Jean-Marc Brondello

Cartilage Tissue Engineering: Towards a Biomaterial-Assisted Mesenchymal Stem Cell Therapy

Injuries to articular cartilage are one of the most challenging issues of musculoskeletal medicine due to the poor intrinsic ability of this tissue for repair. Despite progress in orthopaedic surgery, the lack of efficient modalities of treatment for large chondral defects has prompted research on tissue engineering combining chondrogenic cells, scaffold materials and environmental factors. The aim of this review is to focus on the recent advances made in exploiting the potentials of cell therapy for cartilage engineering. These include: 1) defining the best cell candidates between chondrocytes or multipotent progenitor cells, such as multipotent mesenchymal stromal cells (MSC), in terms of readily available sources for isolation, expansion and repair potential; 2) engineering biocompatible and biodegradable natural or artificial matrix scaffolds as cell carriers, chondrogenic factors releasing factories and supports for defect filling, 3) identifying more specific growth factors and the appropriate scheme of application that will promote both chondrogenic differentiation and then maintain the differentiated phenotype overtime and 4) evaluating the optimal combinations that will answer to the functional demand placed upon cartilage tissue replacement in animal models and in clinics. Finally, some of the major obstacles generally encountered in cartilage engineering are discussed as well as future trends to overcome these limiting issues for clinical applications.

MitoCeption as a new tool to assess the effects of mesenchymal stem/stromal cell mitochondria on cancer cell metabolism and function

Mitochondrial activity is central to tissue homeostasis. Mitochondria dysfunction constitutes a hallmark of many genetic diseases and plays a key role in tumor progression. The essential role of mitochondria, added to their recently documented capacity to transfer from cell to cell, obviously contributes to their current interest. However, determining the proper role of mitochondria in defined biological contexts was hampered by the lack of suitable experimental tools. We designed a protocol (MitoCeption) to directly and quantitatively transfer mitochondria, isolated from cell type A, to recipient cell type B. We validated and quantified the effective mitochondria transfer by imaging, fluorescence-activated cell sorting (FACS) and mitochondrial DNA analysis. We show that the transfer of minute amounts of mesenchymal stem/stromal cell (MSC) mitochondria to cancer cells, a process otherwise occurring naturally in coculture, results in cancer cell enhanced oxidative phosphorylation (OXPHOS) activity and favors cancer cell proliferation and invasion. The MitoCeption technique, which can be applied to different cell systems, will therefore be a method of choice to analyze the metabolic modifications induced by exogenous mitochondria in host cells.

Concerted stimuli regulating osteo-chondral differentiation from stem cells: phenotype acquisition regulated by microRNAs.

AbstractBone and cartilage are being generated de novo through concerted actions of a plethora of signals. These act on stem cells (SCs) recruited for lineage-specific differentiation, with cellular phenotypes representing various functions throughout their life span. The signals are rendered by hormones and growth factors (GFs) and mechanical forces ensuring proper modelling and remodelling of bone and cartilage, due to indigenous and programmed metabolism in SCs, osteoblasts, chondrocytes, as well as osteoclasts and other cell types (eg T helper cells).This review focuses on the concerted action of such signals, as well as the regulatory and/or stabilizing control circuits rendered by a class of small RNAs, designated microRNAs. The impact on cell functions evoked by transcription factors (TFs) via various signalling molecules, also encompassing mechanical stimulation, will be discussed featuring microRNAs as important members of an integrative system. The present approach to cell differentiation in vitro may vastly influence cell engineering for in vivo tissue repair.

Cell Connections by Tunneling Nanotubes: Effects of Mitochondrial Trafficking on Target Cell Metabolism, Homeostasis, and Response to Therapy

Intercellular communications play a major role in tissue homeostasis and responses to external cues. Novel structures for this communication have recently been described. These tunneling nanotubes (TNTs) consist of thin-extended membrane protrusions that connect cells together. TNTs allow the cell-to-cell transfer of various cellular components, including proteins, RNAs, viruses, and organelles, such as mitochondria. Mesenchymal stem cells (MSCs) are both naturally present and recruited to many different tissues where their interaction with resident cells via secreted factors has been largely documented. Their immunosuppressive and repairing capacities constitute the basis for many current clinical trials. MSCs recruited to the tumor microenvironment also play an important role in tumor progression and resistance to therapy. MSCs are now the focus of intense scrutiny due to their capacity to form TNTs and transfer mitochondria to target cells, either in normal physiological or in pathological conditions, leading to changes in cell energy metabolism and functions, as described in this review.

Cellular Senescence is a Common Characteristic Shared by Preneoplasic and Osteo-Arthritic Tissue

Objective: This study aims at highlighting the common signature between cartilaginous tissue in osteoarthritis (OA) and preneoplasic tissues preceding neoplasia and tumour formation and, second, focusing on the molecular mechanisms at the aetiology of both pathologies. Results: Because age is the highest risk factor common for both OA and cancer development, it is tempting to compare the molecular mechanisms occurring at the onset of OA and preneoplasic lesions. Indeed, cellular senescence seems to be a common characteristic. Cellular senescence represents a natural barrier to suppress the unscheduled proliferation of damaged cells acting as a strong tumour suppressor pathway and in OA, it also occurs prematurely in chondrocytes. In this study, we review a number of molecular factors associated with the senescent phenotype. Conclusion: Whereas accumulation of senescent cells in preneoplasic-like lesions leads to tissue degeneration and potentially tumour development; in OA, senescent cells accumulate in a slowly proliferative tissue. This is likely contributing at reducing the risk of cell transformation.

Induction of ASAP (MAP9) contributes to p53 stabilization in response to DNA damage

p53 is a key tumor suppressor that controls DNA damage response and genomic integrity. In response to genotoxic stress, p53 is stabilized and activated, resulting in controlled activation of genes involved in cell cycle arrest, DNA repair and/or apoptosis. ASAP is a centrosome- and spindle-associated protein, the deregulation of which induces severe mitotic defects. We show here that following double-strand break DNA formation, ASAP directly interacts with and stabilizes p53 by enhancing its p300-mediated acetylation and blocking its MDM2-mediated ubiquitination and degradation, leading to an increase of p53 transcriptional activity. Upon DNA damage, ASAP is transiently accumulated before being degraded upon persistent damage. This work links the p53 response with the cytoskeleton and confirms that the DNA-damaging signaling pathway is coordinated by centrosomal proteins. We reveal the existence of a new pathway through which ASAP signals the DNA damage response by regulating the p300-MDM2-p53 loop. These results point out ASAP as a possible target for the design of drugs to sensitize radio-resistant tumors.

Secreted α-Klotho maintains cartilage tissue homeostasis by repressing NOS2 and ZIP8-MMP13 catabolic axis

Progressive loss of tissue homeostasis is a hallmark of numerous age-related pathologies, including osteoarthritis (OA). Accumulation of senescent chondrocytes in joints contributes to the age-dependent cartilage loss of functions through the production of hypertrophy-associated catabolic matrix-remodeling enzymes and pro-inflammatory cytokines. Here, we evaluated the effects of the secreted variant of the anti-aging hormone α-Klotho on cartilage homeostasis during both cartilage formation and OA development. First, we found that α-Klotho expression was detected during mouse limb development, and transiently expressed during in vitro chondrogenic differentiation of bone marrow-derived mesenchymal stem cells. Genome-wide gene array analysis of chondrocytes from OA patients revealed that incubation with recombinant secreted α-Klotho repressed expression of the NOS2 and ZIP8/MMP13 catabolic remodeling axis. Accordingly, α-Klotho expression was reduced in chronically IL1β-treated chondrocytes and in cartilage of an OA mouse model. Finally, in vivo intra-articular secreted α-Kotho gene transfer delays cartilage degradation in the OA mouse model. Altogether, our results reveal a new tissue homeostatic function for this anti-aging hormone in protecting against OA onset and progression.

Inhibitory effect of miR-29a on the chondrogenic differentiation of mesenchymal stem cells

Backgroundand objectives Mesenchymal stem or stromal cells (MSC) are multipotent cells that can differentiate into different lineages, particularly osteoblasts and chondrocytes. The differentiation process of MSC is regulated by various molecules among which Sox9 and Runx2 are key transcription factors leading, respectively to chondrogenesis or osteogenesis. Recently, a new class of regulating factors, namely microRNAs (miRNAs), has been shown to be important for differentiation processes but few miRNAs have been shown to regulate chondrogenesis. The objective of this study is therefore to identify miRNAs involved in the chondrogenic differentiation of MSC. Materials and Methods MiRNA arrays have been done using RNA samples of MSC (day 0) and MSC-derived prechondrocytes (day 3). Analysis of miRNAs, their putative targets and of transcription factors putatively binding to their promoter regions was performed using several prediction softwares, in particular Targetscan. Pre-miRs and antagomiRs were transfected in MSC twice (day 4 and 1) using oligofectamine and chondrogenic differentiation was induced by culture of MSC in micropellets in inductive medium for 21 days. Expression of chondrocyte markers was performed by RT-qPCR. Results Analysis of results from the miRNA arrays together with those from DNA arrays already available in our laboratory indicated that a little number of transcription factors was theoretically able to regulate the majority of the miRNAs that were modulated at day 3 of chondrogenesis. Among the transcription factors, Sox9 and YY1 can putatively bind to the promoter region of miR-29. Using real-time RT-PCR, the authors observed that the expression level of miR-29 progressively and highly decreases during the chondrogenic differentiation. Transfection of Sox9 or YY1 in the Stro-1A MSC cell line significantly reduced the expression of miR-29, while transfection of both factors totally abolished its expression. The effect of gain- and loss-of-function of miR-29 during the chondrogenic differentiation of MSCs by transfecting pre-miRs or antago-miRs confirmed the role of miR-29 during chondrogenesis. Conclusions Our preliminary data show that, during chondrogenesis, miR-29 expression is downregulated, probably through the interaction of Sox9 and YY1 on the miR promoter region. Because miR-29 has been described to regulate different targets (DKK1, sFRP2, Kremen2 and CDK6 whose expression decreases during differentiation), future experiments will investigate whether these target genes are modulated by miR-29.