From osteoarthritic synovium to synovial-derived cells characterization: synovial macrophages are key effector cells.

BACKGROUND: The aim of the study was to characterize synovial cells from OA synovium with low-grade and moderate-grade synovitis and to define the role of synovial macrophages in cell culture. METHODS: Synovial tissue explants were analyzed for the expression of typical markers of synovial fibroblasts (SF), synovial macrophages (SM) and endothelial cells. Synovial cells at passage 1 (p.1) and 5 (p.5) were analyzed for different phenotypical markers by flow cytometric analysis, inflammatory factors by multiplex immunoassay, anabolic and degradative factors by qRT-PCR. P.1 and p.5 synovial cells as different cell models were co-cultured with adipose stem cells (ASC) to define SM effects. RESULTS: Synovial tissue showed a higher percentage of CD68 marker in moderate compared with low-grade synovitis. Isolated synovial cells at p.1 were positive to typical markers of SM (CD14, CD16, CD68, CD80 and CD163) and SF (CD55, CD73, CD90, CD105, CD106), whereas p.5 synovial cells were positive only to SF markers and showed a higher percentage of CD55 and CD106. At p.1 synovial cells released a significantly higher amount of all inflammatory (IL6, CXCL8, CCL2, CCL3, CCL5) and some anabolic (IL10) factors than those of p.5. Moreover, p.1 synovial cells also expressed a higher amount of some degradative factors (MMP13, S100A8, S100A9) than p.5 synovial cells. Co-culture experiments showed that the amount of SM in p.1 synovial cells differently induced or down-modulated some of the inflammatory (IL6, CXCL8, CCL2, CCL3, CCL5) and degradative factors (ADAMTS5, MMP13, S100A8, S100A9). CONCLUSIONS: We found that p.1 (mix of SM and SF) and p.5 (only SF) synovial cells represent two cell models that effectively reproduce the low- or moderate-grade synovitis environment. The presence of SM in culture specifically induces the modulation of the different factors analyzed, confirming that SM are key effector cells.

Epigenetic regulation of human adipose-derived stem cells differentiation.

Adult stem cells have more restricted differentiation potential than embryonic stem cells (ESCs), but upon appropriate stimulation can differentiate into cells of different germ layers. Epigenetic factors, including DNA modifications, take a significant part in regulation of pluripotency and differentiation of ESCs. Less is known about the epigenetic regulation of these processes in adult stem cells. Gene expression profile and location of DNA modifications in adipose-derived stem cells (ADSCs) and their osteogenically differentiated lineages were analyzed using Agilent microarrays. Methylation-specific PCR and restriction-based quantitative PCR were applied for 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) detection in selected loci. The level of DNA modifications in the POU5F1 locus was quantified with deep sequencing. Expression levels of selected genes were assayed by real-time PCR. ADSCs differentiation into osteogenic lineages involved marked changes in both 5mC and 5hmC profiles, but 5hmC changes were more abundant. 5mC losses and 5hmC gains were the main events observed during ADSCs differentiation, and were accompanied by increased expression of TET1 (P = 0.009). In ADSCs, POU5F1 was better expressed than NANOG or SOX2 (P ≤ 0.001). Both 5mC and 5hmC marks were present in the POU5F1 locus, but only hydroxymethylation of specific cytosine showed significant effect on the gene expression. In summary, the data of our study suggest significant involvement of changes in 5hmC profile during the differentiation of human adult stem cells.

Biodistribution of mesenchymal stem/stromal cells in a preclinical setting.

Due to their multi/pluripotency and immunosuppressive properties, mesenchymal stem/stromal cells (MSCs) are important tools for treatment of immune disorders and tissue repair. The increasing uses of MSCs lead to the development of production processes that need to be in accordance with good manufacturing practices (GMP). In Europe, MSCs are somatic cell-therapy products, referred to as advanced-therapy medicinal products (ATMPs), and in the United States MSCs must comply with current good tissue practice requirements. The safety and efficacy of MSCs must be ensured, whatever the cell source, and studies of dose and biodistribution are important aspects of safety testing. Preclinical data on biodistribution and pharmacodynamics are mandatory for approval. It is important to demonstrate that MSCs do not have unwanted homing that could drive to inappropriate differentiation in some organ or to support cancer development as suggested in some experiments. All these aspects should be addressed in a risk-based approach according to recently published guidelines by EMA. In the present article, we summarize the main approaches for labeling and tracking of infused MSCs, report on current animal models, and give an overview of available results on biodistribution.

Production of mesenchymal stromal/stem cells according to good manufacturing practices: a review.

Because of their multi/pluripotency and immunosuppressive properties, mesenchymal stem/stromal cells (MSCs) are important tools for treating immune disorders and for tissue repair. The increasing use of MSCs, their definition as advanced-therapy medicinal products in European regulations, and the US Food and Drug Administration requirements for their production and use imply the use of production processes that should be in accordance with Good Manufacturing Practices (GMPs). Complying with GMPs requires precisely defining the production process (es) as well as the multiple criteria required for a quality final product. Such variables include the environment, staff training and qualification, and controls. Developing processes based on well-defined or completely defined media and operating in closed systems or bioreactors is important and will increase safety and reproducibility. One of the most challenging issues remains implementation of relevant and reproducible controls for safety and efficacy. A linking of researchers, research and development teams, producers, and clinicians is mandatory to achieve GMP-compliant processes with relevant controls for producing well-defined, safe, and efficient MSCs.

Adipose-derived mesenchymal stem cells exert antiinflammatory effects on chondrocytes and synoviocytes from osteoarthritis patients through prostaglandin E2.

OBJECTIVE: To examine the effect of different sources of good manufacturing practice clinical grade adipose-derived mesenchymal stem cells (AD-MSCs) on inflammatory factors in osteoarthritic (OA) chondrocytes and synoviocytes. METHODS: AD-MSCs from infrapatellar Hoffa fat, subcutaneous (SC) hip fat, and SC abdominal fat were cocultured in Transwells with chondrocytes or synoviocytes. Inflammatory factors (interleukin-1ß [IL-1ß], tumor necrosis factor α, IL-6, CXCL1/growth-related oncogene α, CXCL8/IL-8, CCL2/monocyte chemotactic protein 1, CCL3/macrophage inflammatory protein 1α, and CCL5/RANTES) were evaluated by quantitative reverse transcription-polymerase chain reaction or multiplex bead-based immunoassay. The role of different immunomodulators was analyzed. RESULTS: All the inflammatory factors analyzed were down-modulated at the messenger RNA or protein level independently by all 3 AD-MSC sources or by allogeneic AD-MSCs used in coculture with chondrocytes or synoviocytes. Inflammatory factor down-modulation was observed only when AD-MSCs were cocultured with chondrocytes or synoviocytes that produced high levels of inflammatory factors, but no effect was observed in cells that produced low levels of those factors, thus highlighting a dependence of the AD-MSC effect on existing inflammation. The immunomodulators IL-10, IL-1 receptor antagonist, fibroblast growth factor 2, indoleamine 2,3-dioxygenase 1, and galectin 1 were not involved in AD-MSC effects, whereas the cyclooxygenase 2 (COX-2)/prostaglandin E2 (PGE2 ) pathway exerted a role in the mechanism of antiinflammatory AD-MSC action. CONCLUSION: The antiinflammatory effects of AD-MSCs are probably not dependent on AD-MSC adipose tissue sources and donors but rather on the inflammatory status of OA chondrocytes and synoviocytes. AD-MSCs seem to be able to sense and respond to the local environment. Even though a combination of different molecules may be involved in AD-MSC effects, the COX-2/PGE2 pathway may play a role, suggesting that AD-MSCs may be useful for therapies in osteoarticular diseases.

Platelet lysate from whole blood-derived pooled platelet concentrates and apheresis-derived platelet concentrates for the isolation and expansion of human bone marrow mesenchymal stromal cells: production process, content and identification of active components.

BACKGROUND AIMS: The clinical use of human mesenchymal stromal cells (MSC) requires ex vivo expansion in media containing supplements such as fetal bovine serum or, alternatively, human platelet lysate (PL). METHODS: Platelet concentrates were frozen, quarantine stored, thawed and sterile filtered to obtain PL. PL content and its effect on fibroblast-colony-forming unit (CFU-F) formation, MSC proliferation and large-scale expansion were studied. RESULTS: PL contained high levels of basic fibroblast growth factor (bFGF), soluble CD40L (sCD40L), vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), platelet-derived growth factor AA (PDGF-AA), platelet-derived growth factor AB/BB (PDGF-AB/BB), chemokine (C-C) ligand 5 (CCL5; RANTES) transforming growth factor-ß1 (TGF-ß1) and chemokine (C-X-C) ligand 1/2/3 (GRO), with low batch-to-batch variability, and most were stable for up to 14 days. Inhibition of PDGF-BB and bFGF decreased MSC proliferation by about 20% and 50%, respectively. The strongest inhibition (about 75%) was observed with a combination of anti-bFGF + anti-PDGF-BB and anti-bFGF + anti-TGF-ß1 + anti-PDGF-BB. Interestingly, various combinations of recombinant PDGF-BB, bFGF and TGF-ß1 were not sufficient to promote cell proliferation. PL from whole blood-derived pooled platelet concentrates and apheresis platelet concentrates did not differ significantly in their growth-promoting activity on MSC. CONCLUSIONS: PL enhances MSC proliferation and can be regarded as a safe tool for MSC expansion for clinical purposes. \in particular, PDGF-BB and bFGF are essential components for the growth-promoting effect of PL, but are not sufficient for MSC proliferation.

A first approach for the production of human adipose tissue-derived stromal cells for therapeutic use.

Adipose tissue-derived stromal cells (ASCs) are promising tools for the new therapeutic field of regenerative medicine. Many research teams are intent on producing these cells for therapeutic purposes. The cell production must follow strict rules for safety and for constant quality of the cell product to ensure a reliable effect in patients. These rules are grouped under the generic term Good Manufacturing Practices. In this chapter, we describe the general concepts of ASC production for therapeutic use, explaining new terms such as traceability and qualification. We also introduce general requirements for the installation, equipment, material, and staff for the cell production. Then, we outline a general strategy for building a cell culture process. Finally, as an example, we describe the use of CellStack™ chambers and specific tube sets that allow for producing cells beginning with the stromal vascular fraction under near-closed conditions.

A regulatory cross-talk between Vgamma9Vdelta2 T lymphocytes and mesenchymal stem cells.

The physiological functions of human TCRVgamma9Vdelta2(+) gammadelta lymphocytes reactive to non-peptide phosphoantigens contribute to cancer immunosurveillance and immunotherapy. However, their regulation by mesenchymal stem cells (MSC), multipotent and immunomodulatory progenitor cells able to infiltrate tumors, has not been investigated so far. By analyzing freshly isolated TCRVgamma9Vdelta2(+) lymphocytes and primary cell lines stimulated with synthetic phosphoantigen or B-cell lymphoma cell lines in the presence of MSC, we demonstrated that MSC were potent suppressors of gammadelta-cell proliferation, cytokine production and cytolytic responses in vitro. This inhibition was mediated by the COX-2-dependent production of prostaglandin E2 (PGE(2)) and by MSC through EP2 and EP4 inhibitory receptors expressed by Vgamma9Vdelta2 T lymphocytes. COX-2 expression and PGE(2) production by MSC were not constitutive, but were induced by IFN-gamma and TNF-alpha secreted by activated Vgamma9Vdelta2 T cells. This regulatory cross-talk between MSC and Vgamma9Vdelta2 T lymphocytes involving PGE(2) could be of importance for the antitumor and antimicrobial activities of gammadelta T cells.

Mesenchymal Progenitor Cells: Tissue Origin, Isolation and Culture.

SUMMARY: Since the pioneering work of Alexander Friedenstein on multipotent mesenchymal stromal cells (MSCs), a tremendous amount of work has been done to isolate, characterize and culture such cells. Assay of colony forming unit-fibroblasts (CFU-Fs), the hallmark of MSCs, is used to estimate their frequency in tissue. MSCs are adherent cells, so they are easy to isolate, and they show contact inhibition. Thus, several parameters must be taken into account for culture: cell density, number of passages, culture medium, and growth factors used. The purity of the initial material is not a limiting parameter. Similar but not identical cell populations are found in almost all mammal or human tissues. MSCs seem to be very abundant in adipose tissue but at low frequency in blood from umbilical cord or in adult tissue. The culture conditions are very similar, whatever the source of cells. Because of their favorable properties, MSCs are very promising tools for regenerative medicine.

Association of donor TNFRSF6 (FAS) gene polymorphism with acute rejection in renal transplant patients: a case-control study.

BACKGROUND: Genetic factors other than HLA have been reported to be associated with the outcome of organ transplantations. Because binding of FasL to its receptor Fas could play an important role in tubulitis and in the death of graft tubular epithelial cells during kidney allograft rejection, a gene polymorphism recently identified in position -671 in the promoter of the TNFRSF6 gene coding for Fas was investigated in donors. METHODS: A case-control study was performed within a cohort of non-hyperimmunized adult patients who had received cadaveric kidney transplants based on the occurrence or absence of acute cellular rejection in the first 6 months after renal transplantation. Each recipient from the acute rejection group (n = 35) was matched for age (+/- 5 years) and number of HLA-DR mismatches with two recipients within the non-acute rejection group (n = 70). RESULTS: The TNFRSF6-GG genotype was more frequent in donors in the group without rejection episodes. In contrast, patients who received a kidney from a TNFRSF6-A carrier were more likely to experience acute rejection episodes (relative risk nearly 2.1). CONCLUSION: This study suggests that donor TNFRSF6 polymorphism directly or indirectly influences acute kidney rejection episodes.

FasL-transfected endothelial cells decrease the proliferative response of allogeneic PBL.

Graft endothelium has a key role in organ transplantation because it regulates graft infiltration by allogeneic activated T cells. Overexpression of death molecules that could induce apoptosis of alloreactive T cells might be an alternative to the immunosuppressive treatment currently used in graft transplantation. Several studies have shown that immune-privileged sites express Fas ligand (FasL) and induce apoptosis of activated T-cells. We propose that endothelial cells engineered to express FasL could inhibit alloreactive T cell-proliferation by inducing apoptosis. An expression vector was constructed with human FasL cDNA and used to transfect an endothelial cell line (ECV304 cells). We demonstrated that FasL-transfected ECV304 cells were effective in inducing apoptosis of Jurkat T cell lymphoma as an agonist anti-Fas antibody. Using a mixed lymphocyte-endothelial cell culture model we observed that FasL-transfected ECV304 cells which conserved their two principal costimulatory pathways inhibited alloreactive T cell-proliferation by inducing activated T-cell apoptosis. These results suggest that endothelial cells could be interesting candidates to convey a death signal and induce hyporesponsiveness of alloreactive T cells during organ transplantation.