Characterization of the SARS-CoV-2 Neutralization Potential of COVID-19-Convalescent Donors.

The current SARS-CoV-2 pandemic has triggered the development of various SARS-CoV-2 neutralization tests. A wild-type virus (using African green monkey VeroE6 cells), a pseudovirus (using human Caco-2 cells), and a surrogate neutralization test platform were applied to characterize the SARS-CoV-2 neutralization potential of a cohort of 111 convalescent plasma donors over a period of seven months after diagnosis. This allowed an in-depth validation and assay performance analysis of these platforms. More importantly, we found that SARS-CoV-2 neutralization titers were stable or even increased within the observation period, which contradicts earlier studies reporting a rapid waning of Ab titers after three to four months. Moreover, we observed a positive correlation of neutralization titers with increasing age, number of symptoms reported, and the presence of the Rhesus Ag RhD. Validation of the platforms revealed that highest assay performances were obtained with the wild-type virus and the surrogate neutralization platforms. However, our data also suggested that selection of cutoff titers had a strong impact on the evaluation of neutralization potency. When taking strong neutralization potency, as demonstrated by the wild-type virus platform as the gold standard, up to 55% of plasma products had low neutralization titers. However, a significant portion of these products were overrated in their potency when using the surrogate assay with the recommended cutoff titer. In summary, our study demonstrates that SARS-CoV-2 neutralization titers are stable for at least seven months after diagnosis and offers a testing strategy for rapid selection of high-titer convalescent plasma products in a biosafety level 1 environment.

Independent Side-by-Side Validation and Comparison of 4 Serological Platforms for SARS-CoV-2 Antibody Testing.

Highly sensitive and specific platforms for the detection of anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies are becoming increasingly important for evaluating potential SARS-CoV-2 convalescent plasma donors, studying the spread of SARS-CoV-2 infections, and identifying individuals with seroconversion. This study provides a comparative validation of 4 anti-SARS-CoV-2 platforms. A unique feature of the study is the use of a representative cohort of convalescent patients with coronavirus disease 2019 and a mild to moderate disease course. All platforms showed significant correlations with a SARS-CoV-2 plaque reduction neutralization test, with highest sensitivities for the Euroimmun and the Roche platforms, suggesting their preferential use for screening persons at increased risk of SARS-CoV-2 infections.

Validation of Microbiological Testing of Cellular Medicinal Products Containing Antibiotics.

BACKGROUND: The risk of microbial contamination of cellular products can be reduced when cultured in the presence of antibiotics. This however, may impact the sensitivity of microbiological tests. Given that the addition of antibiotics to cell/tissue products does not guarantee sterility but may just reduce the proliferation rate of microorganisms, microbiological testing of medicinal products remains obligatory. Thus, an appropriate method to test for microbial contamination of antibiotic-containing products has to be validated. OBJECTIVES: In the context of microbiological testing of a cellular advance therapy medicinal product, the method was validated and approved by German competent authorities for four different matrices with three matrices containing antibiotics. The paper shall provide help for establishing test methods for other investigational medicinal products which contain antibiotics. METHODS: Matrices were spiked individually with Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Streptococcus pyogenes, Escherichia coli, Clostridium sporogenes, Propionibacterium acnes, Candida albicans, and Aspergillus brasiliensis. Samples were pretreated with penicillinase for 1 h before inoculation and incubation in BacT/ALERT iFA Plus and iFN Plus culture bottles using 3D BacT/ALERT automates. Microorganisms within positive BacT/ALERT bottles were specified. The procedure was performed in two different laboratories to prove robustness of test. RESULTS: All nine tested microorganisms were detected within 14 days of incubation in accordance with requirements of the European Pharmacopoiea in terms of sensitivity, specificity and robustness of the test. Penicillin and streptomycin did not have any influence on specifications defined within the investigational medicinal product dossier. CONCLUSIONS: Culturing cellular products in the presence of antibiotics can serve as an effective method to reduce contamination risk but only if the chosen antibiotics neither have any influence on specifications of the investigational medicinal product nor interfere with microbiological tests. Consequently, cells and tissues primarily contaminated with microorganisms, like placenta, may be considered as a source of cellular therapeutics when cultured for a sufficient time with antibiotics and tested with a validated method. The choice of microorganisms for the validation of the microbiological test should always consider all conceivable scenarios and should not be reduced to minimal criteria defined in European Pharmacopoiea, wrongfully believing to thus save time and effort.

CD90 Is Dispensable for White and Beige/Brown Adipocyte Differentiation.

Brown adipose tissue (BAT) is a thermogenic organ in rodents and humans. In mice, the transplantation of BAT has been successfully used to combat obesity and its comorbidities. While such beneficial properties of BAT are now evident, the developmental and cellular origins of brown, beige, and white adipocytes have remained only poorly understood, especially in humans. We recently discovered that CD90 is highly expressed in stromal cells isolated from human white adipose tissue (WAT) compared to BAT. Here, we studied whether CD90 interferes with brown or white adipogenesis or white adipocyte beiging. We applied flow cytometric sorting of human adipose tissue stromal cells (ASCs), a CRISPR/Cas9 knockout strategy in the human Simpson-Golabi-Behmel syndrome (SGBS) adipocyte model system, as well as a siRNA approach in human approaches supports the hypothesis that CD90 affects brown or white adipogenesis or white adipocyte beiging in humans. Taken together, our findings call the conclusions drawn from previous studies, which claimed a central role of CD90 in adipocyte differentiation, into question.

Biomarkers of bone healing induced by a regenerative approach based on expanded bone marrow-derived mesenchymal stromal cells.

BACKGROUND: Safety and feasibility of a regenerative strategy based on the use of culture-expanded mesenchymal stromal cells (MSCs) have been investigated in phase 2 trials for the treatment of nonunion and osteonecrosis of the femoral head (ONFH). As part of the clinical study, we aimed to evaluate if bone turnover markers (BTMs) could be useful for predicting the regenerative ability of the cell therapy product. MATERIALS AND METHODS: The bone defects of 39 patients (nonunion: n = 26; ONFH: n = 13) were treated with bone marrow-derived MSCs, expanded using a clinical-grade protocol and combined with biphasic calcium phosphate before implantation. Bone formation markers, bone-resorption markers and osteoclast regulatory proteins were measured before treatment (baseline) and after 12 and 24 weeks from surgery. At the same time-points, clinical and radiological controls were performed to evaluate the bone-healing progression. RESULTS: We found that C-Propeptide of Type I Procollagen (CICP) and C-terminal telopeptide of type-I collagen (CTX) varied significantly, not only over time, but also according to clinical results. In patients with a good outcome, CICP increased and CTX decreased, and this trend was observed in both nonunion and ONFH. Moreover, collagen biomarkers were able to discriminate healed patients from non-responsive patients with a good diagnostic accuracy. DISCUSSION: CICP and CTX could be valuable biomarkers for monitoring and predicting the regenerative ability of cell products used to stimulate the repair of refractory bone diseases. To be translated in a clinical setting, these results are under validation in a currently ongoing phase 3 clinical trial.

Translation of a standardized manufacturing protocol for mesenchymal stromal cells: A systematic comparison of validation and manufacturing data.

BACKGROUND: Many data are available on expansion protocols for mesenchymal stromal cells (MSCs) for both experimental settings and manufacturing for clinical trials. However, there is a lack of information on translation of established protocols for Good Manufacturing Practice (GMP) from validation to manufacturing for clinical application. We present the validation and translation of a standardized pre-clinical protocol for isolation and expansion of MSCs for a clinical trial for reconstitution of alveolar bone. METHODS: Key parameters of 22 large-scale expansions of MSCs from bone marrow (BM) for validation were compared with 11 expansions manufactured for the clinical trial "Jaw bone reconstruction using a combination of autologous mesenchymal stromal cells and biomaterial prior to dental implant placement (MAXILLO1)" aimed at reconstruction of alveolar bone. RESULTS: Despite variations of the starting material, the robust protocol led to stable performance characteristics of expanded MSCs. Manufacturing of the autologous advanced therapy medicinal product MAXILLO-1-MSC was possible, requiring 21 days for each product. Transport of BM aspirates and MSCs within 24 h was guaranteed. MSCs fulfilled quality criteria requested by the national competent authority. In one case, the delivered MSCs developed a mosaic in chromosomal finding, showing no abnormality in differentiation capacity, growth behavior or surface marker expression during long-term culture. The proportion of cells with the mosaic decreased in long-term culture and cells stopped growth after 38.4 population doublings. CONCLUSIONS: Clinical use of freshly prepared MSCs, manufactured according to a standardized and validated protocol, is feasible for bone regeneration, even if there was a long local distance between manufacturing center and clinical site. Several parameters, such as colony forming units fibroblasts (CFU-F), percentage of CD34+ cells, cell count of mononuclear cells (MNCs) and white blood cells (WBCs), of the BM may serve as a predictive tool for the yield of MSCs and may help to avoid unnecessary costs for MSC manufacturing due to insufficient cell expansion rates.

Systemic recovery and therapeutic effects of transplanted allogenic and xenogenic mesenchymal stromal cells in a rat blunt chest trauma model.

BACKGROUND: Effective therapy of Acute Lung Injury (ALI) is still a major scientific and clinical problem. To define novel therapeutic strategies for sequelae of blunt chest trauma (TxT) like ALI/Acute Respiratory Distress Syndrome, we have investigated the immunomodulatory and regenerative effects of a single dose of ex vivo expanded human or rat mesenchymal stromal cells (hMSCs/rMSCs) with or without priming, immediately after the induction of TxT in Wistar rats. METHODS: We analyzed the histological score of lung injury, the cell count of the broncho alveolar lavage fluid (BAL), the change in local and systemic cytokine level and the recovery of the administered cells 24 h and 5 days post trauma. RESULTS: The treatment with hMSCs reduced the injury score 24 h after trauma by at least 50% compared with TxT rats without MSCs. In general, TxT rats treated with hMSCs exhibited a lower level of pro-inflammatory cytokines (interleukin [IL]-1B, IL-6) and chemokines (C-X-C motif chemokine ligand 1 [CXCL1], C-C motif chemokine ligand 2 [CCL2]), but a higher tumor necrosis factor alpha induced protein 6 (TNFAIP6) level in the BAL compared with TxT rats after 24 h. Five days after trauma, cytokine levels and the distribution of inflammatory cells were similar to sham rats. In contrast, the treatment with rMSCs did not reveal such therapeutic effects on the injury score and cytokine levels, except for TNFAIP6 level. CONCLUSION: TxT represents a suitable model to study effects of MSCs as an acute treatment strategy after trauma. However, the source of MSCs has to be carefully considered in the design of future studies.

Autologous Mesenchymal Stroma Cells Are Superior to Allogeneic Ones in Bone Defect Regeneration.

The application of autologous mesenchymal stem cells (MSC) for the treatment of bone defects requires two invasive procedures and several weeks of ex vivo cell expansion. To overcome these limitations, the administration of allogeneic MSC may be attractive, because they are anticipated to be immunoprivileged. Because preclinical studies using various animal models are conflicting with respect to the efficacy of allogeneic MSC, we investigated whether autologous and allogeneic human MSC (hMSC) are equally effective in regenerating bone in a humanized mouse model resembling the human immune system. Applying autologous and allogeneic hMSC in critically sized femoral defects, we found that allogeneic hMSC elicited a mild immune response early after implantation, whereas early angiogenic processes were similar in both treatments. At later healing time points, the transplantation of allogeneic hMSC resulted in less bone formation than autologous hMSC, associated with a reduced expression of the osteogenic factor Runx2 and impaired angiogenesis. We found by species-specific staining for collagen-type-1α2 that MSCs of either source did not synthesize new bone matrix, indicating an indirect contribution of transplanted hMSC to bone regeneration. In conclusion, our data suggest that the application of autologous hMSC is superior to that of allogeneic cells for bone defect treatment.

Comparative Analysis of Different Platelet Lysates and Platelet Rich Preparations to Stimulate Tendon Cell Biology: An In Vitro Study.

The poor healing potential of tendons is still a clinical problem, and the use of Platelet Rich Plasma (PRP) was hypothesized to stimulate healing. As the efficacy of PRPs remains unproven, platelet lysate (PL) could be an alternative with its main advantages of storage and characterization before use. Five different blood products were prepared from 16 male donors: human serum, two PRPs (Arthrex, (PRP-ACP); RegenLab (PRP-BCT)), platelet concentrate (apheresis, PC), and PL (freezing-thawing destruction of PC). Additionally, ten commercial allogenic PLs (AlloPL) from pooled donors were tested. The highest concentration of most growth factors was found in AlloPL, whereas the release of growth factors lasted longer in the other products. PRP-ACP, PRP-BCT, and PC significantly increased cell viability of human tenocyte-like cells, whereas PC and AlloPL increased Col1A1 expression and PRP-BCT increased Col3A1 expression. MMP-1, IL-1ß, and HGF expression was significantly increased and Scleraxis expression decreased by most blood products. COX1 expression significantly decreased by PC and AlloPL. No clear positive effects on tendon cell biology could be shown, which might partially explain the weak outcome results in clinical practice. Pooled PL seemed to have the most beneficial effects and might be the future in using blood products for tendon tissue regeneration.

S100A4 and uric acid promote mesenchymal stromal cell induction of IL-10+/IDO+ lymphocytes.

Simple stress or necrotic cell death with subsequent release of damage-associated molecular patterns (DAMPs) is a characteristic feature of most advanced tumors. DAMPs within the tumor microenvironment stimulate tumor-associated cells, including dendritic cells and mesenchymal stromal cells (MSCs). The presence of tumor-infiltrating MSCs is associated with tumor progression and metastasis. Oxidized necrotic material loses its stimulatory capacity for MSCs. As a DAMP, S100A4 is sensitive to oxidation whereas uric acid (UA) acts primarily as an antioxidant. We tested these two biologic moieties separately and in combination for their activity on MSCs. Similar to necrotic tumor material, S100A4 and UA both dose-dependently induced chemotaxis of MSCs with synergistic effects when combined. Substituting for UA, alternative antioxidants (vitamin C, DTT, and N-acetylcysteine) also enhanced the chemotactic activity of S100A4 in a synergistic manner. This emphasizes the reducing potential of UA being, at least in part, responsible for the observed synergy. With regard to MSC proliferation, both S100A4 and UA inhibited MSCs without altering survival or inducing differentiation toward adipo-, osteo-, or chondrocytes. In the presence of S100A4 or UA, MSCs gained an immunosuppressive capability and stably induced IL-10- and IDO-expressing lymphocytes that maintained their phenotype following proliferation. We have thus demonstrated that both S100A4 and UA act as DAMPs and, as such, may play a critical role in promoting some aspects of MSC-associated immunoregulation. Our findings have implications for therapeutic approaches targeting the tumor microenvironment and addressing the immunosuppressive nature of unscheduled cell death within the tumor microenvironment.

Leukemic progenitor cells are susceptible to targeting by stimulated cytotoxic T cells against immunogenic leukemia-associated antigens.

Leukemic stem cells (LSC) might be the source for leukemic disease self-renewal and account for disease relapse after treatment, which makes them a critical target for further therapeutic options. We investigated the role of cytotoxic T-lymphocytes (CTL) counteracting and recognizing LSC. Leukemia-associated antigens (LAA) represent immunogenic structures to target LSC. We enriched the LSC-containing fraction of 20 AML patients and hematopoietic stem cells (HSC) of healthy volunteers. Using microarray analysis and qRT-PCR we detected high expression of several LAA in AML cells but also in LSC. PRAME (p = 0.0085), RHAMM (p = 0.03), WT1 (p = 0.04) and Proteinase 3 (p = 0.04) showed significant differential expression in LSC compared with HSC. PRAME, RHAMM and WT1 are furthermore also lower expressed on leukemic bulk. In contrast, Proteinase 3 indicates a higher expression on leukemic bulk than on LSC. In colony forming unit (CFU) immunoassays, T cells stimulated against various LAA indicated a significant inhibition of CFUs in AML patient samples. The LAA PRAME, RHAMM and WT1 showed highest immunogenic responses with a range up to 58-83%. In a proof of principle xenotransplant mouse model, PRAME-stimulated CTL targeted AML stem cells, reflected by a delayed engraftment of leukemia (p = 0.0159). Taken together, we demonstrated the expression of several LAA in LSC. LAA-specific T cells are able to hamper LSC in immunoassays and in a mouse model, which suggests that immunotherapeutic approaches have the potential to target malignant stem cells.

Standardization of Good Manufacturing Practice-compliant production of bone marrow-derived human mesenchymal stromal cells for immunotherapeutic applications.

BACKGROUND AIMS: Human mesenchymal stem or stromal cells (MSCs) represent a potential resource not only for regenerative medicine but also for immunomodulatory cell therapies. The application of different MSC culture protocols has significantly hampered the comparability of experimental and clinical data from different laboratories and has posed a major obstacle for multicenter clinical trials. Manufacturing of cell products for clinical application in the European Community must be conducted in compliance with Good Manufacturing Practice and requires a manufacturing license. In Germany, the Paul-Ehrlich-Institut as the Federal Authority for Vaccines and Biomedicines is critically involved in the approval process. METHODS: This report summarizes a consensus meeting between researchers, clinicians and regulatory experts on standard quality requirements for MSC production. RESULTS: The strategy for quality control testing depends on the product's cell composition, the manufacturing process and the indication and target patient population. Important quality criteria in this sense are, among others, the immunophenotype of the cells, composition of the culture medium and the risk for malignant transformation, as well as aging and the immunosuppressive potential of the manufactured MSCs. CONCLUSIONS: This position paper intends to provide relevant information to interested parties regarding these criteria to foster the development of scientifically valid and harmonized quality standards and to support approval of MSC-based investigational medicinal products.

T-cell reconstitution after allogeneic stem cell transplantation: assessment by measurement of the sjTREC/ßTREC ratio and thymic naive T cells.

The immune reconstitution after allogeneic hematopoietic stem cell transplantation comprises thymus-dependent and thymus-independent pathways. We wanted to improve the understanding of this complex process using two different measurements at definite checkpoints of T-cell neogenesis. We therefore assessed the thymus-dependent pathway by combining measurements of single joint T-cell receptor excision circles (sjTREC) and ß T-cell receptor excision circles (ßTREC) in an improved quantitative light-cycler hybridization polymerase chain reaction assay. In a subgroup of patients, we additionally assessed the proliferation kinetics of the CD31(+) thymic naïve cell population, which corresponds to recent thymic emigrants by six-color immunostaining. After the establishment of normal values in 22 healthy volunteers, we applied our polymerase chain reaction to 66 patients undergoing allogeneic hematopoietic stem cell transplantation at a median age of 44 years. It took more than 2 years after transplant to restore the pre-transplant thymic proliferation capacity. Only one third of the patients in our longitudinal study reached age-adjusted normal values for both sjTREC and ßTREC at a median follow-up of 558 days, with acute graft-versus-host disease being the most prominent negative factor by univariate analysis. We observed several patterns of sjTREC and ßTREC recovery suggesting different mechanisms of thymic damage in individual patients. In a comparison of CD31(+) thymic naïve cells between volunteers and patients after transplant we found a significantly higher peak proliferation rate within the latter population in the first year after transplantation. The combination of measurements of sjTREC and ßTREC by our simplified polymerase chain reaction assay provides insight about the stage of T-cell development affected by different types of damage and may help to choose the correct therapeutic intervention. Besides the sole thymic T-cell neogenesis, proliferation within the CD31(+) thymic naïve cell compartment contributed to the replenishment of the naïve T-cell pool after transplantation.

Evaluation of Human Mesenchymal Stromal Cells as Carriers for the Delivery of Oncolytic HAdV-5 to Head and Neck Squamous Cell Carcinomas.

Human multipotent mesenchymal stromal cells (hMSCs) are of significant therapeutic interest due to their ability to deliver oncolytic adenoviruses to tumors. This approach is also investigated for targeting head and neck squamous cell carcinomas (HNSCCs). HAdV-5-HexPos3, a recently reported capsid-modified vector based on human adenovirus type 5 (HAdV-5), showed strongly improved infection of both hMSCs and the HNSCC cell line UM-SCC-11B. Given that, we generated life cycle-unmodified and -modified replication-competent HAdV-5-HexPos3 vector variants and analyzed their replication within bone marrow- and adipose tissue-derived hMSCs. Efficient replication was detected for both life cycle-unmodified and -modified vectors. Moreover, we analyzed the migration of vector-carrying hMSCs toward different HNSCCs. Although migration of hMSCs to HNSCC cell lines was confirmed in vitro, no homing of hMSCs to HNSCC xenografts was observed in vivo in mice and in ovo in a chorioallantoic membrane model. Taken together, our data suggest that HAdV-5-HexPos3 is a potent candidate for hMSC-based oncolytic therapy of HNSCCs. However, it also emphasizes the importance of generating optimized in vivo models for the evaluation of hMSC as carrier cells.

A novel approach for large-scale manufacturing of small extracellular vesicles from bone marrow-derived mesenchymal stromal cells using a hollow fiber bioreactor.

Mesenchymal stromal cells (MSCs) are promising therapeutic candidates in a variety of diseases due to having immunomodulatory and pro-regenerative properties. In recent years, MSC-derived small extracellular vesicles (sEVs) have attracted increasing interest as a possible alternative to conventional cell therapy. However, translational processes of sEVs for clinical applications are still impeded by inconsistencies regarding isolation procedures and culture conditions. We systematically compared different methods for sEV isolation from conditioned media of ex vivo expanded bone marrow-derived MSCs and demonstrated considerable variability of quantity, purity, and characteristics of sEV preparations obtained by these methods. The combination of cross flow filtration with ultracentrifugation for sEV isolation resulted in sEVs with similar properties as compared to isolation by differential centrifugation combined with ultracentrifugation, the latter is still considered as gold standard for sEV isolation. In contrast, sEV isolation by a combination of precipitation with polyethylene glycol and ultracentrifugation as well as cross flow filtration and size exclusion chromatography resulted in sEVs with different characteristics, as shown by surface antigen expression patterns. The MSC culture requires a growth-promoting supplement, such as platelet lysate, which contains sEVs itself. We demonstrated that MSC culture with EV-depleted platelet lysate does not alter MSC characteristics, and conditioned media of such MSC cultures provide sEV preparations enriched for MSC-derived sEVs. The results from the systematic stepwise evaluation of various aspects were combined with culture of MSCs in a hollow fiber bioreactor. This resulted in a strategy using cross flow filtration with subsequent ultracentrifugation for sEV isolation. In conclusion, this workflow provides a semi-automated, efficient, large-scale-applicable, and good manufacturing practice (GMP)-grade approach for the generation of sEVs for clinical use. The use of EV-depleted platelet lysate is an option to further increase the purity of MSC-derived sEVs.

Effect of Expansion Media on Functional Characteristics of Bone Marrow-Derived Mesenchymal Stromal Cells.

The therapeutic efficacy of mesenchymal stromal cells (MSCs) has been shown to rely on their immunomodulatory and regenerative properties. In order to obtain sufficient numbers of cells for clinical applications, MSCs have to be expanded ex vivo. Expansion media with xenogeneic-free (XF) growth-promoting supplements like human platelet lysate (PL) or serum- and xenogeneic-free (SF/XF) formulations have been established as safe and efficient, and both groups provide different beneficial qualities. In this study, MSCs were expanded in XF or SF/XF media as well as in mixtures thereof. MSCs cultured in these media were analyzed for phenotypic and functional properties. MSC expansion was optimal with SF/XF conditions when PL was present. Metabolic patterns, consumption of growth factors, and secretome of MSCs differed depending on the type and concentration of supplement. The lactate per glucose yield increased along with a higher proportion of PL. Many factors in the supernatant of cultured MSCs showed distinct patterns depending on the supplement (e.g., FGF-2, TGFß, and insulin only in PL-expanded MSC, and leptin, sCD40L PDGF-AA only in SF/XF-expanded MSC). This also resulted in changes in cell characteristics like migratory potential. These findings support current approaches where growth media may be utilized for priming MSCs for specific therapeutic applications.

Human mesenchymal stem cells respond to native but not oxidized damage associated molecular pattern molecules from necrotic (tumor) material.

Necrosis is a characteristic feature of advanced solid tumors. Released necrotic factors, also referred to as damage associated molecular patterns (DAMPs), are known to critically impact the tumor microenvironment by enhancing angiogenesis or influencing the immune response. We have recently shown that DAMPs can act as chemoattractants and activators of granulocytes. We demonstrate that necrotic material from both normal and tumor cells promotes proliferation and trafficking of human mesenchymal stem cells (MSCs). We characterize the protein high mobility group box 1 (HMGB1) as a crucial member of DAMPs within necrotic material. In addition, we show that DAMPs interfere with expression of indoleamine 2, 3-dioxygenase (IDO) in MSCs. The biological activity of necrotic material toward MSCs is abolished once these DAMPs are oxidized. MSCs found within tumor tissue can act as immunoregulatory cells and are able to promote tumor metastasis, thus playing a crucial role within the tumor microenvironment. Here, we reveal DAMPs to be crucial factors in the setting of MSC biology within the tumor microenvironment. The tumor microenvironment is characterized by reducing and hypoxic conditions that protect DAMPs from oxidation. Based on our results, oxidizing conditions should be considered for therapeutic approaches that target the tumor microenvironment.

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.

Hexon modification of human adenovirus type 5 vectors enables efficient transduction of human multipotent mesenchymal stromal cells.

In adenovirus type 5 (HAdV-5)-derived viral vectors, the fiber protein has been the preferred locale for modifications to alter the natural viral tropism. Hexon, the most abundant capsid protein, has rarely been used for retargeting purposes, likely because the insertion of larger targeting peptides into Hexon often interferes with the assembly of the viral capsid. We previously observed that positively charged molecules enhance the transduction of human multipotent mesenchymal stromal cells (hMSCs)-a cell type of significant interest for clinical development but inefficiently transduced by unmodified HAdV-5-based vectors. As efficient HAdV-5-mediated gene transfer would greatly increase the therapeutic potential of hMSCs, we tested the hypothesis that introducing positively charged amino acids into Hexon might enhance the transduction of hMSCs, enabling efficient expression of selected transgenes. From the constructs that could be rescued as functional virions, one (HAdV-5-HexPos3) showed striking transduction of hMSCs with up to 500-fold increased efficiency. Evaluation of the underlying mechanism identified heparan sulfate proteoglycans (HSPGs) to be essential for virus uptake by the cells. The ease and efficiency of transduction of hMSCs with this vector will facilitate the development of genetically modified hMSCs as therapeutic vehicles in different disciplines, including oncology or regenerative medicine.

Cytokine levels associated with favorable clinical outcome in the CAPSID randomized trial of convalescent plasma in patients with severe COVID-19.

Objectives: To determine the profile of cytokines in patients with severe COVID-19 who were enrolled in a trial of COVID-19 convalescent plasma (CCP). Methods: Patients were randomized to receive standard treatment and 3 CCP units or standard treatment alone (CAPSID trial, ClinicalTrials.gov NCT04433910). The primary outcome was a dichotomous composite outcome (survival and no longer severe COVID-19 on day 21). Time to clinical improvement was a key secondary endpoint. The concentrations of 27 cytokines were measured (baseline, day 7). We analyzed the change and the correlation between serum cytokine levels over time in different subgroups and the prediction of outcome in receiver operating characteristics (ROC) analyses and in multivariate models. Results: The majority of cytokines showed significant changes from baseline to day 7. Some were strongly correlated amongst each other (at baseline the cluster IL-1ß, IL-2, IL-6, IL-8, G-CSF, MIP-1α, the cluster PDGF-BB, RANTES or the cluster IL-4, IL-17, Eotaxin, bFGF, TNF-α). The correlation matrix substantially changed from baseline to day 7. The heatmaps of the absolute values of the correlation matrix indicated an association of CCP treatment and clinical outcome with the cytokine pattern. Low levels of IP-10, IFN-γ, MCP-1 and IL-1ß on day 0 were predictive of treatment success in a ROC analysis. In multivariate models, low levels of IL-1ß, IFN-γ and MCP-1 on day 0 were significantly associated with both treatment success and shorter time to clinical improvement. Low levels of IP-10, IL-1RA, IL-6, MCP-1 and IFN-γ on day 7 and high levels of IL-9, PDGF and RANTES on day 7 were predictive of treatment success in ROC analyses. Low levels of IP-10, MCP-1 and high levels of RANTES, on day 7 were associated with both treatment success and shorter time to clinical improvement in multivariate models. Conclusion: This analysis demonstrates a considerable dynamic of cytokines over time, which is influenced by both treatment and clinical course of COVID-19. Levels of IL-1ß and MCP-1 at baseline and MCP-1, IP-10 and RANTES on day 7 were associated with a favorable outcome across several endpoints. These cytokines should be included in future trials for further evaluation as predictive factors.