cardiomyocyte hypertrophy and induction of cardiac troponin I expression than the neonatal heart. Although human cardiomyocytes at all time points were significantly smaller than the host rat cardiomyocytes,transplanted neonatal rat cardiomyocytes reached adult size and structure by 3 months. Thus,the adult rat heart induces faster maturation than the neonatal heart,and human cardiomyocytes mature more slowly than rat cardiomyocytes. The slower maturation of human cardiomyocytes could be related to environmental mismatch or cell-autonomous factors. View Publication

BACKGROUND AND OBJECTIVES: Interactions between acute myelogenous leukemia (AML) blasts and non-leukemic cells in the bone marrow seem to be important for both disease development and susceptibility to chemotherapy. Recent studies have focused on the endothelial cells,but other non-leukemic cells may also be involved. In the present study we investigated how osteoblasts affect native human AML blasts. DESIGN AND METHODS: AML cells were derived from a large group of consecutive patients. The AML blasts and osteoblastic sarcoma cell lines (Cal72,SJSA-1) were incubated together in different chambers separated by a semipermeable membrane. We investigated effects of co-culture on proliferation,apoptosis and cytokine release. RESULTS: The cross-talk between these two cell populations,achieved via release of soluble mediators,resulted in increased AML blast proliferation,including increased proliferation of clonogenic progenitors,but did not affect spontaneous in vitro apoptosis. Both interleukin (IL) 1-b and granulocyte-macrophage colony-stimulating factor were involved in this growth-enhancing cross-talk,and normal osteoblasts could also increase the AML blast proliferation. Furthermore,co-culture of AML blasts with osteoblastic sarcoma cells as well as normal osteoblasts increased the levels of the pro-angiogenic mediator IL8. INTERPRETATION AND CONCLUSIONS: Our in vitro results suggest that the release of soluble mediators by osteoblasts supports leukemic hematopoiesis through two major mechanisms: (i) direct enhancement of AML blast proliferation; and (ii) enhanced angiogenesis caused by increased IL8 levels. View Publication

Neutrophil apoptosis is essential for the resolution of inflammation but is delayed by several inflammatory mediators. In such terminally differentiated cells it has been uncertain whether these agents can inhibit apoptosis through transcriptional regulation of anti-death (Bcl-X(L),Mcl-1,Bcl2A1) or BH3-only (Bim,Bid,Puma) Bcl2-family proteins. We report that granulocyte/macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor (TNF)-α prevent the normal time-dependent loss of Mcl-1 and Bcl2A1 in neutrophils,and we demonstrate that they cause an NF-κB-dependent increase in Bcl-X(L) transcription/translation. We show that GM-CSF and TNF-α increase and/or maintain mRNA levels for the pro-apoptotic BH3-only protein Bid and that GM-CSF has a similar NF-κB-dependent effect on Bim transcription and BimEL expression. The in-vivo relevance of these findings was indicated by demonstrating that GM-CSF is the dominant neutrophil survival factor in lung lavage from patients with ventilator-associated pneumonia,confirming an increase in lung neutrophil Bim mRNA. Finally GM-CSF caused mitochondrial location of Bim and a switch in phenotype to a cell that displays accelerated caspase-9-dependent apoptosis. This study demonstrates the capacity of neutrophil survival agents to induce a paradoxical increase in the pro-apoptotic proteins Bid and Bim and suggests that this may function to facilitate rapid apoptosis at the termination of the inflammatory cycle. View Publication