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

Intra-Articular Injection of 2 Different Dosages of Autologous and Allogeneic Bone Marrow- and Umbilical Cord-Derived Mesenchymal Stem Cells Triggers a Variable Inflammatory Response of the Fetlock Joint on 12 Sound Experimental Horses

International audienceOsteoarthritis is a significant and costly cause of pain for both humans and horses. The horse has been identified as a suitable modelfor human osteoarthritis. Regenerative therapy with allogeneic mesenchymal stem cells (MSCs) is a promising treatment, but thesafety of this procedure continues to be debated. The aim of this study is to evaluate the safety of intra-articular injections ofallogeneic MSCs on healthy joints by comparing two different dosages and two different tissue sources, namely, bone marrowand umbilical cord blood, with a placebo treatment on the same individuals. We also assessed the influence of autologous versusallogeneic cells for bone marrow-derived MSC treatment. Twelve clinically sound horses were subjected to injections in their 4fetlock joints. Each of the three fetlocks was administered a different MSC type, and the remaining fetlock was injected withphosphate-buffered saline as a control. Six horses received 10 million cells per joint, and the 6 other horses received 20 millioncells per joint. Clinical and ultrasound monitoring revealed that allogeneic bone marrow-derived MSCs induced significantlymore synovial effusion compared to umbilical cord blood-derived MSCs but no significant difference was noted within thesynovial fluid parameters. The administration of 10 million cells in horses triggered significantly more inflammatory signs thanthe administration of 20 million cells. Mesenchymal stem cell injections induced mild to moderate local inflammatory signscompared to the placebo, with individual variability in the sensitivity to the same line of MSCs. Understanding the behavior ofstem cells when injected alone is a step towards the safer use of new strategies in stem cell therapy, where the use of either MSCsecretome or MSCs combined with biomaterials could enhance their viability and metabolic activit

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

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. 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

Chondrogenic Differentiation of Defined Equine Mesenchymal Stem Cells Derived from Umbilical Cord Blood for Use in Cartilage Repair Therapy

International audienceCartilage engineering is a new strategy for the treatment of cartilage damage due to osteoarthritis or trauma in humans. Racehorses are exposed to the same type of cartilage damage and the anatomical, cellular, and biochemical properties of their cartilage are comparable to those of human cartilage, making the horse an excellent model for the development of cartilage engineering. Human mesenchymal stem cells (MSCs) differentiated into chondrocytes with chondrogenic factors in a biomaterial appears to be a promising therapeutic approach for direct implantation and cartilage repair. Here, we characterized equine umbilical cord blood-derived MSCs (eUCB-MSCs) and evaluated their potential for chondrocyte differentiation for use in cartilage repair therapy. Our results show that isolated eUCB-MSCs had high proliferative capacity and differentiated easily into osteoblasts and chondrocytes, but not into adipocytes. A three-dimensional (3D) culture approach with the chondrogenic factors BMP-2 and TGF-β1 potentiated chondrogenic differentiation with a significant increase in cartilage-specific markers at the mRNA level (Col2a1, Acan, Snorc) and the protein level (type II and IIB collagen) without an increase in hypertrophic chondrocyte markers (Col10a1 and Mmp13) in normoxia and in hypoxia. However, these chondrogenic factors caused an increase in type I collagen, which can be reduced using small interfering RNA targeting Col1a2. This study provides robust data on MSCs characterization and demonstrates that eUCB-MSCs have a great potential for cartilage tissue engineering

Characterization and use of equine bone marrow mesenchymal stem cells in horse cartilage engineering

National audiencePURPOSE: Articular cartilage is of great importance for physiological mobility. The structure and the function of this tissue which is characterized by a poor self-repair capacity are frequently disrupted or damaged upon physical trauma or in degenerative osteoarthritis (OA). Given that musculoskeletal disorders are the leading cause of poor performance or early retirement of sports and race horses treating horses for these disorders is relevant. The proposed therapeutic approach which was first developed for human has the potential of being considered as a pre-clinical step for human medicine because the horse is recognized as an excellent model for the study of articular cartilage disorders in humans. This study aims to improve the Autologous Chondrocytes Implantation (ACI) technique by using Mesenchymal Stem cells (MSCs) from bone marrow (easy to collect in horse) as the cell source. Thus MSCs were first characterized before being cultured with chondrogenic conditions in order to find the combination that best enhances and stabilizes the characteristics of the chondrocyte phenotype in order to perform clinical trials in horse. METHODS: MSCs from equine bone marrow were isolated and expanded in monolayer cultures until 4 passages. These cells were characterized by analyzing their proliferative potential their pluripotency (with microenvironmental stimuli) and their capacity to express MSCs specific phenotypic markers defining the MSCs (flow cytometry). In parallel MSCs differentiation in chondrocytes was accomplished via a combinatory approach based on the association of 3D-culture in type I collagen sponges low oxygen tension with chondrogenic factors (BMP-2 TGF-β1) and RNA interference (siRNA to down-regulate type I collagen and HtrA1 protein expression). Finally an extensive analysis at gene and protein levels corresponding to differentiated dedifferentiated and hypertrophic chondrocyte phenotypes was performed. RESULTS: Our results show a very high proliferation potential of MSCs isolated from equine bone marrow. Furthermore the isolated cells satisfy the various criteria of stem cells definition (pluripotency expression of surface markers). In addition siRNAs targeting equine Col1a1 and Htra1 have been functionally validated. Finally we show that the BMP-2 and TGF-β1 combination strongly induces the differentiation of MSCs in chondrocytes. Thus the combined use of specific culture conditions defined within the laboratory with specific growth factors and siRNAs association leads to the in vitro synthesis of a hyaline type neo-cartilage by chondrocytes which present an optimal phenotypic index comparable to the one established with induction of specific differentiation of human MSCs in chondrocytes and close to the one of differentiated/healthy chondrocytes. CONCLUSIONS: These data represent a first step in the development of equine clinical trials which are planed - to better understand the reaction of cartilage tissue after a few weeks of intra-articular implantation and - to make the proof of concept in a large animal model. This approach will allow us to explore the criteria necessary to begin to consider the development of cell therapy for cartilage repair in humans

Characterization and use of Equine Bone Marrow Mesenchymal Stem Cells in Equine Cartilage Engineering. Study of their Hyaline Cartilage Forming Potential when Cultured under Hypoxia within a Biomaterial in the Presence of BMP-2 and TGF-ß1

International audienceArticular cartilage presents a poor capacity for self-repair. Its structure-function are frequently disrupted or damaged upon physical trauma or osteoarthritis in humans. Similar musculoskeletal disorders also affect horses and are the leading cause of poor performance or early retirement of sport- and racehorses. To develop a therapeutic solution for horses, we tested the autologous chondrocyte implantation technique developed on human bone marrow (BM) mesenchymal stem cells (MSCs) on horse BM-MSCs. This technique involves BM-MSC chondrogenesis using a combinatory approach based on the association of 3D-culture in collagen sponges, under hypoxia in the presence of chondrogenic factors (BMP-2 + TGF-β1) and siRNA to knockdown collagen I and HtrA1. Horse BM-MSCs were characterized before being cultured in chondrogenic conditions to find the best combination to enhance, stabilize, the chondrocyte phenotype. Our results show a very high proliferation of MSCs and these cells satisfy the criteria defining stem cells (pluripotency-surface markers expression). The combination of BMP-2 + TGF-β1 strongly induces the chondrogenic differentiation of MSCs and prevents HtrA1 expression. siRNAs targeting Col1a1 and Htra1 were functionally validated. Ultimately, the combined use of specific culture conditions defined here with specific growth factors and a Col1a1 siRNAs (50 nM) association leads to the in vitro synthesis of a hyaline-type neocartilage whose chondrocytes present an optimal phenotypic index similar to that of healthy, differentiated chondrocytes. Our results lead the way to setting up pre-clinical trials in horses to better understand the reaction of neocartilage substitute and to carry out a proof-of-concept of this therapeutic strategy on a large animal model