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

The identification of stem cells within a mixed population of cells is a major hurdle for stem cell biology--in particular,in the identification of induced pluripotent stem (iPS) cells during the reprogramming process. Based on the selective expression of stem cell surface markers,a method to specifically infect stem cells through antibody-conjugated lentiviral particles has been developed that can deliver both visual markers for live-cell imaging as well as selectable markers to enrich for iPS cells. Antibodies recognizing SSEA4 and CD24 mediated the selective infection of the iPS cells over the parental human fibroblasts,allowing for rapid expansion of these cells by puromycin selection. Adaptation of the vector allows for the selective marking of human embryonic stem (hES) cells for their removal from a population of differentiated cells. This method has the benefit that it not only identifies stem cells,but that specific genes,including positive and negative selection markers,regulatory genes or miRNA can be delivered to the targeted stem cells. The ability to specifically target gene delivery to human pluripotent stem cells has broad applications in tissue engineering and stem cell therapies. View Publication

Endothelial cells and macrophages are known to engage in tight and specific interactions that contribute to the modulation of vascular function. Here we show that adult endothelial cells provide critical signals for the selective growth and differentiation of macrophages from several hematopoietic progenitors. The process features the formation of well-organized colonies that exhibit progressive differentiation from the center to the periphery and toward an M2-like phenotype,characterized by enhanced expression of Tie2 and CD206/Mrc1. These colonies are long-lived depending on the contact with the endothelium; removal of the endothelial monolayer results in rapid colony dissolution. We further found that Csf1 produced by the endothelium is critical for the expansion of the macrophage colonies and that blockade of Csf1 receptor impairs colony growth. Functional analyses indicate that these macrophages are capable of accelerating angiogenesis,promoting tumor growth,and effectively engaging in tight associations with endothelial cells in vivo. These findings uncover a critical role of endothelial cells in the induction of macrophage differentiation and their ability to promote further polarization toward a proangiogenic phenotype. This work also highlights some of the molecules underlying the M2-like differentiation,a process that is relevant to the progression of both developmental and pathologic angiogenesis. View Publication

Recent studies demonstrate that brain infiltration of peripheral immune cells and their interaction with brain-resident cells contribute to Parkinson’s disease (PD). However,mechanisms of T cell-brain cell communication are not fully elucidated and models allowing investigation of interaction between T cells and brain-resident cells are required. In this study,we developed a three-dimensional (3D) model composed of stem cell-derived human midbrain organoids (hMO) and peripheral blood T cells. We demonstrated that organoids consist of multiple midbrain-specific cell types,allowing to study T cell motility and interactions with midbrain tissue in a spatially organized microenvironment. We optimized co-culture conditions and demonstrated that T cells infiltrate hMO tissue,leading to neural cell loss. Our work establishes a novel 3D cell co-culture model as a promising tool to investigate the effect of the adaptive immune system on the midbrain and can be used in future studies to address these processes in the context of PD. View Publication

A distinctive feature of both type 1 and type 2 diabetes is the waning of insulin-secreting beta cells in the pancreas. New methods for direct and specific targeting of the beta cells could provide platforms for delivery of pharmaceutical reagents. Imaging techniques such as Positron Emission Tomography (PET) rely on the efficient and specific delivery of imaging reagents,and could greatly improve our understanding of diabetes etiology as well as providing biomarkers for viable beta-cell mass in tissue,in both pancreas and in islet grafts.The DiGeorge Syndrome Critical Region Gene 2 (DGCR2) protein has been suggested as a beta-cell specific protein in the pancreas,but so far there has been a lack of available high-affinity binders suitable for targeted drug delivery or molecular imaging. Affibody molecules belong to a class of small affinity proteins with excellent properties for molecular imaging. Here,we further validate the presence of DGCR2 in pancreatic and stem cell (SC)-derived beta cells,and then describe the generation and selection of several Affibody molecules candidates that target human DGCR2. Using an in-house developed directed evolution method,new DGCR2-binding Affibody molecules were generated and evaluated for thermal stability and affinity. The Affibody molecules variants were further developed as targeting agents for delivering imaging reagents to beta cell. The Affibody molecule ZDGCR2:AM106 displayed nanomolar affinity,suitable stability and biodistribution,with negligible toxicity to islets,qualifying it as a suitable lead candidate for further development as a tool for specific delivery of drugs and imaging reagents to beta cells.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-024-84574-y. View Publication

Leptin is detected in the sera,and leptin receptors are expressed in the cerebrum of mouse embryos,suggesting that leptin plays a role in cerebral development. Compared with the wild type,leptin-deficient (ob/ob) mice had fewer cells at embryonic day (E) 16 and E18 and had fewer 5-bromo-2'-deoxyuridine(+) cells at E14 and E16 in the neuroepithelium. Intracerebroventricular leptin injection in E14 ob/ob embryos increased the number of neuroepithelium cells at E16. In cultured neurosphere cells,leptin treatment increased Hes1 mRNA expression and maintained neural progenitors. Astrocyte differentiation was induced by low-dose (0.1 microg/ml) but not high-dose (1 microg/ml) leptin. High-dose leptin decreased Id mRNA and increased Ngn1 mRNA in neurosphere cells. The neuropeptide Y mRNA level in the cortical plate was lower in ob/ob than the wild type at E16 and E18. These results suggest that leptin maintains neural progenitors and is related to glial and neuronal development in embryos. View Publication

Many studies have demonstrated a role for netrin-1-deleted in colorectal cancer (DCC) interactions in both axon guidance and neuronal migration. Neogenin,a member of the DCC receptor family,has recently been shown to be a chemorepulsive axon guidance receptor for the repulsive guidance molecule (RGM) family of guidance cues [Rajagopalan S,Deitinghoff L,Davis D,Conrad S,Skutella T,Chedotal A,Mueller B,Strittmatter S (2004) Neogenin mediates the action of repulsive guidance molecule. Nat Cell Biol 6:755-762]. Here we show that neogenin is present on neural progenitors,including neurogenic radial glia,in the embryonic mouse forebrain suggesting that neogenin expression is a hallmark of neural progenitor populations. Neogenin-positive progenitors were isolated from embryonic day 14.5 forebrain using flow cytometry and cultured as neurospheres. Neogenin-positive progenitors gave rise to neurospheres displaying a high proliferative and neurogenic potential. In contrast,neogenin-negative forebrain cells did not produce long-term neurosphere cultures and did not possess a significant neurogenic potential. These observations argue strongly for a role for neogenin in neural progenitor biology. In addition,we also observed neogenin on parvalbumin- and calbindin-positive interneuron neuroblasts that were migrating through the medial and lateral ganglionic eminences,suggesting a role for neogenin in tangential migration. Therefore,neogenin may be a multi-functional receptor regulating both progenitor activity and neuroblast migration in the embryonic forebrain. View Publication

A hybrid cell line resulting from the fusion of a Con A-activated normal mouse spleen cell and a transformed mouse T cell (EL-4BU) has been used to prepare and select rat monoclonal antibodies reactive with molecules expressed on the surface of proliferating,as opposed to resting,mouse T cells. In this report,the characterization of two such antigens identified in this way is described. One antigen is a membrane component common to mitogen-activated T and B cells,some bone marrow cells,and various transformed cell lines but is not detectable on either normal thymocytes or the majority of spleen cells by radioimmunoassay or FACS analysis. It has a m.w. of approximately 200,000 daltons under nonreducing conditions and 100,000 daltons under reducing conditions. Antibodies to this antigen precipitate cell-bound transferrin but do not react directly with transferrin itself. It would thus appear that the antigen is the transferrin receptor molecule. The second antigen is not detectable on normal thymocytes,spleen cells,bone marrow cells,or mitogen-stimulated spleen cells but is expressed at high levels on some transformed T cell lines. It,too,appears to be a dimer,with a m.w. of 95,000 daltons under nonreducing conditions,decreasing to 50,000 daltons under reducing conditions. Although the function of the 95,000-dalton antigen is not yet known,its lack of expression on adult T cell populations both before and after activation suggests either a short-lived role at a very early stage of T cell development and/or an association with T cell transformation. View Publication

Sensory neurons sense pathogenic infiltration to drive innate immune responses,but their role in humoral immunity is unclear. Here,using mouse models of Streptococcus pneumoniae infection and Alternaria alternata asthma,we show that sensory neurons are required for B cell recruitment and antibody production. In response to S. pneumoniae,sensory neuron depletion increases bacterial burden and reduces B cell numbers,IgG release,and neutrophil stimulation. Meanwhile,during A. alternata-induced airway inflammation,sensory neuron depletion decreases B cell population sizes,IgE levels,and asthmatic characteristics. Mechanistically,during bacterial infection,sensory neurons preferentially release vasoactive intestinal polypeptide (VIP). In response to asthma,sensory neurons release substance P. Administration of VIP into sensory neuron-depleted mice suppresses bacterial burden,while VIPR1 deficiency increases infection. Similarly,exogenous substance P delivery aggravates asthma in sensory neuron-depleted mice,while substance P deficiency ameliorates asthma. Our data,thus demonstrate that sensory neurons release select neuropeptides which target B cells dependent on the immunogen. Sensory neurons may regulate innate immune cells,but their roles in humoral immunity is still unclear. Here the authors show that bacterial infection and asthma induction induce sensory neuron production of distinct neurotransmitters to dampen B cell responses but differentially target IgG and IgE,respectively,to specifically modulate the symptoms. View Publication

Regulatory T cells (Tregs) are capable of inhibiting the proliferation,activation and function of T cells and play an important role in impeding the immune response to cancer. In chronic lymphocytic leukemia (CLL) a dysfunctional immune response and elevated percentage of effector-like phenotype Tregs have been described. In this study,using the Eµ-TCL1 mouse model of CLL,we evaluated the changes in the Tregs phenotype and their expansion at different stages of leukemia progression. Importantly,we show that Tregs depletion in DEREG mice triggered the expansion of new anti-leukemic cytotoxic T cell clones leading to leukemia eradication. In TCL1 leukemia-bearing mice we identified and characterized a specific Tregs subpopulation,the phenotype of which suggests its role in the formation of an immunosuppressive microenvironment,supportive for leukemia survival and proliferation. This observation was also confirmed by the gene expression profile analysis of these TCL1-specific Tregs. The obtained data on Tregs are consistent with those described so far,however,above all show that the changes in the Tregs phenotype described in CLL result from the formation of a specific,described in this study Tregs subpopulation. In addition,functional tests revealed the ability of Tregs to inhibit T cells that recognize model antigens expressed by leukemic cells. Moreover,inhibition of Tregs with a MALT1 inhibitor provided a therapeutic benefit,both as monotherapy and also when combined with an immune checkpoint inhibitor. Altogether,activation of Tregs appears to be crucial for CLL progression. View Publication

Various systems for differentiating hematopoietic cells from human pluripotent stem cells (PSCs) have been developed,although none have been fully optimized. In this report,we describe the development of a novel three-dimensional system for differentiating hematopoietic cells from PSCs using collagen sponges (CSs) reinforced with poly(ethylene terephthalate) fibers as a scaffold. PSCs seeded onto CSs were differentiated in a stepwise manner with appropriate cytokines under serum-free and feeder-free conditions. This process yielded several lineages of floating hematopoietic cells repeatedly for more than 1 month. On immunohistochemical staining,we detected CD34+ cells and CD45+ cells in the surface and cavities of the CS. Taking advantage of the portability of this system,we were able to culture multiple CSs together floating in medium,making it possible to harvest large numbers of hematopoietic cells repeatedly. Given these findings,we suggest that this novel three-dimensional culture system may be useful in the large-scale culture of PSC-derived hematopoietic cells. View Publication

Ion channels and transporters have increasingly recognized roles in cancer progression through the regulation of cell proliferation,migration,and death. Glioblastoma stem-like cells (GSCs) are a source of tumor formation and recurrence in glioblastoma multiforme,a highly aggressive brain cancer,suggesting that ion channel expression may be perturbed in this population. However,little is known about the expression and functional relevance of ion channels that may contribute to GSC malignancy. Using RNA sequencing,we assessed the enrichment of ion channels in GSC isolates and non-tumor neural cell types. We identified a unique set of GSC-enriched ion channels using differential expression analysis that is also associated with distinct gene mutation signatures. In support of potential clinical relevance,expression of selected GSC-enriched ion channels evaluated in human glioblastoma databases of The Cancer Genome Atlas and Ivy Glioblastoma Atlas Project correlated with patient survival times. Finally,genetic knockdown as well as pharmacological inhibition of individual or classes of GSC-enriched ion channels constrained growth of GSCs compared to normal neural stem cells. This first-in-kind global examination characterizes ion channels enriched in GSCs and explores their potential clinical relevance to glioblastoma molecular subtypes,gene mutations,survival outcomes,regional tumor expression,and experimental responses to loss-of-function. Together,the data support the potential biological and therapeutic impact of ion channels on GSC malignancy and provide strong rationale for further examination of their mechanistic and therapeutic importance. View Publication