Delphine Logeart-Avramoglou

Optimization of a gene electrotransfer method for mesenchymal stem cell transfection

Gene electrotransfer is an efficient and reproducible nonviral gene transfer technique useful for the nonpermanent expression of therapeutic transgenes. The present study established optimal conditions for the electrotransfer of reporter genes into mesenchymal stem cells (MSCs) isolated from rat bone marrow by their selective adherence to tissue-culture plasticware. The electrotransfer of the lacZ reporter gene was optimized by adjusting the pulse electric field intensity, electric pulse type, electropulsation buffer conductivity and electroporation temperature. LacZ electrotransfection into MSCs was optimal at 1500 V cm−1 with pre-incubation in Spinners minimum essential medium buffer at 22 °C. Under these conditions β-galactosidase expression was achieved in 29±3% of adherent cells 48 h post transfection. The kinetics of β-galactosidase activity revealed maintenance of β-galactosidase production for at least 10 days. Moreover, electroporation did not affect the MSC potential for multidifferentiation; electroporated MSCs differentiated into osteoblastic, adipogenic and chondrogenic lineages to the same extent as cells that were not exposed to electric pulses. Thus, this study demonstrates the feasibility of efficient transgene electrotransfer into MSCs while preserving cell viability and multipotency.

Carboxymethyl benzylamide sulfonate dextrans (CMDBS), a family of biospecific polymers endowed with numerous biological properties: a review.

The functionalized dextrans termed carboxymethyl benzylamide sulfonate dextran (CMDBS) represent a family encompassing a wide range of polymers. These soluble macromolecular compounds, which are substituted with specific chemical functional groups, are designed to interact with living systems. By analogy with glycosaminoglycan heparin, a natural highly charged anionic polysaccharide that exerts a variety of biological effects, we postulated that CMDBS compounds also possess binding sites capable of specific interactions with biological constituents, depending on the overall composition of the polymer. The synthesis and heparin-like properties of these CMDBS have been extensively investigated. Thus, it appears that dextran derivatives can mimic the action of heparin in regard to its interactions with antithrombin and serine proteases involved in blood coagulation. Other derivatives interact with various components of the immune system or with adhesive proteins such as fibronectin in modulating the proliferation of Staphylococcus aureus. Because they are able to stimulate wound healing in various in vivo models, these polysaccharides may also constitute a family of tissue repair agents because of their protecting and potentiating effects with heparin binding growth factors. Moreover, dextran derivatives in contact with cells such as endothelial cells, smooth muscle cells, or tumoral cells can affect both cell proliferation and metabolism. It appears that these bioactive polymers are also efficient tools to investigate the precise mechanism of action of individual biological activities by contrasting their mode of action to that of heparin. In addition to their numerous biological properties and biospecificity, functionalized dextrans are relatively simple to manufacture and exempt of donor contaminant, which make them attractive in a variety of clinical applications.

Oxygen Tension Regulates Human Mesenchymal Stem Cell Paracrine Functions

Mesenchymal stem cells (MSCs) have captured the attention and research endeavors of the scientific world because of their differentiation potential. However, there is accumulating evidence suggesting that the beneficial effects of MSCs are predominantly due to the multitude of bioactive mediators secreted by these cells. Because the paracrine potential of MSCs is closely related to their microenvironment, the present study investigated and characterized select aspects of the human MSC (hMSC) secretome and assessed its in vitro and in vivo bioactivity as a function of oxygen tension, specifically near anoxia (0.1% O2) and hypoxia (5% O2), conditions that reflect the environment to which MSCs are exposed during MSC‐based therapies in vivo. In contrast to supernatant conditioned media (CM) obtained from hMSCs cultured at either 5% or 21% of O2, CM from hMSCs cultured under near anoxia exhibited significantly (p < .05) enhanced chemotactic and proangiogenic properties and a significant (p < .05) decrease in the inflammatory mediator content. An analysis of the hMSC secretome revealed a specific profile under near anoxia: hMSCs increase their paracrine expression of the angiogenic mediators vascular endothelial growth factor (VEGF)‐A, VEGF‐C, interleukin‐8, RANTES, and monocyte chemoattractant protein 1 but significantly decrease expression of several inflammatory/immunomodulatory mediators. These findings provide new evidence that elucidates aspects of great importance for the use of MSCs in regenerative medicine and could contribute to improving the efficacy of such therapies.

Proangiogenic and Prosurvival Functions of Glucose in Human Mesenchymal Stem Cells upon Transplantation

A major limitation in the development of cellular therapies using human mesenchymal stem cells (hMSCs) is cell survival post‐transplantation. In this study, we challenged the current paradigm of hMSC survival, which assigned a pivotal role to oxygen, by testing the hypothesis that exogenous glucose may be key to hMSC survival. We demonstrated that hMSCs could endure sustained near‐anoxia conditions only in the presence of glucose. In this in vitro cell model, the protein expressions of Hif‐1α and angiogenic factors were upregulated by the presence of glucose. Ectopically implanted tissue constructs supplemented with glucose exhibited four‐ to fivefold higher viability and were more vascularized compared to those without glucose at day 14. These findings provided the first direct in vitro and in vivo demonstration of the proangiogenic and prosurvival functions of glucose in hMSC upon transplantation and identified glucose as an essential component of the ideal scaffold for transplanting stem cells. STEM CELLS2013;31:526–535

Interaction of specifically chemically modified dextrans with transforming growth factor β1: potentiation of its biological activity

Transforming growth factor beta (TGFbeta), a potent multifunctional cytokine, is well known to demonstrate heparin binding ability. This study investigated the binding capacity of heparin-like family of chemically modified dextrans to TGFbeta1. Dextran derivatives with various substitution contents in carboxymethyl, benzylamide and sulfate groups were evaluated using a gel mobility shift assay. This structure-function study indicated that a synergistic role of benzylamide and sulfate substituents resulted in an optimal interaction with the growth factor. The effect of these polymers on the biological response of TGFbeta1 was assessed using mink lung epithelial cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct (PAI/Luc). When the growth factor was mixed with 250 microg/mL of carboxymethyl-benzylamide-dextran (DCMB) or carboxymethyl-benzylamide-sulfate-dextran (DCMBSu), the luciferase gene expression was enhanced. Only polymers exhibiting TGFbeta1 binding demonstrated a biological potentiating effect. However, this effect was strongly amplified as the cell plating time increased (35-fold increase with a 2 days plating time versus 1.1-fold increase with a 4 hr plating time at a 0.25 ng/mL concentration of TGFbeta1). TGFbeta1 induced the PAI/Luc construct in a dose-dependent fashion but its effect diminished when added to cells previously cultured for 24 and 48 hr. The results indicated that the potentiating effect required a complex formation between TGFbeta1 and polymers, the action of which seeming to locally maintain TGFbeta1 in an active form. TGFbeta isoforms playing a key role in the process of bone repair, specifically designed functionalized dextrans could potentiate the in vivo TGFbeta1 biological effect and be used in the field of wound healing applications.

Enhancement of the biological activity of BMP‐2 by synthetic dextran derivatives

In the present study, we explored the binding capacity of synthetic heparin-like dextran derivatives to recombinant human bone morphogenetic protein 2 (BMP-2), a heparin-binding osteoinductive growth factor. Affinity electrophoresis analysis provided evidence that carboxymethylated dextran polymers grafted with high amounts of benzylamide groups (named DMCB) interact with BMP-2. The capability of such polysaccharides to potentiate the growth factor biological activity was then investigated. In vitro, DMCB dose-dependently promoted osteoblast differentiation induced by BMP-2 in C2C12 myoblasts more efficiently than heparin. A screening study provided evidence that the potentiating effects of the dextran derivatives on the BMP-2-induced alkaline phosphatase activity improved with their benzylamide groups content and, therefore, with their affinity for the growth factor. The biological activity of BMP-2 was monitored in the culture medium after 6 days using C2C12 cells (containing a BMP sensitive luciferase reporter gene). Like heparin, DMCB sustained the biological activity of the growth factor; this result suggests that the formation of the BMP-2/DMCB complex may protect the protein from being inactivated. In rats in vivo, DMCB also stimulated ectopic calcification mediated by BMP-2. These data indicate that dextran-based polysaccharides prolong the half-life of the growth factor and promote its biological activity.

Animal Models for Bone Tissue Engineering Purposes

To assess the efficacy of engineered tissues, it is necessary to have (1) appropriate large animal models that mimic the clinical setting and (2) relevant methods of monitoring the biofuntionality of these tissues. However, developing these tissue constructs is a step-by-step process in which numerous variables such as scaffold design, source of stem cells and mode of growth factor application have to be optimized. After an in vitro optimization phase, the use of small animal models to optimize these various parameters and sort out any teething problems is recommended before launching into large animal models. Depending on the experimental aims, engineered tissues can be transplanted into either ectopic sites (subcutaneously or intramuscularly) or orthotopic sites. In all these experimental studies, non invasive imaging methods (X-ray, magnetic resonance, in vivo fluorescence, ultrasound imaging methods, etc.) as well as detailed quantitative molecular and histological analyses have been used to monitor the in vivo behavior of the engineered constructs. In this chapter we take stock of the present state of the art in this field.

The pH in the microenvironment of human mesenchymal stem cells is a critical factor for optimal osteogenesis in tissue-engineered constructs.

The present study aimed at elucidating the effect of local pH in the extracellular microenvironment of tissue-engineered (TE) constructs on bone cell functions pertinent to new tissue formation. To this aim, we evaluated the osteogenicity process associated with bone constructs prepared from human Bone marrow-derived mesenchymal stem cells (hBMSC) combined with 45S5 bioactive glass (BG), a material that induces alkalinization of the external medium. The pH measured in cell-containing BG constructs was around 8.0, that is, 0.5 U more alkaline than that in two other cell-containing materials (hydroxyapatite/tricalcium phosphate [HA/TCP] and coral) constructs tested. When implanted ectopically in mice, there was no de novo bone tissue in the BG cell-containing constructs, in contrast to results obtained with either HA/TCP or coral ceramics, which consistently promoted the formation of ectopic bone. In addition, the implanted 50:50 composites of both HA/TCP:BG and coral:BG constructs, which displayed a pH of around 7.8, promoted 20-30-fold less amount of bone tissue. Interestingly, hBMSC viability in BG constructs was not affected compared with the other two types of material constructs tested both in vitro and in vivo. Osteogenic differentiation (specifically, the alkaline phosphatase [ALP] activity and gene expression of RUNX2, ALP, and BSP) was not affected when hBMSC were maintained in moderate alkaline pH (≤7.90) external milieu in vitro, but was dramatically inhibited at higher pH values. The formation of mineralized nodules in the extracellular matrix of hBMSC was fully inhibited at alkaline (>7.54) pH values. Most importantly, there is a pH range (specifically, 7.9-8.27) at which hBMSC proliferation was not affected, but the osteogenic differentiation of these cells was inhibited. Altogether, these findings provided evidence that excessive alkalinization in the microenvironment of TE constructs (resulting, for example, from material degradation) affects adversely the osteogenic differentiation of osteoprogenitor cells.

Desferrioxamine-driven upregulation of angiogenic factor expression by human bone marrow stromal cells

Bone marrow stromal cells (BMSCs) are the subject of intense research because of their biological properties and potential use for the repair of damaged tissues. Success of BMSC‐based therapies, however, relies on a number of methodological improvements, including the establishment of a vascular network providing nutrients and oxygen to the transplanted cells and ensuring their immediate survival and long‐term functionality. We described a method to enhance the autocrine expression of angiogenic factors by BMSCs. For this purpose, human BMSCs were treated with desferrioxamine (DFX). No PDGF‐BB, VEGF‐R1 or ‐R2 mRNA expression was detected under any of the conditions tested. mRNA and protein expression levels of TGFβ1 were similar in BMSCs, whether they were exposed to DFX (50 µM) or to control conditions under normoxia for 48 h. In comparison with the results obtained with control conditions under normoxia, exposure of BMSCs to DFX for 48 h resulted in upregulation of bFGF at the protein (26‐fold) but not at the mRNA levels and VEGF at both the mRNA (1.5‐fold) and protein levels (4.5‐fold). In comparison with the results obtained with control conditions under hypoxia, DFX induced a 50% increase in VEGF secretion but led to the same level of hypoxia inducible factor‐1α protein expression (a transduction factor involved in angiogenic factor expression and known to be activated by DFX). Exposure of BMSCs to DFX resulted in oversecretion of angiogenic factors, suggesting that DFX‐treated BMSCs could be used to supply angiogenic factors. Copyright

Secondary Structure of rhBMP-2 in a Protective Biopolymeric Carrier Material

Efficient delivery of growth factors is one of the great challenges of tissue engineering. Polyelectrolyte multilayer films (PEM) made of biopolymers have recently emerged as an interesting carrier for delivering recombinant human bone morphogenetic protein 2 (rhBMP-2 noted here BMP-2) to cells in a matrix-bound manner. We recently showed that PEM made of poly(l-lysine) and hyaluronan (PLL/HA) can retain high and tunable quantities of BMP-2 and can deliver it to cells to induce their differentiation in osteoblasts. Here, we investigate quantitatively by Fourier transform infrared spectroscopy (FTIR) the secondary structure of BMP-2 in solution as well as trapped in a biopolymeric thin film. We reveal that the major structural elements of BMP-2 in solution are intramolecular β-sheets and unordered structures as well as α-helices. Furthermore, we studied the secondary structure of rhBMP-2 trapped in hydrated films and in dry films since drying is an important step for future applications of these bioactive films onto orthopedic biomaterials. We demonstrate that the structural elements were preserved when BMP-2 was trapped in the biopolymeric film in hydrated conditions and, to a lesser extent, in dry state. Importantly, its bioactivity was maintained after drying of the film. Our results appear highly promising for future applications of these films as coatings of biomedical materials, to deliver bioactive proteins while preserving their bioactivity upon storage in dry state.