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

SUMMARY Circular RNAs (circRNAs) are stable RNAs present in cell-free RNA,which may comprise cellular debris and pathogen genomes. Here we investigate the phenomenon and mechanism of cellular uptake and intracellular fate of exogenous circRNAs. Human myeloid cells and B cells selectively internalize extracellular circRNAs. Macrophage uptake of circRNA is rapid,energy-dependent,and saturable. CircRNA uptake can lead to translation of encoded sequences and antigen presentation. The route of internalization influences immune activation after circRNA uptake,with distinct gene expression programs depending on the route of RNA delivery. Genome-scale CRISPR screens and chemical inhibitor studies nominate macrophage scavenger receptor MSR1,toll-like receptors,and mTOR signaling as key regulators of receptor-mediated phagocytosis of circRNAs,a dominant pathway to internalize circRNAs in parallel to macropinocytosis. These results suggest that cell-free circRNA serves as an “eat me” signal and danger-associated molecular pattern,indicating orderly pathways of recognition and disposal. eTOC Blurb: Amaya et. al. explores how cells take up extracellular circular RNAs (CircRNAs) and their impact on immune signaling. Macrophages readily internalize circRNAs,and this study identifies the specific receptors and signaling pathways governing circRNA internalization,highlighting their role as signaling molecules for immune recognition and disposal. Graphical Abstract View Publication

We asked whether the hypoxia-regulated factor,insulin-like growth factor binding protein-3 (IGFBP3),could modulate stem cell factor receptor (c-kit+),stem cell antigen-1 (sca-1+),hematopoietic stem cell (HSC),or CD34+ endothelial precursor cell (EPC) function. Exposure of CD34+ EPCs to IGFBP3 resulted in rapid differentiation into endothelial cells and dose-dependent increases in cell migration and capillary tube formation. IGFBP3-expressing plasmid was injected into the vitreous of neonatal mice undergoing the oxygen-induced retinopathy (OIR) model. In separate studies,GFP-expressing HSCs were transfected with IGFBP3 plasmid and injected into the vitreous of OIR mice. Administering either IGFBP3 plasmid alone or HSCs transfected with the plasmid resulted in a similar reduction in areas of vasoobliteration,protection of the developing vasculature from hyperoxia-induced regression,and reduction in preretinal neovascularization compared to control plasmid or HSCs transfected with control plasmid. In conclusion,IGFBP3 mediates EPC migration,differentiation,and capillary formation in vitro. Targeted expression of IGFBP3 protects the vasculature from damage and promotes proper vascular repair after hyperoxic insult in the OIR model. IGFBP3 expression may represent a physiological adaptation to ischemia and potentially a therapeutic target for treatment of ischemic conditions. View Publication

Cdx1,Cdx2,and Cdx4 comprise the caudal-like Cdx gene family in mammals,whose homologues regulate hematopoietic development in zebrafish. Previously,we reported that overexpression of Cdx4 enhances hematopoietic potential from murine embryonic stem cells (ESCs). Here we compare the effect of ectopic Cdx1,Cdx2,and Cdx4 on the differentiation of murine ESC-derived hematopoietic progenitors. The 3 Cdx genes differentially influence the formation and differentiation of hematopoietic progenitors within a CD41(+)c-kit(+) population of embryoid body (EB)-derived cells. Cdx1 and Cdx4 enhance,whereas Cdx2 strongly inhibits,the hematopoietic potential of CD41(+)ckit(+) EB-derived cells,changes that are reflected by effects on hematopoietic lineage-specific and Hox gene expression. When we subject stromal cell and colony assay cultures of EB-derived hematopoietic progenitors to ectopic expression of Cdx genes,Cdx4 dramatically enhances,whereas Cdx1 and Cdx2 both inhibit hematopoietic activity,probably by blocking progenitor differentiation. These data demonstrate distinct effects of Cdx genes on hematopoietic progenitor formation and differentiation,insights that we are using to facilitate efforts at in vitro culture of hematopoietic progenitors from ESC. The behavior of Cdx genes in vitro suggests how derangement of these developmental regulators might contribute to leukemogenesis. View Publication

Irradiation with X-rays has been widely utilized in the clinical treatment of solid tumors and certain hematopoietic malignancies. However,this method fails to completely distinguish between malignant and normal cells. Prolonged or repeated exposure to radiation,whether due to occupational hazards or therapeutical interventions,can cause damage to normal tissues,particularly impacting the hematopoietic system. Therefore,it is important to investigate the effects of total body irradiation on the hematopoietic system of mice and to compare the inhibitory effects of various doses of irradiation on this system. In this study,we primarily employed flow cytometry to analyze mature lineage cells in the peripheral blood,as well as immature hematopoietic stem and progenitor cells (HSPCs) in the bone marrow and spleen. Additionally,we evaluated the multilineage differentiation capacity of HSPCs through colony-forming cell assays. Our results indicated that peripheral B and T cells demonstrated increased sensitivity to irradiation,with significant cell death observed 1-day post-irradiation. Common lymphoid progenitor cells exhibited greater radiotolerance compared to other progenitor cell types,enabling them to maintain a certain population even at elevated doses. Moreover,notable differences were observed between intramedullary and extramedullary hematopoietic stem cells and common lymphoid progenitor cells regarding the extent of damage and recovery rate following irradiation. The multilineage differentiation capacity of HSPCs was also compromised during radiation exposure. In conclusion,different types of mature blood cells,along with immature HSPCs,exhibited varying degrees of sensitivity and tolerance to irradiation,resulting in distinct alterations in cell percentages and numbers. View Publication

Expansion of Human Hematopoietic Stem Cells (HSCs) is a rapidly advancing field showing great promise for clinical applications. Recent evidence has implicated the nervous system and glial family ligands (GFLs) as potential drivers of hematopoietic survival and self-renewal in the bone marrow niche,but how to apply this to HSC maintenance and expansion is yet to be explored. We demonstrate a role for the GFL receptor,RET,at the cell surface of HSCs,in mediating sustained cellular growth,resistance to stress and improved cell survival throughout in vitro expansion. HSCs treated with the key RET ligand/co-receptor complex,GDNF/GFRa1,show improved progenitor function at primary transplantation and improved long-term HSC function at secondary transplantation. Finally,we demonstrate that RET drives a multi-faceted intracellular signalling pathway,including key signalling intermediates AKT,ERK1/2,NFkB and p53,responsible for a wide range of cellular and genetic responses which improve cell growth and survival under culture conditions. View Publication

Anti-obesity medications (AOMs) have become one of the most prescribed drugs in human medicine. While AOMs are known to impact adult neurogenesis in the hypothalamus,their effects on the functional maturation of hypothalamic neurons remain unexplored. Given that AOMs target neurons in the Medial Basal Hypothalamus (MBH),which play a crucial role in regulating energy homeostasis,we hypothesized that AOMs might influence the functional maturation of these neurons,potentially rewiring the MBH. To investigate this,we exposed hypothalamic neurons derived from human induced pluripotent stem cells (hiPSCs) to Semaglutide and lipidized prolactin-releasing peptide (LiPR),two anti-obesity compounds. Contrary to our expectations,treatment with Semaglutide or LiPR during neuronal maturation did not affect the proportion of anorexigenic,Pro-opiomelanocortin-expressing (POMC+) neurons. Additionally,LiPR did not alter the morphology of POMC+ neurons or the expression of selected genes critical for the metabolism or development of anorexigenic neurons. Furthermore,LiPR did not impact the proportion of adult-generated POMC+ neurons in the mouse MBH. Taken together,these results suggest that AOMs do not influence the functional maturation of anorexigenic hypothalamic neurons. View Publication

We report a system for Cre-regulated expression of RNA interference in vivo. Expression cassettes comprise selectable and FACS-sortable markers in tandem with additional marker genes and shRNAs in the antisense orientation. The cassettes are flanked by tandem LoxP sites arranged so that Cre expression inverts the marker-shRNA construct,allowing its regulated expression (and,at the same time,deletes the original selection/marker genes). The cassettes can be incorporated into retroviral or lentiviral vectors and delivered to cells in culture or used to generate transgenic mice. We describe cassettes incorporating various combinations of reporter genes,miRNA-based RNAi (including two shRNA constructs at once),and oncogenes and demonstrate the delivery of effective RNA interference in cells in culture,efficient transduction into hematopoietic stem cells with cell-type-specific knockdown in their progeny,and rapid generation of regulated shRNA knockdown in transgenic mice. These vector systems allow regulated combinatorial manipulation (both overexpression and loss of function) of gene expression in multiple systems in vitro and in vivo. View Publication

Stem cell therapies hold great promise for repairing tissues damaged due to disease or injury. However,a major obstacle facing this field is the difficulty in identifying cells of a desired phenotype from the heterogeneous population that arises during stem cell differentiation. Conventional fluorescence flow cytometry and magnetic cell purification require exogenous labeling of cell surface markers which can interfere with the performance of the cells of interest. Here,we describe a non-genetic,label-free cell cytometry method based on electrophysiological response to stimulus. As many of the cell types relevant for regenerative medicine are electrically-excitable (e.g. cardiomyocytes,neurons,smooth muscle cells),this technology is well-suited for identifying cells from heterogeneous stem cell progeny without the risk and expense associated with molecular labeling or genetic modification. Our label-free cell cytometer is capable of distinguishing clusters of undifferentiated human induced pluripotent stem cells (iPSC) from iPSC-derived cardiomyocyte (iPSC-CM) clusters. The system utilizes a microfluidic device with integrated electrodes for both electrical stimulation and recording of extracellular field potential (FP) signals from suspended cells in flow. The unique electrode configuration provides excellent rejection of field stimulus artifact while enabling sensitive detection of FPs with a noise floor of 2 $$V(rms). Cells are self-aligned to the recording electrodes via hydrodynamic flow focusing. Based on automated analysis of these extracellular signals,the system distinguishes cardiomyocytes from non-cardiomyocytes. This is an entirely new approach to cell cytometry,in which a cell's functionality is assessed rather than its expression profile or physical characteristics. View Publication

Since the 1960s,the stem cells have been extensively studied including embryonic stem cells,neural stem cells,bone marrow hematopoietic stem cells,and mesenchymal stem cells. In the recent years,several stem cells have been initially used in the treatment of diseases,such as in bone marrow transplant. At the same time,isolation and culture experimental technologies for stem cell research have been widely developed in recent years. In addition,molecular imaging technologies including optical molecular imaging,positron emission tomography,single-photon emission computed tomography,and computed tomography have been developed rapidly in recent the 10 years and have also been used in the research on disease mechanism and evaluation of treatment of disease related with stem cells. This paper will focus on recent typical isolation,culture,and observation techniques of stem cells followed by a concise introduction. Finally,the current challenges and the future applications of the new technologies in stem cells are given according to the understanding of the authors,and the paper is then concluded. View Publication

Mechanisms that maintain cancer stem cells are crucial to tumour progression. The ID2 protein supports cancer hallmarks including the cancer stem cell state. HIFα transcription factors,most notably HIF2α (also known as EPAS1),are expressed in and required for maintenance of cancer stem cells (CSCs). However,the pathways that are engaged by ID2 or drive HIF2α accumulation in CSCs have remained unclear. Here we report that DYRK1A and DYRK1B kinases phosphorylate ID2 on threonine 27 (Thr27). Hypoxia downregulates this phosphorylation via inactivation of DYRK1A and DYRK1B. The activity of these kinases is stimulated in normoxia by the oxygen-sensing prolyl hydroxylase PHD1 (also known as EGLN2). ID2 binds to the VHL ubiquitin ligase complex,displaces VHL-associated Cullin 2,and impairs HIF2α ubiquitylation and degradation. Phosphorylation of Thr27 of ID2 by DYRK1 blocks ID2-VHL interaction and preserves HIF2α ubiquitylation. In glioblastoma,ID2 positively modulates HIF2α activity. Conversely,elevated expression of DYRK1 phosphorylates Thr27 of ID2,leading to HIF2α destabilization,loss of glioma stemness,inhibition of tumour growth,and a more favourable outcome for patients with glioblastoma. View Publication