Cardiosphere-derived cells suppress allogeneic lymphocytes by production of PGE2 acting via the EP4 receptor

derived cells (CDCs) are a cardiac progenitor cell population, which have been shown to possess cardiac regenerative properties and can improve heart function in a variety of cardiac diseases. Studies in large animal models have predominantly focussed on using autologous cells for safety, however allogeneic cell banks would allow for a practical, cost-effective and efficient use in a clinical setting. The aim of this work was to determine the immunomodulatory status of these cells using CDCs and lymphocytes from 5 dogs. CDCs expressed MHC I but not MHC II molecules and in mixed lymphocyte reactions demonstrated a lack of lymphocyte proliferation in response to MHC-mismatched CDCs. Furthermore, MHC-mismatched CDCs suppressed lymphocyte proliferation and activation in response to Concanavalin A. Transwell experiments demonstrated that this was predominantly due to direct cell-cell contact in addition to soluble mediators whereby CDCs produced high levels of PGE2 under inflammatory conditions. This led to down-regulation of CD25 expression on lymphocytes via the EP4 receptor. Blocking prostaglandin synthesis restored both, proliferation and activation (measured via CD25 expression) of stimulated lymphocytes. We demonstrated for the first time in a large animal model that CDCs inhibit proliferation in allo-reactive lymphocytes and have potent immunosuppressive activity mediated via PGE2

Isolation and characterization of resident endogenous c-Kit⁺ cardiac stem cells from the adult mouse and rat heart

This protocol describes the isolation of endogenous c-Kit (also known as CD117)-positive (c-Kit⁺), CD45-negative (CD45⁻) cardiac stem cells (eCSCs) from whole adult mouse and rat hearts. The heart is enzymatically digested via retrograde perfusion of the coronary circulation, resulting in rapid and extensive breakdown of the whole heart. Next, the tissue is mechanically dissociated further and cell fractions are separated by centrifugation. The c-Kit⁺ CD45⁻ eCSC population is isolated by magnetic-activated cell sorting technology and purity and cell numbers are assessed by flow cytometry. This process takes ∼4 h for mouse eCSCs or 4.5 h for rat eCSCs. We also describe how to characterize c-Kit⁺ CD45⁻ eCSCs. The c-Kit⁺ CD45⁻eCSCs exhibit the defining characteristics of stem cells: they are self-renewing, clonogenic and multipotent. This protocol also describes how to differentiate eCSCs into three main cardiac lineages: functional, beating cardiomyocytes, smooth muscle, and endothelial cells. These processes take 17-20 d

Melanoma Spheroids Grown Under Neural Crest Cell Conditions Are Highly Plastic Migratory/Invasive Tumor Cells Endowed with Immunomodulator Function

International audienceBACKGROUND: The aggressiveness of melanoma tumors is likely to rely on their well-recognized heterogeneity and plasticity. Melanoma comprises multi-subpopulations of cancer cells some of which may possess stem cell-like properties. Although useful, the sphere-formation assay to identify stem cell-like or tumor initiating cell subpopulations in melanoma has been challenged, and it is unclear if this model can predict a functional phenotype associated with aggressive tumor cells. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the molecular and functional phenotypes of melanoma spheroids formed in neural crest cell medium. Whether from metastatic or advanced primary tumors, spheroid cells expressed melanoma-associated markers. They displayed higher capacity to differentiate along mesenchymal lineages and enhanced expression of SOX2, NANOG, KLF4, and/or OCT4 transcription factors, but not enhanced self-renewal or tumorigenicity when compared to their adherent counterparts. Gene expression profiling attributed a neural crest cell signature to these spheroids and indicated that a migratory/invasive and immune-function modulating program could be associated with these cells. In vitro assays confirmed that spheroids display enhanced migratory/invasive capacities. In immune activation assays, spheroid cells elicited a poorer allogenic response from immune cells and inhibited mitogen-dependent T cells activation and proliferation more efficiently than their adherent counterparts. Our findings reveal a novel immune-modulator function of melanoma spheroids and suggest specific roles for spheroids in invasion and in evasion of antitumor immunity. CONCLUSION/SIGNIFICANCE: The association of a more plastic, invasive and evasive, thus a more aggressive tumor phenotype with melanoma spheroids reveals a previously unrecognized aspect of tumor cells expanded as spheroid cultures. While of limited efficiency for melanoma initiating cell identification, our melanoma spheroid model predicted aggressive phenotype and suggested that aggressiveness and heterogeneity of melanoma tumors can be supported by subpopulations other than cancer stem cells. Therefore, it could be constructive to investigate melanoma aggressiveness, relevant to patients and clinical transferability

Yeast Gis2 and its human ortholog CNBP are novel components of stress-induced RNP granules.

Although a CCTG expansion in the gene encoding the zinc knuckle protein CNBP causes a common form of muscular dystrophy, the function of both human CNBP and its putative budding yeast ortholog Gis2 remain poorly understood. Here we report the protein interactions of Gis2 and the subcellular locations of both Gis2 and CNBP. We found that Gis2 exhibits RNA-dependent interactions with two proteins involved in mRNA recognition, the poly(A) binding protein and the translation initiation factor eIF4G. We show that Gis2 is a component of two large RNA-protein granules, processing bodies and stress granules, which contain translationally repressed mRNAs. Consistent with a functional ortholog, CNBP also associates with the poly(A) binding protein and accumulates in stress granules during arsenite treatment of human cells. These results implicate both Gis2 and CNBP in mRNA handling during stress

Gis2 associates with proteins involved in translation initiation.

<p>(A) After tandem affinity purification, eluates from an untagged strain and a strain expressing Gis2-TAP were fractionated in a SDS-polyacrylamide gel and proteins visualized by silver staining. Lane 1, molecular size markers. Sizes are in kDa. (B) Lysates of an untagged strain and a strain expressing Gis2-GFP were subjected to immunoprecipitation with anti-GFP antibodies. Prior to immunoprecipitation, Gis2-GFP lysates were incubated with the indicated amounts of RNase A. Proteins in immunoprecipitates were detected by Western blotting with antibodies against Pab1, eIF4G1, and eIF4G2. The efficiency of immunoprecipitation was determined by re-probing with anti-GFP. As a negative control, the blot was reprobed to detect Pgk1. (C) Lysates of untagged and <i>Gis2-(FLAG</i>)<i>₃</i> strains expressing Pab1-GFP, eIF4G1-GFP or eIF4G2-GFP were subjected to immunoprecipitation with anti-GFP antibodies. After Western blotting, Gis2-(FLAG)₃ was detected with anti-FLAG antibodies. To examine immunoprecipitation efficiency, Pab1-GFP, eIF4G1-GFP and eIF4G2-GFP were detected with anti-GFP antibodies. Pgk1 was detected as a negative control.</p

Gis2 accumulates in P-bodies and stress granules during stationary phase.

<p>(A and B) Yeast strains expressing chromosomal Gis2-mCh and the indicated (A) P-body or (B) stress granule markers were grown for 4 days in glucose-containing media and examined using confocal microscopy. Scale bar, 5 µm.</p

Cycloheximide enhances the rate of disappearance of CNBP-positive foci.

<p>(A and B) To induce P-bodies and stress granules, HeLa cells expressing RFP-DCP1a and RFP-RCK and untransfected cells were incubated with 500 µM arsenite for 30 minutes. Following arsenite treatment, cells were allowed to recover in fresh medium for 45 minutes in the (A) absence or (B) presence of cycloheximide. After fixation, transfected cells were subjected to immunofluorescence to detect CNBP (top and middle panels) or CNBP and TIAR (bottom panels) and examined using confocal microscopy.</p

A small fraction of Gis2 sediments with polyribosomes.

<p>(A and B) <i>GIS2-GFP</i> cell lysates were prepared in the presence (A) or absence (B) of cycloheximide and fractionated in 15–50% sucrose gradients. Fractions were collected while monitoring OD₂₅₄. Proteins were subjected to Western blotting to detect Gis2-GFP, Pab1, eIF4G1, eIF4G2 and ribosomal proteins L1A and L1B. (C and D) <i>GIS2-GFP</i> cell lysates prepared in the presence of cycloheximide were either untreated (C) or incubated with 5 U/µl micrococcal nuclease (D) prior to sedimentation. Fractions from each gradient were analyzed in two gels as indicated by the lines.</p