Immunophenotyping Reveals the Diversity of Human Dental Pulp Mesenchymal Stromal Cells In vivo and Their Evolution upon In vitro Amplification

International audienceMesenchymal stromal/stem cells (MSCs) from human dental pulp (DP) can be expanded in vitro for cell-based and regenerative dentistry therapeutic purposes. However, their heterogeneity may be a hurdle to the achievement of reproducible and predictable therapeutic outcomes. To get a better knowledge about this heterogeneity, we designed a flow cytometric strategy to analyze the phenotype of DP cells in vivo and upon in vitro expansion with stem cell markers. We focused on the CD31 − cell population to exclude endothelial and leukocytic cells. Results showed that the in vivo CD31 − DP cell population contained 1.4% of CD56 + , 1.5% of CD146 + , 2.4% of CD271 + and 6.3% of MSCA-1 + cells but very few Stro-1 + cells (≤1%). CD56 + , CD146 + , CD271 + , and MSCA-1 + cell subpopulations expressed various levels of these markers. CD146 + MSCA-1 + , CD271 + MSCA-1 + , and CD146 + CD271 + cells were the most abundant DP-MSC populations. Analysis of DP-MSCs expanded in vitro with a medicinal manufacturing approach showed that CD146 was expressed by about 50% of CD56 + , CD271 + , MSCA-1 + , and Stro-1 + cells, and MSCA-1 by 15-30% of CD56 + , CD146 + , CD271 + , and Stro-1 + cells. These ratios remained stable with passages. CD271 and Stro-1 were expressed by <1% of the expanded cell populations. Interestingly, the percentage of CD56 + cells strongly increased from P1 (25%) to P4 (80%) both in all sub-populations studied. CD146 + CD56 + , MSCA-1 + CD56 + , and CD146 + MSCA-1 + cells were the most abundant DP-MSCs at the end of P4. These results established that DP-MSCs constitute a heterogeneous mixture of cells in pulp tissue in vivo and in culture, and that their phenotype is modified upon in vitro expansion. Further studies are needed to determine whether co-expression of specific MSC markers confers DP cells specific properties that could be used for the regeneration of human tissues, including the dental pulp, with standardized cell-based medicinal products

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

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

Control of Collagen Production in Mouse Chondrocytes by Using a Combination of Bone Morphogenetic Protein-2 and Small Interfering RNA Targeting Col1a1 for Hydrogel-Based Tissue-Engineered Cartilage

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Presence of type IIB procollagen in mouse articular cartilage and growth plate is revealed by immuno-histochemical analysis with a novel specific antibody

Type II collagen is the major fibrillar collagen in cartilage. It is synthesized in the form of precursors (procollagens) containing N- and C-terminal propeptides. The two main isoforms of type II procollagen protein are type IIA and type IIB procollagens, generated in a developmentally regulated manner by differential splicing of the primary gene transcript. Isoform IIA contains exon 2 and is produced mainly by chondroprogenitor cells while isoform IIB lacks exon 2 and is produced by differentiated chondrocytes. Thus, expression of IIA and IIB isoforms are reliable markers for identifying the differentiation status of chondrocytes but their biological function in the context of skeletal development is still not yet fully understood. Specific antibodies against IIA and IIB procollagen isoforms are already available. In this study, a synthetic peptide spanning the junction between exon 1 and exon 3 of the murine sequence was used as an immunogen to generate a novel rabbit polyclonal antibody directed against procollagen IIB. Characterization of this antibody by Western-blotting analysis of murine cartilage extracts and ELISA tests demonstrated its specificity to the type IIB isoform. Furthermore, by immunohistochemical studies, this antibody allowed the detection of procollagen IIB in embryonic cartilage as well as in articular cartilage and growth plate of young adult mice. Interestingly, this is the first antibody that has allowed the detection of procollagen IIB at both the intra- and extracellular level. This antibody therefore represents an interesting new tool for monitoring the spatial and temporal distribution of IIB isoforms in skeletal tissues of mouse models and for tracking the trafficking and processing of type IIB procollagen

Combination of bioactive factors and IEIK13 self-assembling peptide hydrogel promotes cartilage matrix production by human nasal chondrocytes

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Evaluation of the biocompatibility and stability of allogeneic tissue-engineered cartilage in humanized mice

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Analysis of collagen expression during chondrogenic induction of human bone marrow mesenchymal stem cells

International audienceAdult mesenchymal stem cells (MSCs) are currently being investigated as an alternative to chondrocytes for repairing cartilage defects. As several collagen types participate in the formation of cartilage-specific extracellular matrix, we have investigated their gene expression levels during MSC chondrogenic induction. Bone marrow MSCs were cultured in pellet in the presence of BMP-2 and TGF-β3 for 24 days. After addition of FGF-2, at the fourth passage during MSC expansion, there was an enhancing effect on specific cartilage gene expression when compared to that without FGF-2 at day 12 in pellet culture. A switch in expression from the pre-chondrogenic type IIA form to the cartilage-specific type IIB form of the collagen type II gene was observed at day 24. A short-term addition of FGF-2 followed by a treatment with BMP-2/TGF-β3 appears sufficient to accelerate chondrogenesis with a particular effect on the main cartilage collagens

Phenotypic Identification of Dental Pulp Mesenchymal Stem/Stromal Cells Subpopulations with Multiparametric Flow Cytometry

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Evaluation of the biocompatibility and stability of allogeneic tissue-engineered cartilage in humanized mice

International audienceArticular cartilage (AC) has poor capacities of regeneration and lesions often lead to osteoarthritis. Current AC reconstruction implies autologous chondrocyte implantation which requires tissue sampling and grafting. An alternative approach would be to use scaffolds containing off-the-shelf allogeneic human articular chondrocytes (HACs). To investigate tolerance of allogeneic HACs by the human immune system, we developed a humanized mouse model implanted with allogeneic cartilage constructs generated in vitro. A prerequisite of the study was to identify a scaffold that would not provoke inflammatory reaction in host. Therefore, we first compared the response of hu-mice to two biomaterials used in regenerative medicine, collagen sponge and agarose hydrogel. Four weeks after implantation in hu-mice, acellular collagen sponges, but not acellular agarose hydrogels, showed positive staining for CD3 (T lymphocytes) and CD68 (macrophages), suggesting that collagen scaffold elicits weak inflammatory reaction. These data led us to deepen our evaluation of the biocompatibility of allogeneic tissue-engineered cartilage by using agarose as scaffold. Agarose hydrogels were combined with allogeneic HACs to reconstruct cartilage in vitro. Particular attention was paid to HLA-A2 compatibility between HACs to be grafted and immune human cells of hu-mice: HLA-A2+ or HLA-A2- HACs agarose hydrogels were cultured in the presence of a chondrogenic cocktail and implanted in HLA-A2+ hu-mice. After four weeks implantation and regardless of the HLA-A2 phenotype, chondrocytes were well-differentiated and produced cartilage matrix in agarose. In addition, no sign of T-cell or macrophage infiltration was seen in the cartilaginous constructs and no significant increase in subpopulations of T lymphocytes and monocytes was detected in peripheral blood and spleen. We show for the first time that humanized mouse represents a useful model to investigate human immune responsiveness to tissue-engineered cartilage and our data together indicate that allogeneic cartilage constructs can be suitable for cartilage engineering