Comparison of the Chondrogenic Potential of Mesenchymal Stem Cells Derived from Bone Marrow and Umbilical Cord Blood Intended for Cartilage Tissue Engineering

International audienceOsteoarthritis (OA) remains incurable in humans or horses and mesenchymal stromal/stem cells (MSCs) represent an attractive solution for producing a neocartilage substitute. However, the best MSC source still needs to be identified. This study compared the chondrogenic potential of equine MSCs derived from bone marrow (BM) and umbilical cord blood (UCB), at their undifferentiated status to check if one cell source is better proned, and after chondrogenic-induced differentiation. Chondrogenesis was induced by culture in collagen scaffold with BMP-2 + TGF-ß1 in hypoxia or normoxia. MSCs chondrogenic potential was evaluated using the mRNA and corresponding protein levels for osteogenic, hypertrophic and chondrogenic markers. MSCs characterization demonstrated that BM- and UCB-MSCs differ in proliferation and tripotencies. At undifferentiated status, they also showed differences in their expression of osteogenic, chondrogenic and hypertrophic markers. Upon chondrogenesis induction, both MSCs sources exhibited increased chondrogenic expression and produce an extracellular matrix (ECM) of better quality in hypoxia, although collagen I remained expressed. UCB-MSCs produced higher amounts of collagen II, particularly its IIB isoform, than BM-MSCs, but also collagen I and Htra1, regardless of the oxygen condition. Finally, immunohistochemistry revealed that the BM-MSCs synthesized an ECM of higher quality, regarding the more homogenous distribution of type IIB collagen, compared to UCB-MSCs. Considering collagen I as the major undesirable component in the neo-synthesis of in vitro cartilage, we recommend using BM-MSCs for horse cartilage engineering

Improvement of the Chondrocyte-Specific Phenotype upon Equine Bone Marrow Mesenchymal Stem Cell Differentiation: Influence of Culture Time, Transforming Growth Factors and Type I Collagen siRNAs on the Differentiation Index

Articular 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. Similar musculoskeletal disorders also affect horses and 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. This study aims to improve the autologous chondrocyte implantation technique by using equine mesenchymal stem cells (MSCs) from bone marrow differentiated into chondrocytes that can be implanted in the chondral lesion. The optimized protocol relies on culture under hypoxia within type I/III collagen sponges. Here, we explored three parameters that influence MSC differentiation: culture times, growth factors and RNA interference strategies. Our results suggest first that an increase in culture time from 14 to 28 or 42 days lead to a sharp increase in the expression of chondrocyte markers, notably type II collagen (especially the IIB isoform), along with a concomitant decrease in HtrA1 expression. Nevertheless, the expression of type I collagen also increased with longer culture times. Second, regarding the growth factor cocktail, TGF-β3 alone showed promising result but the previously tested association of BMP-2 and TGF-β1 better limits the expression of type I collagen. Third, RNA interference targeting Col1a2 as well as Col1a1 mRNA led to a more significant knockdown, compared with a conventional strategy targeting Col1a1 alone. This chondrogenic differentiation strategy showed a strong increase in the Col2a1:Col1a1 mRNA ratio in the chondrocytes derived from equine bone marrow MSCs, this ratio being considered as an index of the functionality of cartilage. These data provide evidence of a more stable chondrocyte phenotype when combining Col1a1 and Col1a2 siRNAs associated to a longer culture time in the presence of BMP-2 and TGF-β1, opening new opportunities for preclinical trials in the horse. In addition, because the horse is an excellent model for human articular cartilage disorders, the equine therapeutic approach developed here can also serve as a preclinical step for human medicine

P608 : Déficit en cholestérol et altération de la voie Sonic Hedgehog dans la trisomie 18

International audienc

P608 : Déficit en cholestérol et altération de la voie Sonic Hedgehog dans la trisomie 18

International audienc

The secretome of equine bone marrow-derived mesenchymal stem cells enhanced regenerative phenotype of equine articular chondrocytes

International audienceEquine osteoarthritis (OA) leads to cartilage degradation with impaired animal well-being, premature cessation of sport activity, and financial losses. Many hopes lie on mesenchymal stem cell (MSC)-based therapies to repair cartilage, but these techniques face limitations inherent to cell itself. Soluble mediators and extracellular vesicles (EVs) secreted by MSCs are the alternative to overcome those limitations while preserving MSC restorative properties (Cosenza et al., 2017). The aim of this study was to assess the effect of equine bone marrow MSC secretome on equine articular chondrocytes (eACs) by indirect co-culture and/or MSC-conditioned media (CM) containing EVs. The expression of healthy cartilage/OA, and proliferation markers was evaluated in eACs cultured in monolayers or as 3D organoids. In vitro repair experiments with MSC-CM were made to evaluate the proliferation and migration of eACs. Our results demonstrated that MSC secretome influences eAC phenotype by increasing cartilage functionality markers such as Prg4, collagens and proliferation associated molecule in a greater way than MSCs. Cell migration was also enhanced, which could delay OA final outcomes. To confirm the presence of nanosized EVs in MSC-CM, nanoparticle tracking assay, transmission electron microscopy and exosome-specific markers detection were assessed. Lastly, fluorescence staining revealed effective uptake of exosomes by eAC. This study makes acellular therapy an appealing strategy to improve equine OA treatments. However, MSC secretome contains a wide variety of soluble mediators and small EVs, such as exosomes, and further investigation must be performed to understand the mechanisms occurring behind these promising effects

The secretome of equine bone marrow-derived mesenchymal stem cells enhanced regenerative phenotype of equine articular chondrocytes

International audienceEquine osteoarthritis (OA) is a sequential disease which leads to cartilage degradation and painful bone frictions. It induces impaired animal well-being, premature cessation of sport activity, and financial losses. Fibrocartilage synthesis occurring during cartilage destruction is a physiological response, allowing bone protection but reducing tissue mechanical resistance. To date, there is a lack of curative therapies. Mesenchymal stem cell (MSC)-based therapies are promising for cartilage repair, but face limitations inherent to cell itself which can be overcome using their secretomethrough acellular therapy approaches.To understand the effects of equine bone marrow (BM-)MSC secretome on equine articular chondrocytes (eAC) phenotype, indirect co-culture experiments were first performed. Then, we wantedto recapitulate the effects we observed in co-culture on eAC using the MSC-conditioned medium (CM) to make acellular therapy conceivable. We assessed hyaline cartilage and fibrocartilage markers at the transcription and protein levels, and evaluated eAC migratory capacities, which are of interest during OA therapy to favor the filling of the cartilage defects. To optimize immunomodulation properties of MSC secretome for future experiments, MSC priming relevance with interleukin (IL)1-β was evaluated. Suspected to be the principal vectors of the effects observed, exosomes were isolated through chemical precipitation and then characterized to confirm their nature

Bone Marrow MSC Secretome Increases Equine Articular Chondrocyte Collagen Accumulation and Their Migratory Capacities

International audienceEquine osteoarthritis (OA) leads to cartilage degradation with impaired animal well-being, premature cessation of sport activity, and financial losses. Mesenchymal stem cell (MSC)-based therapies are promising for cartilage repair, but face limitations inherent to the cell itself. Soluble mediators and extracellular vesicles (EVs) secreted by MSCs are the alternatives to overcome those limitations while preserving MSC restorative properties. The effect of equine bone marrow MSC secretome on equine articular chondrocytes (eACs) was analyzed with indirect co-culture and/or MSC-conditioned media (CM). The expression of healthy cartilage/OA and proliferation markers was evaluated in eACs (monolayers or organoids). In vitro repair experiments with MSC-CM were made to evaluate the proliferation and migration of eACs. The presence of nanosized EVs in MSC-CM was appraised with nanoparticle tracking assay and transmission electron microscopy. Our results demonstrated that the MSC secretome influences eAC phenotype by increasing cartilage functionality markers and cell migration in a greater way than MSCs, which could delay OA final outcomes. This study makes acellular therapy an appealing strategy to improve equine OA treatments. However, the MSC secretome contains a wide variety of soluble mediators and small EVs, such as exosomes, and further investigation must be performed to understand the mechanisms occurring behind these promising effects

The secretome of equine bone marrow-derived mesenchymal stem cells enhanced regenerative phenotype of equine articular chondrocytes

National audienceEquine osteoarthritis (OA) leads to cartilage degradation with impaired animal well-being, premature cessation of sport activity, and financial losses. Many hopes lie on mesenchymal stem cell (MSC)-based therapies to repair cartilage, but these techniques face limitations inherent to cell itself. Soluble mediators and extracellular vesicles (EVs) secreted by MSCs are the alternative to overcome those limitations while preserving MSC restorative properties (Cosenza et al., 2017). The aim of this study was to assess the effect of equine bone marrow MSC secretome on equine articular chondrocytes (eACs) by indirect co-culture and/or MSC-conditioned media (CM) containing EVs. The expression of healthy cartilage/OA, and proliferation markers was evaluated in eACs cultured in monolayers or as 3D organoids. In vitro repair experiments with MSC-CM were made to evaluate the proliferation and migration of eACs. Our results demonstrated that MSC secretome influences eAC phenotype by increasing cartilage functionality markers such as Prg4, collagens and proliferation associated molecule in a greater way than MSCs. Cell migration was also enhanced, which could delay OA final outcomes. To confirm the presence of nanosized EVs in MSC-CM, nanoparticle tracking assay, transmission electron microscopy and exosome-specific markers detection were assessed. Lastly, fluorescence staining revealed effective uptake of exosomes by eAC. This study makes acellular therapy an appealing strategy to improve equine OA treatments. However, MSC secretome contains a wide variety of soluble mediators and small EVs, such as exosomes, and further investigation must be performed to understand the mechanisms occurring behind these promising effects

Resource-Based Data Availability for Erbb2-Driven Breast Cancer in Asian Women: Experts' Opinion

International audienceArticular cartilage is frequently altered upon trauma or in osteoarthritis (OA), a degenerative disease that is currently incurable. Cartilage tissue engineering/cell therapy offer new insights to cure these articular disorders. Since the first generation of cartilage tissue engineering led to a poor quality cartilage rich in the hyaline cartilage atypical type I collagen, it has been followed with improved generations. One way to improve these strategies was to diversify the cell type used. Mesenchymal stem cells (MSC) are considered as an interesting cell type in order to produce a hyaline cartilage substitute. This study aimed to compare umbilical cord blood (UCB) and bone marrow (BM) derived MSC. BM and UCB MSC were isolated and then amplified in monolayer culture. We characterized MSC by assessing their proliferative and multipotence capacities, and the presence of cluster of differentiation (CD) characteristic of MSC. Then, we compared MSC at their basal state and after a chondrogenic differentiation, which consisted of culture in hypoxia or normoxia in a biomaterial, with chondrogenic factors (BMP-2, TGF-ß1). Criteria used to compare MSC were the mRNA level and protein amount of several osteogenic (osteocalcin/Runx2), hypertrophic (type X collagen) and chondrogenic markers (type II collagen). Furthermore, we analysed the extracellular matrix(ECM) composition/structure by immunochemistry

RNA interference and BMP-2 stimulation allows equine chondrocytes redifferentiation in 3D-Hypoxia cell culture model: application for matrix-induced autologous chondrocyte implantation

As in humans, osteoarthritis (OA) causes considerable economic loss to the equine industry. New hopes for cartilage repair have emerged with the matrix-associated autologous chondrocyte implantation (MACI). Nevertheless, its limitation is due to the dedifferentiation occurring during the chondrocyte amplification phase, leading to the loss of its capacity to produce a hyaline extracellular matrix (ECM). To enhance the MACI therapy efficiency, we have developed a strategy for chondrocyte redifferentiation, and demonstrated its feasibility in the equine model. Thus, to mimic the cartilage microenvironment, the equine dedifferentiated chondrocytes were cultured in type I/III collagen sponges for 7 days under hypoxia in the presence of BMP-2. In addition, chondrocytes were transfected by siRNA targeting Col1a1 and Htra1 mRNAs, which are overexpressed during dedifferentiation and OA. To investigate the quality of the neo-synthesized ECM, specific and atypical cartilage markers were evaluated by RT-qPCR and Western blot. Our results show that the combination of 3D hypoxia cell culture, BMP-2 (Bone morphogenetic protein-2), and RNA interference, increases the chondrocytes functional indexes (Col2a1/Col1a1, Acan/Col1a1), leading to an effective chondrocyte redifferentiation. These data represent a proof of concept for this process of application, in vitro, in the equine model, and will lead to the improvement of the MACI efficiency for cartilage tissue engineering therapy in preclinical/clinical trials, both in equine and human medicine