A standardized procedure to obtain mesenchymal stem/stromal cells from minimally manipulated dental pulp and Wharton’s jelly samples

Transplantation of mesenchymal stem/stromal cells (MSCs) has emerged as an effective method to treat diseased or damaged organs and tissues, and hundreds of clinical trials using MSCs are currently under way to demonstrate the validity of such a therapeutic approach. However, most MSCs used for clinical trials are prepared in research laboratories with insufficient manufacturing quality control.In particular, laboratories lack standardized procedures for in vitro isolation of MSCs from tissue samples, resulting in heterogeneous populations of cells and variable experimental and clinical results. MSCs are now referred to as Human Cellular Tissue-based Products or Advanced Therapy Medicinal Products, and guidelines from the American Code of Federal Regulation of the Food and Drug Administration (21 CFR Part 1271) and from the European Medicines Agency (European Directive 1394/2007) define requirements for appropriate production of these cells. These guidelines, commonly called “Good Manufacturing Practices” (GMP), include recommendations about laboratory cell culture procedures to ensure optimal reproducibility, efficacy and safety of the final medicinal product. In particular, the Food and Drug Administration divides ex vivo cultured cells into “minimally” and “more than minimally” manipulated samples, in function of the use or not of procedures “that might alter the biological features of the cells”. Today, minimal manipulation conditions have not been defined for the collection and isolation of MSCs (Torre et al. 2015)(Ducret et al. 2015).Most if not all culture protocols that have been reported so far are unsatisfactory, because of the use of xeno- or allogeneic cell culture media, enzymatic treatment and long-term cell amplification that are known to alter the quality of MSCs. The aim of this study was to describe a standardized procedure for recovering MSCs with minimal handling from two promising sources, the dental pulp (DP) and the Wharton’s jelly (WJ) of the umbilical cord. The quality and homogeneity of the expanded cell populations were assessed by using flow cytometry with criteria that go beyond the International Society of Cellular Therapy (ISCT) guidelines for MSC characterization

Expression of Semaphorin-3A and its receptors in endochondral ossification: potential role in skeletal development and innervation.

Bone tissue is densely innervated, and there is increasing evidence for a neural control of bone metabolism. Semaphorin-3A is a very important regulator of neuronal targeting in the peripheral nervous system as well as in angiogenesis, and knockout of the Semaphorin-3A gene induces abnormal bone and cartilage development. We analyzed the spatial and temporal expression patterns of Semaphorin-3A signaling molecules during endochondral ossification, in parallel with the establishment of innervation. We show that osteoblasts and chondrocytes differentiated in vitro express most members of the Semaphorin-3A signaling system (Semaphorin-3A, Neuropilin-1, and Plexins-A1 and -A2). In vitro, osteoclasts express most receptor chains but not the ligand. In situ, these molecules are all expressed in the periosteum and by resting, prehypertrophic and hypertrophic chondrocytes in ossification centers before the onset of neurovascular invasion. They are detected later in osteoblasts and also osteoclasts, with differences in intensity and regional distribution. Semaphorin-3A and Neuropilin-1 are also expressed in the bone marrow. Plexin-A3 is not expressed by bone cell lineages in vitro. It is detected early in the periosteum and hypertrophic chondrocytes. After the onset of ossification, this chain is restricted to a network of cell processes in close vicinity to the cells lining the trabeculae, similar to the pattern observed for neural markers at the same stages. After birth, while the density of innervation decreases, Plexin-A3 is strongly expressed by blood vessels on the ossification front. In conclusion, Semaphorin-3A signaling is present in bone and seems to precede or coincide at the temporal but also spatial level with the invasion of bone by blood vessels and nerve fibers. Expression patterns suggest Plexin-A3/Neuropilin-1 as a candidate receptor in target cells for the regulation of bone innervation by Semaphorin-3A

Improvement of the chondrocyte-specific phenotype upon equine bone marrow mesenchymal stem cell differentiation. Influence of TGF-ß1 or TGF-ß3, associated with BMP-2 and type I collagen siRNAs

International audienceArticular cartilage is a tissue characterized by its poor intrinsic capacity for self-repair. This tissue is frequently altered upon trauma or in osteoarthritis (OA), a degenerative disease that is currently incurable. Consequently, cartilage markers, such as type II collagen, are degraded whereas atypic molecules, such as type I collagen, are newly synthetized. Another essential phenomenon occurring in OA is the upregulation of HtrA1, a serine protease targeting upstream receptors of signalling pathways involved in the synthesis of articular cartilage markers. OA incurs considerable economic loss for the equine sector. In the view to develop new therapies for humans and horses, significant progress in tissue engineering has led to the emergence of new generations of cartilage therapy. Matrix-associated autologous chondrocyte implantation is an advanced 3D cell-based therapy that holds promise for cartilage repair. The aim of this study is to improve the autologous chondrocyte implantation strategy by enhancing the chondrogenic differentiation of mesenchymal stem cells (MSCs) in order to increase the type II collagen/ type I collagen ratio

ZAK beta is activated by cellular compression and mediates contraction-induced MAP kinase signaling in skeletal muscle

Mechanical inputs give rise to p38 and JNK activation, which mediate adaptive physiological responses in various tissues. In skeletal muscle, contraction-induced p38 and JNK signaling ensure adaptation to exercise, muscle repair, and hypertrophy. However, the mechanisms by which muscle fibers sense mechanical load to activate this signaling have remained elusive. Here, we show that the upstream MAP3K ZAK beta is activated by cellular compression induced by osmotic shock and cyclic compression in vitro, and muscle contraction in vivo. This function relies on ZAKO's ability to recognize stress fibers in cells and Z-discs in muscle fibers when mechanically perturbed. Consequently, ZAK-deficient mice present with skeletal muscle defects characterized by fibers with centralized nuclei and progressive adaptation towards a slower myosin profile. Our results highlight how cells in general respond to mechanical compressive load and how mechanical forces generated during muscle contraction are translated into MAP kinase signaling.Peer reviewe

Tendon collagen fibrillogenesis is a multistep assembly process as revealed by quick-freezing and freeze-substitution.

International audienceThe ultrastructure of chick embryo tendons has been examined after quick-freezing by liquid helium and freeze-substitution. Several stages of collagen assemblies were observed: intracellular packing of SLS-like aggregates surrounded by membrane containing areas with a clathrin coat; fine non cross-striated filaments connecting the cell membrane at 1 pole of the cells and collagen fibrils; tufts of filaments directly linked to collagen fibrils. This study reveals that some stages are more constant and abundant than supposed (the intracellular SLS-like aggregates) and that other extracellular assemblies that were hypothesized but usually badly preserved by conventional electron microscopy are clearly captured by the method.The ultrastructure of chick embryo tendons has been examined after quick-freezing by liquid helium and freeze-substitution. Several stages of collagen assemblies were observed: intracellular packing of SLS-like aggregates surrounded by membrane containing areas with a clathrin coat; fine non cross-striated filaments connecting the cell membrane at 1 pole of the cells and collagen fibrils; tufts of filaments directly linked to collagen fibrils. This study reveals that some stages are more constant and abundant than supposed (the intracellular SLS-like aggregates) and that other extracellular assemblies that were hypothesized but usually badly preserved by conventional electron microscopy are clearly captured by the method

Gene expression analysis in cartilage by in situ hybridization.

International audienceIn situ hybridization allows detection and localization of specific nucleic acid sequences directly within a cell or tissue. We present an in situ hybridization protocol using double-stranded DNA or single-stranded RNA probes labeled with [32P] to localize and visualize the temporal and spatial distribution of cartilage-characteristic mRNAs. Probes labeled with this high-energy isotope provide good resolution at the tissue level with relatively low background; as a result of the probes that can be obtained that have a higher specificity to emulsion activity, very short exposure times are required.In situ hybridization allows detection and localization of specific nucleic acid sequences directly within a cell or tissue. We present an in situ hybridization protocol using double-stranded DNA or single-stranded RNA probes labeled with [32P] to localize and visualize the temporal and spatial distribution of cartilage-characteristic mRNAs. Probes labeled with this high-energy isotope provide good resolution at the tissue level with relatively low background; as a result of the probes that can be obtained that have a higher specificity to emulsion activity, very short exposure times are required

RNA extraction from cartilage.

International audienceThe direct isolation of RNA from cartilage has often proved difficult owing to a number of factors. Cartilage has a low cell content and contains an extracellular matrix rich in proteoglycans, which copurify with the RNA as they are large and negatively charged macromolecules. In our laboratory, we are interested in searching for genes differentially expressed in chondrocytes in diverse in vivo situations, for instance during maturation of chondrocytes in the growth plate or during cartilage degeneration. We found that treatment by proteinase K in 1 M guanidinium isothiocyanate prior to cesium trifluoroacetate ultracentrifugation was crucial to increase the yield and purity of RNA extracted from cartilage matrix. This protocol indeed led to reproducible patterns of differential display reverse transcriptase-polymerase chain reaction (RT-PCR) and should be useful for identifying genes differentially expressed by chondrocytes in situ.The direct isolation of RNA from cartilage has often proved difficult owing to a number of factors. Cartilage has a low cell content and contains an extracellular matrix rich in proteoglycans, which copurify with the RNA as they are large and negatively charged macromolecules. In our laboratory, we are interested in searching for genes differentially expressed in chondrocytes in diverse in vivo situations, for instance during maturation of chondrocytes in the growth plate or during cartilage degeneration. We found that treatment by proteinase K in 1 M guanidinium isothiocyanate prior to cesium trifluoroacetate ultracentrifugation was crucial to increase the yield and purity of RNA extracted from cartilage matrix. This protocol indeed led to reproducible patterns of differential display reverse transcriptase-polymerase chain reaction (RT-PCR) and should be useful for identifying genes differentially expressed by chondrocytes in situ

Expression of simian virus 40 large T (tumor) oncogene in mouse chondrocytes induces cell proliferation without loss of the differentiated phenotype.

We have infected primary embryonic mouse limb chondrocytes with a retrovirus carrying simian virus 40 early regions and have obtained a monoclonal mouse chondrocyte line, MC615, that was able to grow on culture dishes for at least 7 months and 20 passages. MC615 cells show expression of simian virus 40 large T (tumor) antigen and express markers characteristic of cartilage in vivo, such as types II, IX, and XI collagen, as well as cartilage aggrecan and link protein. These data show that cell growth induced by large T oncogene expression does not prevent the maintenance of the chondrocytic phenotype

[Differentiation of adult human mesenchymal stem cells: Chondrogenic effect of BMP-2.]

International audienceArticular cartilage is essential for the motion of the skeleton. However, this tissue is unable to spontaneously repair once injured, since it is avascular and aneural. Numerous repair strategies are developed, but they do not lead to a functional tissue and research into cartilage repair focuses now on tissue engineering technics. Adult mesenchymal stem cells (MSC), present in various tissues, have the potential to differentiate into chondrocytes in vitro in response to specific growth factors. The members of the transforming growth factor beta, among them the bone morphogenetic protein (BMP)-2, appear very promising inducers in this context. BMP-2 favours chondrogenic expression, in particular expression of type IIB collagen, the cartilage-specific isoform of this collagen. Therefore, collagen type IIB is a good indicator of the differentiation state of MSC. However, since BMP-2 has also osteogenic properties, it is critical to differentially control chondrogenic and osteogenic properties of BMP-2 when used with MSC. Strategies for this control are presented in this review. Most likely, this is the combination of growth factors such as BMP-2 with biomaterials that will lead to the successful use of MSC for cartilage repair.Articular cartilage is essential for the motion of the skeleton. However, this tissue is unable to spontaneously repair once injured, since it is avascular and aneural. Numerous repair strategies are developed, but they do not lead to a functional tissue and research into cartilage repair focuses now on tissue engineering technics. Adult mesenchymal stem cells (MSC), present in various tissues, have the potential to differentiate into chondrocytes in vitro in response to specific growth factors. The members of the transforming growth factor beta, among them the bone morphogenetic protein (BMP)-2, appear very promising inducers in this context. BMP-2 favours chondrogenic expression, in particular expression of type IIB collagen, the cartilage-specific isoform of this collagen. Therefore, collagen type IIB is a good indicator of the differentiation state of MSC. However, since BMP-2 has also osteogenic properties, it is critical to differentially control chondrogenic and osteogenic properties of BMP-2 when used with MSC. Strategies for this control are presented in this review. Most likely, this is the combination of growth factors such as BMP-2 with biomaterials that will lead to the successful use of MSC for cartilage repair