Anti-inflammatory effect of interleukin-6 highly enriched in secretome of two clinically relevant sources of mesenchymal stromal cells.

Despite several advances in the field of regenerative medicine, clinical management of extensive skin wounds or burns remains a major therapeutic issue. During the past few years, Mesenchymal Stromal Cells (MSCs) have emerged as a novel therapeutic tool to promote tissue repair through their anti-inflammatory, pro-trophic and pro-remodeling effects. They exert their biological activity mainly via the secretion of soluble bioactive molecules such as cytokines, growth factors, proteins and microRNAs which can be encapsulated within extracellular vesicles (EV). The recent discovery of their high plasticity to external stimuli has fostered the development of new targeted therapies known as priming strategies, to enhance their potential. Our team recently showed that Interleukin-1ß (IL-1ß)-primed gingival MSCs promote wound healing and epidermal engraftment in vitro, and in vivo through their secreted products that contain extracellular vesicles. In the present work, we investigated whether two common sources of MSCs, gingiva and bone marrow, could respond similarly to IL-1ß to favor pro-healing capabilities of their secretome. We showed that both primed-MSC sources, or their related secreted products, are able to reduce inflammation in LPS-challenged human monocytic THP-1 cell line. IL-1ß priming enhanced MSC secretion of wound healing-related growth factors, cytokines and miRNAs in both sources. Among them, interleukin 6 was shown to be involved in the anti-inflammatory effect of MSC secreted products. Overall, these results underline the pro-healing properties of both MSC sources and their secretome upon IL-1ß priming and their potential to improve the current medical treatment of severe wounds.

Development of extracellular vesicle-based medicinal products: A position paper of the group "Extracellular Vesicle translatiOn to clinicaL perspectiVEs - EVOLVE France".

Extracellular vesicles (EV) are emergent therapeutic effectors that have reached clinical trial investigation. To translate EV-based therapeutic to clinic, the challenge is to demonstrate quality, safety, and efficacy, as required for any medicinal product. EV research translation into medicinal products is an exciting and challenging perspective. Recent papers, provide important guidance on regulatory aspects of pharmaceutical development, defining EVs for therapeutic applications and critical considerations for the development of potency tests. In addition, the ISEV Task Force on Regulatory Affairs and Clinical Use of EV-based Therapeutics as well as the Exosomes Committee from the ISCT are expected to contribute in an active way to the development of EV-based medicinal products by providing update on the scientific progress in EVs field, information to patients and expert resource network for regulatory bodies. The contribution of our work group "Extracellular Vesicle translatiOn to clinicaL perspectiVEs - EVOLVE France", created in 2020, can be positioned in complement to all these important initiatives. Based on complementary scientific, technical, and medical expertise, we provide EV-specific recommendations for manufacturing, quality control, analytics, non-clinical development, and clinical trials, according to current European legislation. We especially focus on early phase clinical trials concerning immediate needs in the field. The main contents of the investigational medicinal product dossier, marketing authorization applications, and critical guideline information are outlined for the transition from research to clinical development and ultimate market authorization.

Self-Assembled Collagen Microparticles by Aerosol as a Versatile Platform for Injectable Anisotropic Materials.

Extracellular matrices (ECM) rich in type I collagen exhibit characteristic anisotropic ultrastructures. Nevertheless, working in vitro with this biomacromolecule remains challenging. When processed, denaturation of the collagen molecule is easily induced in vitro avoiding proper fibril self-assembly and further hierarchical order. Here, an innovative approach enables the production of highly concentrated injectable collagen microparticles, based on collagen molecules self-assembly, thanks to the use of spray-drying process. The versatility of the process is shown by performing encapsulation of secretion products of gingival mesenchymal stem cells (gMSCs), which are chosen as a bioactive therapeutic product for their potential efficiency in stimulating the regeneration of a damaged ECM. The injection of collagen microparticles in a cell culture medium results in a locally organized fibrillar matrix. The efficiency of this approach for making easily handleable collagen microparticles for encapsulation and injection opens perspectives in active tissue regeneration and 3D bioprinted scaffolds.

IL-1ß-Primed Mesenchymal Stromal Cells Improve Epidermal Substitute Engraftment and Wound Healing via Matrix Metalloproteinases and Transforming Growth Factor-ß1.

Since the 1980s, deep and extensive skin wounds and burns are treated with autologous split-thickness skin grafts, or cultured epidermal autografts, when donor sites are limited. However, the clinical use of cultured epidermal autografts often remains unsatisfactory because of poor engraftment rates, altered wound healing, and reduced skin functionality. In the past few decades, mesenchymal stromal cells (MSCs) have raised much attention because of their anti-inflammatory, protrophic, and pro-remodeling capacities. More specifically, gingival MSCs have been shown to possess enhanced wound healing properties compared with other tissue sources. Growing evidence also indicates that MSC priming could potentiate therapeutic effects in diverse in vitro and in vivo models of skin trauma. In this study, we found that IL-1ß-primed gingival MSCs promoted cell migration, dermal-epidermal junction formation, and inflammation reduction in vitro, as well as improved epidermal substitute engraftment in vivo. IL-1ß-primed gingival MSCs had different secretory profiles from naive gingival MSCs, characterized by an overexpression of transforming growth factor-ß and matrix metalloproteinase (MMP) pathway agonists. Eventually, MMP-1, MMP-9, and transforming growth factor-ß1 appeared to be critically involved in IL-1ß-primed gingival MSC mechanisms of action.

Influence of fibrin matrices and their released factors on epidermal substitute phenotype and engraftment.

Cultured epithelial autografts (CEAs) represent a life-saving surgical technique for full-thickness skin burns covering more than 60% total body surface area. However, CEAs present numerous drawbacks leading to heavy cosmetic and functional sequelae. In our previous study, we showed that human plasma-based fibrin matrices (hPBM) could improve the reparative potential of CEAs. Therefore, in the present work, we sought to investigate the role of hPBM compared with fibrin from purified fibrinogen (FPF) or plastic support on epidermal substitute formation and engraftment. The use of hPBM for epidermal substitute culture improved keratinocyte migration, proliferation, and epidermal substitute organization to a better extent than FPF in vitro. Both fibrin matrices favored greater dermal-epidermal junction protein deposition and prevented their degradation. Keratinocyte differentiation was also decreased using both fibrin matrices. Basement membrane protein deposition was mainly influenced by matrix whereas growth factors released from fibrin especially by hPBM were shown to enhance in vitro keratinocyte migration, proliferation, and epidermal substitute organization. Ultimately, epidermal substitutes grown on hPBM displayed better engraftment rates than those cultured on FPF or on plastic support in a NOD-SCID model of acute wound with the formation of a functional dermal-epidermal junction. Together, these results show the positive impact of fibrin matrices and their released growth factor on epidermal substitute phenotype and grafting efficiency. Fibrin matrices, and especially hPBM, may therefore be of interest to favor the treatment of full-thickness burn patients.

Mesenchymal Stromal Cell Preconditioning: The Next Step Toward a Customized Treatment For Severe Burn.

Over the last century, the clinical management of severe skin burns significantly progressed with the development of burn care units, topical antimicrobials, resuscitation methods, early eschar excision surgeries, and skin grafts. Despite these considerable advances, the present treatment of severe burns remains burdensome, and patients are highly susceptible to skin engraftment failure, infections, organ dysfunction, and hypertrophic scarring. Recent researches have focused on mesenchymal stromal cell (MSC) therapy and hold great promises for tissue repair, as reported in several animal studies and clinical cases. In the present review, we will provide an up-to-date outlook of the pathophysiology of severe skin burns, clinical treatment modalities and current limitations. We will then focus on MSCs and their potential in the burn wound healing both in in vitro and in vivo studies. A specific attention will be paid to the cell preconditioning approach, as a means of improving the MSC efficacy in the treatment of major skin burns. In particular, we will debate how several preconditioning cues would modulate the MSC properties to better match up with the burn pathophysiology in the course of the cell therapy. Finally, we will discuss the clinical interest and feasibility of a MSC-based therapy in comparison to their paracrine derivatives, including microvesicles and conditioned media for the treatment of major skin burn injuries.

Biotechnological Management of Skin Burn Injuries: Challenges and Perspectives in Wound Healing and Sensory Recovery.

Many wound management protocols have been developed to improve wound healing after burn with the primordial aim to restore the barrier function of the skin and also provide a better esthetic outcome. Autologous skin grafts remain the gold standard in the treatment of skin burn, but this treatment has its limitation especially for patients presenting limited donor sites due to extensive burn areas. Deep burn injuries also alter the integrity of skin-sensitive innervation and have an impact on patient's quality of life by compromising perceptions of touch, temperature, and pain. Thus, patients can suffer from long-term disabilities ranging from cutaneous sensibility loss to chronic pain. The cellular mechanisms involved in skin reinnervation following injury are not elucidated yet. Depending on the depth of the burn, nerve sprouting can occur from the wound bed or the surrounding healthy tissue, but somehow this process fails to provide correct reinnervation of the wound during scarring. In addition, several clinical observations indicate that damage to the peripheral nervous system influences wound healing, resulting in delayed wound healing or chronic wounds, underlining the role of innervation and neuromediators for normal cutaneous tissue repair development. Promising tissue engineering strategies, including the use of biomaterials, skin substitutes, and stem cells, could provide novel alternative treatments in wound healing and help in improving patient's sensory recovery.

Bioengineering a human plasma-based epidermal substitute with efficient grafting capacity and high content in clonogenic cells.

UNLABELLED: Cultured epithelial autografts (CEAs) produced from a small, healthy skin biopsy represent a lifesaving surgical technique in cases of full-thickness skin burn covering >50% of total body surface area. CEAs also present numerous drawbacks, among them the use of animal proteins and cells, the high fragility of keratinocyte sheets, and the immaturity of the dermal-epidermal junction, leading to heavy cosmetic and functional sequelae. To overcome these weaknesses, we developed a human plasma-based epidermal substitute (hPBES) for epidermal coverage in cases of massive burn, as an alternative to traditional CEA, and set up critical quality controls for preclinical and clinical studies. In this study, phenotypical analyses in conjunction with functional assays (clonal analysis, long-term culture, or in vivo graft) showed that our new substitute fulfills the biological requirements for epidermal regeneration. hPBES keratinocytes showed high potential for cell proliferation and subsequent differentiation similar to healthy skin compared with a well-known reference material, as ascertained by a combination of quality controls. This work highlights the importance of integrating relevant multiparameter quality controls into the bioengineering of new skin substitutes before they reach clinical development. SIGNIFICANCE: This work involves the development of a new bioengineered epidermal substitute with pertinent functional quality controls. The novelty of this work is based on this quality approach.

MRAS GTPase is a novel stemness marker that impacts mouse embryonic stem cell plasticity and Xenopus embryonic cell fate.

Pluripotent mouse embryonic stem cells (mESCs), maintained in the presence of the leukemia inhibitory factor (LIF) cytokine, provide a powerful model with which to study pluripotency and differentiation programs. Extensive microarray studies on cultured cells have led to the identification of three LIF signatures. Here we focus on muscle ras oncogene homolog (MRAS), which is a small GTPase of the Ras family encoded within the Pluri gene cluster. To characterise the effects of Mras on cell pluripotency and differentiation, we used gain- and loss-of-function strategies in mESCs and in the Xenopus laevis embryo, in which Mras gene structure and protein sequence are conserved. We show that persistent knockdown of Mras in mESCs reduces expression of specific master genes and that MRAS plays a crucial role in the downregulation of OCT4 and NANOG protein levels upon differentiation. In Xenopus, we demonstrate the potential of Mras to modulate cell fate at early steps of development and during neurogenesis. Overexpression of Mras allows gastrula cells to retain responsiveness to fibroblast growth factor (FGF) and activin. Collectively, these results highlight novel conserved and pleiotropic effects of MRAS in stem cells and early steps of development.

Glycosaminoglycan mimetic associated to human mesenchymal stem cell-based scaffolds inhibit ectopic bone formation, but induce angiogenesis in vivo.

Tissue engineering approaches to stimulate bone formation currently combine bioactive scaffolds with osteocompetent human mesenchymal stem cells (hMSC). Moreover, osteogenic and angiogenic factors are required to promote differentiation and survival of hMSC through improved vascularization through the damaged extracellular matrix (ECM). Glycosaminoglycans (GAGs) are ECM compounds acting as modulators of heparin-binding protein activities during bone development and regenerative processes. GAG mimetics have been proposed as ECM stabilizers and were previously described for their positive effects on bone formation and angiogenesis after local treatment. Here, we developed a strategy associating the GAG mimetic [OTR4120] with bone substitutes to optimize stem cell-based therapeutic products. We showed that [OTR4120] was able to potentiate proliferation, migration, and osteogenic differentiation of hMSC in vitro. Its link to tricalcium phosphate/hydroxyapatite scaffolds improved their colonization by hMSC. Surprisingly, when these combinations were tested in an ectopic model of bone formation in immunodeficient mice, the GAG mimetics inhibit bone formation induced by hMSC and promoted an osteoclastic activity. Moreover, the inflammatory response was modulated, and the peri-implant vascularization stimulated. All together, these findings further support the ability of GAG mimetics to organize the local ECM to coordinate the host response toward the implanted biomaterial, and to inhibit the abnormal bone formation process on a subcutaneous ectopic site.

A new platelet cryoprecipitate glue promoting bone formation after ectopic mesenchymal stromal cell-loaded biomaterial implantation in nude mice.

INTRODUCTION: This study investigated the promising effect of a new Platelet Glue obtained from Cryoprecipitation of Apheresis Platelet products (PGCAP) used in combination with Mesenchymal Stromal Cells (MSC) loaded on ceramic biomaterials to provide novel strategies enhancing bone repair. METHODS: PGCAP growth factor content was analyzed by ELISA and compared to other platelet and plasma-derived products. MSC loaded on biomaterials (65% hydroxyapatite/35% beta-TCP or 100% beta-TCP) were embedded in PGCAP and grown in presence or not of osteogenic induction medium for 21 days. Biomaterials were then implanted subcutaneously in immunodeficient mice for 28 days. Effect of PGCAP on MSC was evaluated in vitro by proliferation and osteoblastic gene expression analysis and in vivo by histology and immunohistochemistry. RESULTS: We showed that PGCAP, compared to other platelet-derived products, allowed concentrating large amount of growth factors and cytokines which promoted MSC and osteoprogenitor proliferation. Next, we found that PGCAP improves the proliferation of MSC and osteogenic-induced MSC. Furthermore, we demonstrated that PGCAP up-regulates the mRNA expression of osteogenic markers (Collagen type I, Osteonectin, Osteopontin and Runx2). In vivo, type I collagen expressed in ectopic bone-like tissue was highly enhanced in biomaterials embedded in PGCAP in the absence of osteogenic pre-induction. Better results were obtained with 65% hydroxyapatite/35% beta-TCP biomaterials as compared to 100% beta-TCP. CONCLUSIONS: We have demonstrated that PGCAP is able to enhance in vitro MSC proliferation, osteoblastic differentiation and in vivo bone formation in the absence of osteogenic pre-induction. This clinically adaptable platelet glue could be of interest for improving bone repair.

The FunGenES database: a genomics resource for mouse embryonic stem cell differentiation.

Embryonic stem (ES) cells have high self-renewal capacity and the potential to differentiate into a large variety of cell types. To investigate gene networks operating in pluripotent ES cells and their derivatives, the "Functional Genomics in Embryonic Stem Cells" consortium (FunGenES) has analyzed the transcriptome of mouse ES cells in eleven diverse settings representing sixty-seven experimental conditions. To better illustrate gene expression profiles in mouse ES cells, we have organized the results in an interactive database with a number of features and tools. Specifically, we have generated clusters of transcripts that behave the same way under the entire spectrum of the sixty-seven experimental conditions; we have assembled genes in groups according to their time of expression during successive days of ES cell differentiation; we have included expression profiles of specific gene classes such as transcription regulatory factors and Expressed Sequence Tags; transcripts have been arranged in "Expression Waves" and juxtaposed to genes with opposite or complementary expression patterns; we have designed search engines to display the expression profile of any transcript during ES cell differentiation; gene expression data have been organized in animated graphs of KEGG signaling and metabolic pathways; and finally, we have incorporated advanced functional annotations for individual genes or gene clusters of interest and links to microarray and genomic resources. The FunGenES database provides a comprehensive resource for studies into the biology of ES cells.

The LIF cytokine: towards adulthood.

The aim of this article is to recapitulate the key features of leukaemia inhibitory factor cytokine (LIF), to review its numerous physiological effects and to comment on the most recent data. We will also present results of transcriptome analyses, which have highlighted different categories of LIF targets, identified in murine embryonic stem (ES) cells and early derivatives. We hope to stimulate new research fields on this puzzling cytokine, which, forty years after its discovery, has still not disclosed all its secrets.

Three LIF-dependent signatures and gene clusters with atypical expression profiles, identified by transcriptome studies in mouse ES cells and early derivatives.

BACKGROUND: Mouse embryonic stem (ES) cells remain pluripotent in vitro when grown in the presence of the cytokine Leukaemia Inhibitory Factor (LIF). Identification of LIF targets and of genes regulating the transition between pluripotent and early differentiated cells is a critical step for understanding the control of ES cell pluripotency. RESULTS: By gene profiling studies carried out with mRNAs from ES cells and their early derivatives treated or not with LIF, we have identified i) LIF-dependent genes, highly expressed in pluripotent cells, whose expression level decreases sharply upon LIF withdrawal [Pluri genes], ii) LIF induced genes [Lifind genes] whose expression is differentially regulated depending upon cell context and iii) genes specific to the reversible or irreversible committed states. In addition, by hierarchical gene clustering, we have identified, among eight independent gene clusters, two atypical groups of genes, whose expression level was highly modulated in committed cells only. Computer based analyses led to the characterization of different sub-types of Pluri and Lifind genes, and revealed their differential modulation by Oct4 or Nanog master genes. Individual knock down of a selection of Pluri and Lifind genes leads to weak changes in the expression of early differentiation markers, in cell growth conditions in which these master genes are still expressed. CONCLUSION: We have identified different sets of LIF-regulated genes depending upon the cell state (reversible or irreversible commitment), which allowed us to present a novel global view of LIF responses. We are also reporting on the identification of genes whose expression is strictly regulated during the commitment step. Furthermore, our studies identify sub-networks of genes with a restricted expression in pluripotent ES cells, whose down regulation occurs while the master knot (composed of OCT4, SOX2 and NANOG) is still expressed and which might be down-regulated together for driving cells towards differentiation.