PDF file - 67K, Effect of GDF15 on T308 Akt phosphorylation of MOLP-6 stroma-dependent cells in culture condition with 10% fetal calf serum

PDF file - 64K, Baseline phosphorylation of pT308 Akt in MOLP-6 and MM1.S cells

PDF file - 90K, Effect of GDF15 on ERK1/2 phosphorylation in MOLP-6 and MM1.S cells

PDF file - 67K, Transfection of BM-MSC with plasmids encoding shRNA targeting GDF15 and co-culture with MOLP-6 cells

PDF file - 44K, Concentration of GDF15 measured by ELISA in 72 hours culture supernatants of primary BM-MSCs and MM cells from 3 patients, and MM cell lines MOLP-6 and MM1.S

PDF file - 84K, Baseline phosphorylation of pT308 Akt in MOLP-6 and MM1.S cells

Bone marrow (BM) mesenchymal stromal cells (MSCs) are abnormal in multiple myeloma (MM) and play a critical role by promoting growth, survival, and drug resistance of MM cells. We observed higher Toll-like receptor 4 (TLR4) gene expression in MM MSCs than in MSCs from healthy donors. At the clinical level, we highlighted that TLR4 expression in MM MSCs evolves in parallel with the disease stage. Thus, we reasoned that the TLR4 axis is pivotal in MM by increasing the protumor activity of MSCs. Challenging primary MSCs with TLR4 agonists increased the expression of CD54 and interleukin-6 (IL-6), 2 factors directly implicated in MM MSC-MM cell crosstalk. Then, we evaluated the therapeutic efficacy of a TLR4 antagonist combined or not with conventional treatment in vitro with MSC-MM cell coculture and in vivo with the Vk*MYC mouse model. Selective inhibition of TLR4 specifically reduced the MM MSC ability to support the growth of MM cells in an IL-6-dependent manner and delayed the development of MM in the Vk*MYC mouse model by altering the early disease phase in vivo. For the first time, we demonstrate that specific targeting of the pathological BM microenvironment via TLR4 signaling could be an innovative approach to alter MM pathology development.

EDITORIAL article Front. Cell Dev. Biol., 05 May 2022Sec. Stem Cell Research https://doi.org/10.3389/fcell.2022.909550

Multiple myeloma (MM) is an incurable B cell neoplasia characterized by the accumulation of tumor plasma cells within the bone marrow (BM). As a consequence, bone osteolytic lesions develop in 80% of patients and remain even after complete disease remission. We and others had demonstrated that BM-derived mesenchymal stromal cells (MSCs) are abnormal in MM and thus cannot be used for autologous treatment to repair bone damage. Adipose stromal cells (ASCs) represent an interesting alternative to MSCs for cellular therapy. Thus, in this study, we wondered whether they could be a good candidate in repairing MM bone lesions. For the first time, we present a transcriptomic, phenotypic, and functional comparison of ASCs from MM patients and healthy donors (HDs) relying on their autologous MSC counterparts. In contrast to MM MSCs, MM ASCs did not exhibit major abnormalities. However, the changes observed in MM ASCs and the supportive property of ASCs on MM cells question their putative and safety uses at an autologous or allogenic level.

Clinical-grade mesenchymal stromal cells (MSCs) can be expanded from bone marrow and adipose tissue to treat inflammatory diseases and degenerative disorders. However, the influence of their tissue of origin on their functional properties, including their immunosuppressive activity, remains unsolved. In this study, we produced paired bone marrow-derived mesenchymal stromal cell (BM-MSC) and adipose-derived stromal cell (ASC) batches from 14 healthy donors. We then compared them using transcriptomic, phenotypic, and functional analyses and validated our results on purified native MSCs to infer which differences were really endowed by tissue of origin. Cultured MSCs segregated together owing to their tissue of origin based on their gene expression profile analyzed using differential expression and weighted gene coexpression network analysis. This translated into distinct immune-related gene signatures, phenotypes, and functional cell interactions. Importantly, sorted native BM-MSCs and ASCs essentially displayed the same distinctive patterns than their in vitro-expanded counterparts. As a whole, ASCs exhibited an immune profile consistent with a stronger inhibition of immune response and a lower immunogenicity, supporting the use of adipose tissue as a valuable source for clinical applications.

Controlling microarchitecture in polymer scaffolds is a priority in material design for soft tissue applications. This paper reports for the first time the elaboration of alginate foam-based scaffolds for mesenchymal stem cell (MSC) delivery and a comparative study of various surfactants on the final device performance. The use of surfactants permitted to obtain highly interconnected porous scaffolds with tunable pore size on surface and in cross-section. Their mechanical properties in compression appeared to be adapted to soft tissue engineering. Scaffold structures could sustain MSC proliferation over 14 days. Paracrine activity of scaffold-seeded MSCs varied with the scaffold structure and growth factors release was globally improved in comparison with control alginate scaffolds. Our results provide evidence that exploiting different surfactant types for alginate foam preparation could be an original method to obtain biocompatible scaffolds with tunable architecture for soft tissue engineering.

Background The MESAMI 1 trial was a bicentric pilot study designed to test the feasibility and safety of intramyocardially injected autologous bone marrow-derived mesenchymal stromal cells (MSCs) for the treatment of ischemic cardiomyopathy. Methods and results The study included 10 patients with chronic myocardial ischemia, left ventricular (LV) ejection fractions (EFs) of ≤35%, and reversible perfusion defects who were on stable optimal medical therapy and were not candidates for revascularization. MSCs (mean: 61.5 × 106 cells per patient) were injected into 10–16 viable sites at the border of the LV scar via a NOGA-guided catheter. Both primary endpoints, feasibility (successful harvest, expansion, and injection of autologous MSCs) and safety (absence of severe adverse events [SAEs]) were met in all 10 patients at the 1-month follow-up time point, and none of the SAEs reported during the full 2-year follow-up period were attributable to the study intervention. The results of secondary efficacy endpoint analyses identified significant improvements from baseline to Month 12 in LVEF (29.4 ± 2.0% versus 35.7 ± 2.5%; p = 0.003), LV end-systolic volume (167.8 ± 18.8 mL versus 156.1 ± 28.6 mL; p = 0.04), 6-min walk test and NYHA functional class. Conclusions Our results suggest that autologous MSCs can be safely administered to the hearts of patients with severe, chronic, reversible myocardial ischemia and impaired cardiac function and may be associated with improvements in cardiac performance, LV remodeling, and patient functional status. A randomized, double blind, multicenter, placebo-controlled clinical trial (MESAMI 2) will evaluate the efficacy of this treatment approach in a larger patient population. Clinical Trial Registration: Unique identifier: NCT01076920.

Event Abstract Back to Event Design of biopolymer-based 3D scaffolds for cardiac mesenchymal stem cell therapy Raya Bushkalova1, 2, Caroline Ceccaldi1, 2, Christophe Tenailleau3, Benjamin Duployer3, Philippe Bourin4, Daniel Cussac1, Angelo Parini1, 5, Brigitte Sallerin1, 5 and Sophie Girod Fullana2 1 UMR Inserm/UPS 1048, I2MC, France 2 Université De Toulouse, CIRIMAT, UPS-INPT-CNRS, Faculté De Pharmacie, France 3 Université De Toulouse, CIRIMAT, UPS-INPT-CNRS, France 4 Etablissement Français Du Sang, Laboratoire De Thérapie Cellulaire, France 5 CHU Toulouse, Pôle Pharmacie Toulouse, France INTRODUCTION Since the past decades, there is a growing interest in the use of bone marrow mesenchymal stem cells (MSCs) to regenerate tissues after acute and chronic diseases through the secretion of paracrine factors[1]. In the case of cardiac ischemia, MSCs delivery to the targeted organ via a 3D biomimetic scaffold may present several advantages in comparison with direct cell injection, including cell retention on the injury site and improved viability and secretion. The success of this therapeutic strategy lies on the scaffold’s design, as its biocompatibility and architecture influence host’s reaction and implanted cells fate. Our group already showed the interest of using alginate to design tailored scaffolds for soft tissues cell therapy[2][3]. In this context, we describe here the design of biopolymer based-scaffolds specifically tailored for MSC immobilization and improvement of their therapeutic effects on heart. EXPERIMENTAL METHODS Biopolymer based 3D scaffolds were produced by mixing alginate solutions either with chitosan to form complexes of polyelectrolytes of opposite charge (PEC)[4], or with a porogen and various surfactants to generate foams. After cross-linking, rinsing and lyophilisation steps, macroporous scaffolds were obtained and characterized by Fourier-Transformed Infra-Red (FTIR) and/or Raman spectroscopy . Their 3D-architecture was assessed by scanning electron microscopy (SEM), micro-computed tomography (micro-CT) and Raman or fluorescence coupled confocal microscopy. Their mechanical behaviour and rheological properties were also studied in order to evaluate their accordance with cardiac tissue characteristics. Cell distribution through the thickness of scaffolds and quantification of cell metabolic activity was evidenced by the Live/Dead® and the AlamarBlue® assays, respectively. Finally, cell secretion function was investigated by the quantification of HGF, FGF-2 and VEGF released in the supernatant of MSCs-loaded scaffolds. RESULTS AND DISCUSSION Whatever the formulation tested, highly interconnected porous scaffolds were obtained, with pore sizes suitable for 3D cell culture (100-250 µm on surface and in cross-section, determined by micro-CT). All scaffolds exhibited storage moduli higher than their loss moduli, thus confirming a well-structured polymeric network. Their mechanical properties were in the range of the elastic moduli of soft tissues (scaffolds: 10-27 kPa; soft tissues: 1-20 kPa). FTIR analysis confirmed the efficacy of the rinsing steps, warranty of alginate scaffolds’ preserved biocompatibility. The generated porosity allowed an efficient cell seeding in-depth , and the metabolic activity of the seeded MSCs was maintained during 14 days showing the good in vitro biocompatibility of all the scaffolds. In addition, the MSC secretion level measurements revealed that growth factors release was globally improved in comparison with control alginate scaffolds (VEGF: p<0.05; HGF and FGF2: p>0.05) and vary according to the scaffold design. CONCLUSION Our work presents for the first time a comparative study of two "green" design strategies (foam based- scaffolds and PEC-based-scaffolds) to generate highly porous scaffolds with tunable properties for cardiac tissue engineering with MSCs. By varying the operating conditions, various microarchitectures were generated, together with different secretion profiles of the seeded MSCs, which could give rise to different biological effects in vivo. Associating MSCs with the designed biopolymer -based scaffolds appears as a promising strategy to improve cardiac cell therapy and may be a tool to study microenvironment influence on stem cells behavior. Région Midi-Pyrénées; INSERM; CNRSReferences:[1] Dvir-Ginzberg et al. 2008[2] Trouche et al., Cell Transplant 2010[3] Ceccaldi et al., Cell Transplant, 2012[4] Ceccaldi et al., Acta Biomaterialia, 2014 Keywords: 3D scaffold, biomacromolecule, Heart repair, Cell functionality Conference: 10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016. Presentation Type: Poster Topic: Biomaterials for cardiovascular applications, vascular grafts and embolic devices Citation: Bushkalova R, Ceccaldi C, Tenailleau C, Duployer B, Bourin P, Cussac D, Parini A, Sallerin B and Girod Fullana S (2016). Design of biopolymer-based 3D scaffolds for cardiac mesenchymal stem cell therapy. Front. Bioeng. Biotechnol. Conference Abstract: 10th World Biomaterials Congress. doi: 10.3389/conf.FBIOE.2016.01.00734 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Mar 2016; Published Online: 30 Mar 2016. Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Raya Bushkalova Caroline Ceccaldi Christophe Tenailleau Benjamin Duployer Philippe Bourin Daniel Cussac Angelo Parini Brigitte Sallerin Sophie Girod Fullana Google Raya Bushkalova Caroline Ceccaldi Christophe Tenailleau Benjamin Duployer Philippe Bourin Daniel Cussac Angelo Parini Brigitte Sallerin Sophie Girod Fullana Google Scholar Raya Bushkalova Caroline Ceccaldi Christophe Tenailleau Benjamin Duployer Philippe Bourin Daniel Cussac Angelo Parini Brigitte Sallerin Sophie Girod Fullana PubMed Raya Bushkalova Caroline Ceccaldi Christophe Tenailleau Benjamin Duployer Philippe Bourin Daniel Cussac Angelo Parini Brigitte Sallerin Sophie Girod Fullana Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

UNLABELLED:: Osteoarthritis (OA) is the most widespread musculoskeletal disorder in adults. It leads to cartilage damage associated with subchondral bone changes and synovial inflammation, causing pain and disability. The present study aimed at evaluating the safety of a dose-escalation protocol of intra-articular injected adipose-derived stromal cells (ASCs) in patients with knee OA, as well as clinical efficacy as secondary endpoint. A bicentric, uncontrolled, open phase I clinical trial was conducted in France and Germany with regulatory agency approval for ASC expansion procedure in both countries. From April 2012 to December 2013, 18 consecutive patients with symptomatic and severe knee OA were treated with a single intra-articular injection of autologous ASCs. The study design consisted of three consecutive cohorts (six patients each) with dose escalation: low dose (2 × 10(6) cells), medium dose (10 × 10(6)), and high dose (50 × 10(6)). The primary outcome parameter was safety evaluated by recording adverse events throughout the trial, and secondary parameters were pain and function subscales of the Western Ontario and McMaster Universities Arthritis Index. After 6 months of follow-up, the procedure was found to be safe, and no serious adverse events were reported. Four patients experienced transient knee joint pain and swelling after local injection. Interestingly, patients treated with low-dose ASCs experienced significant improvements in pain levels and function compared with baseline. Our data suggest that the intra-articular injection of ASCs is a safe therapeutic alternative to treat severe knee OA patients. A placebo-controlled double-blind phase IIb study is being initiated to assess clinical and structural efficacy. SIGNIFICANCE:Although this phase I study included a limited number of patients without a placebo arm, it showed that local injection of autologous adipose-derived stem cells was safe and well tolerated in patients with knee osteoarthritis. This study also provides encouraging preliminary evidence of efficacy. Larger and controlled long-term studies are now mandatory to confirm whether this new strategy of cell therapy can improve pain and induce structural benefit in osteoarthritis.

The aim of this work was to understand whether the nature of breast cancer cells could modify the nature of the dialog of mesenchymal stem cells (MSCs) with cancer cells. By treating MSCs with the conditioned medium of metastatic Estrogen-receptor (ER)-negative MDA-MB-231, or non-metastatic ER-positive MCF-7 breast cancer cells, we observed that a number of chemokines were produced at higher levels by MSCs treated with MDA-MB-231 conditioned medium (CM). MDA-MB-231 cells were able to induce NF-κB signaling in MSC cells. This was shown by the use of a NF-kB chemical inhibitor or an IκB dominant negative mutant, nuclear translocation of p65 and induction of NF-κB signature. Our results suggest that MDA-MB-231 cells exert their effects on MSCs through the secretion of IL-1β, that activates MSCs and induces the same chemokines as the MDA-MB-231CM. In addition, inhibition of IL-1β secretion in the MDA-MB-231 cells reduces the induced production of a panel of chemokines by MSCs, as well the motility of MDA-MB-231 cells. Our data suggest that aggressive breast cancer cells secrete IL-1β, which increases the production of chemokines by MSCs.

Bone marrow mesenchymal stem cells ( MSC s) are plastic adherent cells that can differentiate into various tissue lineages, including osteoblasts, adipocytes and chondrocytes. However, this progenitor property is not shared by all cells within the MSC population. In addition, MSC s vary in their proliferation capacity and expression of markers. Because of heterogeneity of CD 146 expression in the MSC population, we compared CD 146 −/Low and CD 146 High cells under clonal conditions and after sorting of the non‐clonal cell population to determine whether this expression is associated with specific functions. CD 146 −/Low and CD 146 High bone marrow MSC s did not differ in colony‐forming unit‐fibroblast number, osteogenic, adipogenic and chondrogenic differentiation or in vitro haematopoietic‐supportive activity. However, CD 146 −/Low clones proliferated slightly but significantly faster than did CD 146 High clones. In addition, a strong expression of CD 146 molecule was associated with a commitment to a vascular smooth muscle cell ( VSMC ) lineage characterized by a strong up‐regulation of calponin‐1 and SM 22α expression and an ability to contract collagen matrix. Thus, within a bone marrow MSC population, certain subpopulations characterized by high expression of CD 146, are committed towards a VSMC lineage.

Successful preliminary studies have encouraged a more translational phase for stem cell research. Nevertheless, advances in the culture of human bone marrow-derived mesenchymal stromal/stem cells (hBM-MSC) and osteoconductive qualities of combined biomaterials can be undermined if necessary cell transportation procedures prove unviable. We aimed at evaluating the effect of transportation conditions on cell function, including the ability to form bone in vivo, using procedures suited to clinical application. hBM-MSC expanded in current Good Manufacturing Practice (cGMP) facilities (cGMP-hBM-MSC) to numbers suitable for therapy were transported overnight within syringes and subsequently tested for viability. Scaled-down experiments mimicking shipment for 18h at 4 degrees C tested the influence of three different clinical-grade transportation buffers (0.9% saline alone or with 4% human serum albumin [HSA] from two independent sources) compared with cell maintenance medium. Cell viability after shipment was >80% in all cases, enabling evaluation of (1) adhesion to plastic flasks and hydroxyapatite tricalcium phosphate osteoconductive biomaterial (HA/-TCP 3D scaffold); (2) proliferation rate; (3) ex vivo osteogenic differentiation in contexts of 2D monolayers on plastic and 3D HA/-TCP scaffolds; and (4) in vivo ectopic bone formation after subcutaneous implantation of cells with HA/-TCP scaffold into NOD/SCID mice. Von Kossa staining was used to assess ex vivo osteogenic differentiation in 3D cultures, providing a quantifiable test of 3D biomineralization ex vivo as a rapid, cost-effective potency assay. Near-equivalent capacities for cell survival, proliferation, and osteogenic differentiation were found for all transportation buffers. Moreover, cGMP-hBM-MSC transported from a production facility under clinical-grade conditions of 4% HSA in 0.9% saline to a destination 18h away showed prompt adhesion to HA/-TCP 3D scaffold and subsequent in vivo bone formation. A successfully validated transportation protocol extends the applicability of fresh stem cells involving multicentric trials for regenerative medicine.

The aim of this study was to assess the immune modulatory properties of human mesenchymal stromal cells obtained from bone marrow (BM-MSCs), fat (ASCs), and cord blood (CB-MSCs) in the presence of a hydroxyapatite and tricalcium-phosphate (HA/TCP) biomaterial as a scaffold for MSC delivery. In resting conditions, a short-term culture with HA/TCP did not modulate the anti-apoptotic and suppressive features of the various MSC types toward T, B, and NK cells; in addition, when primed with inflammatory cytokines, MSCs similarly increased their suppressive capacities in the presence or absence of HA/TCP. The long-term culture of BM-MSCs with HA/TCP induced an osteoblast-like phenotype with upregulation of OSTERIX and OSTEOCALCIN, similar to what was obtained with dexamethasone and, to a higher extent, with bone morphogenetic protein 4 (BMP-4) treatment. MSC-derived osteoblasts did not trigger immune cell activation, but were less efficient than undifferentiated MSCs in inhibiting stimulated T and NK cells. Interestingly, their suppressive machinery included not only the activation of indoleamine-2,3 dioxygenase (IDO), which plays a central role in T-cell inhibition, but also cyclooxygenase-2 (COX-2) that was not significantly involved in the immune modulatory effect of human undifferentiated MSCs. Since COX-2 is significantly involved in bone healing, its induction by HA/TCP could also contribute to the therapeutic activity of MSCs for bone tissue engineering.

Human adipose tissue-derived stromal cells (ASC) are used in various clinical trials. Transport or storage of cell products may be required, our aim was to define good-manufacturing practice (GMP) preservation protocols. This study focused on the characterization of fresh or cryopreserved cells during cold storage in liquid solution.