Transcription factor-based cellular reprogramming has opened the way to converting somatic cells to a pluripotent state, but has faced limitations resulting from the requirement for transcription factors and the relative inefficiency of the process. We show here that expression of the miR302/367 cluster rapidly and efficiently reprograms mouse and human somatic cells to an iPS state without a requirement for exogenous transcription factors. This miRNA-based reprogramming approach is two orders of magnitude more efficient than standard Oct4/Sox2/Klf4/Myc-mediated methods. Mouse and human miR302/367 iPS cells display similar characteristics to Oct4/Sox2/Klf4/Myc-iPS cells, including pluripotency marker expression, teratoma formation, and, for mouse cells, chimera contribution and germline contribution. We found that miR367 expression is required for miR302/367-mediated reprogramming and activates Oct4 gene expression, and that suppression of Hdac2 is also required. Thus, our data show that miRNA and Hdac-mediated pathways can co-operate in a powerful way to reprogram somatic cells to pluripotency.
BackgroundThe ability to properly model intravascular steps in metastasis is essential in identifying key physical, cellular, and molecular determinants that can be targeted therapeutically to prevent metastatic disease. Research on the vascular microenvironment has been hindered by challenges in studying this compartment in metastasis under conditions that reproduce in vivo physiology while allowing facile experimental manipulation.Methodology/Principal FindingsWe present a microfluidic vasculature system to model interactions between circulating breast cancer cells with microvascular endothelium at potential sites of metastasis. The microfluidic vasculature produces spatially-restricted stimulation from the basal side of the endothelium that models both organ-specific localization and polarization of chemokines and many other signaling molecules under variable flow conditions. We used this microfluidic system to produce site-specific stimulation of microvascular endothelium with CXCL12, a chemokine strongly implicated in metastasis.Conclusions/SignificanceWhen added from the basal side, CXCL12 acts through receptor CXCR4 on endothelium to promote adhesion of circulating breast cancer cells, independent of CXCL12 receptors CXCR4 or CXCR7 on tumor cells. These studies suggest that targeting CXCL12-CXCR4 signaling in endothelium may limit metastases in breast and other cancers and highlight the unique capabilities of our microfluidic device to advance studies of the intravascular microenvironment in metastasis.
The plasticity of differentiated cells in adult tissues undergoing repair is an area of intense research. Pulmonary alveolar Type II cells produce surfactant and function as progenitors in the adult, demonstrating both self-renewal and differentiation into gas exchanging Type I cells. In vivo, Type I cells are thought to be terminally differentiated and their ability to give rise to alternate lineages has not been reported. Here, we show that Hopx becomes restricted to Type I cells during development. However, unexpectedly, lineage-labeled Hopx+ cells both proliferate and generate Type II cells during adult alveolar regrowth following partial pneumonectomy. In clonal 3D culture, single Hopx+ Type I cells generate organoids composed of Type I and Type II cells, a process modulated by TGFβ signaling. These findings demonstrate unanticipated plasticity of Type I cells and a bi-directional lineage relationship between distinct differentiated alveolar epithelial cell types in vivo and in single cell culture.
Summary Progenitor cells differentiate into specialized cell types through coordinated expression of lineage-specific genes and modification of complex chromatin configurations. We demonstrate that a histone deacetylase (Hdac3) organizes heterochromatin at the nuclear lamina during cardiac progenitor lineage restriction. Specification of cardiomyocytes is associated with reorganization of peripheral heterochromatin and, independent of deacetylase activity, Hdac3 tethers peripheral heterochromatin containing lineage-relevant genes to the nuclear lamina. Deletion of Hdac3 in cardiac progenitor cells releases genomic regions from the nuclear periphery, leading to precocious cardiac gene expression and differentiation into cardiomyocytes; in contrast, restricting Hdac3 to the nuclear periphery rescues myogenesis in progenitors otherwise lacking Hdac3. Our results suggest that availability of genomic regions for activation by lineage-specific factors is regulated in part through dynamic chromatin-nuclear lamina interactions and that competence of a progenitor cell to respond to differentiation signals may depend upon coordinated movement of responding gene loci away from the nuclear periphery.
Cardiac progenitor cells are multipotent and give rise to cardiac endothelium, smooth muscle, and cardiomyocytes. Here, we define and characterize the cardiomyoblast intermediate that is committed to the cardiomyocyte fate, and we characterize the niche signals that regulate commitment. Cardiomyoblasts express Hopx, which functions to coordinate local Bmp signals to inhibit the Wnt pathway, thus promoting cardiomyogenesis. Hopx integrates Bmp and Wnt signaling by physically interacting with activated Smads and repressing Wnt genes. The identification of the committed cardiomyoblast that retains proliferative potential will inform cardiac regenerative therapeutics. In addition, Bmp signals characterize adult stem cell niches in other tissues where Hopx-mediated inhibition of Wnt is likely to contribute to stem cell quiescence and to explain the role of Hopx as a tumor suppressor.
Chemokine CXCL12 is proposed to promote multiple steps in growth of primary tumors and progression to metastatic disease in more than 20 different cancers. Functions of CXCL12 previously were believed to be controlled only by receptor CXCR4, but CXCR7 was recently identified as a second receptor for this chemokine. CXCR7 increases tumor formation and metastasis in mouse models, suggesting that this receptor may also be a key target for blocking effects of CXCL12 in cancer. To image activation of CXCR7 in intact cells and living mice, we tested the hypothesis that binding of chemokine ligands to CXCR7 recruits beta-arrestins, a family of cytosolic adapter proteins that interact with many activated chemokine and related seven-transmembrane receptors. Using firefly luciferase protein fragment complementation, we established that chemokine ligands CXCL12 and CXCL11 significantly increase association of CXCR7 and beta-arrestins with preferential interaction of the receptor with beta-arrestin 2. The magnitude of interactions between CXCR7 and beta-arrestin 2 increased over time after treatment with ligands, contrasting with transient association of beta-arrestin 2 and CXCR4. beta-Arrestin 2 increased uptake of CXCL12 in cells expressing CXCR7, emphasizing the functional relevance of the interaction between CXCR7 and beta-arrestin 2. In an orthotopic xenograft model of human breast cancer, we used bioluminescence imaging to quantify changes in the association of CXCR7 and beta-arrestin 2. These studies demonstrate ligand-dependent interactions of CXCR7 with beta-arrestin 2 that promote accumulation of chemokines and establish an imaging assay for the dynamic regulation of CXCR7 by chemokines and candidate therapeutic agents in cell-based assays and living mice.
Seven-transmembrane (G-protein coupled) receptors are key regulators of normal physiology and a large number of diseases, and this family of receptors is the target for almost half of all drugs. Cell culture models suggest that homodimerization and heterodimerization of 7-transmembrane receptors regulate processes including specificity of ligand binding and activation of downstream signaling pathways, making receptor dimerization a critical determinant of receptor biology and a promising new therapeutic target. To monitor receptor dimerization in cell-based assays and living animals, we developed a protein fragment complementation assay based on firefly luciferase to investigate dimerization of chemokine receptors CXCR4 and CXCR7, two 7-transmembrane receptors with central functions in normal development, cancer, and other diseases. Treatment with chemokine ligands and pharmacologic agents produced time- and dose-dependent changes in reporter signal. Chemokines regulated reporter bioluminescence for CXCR4 or CXCR7 homodimers without affecting signals from receptor heterodimers. In a tumor xenograft model of breast cancer, we used bioluminescence imaging to measure changes in receptor homodimerization in response to pharmacologic agents. This technology should be valuable for analyzing function and therapeutic modulation of receptor dimerization in intact cells and living mice.
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