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

Hypoxia potentiates the BMP-2 driven COL2A1 stimulation in human articular chondrocytes via p38 MAPK

International audienc

Chondrogenic potential of bone marrow- and adipose tissue-derived adult human mesenchymal stem cells.

International audienceRegarding cartilage repair, tissue engineering is currently focusing on the use of adult mesenchymal stem cells (MSC) as an alternative to autologous chondrocytes. The potential of stem cells from various tissues to differentiate towards the chondrogenic phenotype has been investigated and it appears that the most common and studied sources are bone marrow (BM) and adipose tissue (AT) for historical and easy access reasons. In addition to three dimensional environment, the presence of member(s) of the transforming growth factor (TGF-β family and low oxygen tension have been reported to promote the in vitro differentiation of MSCs. Our work aimed at characterizing and comparing the degree of chondrogenic differentiation of MSCs isolated from BM and AT cultured in the same conditions. We also further aimed at and at determining whether hypoxia (2% oxygen) could affect the chondrogenic potential of AT-MSCs. Cells were first expanded in the presence of FGF-2, then harvested and centrifuged to allow formation of cell pellets, which were cultured in the presence of TGF-β3 and/or Bone Morphogenetic Protein-2 (BMP-2) and with 2 or 20% oxygen tension, for 24 days. Markers of the chondrocyte (COL2A1, AGC1, Sox9) and hypertrophic chondrocyte (COL10A1, MMP-13) were monitored by real-time PCR and/or by immunohistological staining. Our data show that BMP-2/TGF-β3 combination is the best culture condition to induce the chondrocyte phenotype in pellet cultures of BM and AT-MSCs. Particularly, a switch in the expression of the pre-chondrogenic type IIA form to the cartilage-specific type IIB form of COL2A1 was observed. A parallel increase in gene expression of COL10A1 and MMP-13 was also recorded. However when AT-MSCs were cultured in hypoxia, the expression of markers of hypertrophic chondrocytes decreased when BMP-2/TGF-β3 were present in the medium. Thus it seems that hypoxia participates to the control of AT-MSCs chondrogenesis. Altogether, these cellular model systems will help us to investigate further the potential of different adult stem cells for cartilage engineering