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

This study presents a comprehensive transcriptomic analysis of feeder-free extended pluripotent stem cells (ffEPSCs) and their parental human embryonic stem cells (ESCs),providing new insights into understanding human early development and cellular heterogeneity of pluripotency. Leveraging Smart-seq2-based single-cell RNA sequencing (scRNA-seq),we have compared gene expression profiles between ESCs and ffEPSCs and uncovered distinct subpopulations within both groups. Through pseudotime analysis,we have mapped the transition process from ESCs to ffEPSCs,revealing critical molecular pathways involved in the shift from a primed pluripotency to an extended pluripotent state. Additionally,we have employed repeat sequence analysis based on the latest T2T database and identified the stage-specific repeat elements contributing to regulating pluripotency and developmental transitions. This dataset deepens our understanding on early pluripotency and highlights the role of repeat sequences in early embryonic development. Our findings thus offer valuable resources for researchers in stem cell biology,pluripotency,early embryonic development,and potential cell therapy and regenerative medical applications. View Publication

The differentiation and specificity of human CD4+ T follicular helper cells (TFH cells) after influenza vaccination have been poorly defined. Here we profiled blood and draining lymph node (LN) samples from human volunteers for over 2 years after two influenza vaccines were administered 1 year apart to define the evolution of the CD4+ TFH cell response. The first vaccination induced an increase in the frequency of circulating TFH (cTFH) and LN TFH cells at week 1 postvaccination. This increase was transient for cTFH cells,whereas the LN TFH cells further expanded during week 2 and remained elevated in frequency for at least 3 months. We observed several distinct subsets of TFH cells in the LN,including pre-TFH cells,memory TFH cells,germinal center (GC) TFH cells and interleukin-10+ TFH cell subsets beginning at baseline and at all time points postvaccination. The shift toward a GC TFH cell phenotype occurred with faster kinetics after the second vaccine compared to the first vaccine. We identified several influenza-specific TFH cell clonal lineages,including multiple responses targeting internal influenza virus proteins,and found that each TFH cell state was attainable within a clonal lineage. Thus,human TFH cells form a durable and dynamic multitissue network. Schattgen et al. profiled the subsets and clonality of CD4+ TFH cells in the blood and lymph nodes of human volunteers who received two influenza vaccines 1 year apart to characterize their dynamics and clonal evolution over 2 years. View Publication

During kidney development,nephron epithelia arise de novo from fate-committed mesenchymal progenitors through a mesenchymal-to-epithelial transition (MET). Downstream of fate specification,transcriptional mechanisms that drive establishment of epithelial morphology are poorly understood. We used human iPSC-derived renal organoids,which recapitulate nephrogenesis,to investigate mechanisms controlling renal MET. Multi-ome profiling via snRNA-seq and ATAC-seq of organoids identified dynamic changes in gene expression and chromatin accessibility driven by activators and repressors throughout MET. CRISPR interference identified that paired box 8 (PAX8) is essential for initiation of MET in human renal organoids,contrary to in vivo mouse studies,likely by activating a cell-adhesion program. While Wnt/β-catenin signaling specifies nephron fate,we find that it must be attenuated to allow hepatocyte nuclear factor 1-beta (HNF1B) and TEA-domain (TEAD) transcription factors to drive completion of MET. These results identify the interplay between fate commitment and morphogenesis in the developing human kidney,with implications for understanding both developmental kidney diseases and aberrant epithelial plasticity following adult renal tubular injury. View Publication

In recent years,Raman spectroscopy has garnered growing interest in the field of biomedical research. It offers a non-invasive and label-free approach to defining the molecular fingerprint of immune cells. We utilized Raman spectroscopy on optically trapped immune cells to investigate their molecular compositions. While numerous immune cell types have been studied in the past,the characterization of living human CD3/CD28-stimulated T cell subsets remains incomplete. In this study,we demonstrate the capability of Raman spectroscopy to readily distinguish between naïve and stimulated CD4 and CD8 cells. Additionally,we compared these cells with monocytes and discovered remarkable similarities between stimulated T cells and monocytes. This paper contributes to expanding our knowledge of Raman spectroscopy of immune cells and serves as a launching point for future clinical applications. View Publication

Maturation of human pluripotent stem (hPS) cell-derived cardiomyocytes is critical for their use as a model system. Here we mimic human heart maturation pathways in the setting of hPS cell-derived cardiac organoids (hCOs). Specifically,transient activation of 5′ AMP-activated protein kinase and estrogen-related receptor enhanced cardiomyocyte maturation,inducing expression of mature sarcomeric and oxidative phosphorylation proteins,and increasing metabolic capacity. hCOs generated using the directed maturation protocol (DM-hCOs) recapitulate cardiac drug responses and,when derived from calsequestrin 2 ( CASQ2 ) and ryanodine receptor 2 ( RYR2 ) mutant hPS cells exhibit a pro-arrhythmia phenotype. These DM-hCOs also comprise multiple cell types,which we characterize and benchmark to the human heart. Modeling of cardiomyopathy caused by a desmoplakin ( DSP ) mutation resulted in fibrosis and cardiac dysfunction and led to identifying the bromodomain and extra-terminal inhibitor INCB054329 as a drug mitigating the desmoplakin-related functional defect. These findings establish DM-hCOs as a versatile platform for applications in cardiac biology,disease and drug screening. Subject terms: Tissue engineering,Differentiation,Cardiomyopathies View Publication

CD24 expression on pro-B cells plays a role in B cell selection and development in the bone marrow. We previously detected higher CD24 expression and frequency within IgD+ na{\{i}}ve and memory B cells in patients with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) compared with age-matched healthy controls (HC). Here we investigated the relationship between CD24 expression and B cell maturation. In vitro stimulation of isolated B cells in response to conventional agonists were used to follow the dynamics of CD24 positivity during proliferation and differentiation (or maturation). The relationship between CD24 expression to cycles of proliferation and metabolism in purified B cells from HC was also investigated using phospho-flow (phosphorylation of AMPK-pAMPK) 1proton nuclear magnetic resonance and Mitotracker Far-red (Mitochondrial mass-MM). In vitro in the absence of stimulation there was an increased percentage of CD24+ viable B cells in ME/CFS patients compared to HC (p {\textless} 0.05) following 5 days culture. Following stimulation with B cell agonists percentage of CD24+B cells in both na{\"{i}}ve and memory B cell populations decreased. P {\textless} 0.01). There was a negative relationship between percentage of CD24+B cells with MM (R2 = 0.76; p {\textless} 0.01) which was subsequently lost over sequential cycles of proliferation. There was a significant correlation between CD24 expression on B cells and the usage of glucose and secretion of lactate in vitro. Short term ligation of the B cell receptor with anti-IgM antibody significantly reduced the viability of CD24+ memory B cells compared to those cross-linked by anti-IgD or anti-IgG antibody. A clear difference was found between na{\""{i}}ve and memory B cells with respect to CD24 expression and pAMPK most notably a strong positive association in IgD+IgM+ memory B cells. In vitro findings confirmed dysregulation of CD24-expressing B cells from ME/CFS patients previously suggested by immunophenotype studies of B cells from peripheral blood. CD24-negative B cells underwent productive proliferation whereas CD24+ B cells were either unresponsive or susceptible to cell death upon BCR-engagement alone. We suggest that CD24 expression may reflect variations in energy metabolism on different B cell subsets.""" View Publication

Previous studies from this laboratory have demonstrated that fibroblast growth factor 1 together with a number of co-activator molecules (dopamine,TPA,IBMX/forskolin),will induce the expression of the catecholamine biosynthetic enzyme tyrosine hydroxylase (TH) in 10% of human neurons (hNTs) derived from the NT2 cell line [10]. In the present study,we found that TH induction was increased to nearly 75% in hNTs when cells were permitted to age 2 weeks in culture prior to treatment with the differentiation cocktail. This high level of TH expression was sustained 7 days after removal of the differentiating agents from the media. Moreover,the induced TH present in these cells was enzymatically active,resulting in the production of low levels of dopamine (DA) and its metabolite DOPAC. These findings suggest that hNTs may provide an important tissue culture model for the study of factors regulating TH gene expression in human neurons. Moreover,hNTs may serve,in vivo,as a source of human DA neurons for use in transplantation therapies. View Publication

The human blood group i and I antigens are determined by linear and branched poly-N-acetyllactosamine structures,respectively. In erythrocytes,the fetal i antigen is converted to the adult I antigen by I-branching beta-1,6-N-acetylglucosaminyltransferase (IGnT) during development. Dysfunction of the I-branching enzyme may result in the adult i phenotype in erythrocytes. However,the I gene responsible for blood group I antigen has not been fully confirmed. We report here a novel human I-branching enzyme,designated IGnT3. The genes for IGnT1 (reported in 1993),IGnT2 (also presented in this study),and IGnT3 consist of 3 exons and share the second and third exons. Bone marrow cells preferentially expressed IGnT3 transcript. During erythroid differentiation using CD34(+) cells,IGnT3 was markedly up-regulated with concomitant decrease in IGnT1/2. Moreover,reticulocytes expressed the IGnT3 transcript,but IGnT1/2 was below detectable levels. By molecular genetic analyses of an adult i pedigree,individuals with the adult i phenotype were revealed to have heterozygous alleles with mutations in exon 2 (1006GtextgreaterA; Gly336Arg) and exon 3 (1049GtextgreaterA; Gly350Glu),respectively,of the IGnT3 gene. Chinese hamster ovary (CHO) cells transfected with each mutated IGnT3 cDNA failed to express I antigen. These findings indicate that the expression of the blood group I antigen in erythrocytes is determined by a novel IGnT3,not by IGnT1 or IGnT2. View Publication

BACKGROUND Haemolytic infection lyses red blood cells,releasing haemoglobin (Hb) into the plasma. Although recent studies showed that immune cells recognize redox-active cytotoxic extracellular Hb (metHb) bound to pathogen-associated molecular patterns (PAMPs),currently available information is limited to experiments performed in defined conditions using single cell lines. Therefore,a systemic approach targeting primary whole blood cells is required to better understand the cellular immune defence against metHb and PAMPs,when under a haemolytic infection. METHODS We investigated how human white blood cells,including neutrophils,respond to metHb and lipoteichoic acid (LTA) by measuring reactive oxygen species (ROS),signalling mediators (ERK and p38),NF-κB,cytokines,elastase secretion and cell activation markers. FINDINGS metHb activates NF-κB in TLR2-expressing HEK293 cells but not in normal or TLR9-expressing HEK293 cells. Treatment of isolated neutrophils with metHb increased production of ROS and expressions of IL-8,TNFα,and CD11b,which were further enhanced by metHb + LTA complex. While LTA stimulated the survival of neutrophils,it caused apoptotic cell death when complexed with metHb. The activation of neutrophils by metHb + LTA was subdued by the presence of other types of white blood cells. INTERPRETATION metHb and metHb + LTA complex are ligands of TLR2,inducing an unconventional TLR signalling pathway. Neutrophils are a highly sensitive cell type to metHb + LTA complex. During a haemolytic infection,white blood cells in the vicinity crosstalk to modulate neutrophil TLR-signalling induced by metHb and LTA. View Publication

Short bowel syndrome (SBS) is characterized by poor nutrient absorption due to a deficit of healthy intestine. Current treatment practices rely on providing supportive medical therapy with parenteral nutrition; while life saving,such interventions are not curative and are still associated with significant co-morbidities. As approaches to lengthen remaining intestinal tissue have been met with only limited success and intestinal transplants have poor survival outcomes,new approaches to treating SBS are necessary. Human intestine derived from embryonic stem cells (hESCs) or induced pluripotent stem cells (iPSCs),called human intestinal organoids (HIOs),have the potential to offer a personalized and scalable source of intestine for regenerative therapies. However,given that HIOs are small three-dimensional structures grown in vitro,methods to generate usable HIO-derived constructs are needed. We investigated the ability of hESCs or HIOs to populate acellular porcine intestinal matrices and artificial polyglycolic/poly L lactic acid (PGA/PLLA) scaffolds,and examined the ability of matrix/scaffolds to thrive when transplanted in vivo. Our results demonstrate that the acellular matrix alone is not sufficient to instruct hESC differentiation towards an endodermal or intestinal fate. We observed that while HIOs reseed acellular porcine matrices in vitro,the HIO-reseeded matrices do not thrive when transplanted in vivo. In contrast,HIO-seeded PGA/PLLA scaffolds thrive in vivo and develop into tissue that looks nearly identical to adult human intestinal tissue. Our results suggest that HIO-seeded PGA/PLLA scaffolds are a promising avenue for developing the mucosal component of tissue engineered human small intestine,which need to be explored further to develop them into fully functional tissue. View Publication

Zika virus (ZIKV) is an emerging mosquito-transmitted flavivirus that can cause severe disease,including congenital birth defects during pregnancy(1). To develop candidate therapeutic agents against ZIKV,we isolated a panel of human monoclonal antibodies (mAbs) from subjects with prior ZIKV infection. A subset of mAbs recognized diverse epitopes on the envelope (E) protein and exhibited potently neutralizing activity. One of the most inhibitory mAbs,ZIKV-117,broadly neutralized infection of ZIKV strains corresponding to African,Asian,and American lineages. Epitope mapping studies revealed that ZIKV-117 recognized a unique quaternary epitope on the E protein dimer-dimer interface. We evaluated the therapeutic efficacy of ZIKV-117 in pregnant and non-pregnant mice. mAb treatment markedly reduced tissue pathology,placental and fetal infection,and mortality in mice. Thus,neutralizing human mAbs can protect against maternal-fetal transmission,infection and disease,and reveal important determinants for structure-based rational vaccine design efforts. View Publication