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

Cancer stem cells (CSCs) have been identified in a growing number of malignancies and are functionally defined by their ability to undergo self-renewal and produce differentiated progeny. These properties allow CSCs to recapitulate the original tumor when injected into immunocompromised mice. CSCs within an epithelial malignancy were first described in breast cancer and found to display specific cell surface antigen expression (CD44+CD24(low/�?�)). Since then,CSCs have been identified in an increasing number of other human malignancies using CD44 and CD24 as well as a number of other surface antigens. Physiologic properties,including aldehyde dehydrogenase (ALDH) activity,have also been used to isolate CSCs from malignant tissues. Recently,we and others identified CSCs from pancreatic adenocarcinoma based on ALDH activity and the expression of the cell surface antigens CD44 and CD24,and CD133. These highly tumorigenic populations may or may not be overlapping and display other functions. We found that ALDH+ and CD44+CD24+ pancreatic CSCs are similarly tumorigenic,but ALDH+ cells are relatively more invasive. In this protocol we describe a method to isolate viable pancreatic CSCs from low-passage human xenografts. Xenografted tumors are harvested from mice and made into a single-cell suspension. Tissue debris and dead cells are separated from live cells and then stained using antibodies against CD44 and CD24 and using the ALDEFLUOR reagent,a fluorescent substrate of ALDH. CSCs are then isolated by fluorescence activated cell sorting. Isolated CSCs can then be used for analytical or functional assays requiring viable cells. View Publication

HIV elite controllers (EC) are a rare group of HIV-infected patients who are able to maintain undetectable viral loads during a long period of time in the absence of antiretroviral treatment. Adaptive immunity and host genetic factors,although implicated,do not entirely explain this phenomenon. On the other hand,plasmacytoid dendritic cells (pDCs) are the principal type I interferon (IFN) producers in response to viral infection,and it is unknown whether pDCs are involved in the control of HIV infection in EC. In our study,we analyzed peripheral pDC levels and IFN-α production by peripheral blood mononuclear cells (PBMCs) in EC compared to other groups of HIV-infected patients,the ability of pDCs to reduce HIV production in vitro,and the mechanisms potentially involved. We showed preserved pDC counts and IFN-α production in EC. We also observed a higher capacity of pDCs from EC to reduce HIV production and to induce T cell apoptosis,whereas pDCs from viremic patients barely responded without previous Toll-like receptor 9 (TLR-9) stimulus. The preserved functionality of pDCs from EC to reduce viral production may be one of the mechanisms involved in the control of HIV viremia in these subjects. These results demonstrate the importance of innate immunity in HIV pathogenesis,and an understanding of pDC mechanisms would be helpful for the design of new therapies. View Publication

Reversing brain degeneration and trauma lesions will depend on cell therapy. Our previous work identified neural precursor cells derived from the skeletal muscle of Nestin-GFP transgenic mice,but their identity,origin,and potential survival in the brain are only vaguely understood. In this work,we show that Nestin-GFP+ progenitor cells share morphological and molecular markers with NG2-glia,including NG2,PDGFRα,O4,NGF receptor (p75),glutamate receptor-1(AMPA),and A2B5 expression. Although these cells exhibit NG2,they do not express other pericyte markers,such as α-SMA or connexin-43,and do not differentiate into the muscle lineage. Patch-clamp studies displayed outward potassium currents,probably carried through Kir6.1 channels. Given their potential therapeutic application,we compared their abundance in tissues and concluded that skeletal muscle is the richest source of predifferentiated neural precursor cells. We found that these cells migrate toward the neurogenic subventricular zone displaying their typical morphology and nestin-GFP expression two weeks after brain injection. For translational purposes,we sought to identify these neural progenitor cells in wild-type species by developing a DsRed expression vector under Nestin-Intron II control. This approach revealed them in nonhuman primates and aging rodents throughout the lifespan. View Publication

Inductive signals and transcription factors involved in motor neuron generation have been identified,raising the question of whether these developmental insights can be used to direct stem cells to a motor neuron fate. We show that developmentally relevant signaling factors can induce mouse embryonic stem (ES) cells to differentiate into spinal progenitor cells,and subsequently into motor neurons,through a pathway recapitulating that used in vivo. ES cell-derived motor neurons can populate the embryonic spinal cord,extend axons,and form synapses with target muscles. Thus,inductive signals involved in normal pathways of neurogenesis can direct ES cells to form specific classes of CNS neurons. View Publication

A new method of spatially controlled gene regulation in 3D-cultured human embryonic stem cells is developed using hollow gold nanoshells (HGNs) and near-infrared (NIR) light. Targeted cell(s) are discriminated from neighboring cell(s) by focusing NIR light emitted from a two-photon microscope. Irradiation of cells that have internalized HGNs releases surface attached siRNAs and leads to concomitant gene downregulation. View Publication

Induced pluripotent stem cells (iPSCs) are being pursued as a source of cells for autologous therapies,many of which will be aimed at aged patients. To explore the impact of age on iPSC quality,we produced iPSCs from blood cells of 16 donors aged 21-100. We find that iPSCs from older donors retain an epigenetic signature of age,which can be reduced through passaging. Clonal expansion via reprogramming also enables the discovery of somatic mutations present in individual donor cells,which are missed by bulk sequencing methods. We show that exomic mutations in iPSCs increase linearly with age,and all iPSC lines analyzed carry at least one gene-disrupting mutation,several of which have been associated with cancer or dysfunction. Unexpectedly,elderly donors (textgreater90 yrs) harbor fewer mutations than predicted,likely due to a contracted blood progenitor pool. These studies establish that donor age is associated with an increased risk of abnormalities in iPSCs and will inform clinical development of reprogramming technology. View Publication

The spinal cord consists of multiple neuronal cell types that are critical to motor control and arise from distinct progenitor domains in the developing neural tube. Excitatory V2a interneurons in particular are an integral component of central pattern generators that control respiration and locomotion; however,the lack of a robust source of human V2a interneurons limits the ability to molecularly profile these cells and examine their therapeutic potential to treat spinal cord injury (SCI). Here,we report the directed differentiation of CHX10(+) V2a interneurons from human pluripotent stem cells (hPSCs). Signaling pathways (retinoic acid,sonic hedgehog,and Notch) that pattern the neural tube were sequentially perturbed to identify an optimized combination of small molecules that yielded ∼25% CHX10(+) cells in four hPSC lines. Differentiated cultures expressed much higher levels of V2a phenotypic markers (CHX10 and SOX14) than other neural lineage markers. Over time,CHX10(+) cells expressed neuronal markers [neurofilament,NeuN,and vesicular glutamate transporter 2 (VGlut2)],and cultures exhibited increased action potential frequency. Single-cell RNAseq analysis confirmed CHX10(+) cells within the differentiated population,which consisted primarily of neurons with some glial and neural progenitor cells. At 2 wk after transplantation into the spinal cord of mice,hPSC-derived V2a cultures survived at the site of injection,coexpressed NeuN and VGlut2,extended neurites textgreater5 mm,and formed putative synapses with host neurons. These results provide a description of V2a interneurons differentiated from hPSCs that may be used to model central nervous system development and serve as a potential cell therapy for SCI. View Publication

Adult-derived hippocampal progenitors generate neurons,astrocytes,and oligodendrocytes in vitro and following grafting into the adult brain. Although these progenitors have a considerable capacity for in vitro self renewal,it is not known if each lineage is generated by separate committed precursors or by multipotent stem cells. By genetic marking,we have followed individual cells through the process of proliferative expansion,commitment,and differentiation. All three lineages are generated by single marked cells and the relative proportions of each lineage can be strongly influenced by environmental cues. Differentiation is accompanied by a characteristic progression of lineage-specific markers and can be potentiated by retinoic acid,elevated cyclic AMP,or neurotrophic factors. The ability to genetically mark and clone normal diploid hippocampal progenitors provides the first definitive evidence that multipotent neural stem cells exist outside of the adult striatal subventricular zone and supports the hypothesis that FGF-2-responsive neural stem cells may be broadly distributed in the adult brain. View Publication