Chondrogenesis is a process involving stem-cell differentiation through the coordinated effects of growth/differentiation factors and extracellular matrix (ECM) components. Recently, mesenchymal stem cells (MSCs) were found within the cartilage, which constitutes a specific niche composed of ECM proteins with unique features. Therefore, we hypothesized that the induction of MSC differentiation towards chondrocytes might be induced and/or influenced by molecules from the microenvironment. Using microarray analysis, we previously identified genes that are regulated during MSC differentiation towards chondrocytes. In this study, we wanted to precisely assess the differential expression of genes associated with the microenvironment using a large-scale real-time PCR assay, according to the simultaneous detection of up to 384 mRNAs in one sample. Chondrogenesis of bone-marrow-derived human MSCs was induced by culture in micropellet for various periods of time. Total RNA was extracted and submitted to quantitative RT-PCR. We identified molecules already known to be involved in attachment and cell migration, including syndecans, glypicans, gelsolin, decorin, fibronectin, and type II, IX and XI collagens. Importantly, we detected the expression of molecules that were not previously associated with MSCs or chondrocytes, namely metalloproteases (MMP-7 and MMP-28), molecules of the connective tissue growth factor (CTGF); cef10/cyr61 and nov (CCN) family (CCN3 and CCN4), chemokines and their receptors chemokine CXC motif ligand (CXCL1), Fms-related tyrosine kinase 3 ligand (FlT3L), chemokine CC motif receptor (CCR3 and CCR4), molecules with A Disintegrin And Metalloproteinase domain (ADAM8, ADAM9, ADAM19, ADAM23, A Disintegrin And Metalloproteinase with thrombospondin type 1 motif ADAMTS-4 and ADAMTS-5), cadherins (4 and 13) and integrins (α4, α7 and β5). Our data suggest that crosstalk between ECM components of the microenvironment and MSCs within the cartilage is responsible for the differentiation of MSCs into chondrocytes.α-MEM = α-minimum essential medium; ADAM = A Disintegrin And Metalloproteinase molecule; ADAMTS = A Disintegrin And Metalloproteinase with thrombospondin type 1 motif; ALCAM = Activated leukocyte cell adhesion molecule; b-FGF = basic fibroblast growth factor; BSA = bovine serum albumin; CAM = cell-adhesion molecule; CCL = chemokine CC motif ligand; CCN = CTGF; cef10/cyr61 and nov; CCR = chemokine CC motif receptor; COMP = cartilage oligomeric matrix protein; Ct = threshold cycle; CTGF = connective tissue growth factor; CXCL = chemokine CXC motif ligand; CXCR = chemokine CXC motif receptor; CYR = cysteine-rich angiogenic inducer; DMEM = Dulbecco's modified Eagle's medium; ECM = extracellular matrix; EDTA = ethylene diamine tetracetic acid; FACS = fluorescence-activated cell sorter; FBS = fetal bovine serum; FlT3L = Fmsrelated tyrosine kinase 3 ligand; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; hBMP = human bone morphogenetic protein; Hg = Hedgehog; ICAM = intercellular cell adhesion molecu...
The mechanism of cell entry and intracellular fate of a gene transfer vector composed of a receptor-targeting, DNA-condensing peptide, RGD-oligolysine, a luciferase encoding plasmid DNA (pDNA) and a cationic liposome was examined. We demonstrate by confocal microscopy, electron microscopy and subcellular fractionation that the major mechanism of entry of the vector is endocytic. The vector complex rapidly (5 min) internalizes into early endosomes, then late endosomes and lysosomes. Entry involves, at least in part, clathrin-coated pit-mediated endocytosis since different conditions or drugs known to influence this pathway modify both uptake of pDNA and its expression. The observed increase in expression with addition of a lip some
IntroductionEfficient gene transfer into T lymphocytes may allow the treatment of several genetic dysfunctions of the hematopoietic system, such as severe combined immunodeficiency, 1,2 and the development of novel therapeutic strategies for diseases such as cancers and acquired immunodeficiency syndrome. 3 To reach this goal, it is essential to preserve the functional properties of the transduced cells, that is, their capacity to appropriately react on stimulation of the immune system. Therefore, only minimal ex vivo manipulation of the cells should be performed during gene transfer because growth factor/cytokine combinations, used to force cell proliferation, often lead to skewing of cell populations and may alter their ability to respond to novel antigens. 4,5 Ideally, gene delivery should be best achieved in vivo to minimize contacts of target cells with nonphysiologic cell culture reagents that may induce differentiation or proliferation.Vectors derived from retroviruses are probably among the most suitable tools to achieve a long-term gene transfer because they allow stable integration of a transgene and its propagation in daughter cells. To date, vectors derived from oncoretroviruses such as murine leukemia viruses (MLVs) have been widely used for gene transfer into human T cells, 6 essentially because of the simplicity of their manipulation. Perhaps one of the most important drawbacks associated with the use of such vectors is their inability to transduce nonproliferating target cells. Indeed, following internalization of the vector into the target cell cytoplasm and reverse transcription, transport of the preintegration complex to the nucleus requires the breakdown of the nuclear membranes during mitosis. 7,8 This provides a formidable barrier to the use of MLV-based vectors in the many gene therapy protocols for which target cells are quiescent or for which induction of cell proliferation is to be avoided. Thus, the recent emergence of lentiviral vectors may provide a valuable alternative to overcome this problem owing to the lentivirus mechanism that allows mitosis-independent nuclear import of the preintegration complex and infection of nonproliferating cells. [9][10][11] Several studies have now established the capacity of these vectors derived from human immunodeficiency virus 1 (HIV-1) to transduce various types of nonproliferating cells both in vitro and in vivo. 12 However, some cell types that are important gene therapy targets are refractory to gene transfer with lentiviral vectors, despite the most recent improvements brought into their structures. [13][14][15][16] This includes, in particular, early progenitor hematopoietic stem cells in G 0 , 17 monocytes, 18,19 and resting T lymphocytes. 14 That the parental virus, HIV-1, can enter into resting T lymphocytes but does not replicate, [20][21][22][23][24] has been attributed to multiple post-entry blocks. This includes, in particular, (1) For personal use only. on May 12, 2018. by guest www.bloodjournal.org From lack of adenosine triphosphat...
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