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Recent years have witnessed the introduction of ex vivo expanded dermal fibroblasts for several cell therapy and tissue-engineering applications, including the treatment of facial scars and burns, representing a promising cell type for regenerative medicine. We tested different in-house produced human platelet lysate (HPL) solutions against fetal bovine serum as supplements for in vitro fibroblast expansion by comparing cell yield, molecular marker expression, extracellular matrix (ECM) generation, genomic stability and global gene expression. Our in-house produced HPL supported fibroblast growth at levels similar to those for FBS and commercial HPL products and was superior to AB human serum. Cells grown in HPL maintained a fibroblast phenotype (VIM+, CD44+, CD13+, CD90+), ECM generation capacity (FN+, COL1+) and a normal karyotype, although gene expression profiling revealed changes related to cell metabolism, adhesion and cellular senescence. The HPL manufacturing process was validated within a GMP compliant system and the solution was stable at -80ºC and -20ºC for 2 years. Dermal fibroblasts expanded in vitro with HPL maintain a normal karyotype and expression of fibroblast markers, with only minor changes in their global gene expression profile. Our in-house produced GMP-HPL is an efficient, safe and economical cell culture supplement that can help increase the healthcare activity of blood transfusion centers through the re-use of transfusional plasma and platelets approaching their expiration date. Currently, our HPL solution is approved by the Spanish Agency of Medicines and Medical Devices and is being used in the manufacture of cell therapy products.Abbreviations: AB plasma: plasma group AB; ABHS: AB Human Serum; ABHS+GF: AB Human Serum supplemented with growth factors; ANOVA: Analysis of variance; ATMPs: Advanced Therapies for Medicinal Products; CPE: cytopathic effect; DEGs: Differentially expressed genes; DMEM: Dulbecco's modified Eagle's Medium; ECM: Extracellular matrix; ELISA: enzyme-linked immunosorbent assay; FBS: Fetal bovine serum; FDR: False discovery rate; FGF: Fibroblast growth factor; GMP: Good manufacturing practice; HPL: Human platelet lysate; HPL-CM: commercial human platelet lysate; MSCs: mesenchymal stem cells; NEAA: non-essential amino acids; P/S: penicillin/streptomycin; PBS: phosphate buffered saline; PC: leukodepleted platelet concentrate; PCR: polymerase chain reaction; PDGF: Platelet-derived growth factor; PDGFRA: Platelet-derived growth factor receptor alpha; qPCR: quantitative polymerase chain reaction; RNA: Ribonucleic acid; RT: Room temperature; TAC: Transcriptome analysis console; TGF-β: Transforming growth factor beta., This study has been funded by the Instituto de Salud Carlos III through the project “DTS17/00137” (Co-funded by European Regional Development Fund/European Social Fund “A way to make Europe”).

Anti-CD19 chimeric antigen receptor (CAR)-T cells have achieved impressive outcomes for the treatment of relapsed and refractory B-lineage neoplasms.However, important limitations still remain due to severe adverse events (i.e., cytokine release syndrome and neuroinflammation) and relapse of 40%–50%of the treated patients.MostCAR-Tcells are generated using retroviral vectors with strong promoters that lead to high CAR expression levels, tonic signaling, premature exhaustion, and overstimulation, reducing efficacy and increasing side effects. Here, we show that lentiviral vectors (LVs) expressing the transgene through a WAS gene promoter (AW-LVs) closely mimic the T cell receptor (TCR)/CD3 expression kinetic upon stimulation. These AW-LVs can generate improved CAR-T cells as a consequence of theirmoderate andTCR-like expression profile. Compared with CAR-T cells generated with human elongation factor a (EF1a)-driven-LVs, AW-CAR-T cells exhibited lower tonic signaling, higher proportion of naive and stem cell memory T cells, less exhausted phenotype, and milder secretion of tumor necrosis factor alpha (TNF-a) and interferon (IFN)-ɣ after efficient destruction of CD19+ lymphoma cells, both in vitro and in vivo.Moreover, we also showed their improved efficiency using an in vitro CD19+ pancreatic tumor model. We finally demonstrated the feasibility of large-scale manufacturing ofAW-CAR-T cells in good manufacturing practice (GMP)-like conditions. Based on these data, we propose the use of AW-LVs for the generation of improved CAR-T products., Spanish ISCIII Health Research Fund, European Commission PI15/02015 PI18/00337 PI21/00298 RD21/0017/0004 PI18/00330 PI17/00672, CSyF of the Junta de Andalucia FEDER/European Cohesion Fund (FSE) for Andalusia 2016000073391-TRA 2016000073332-TRA PI-57069 PA IDI-Bio326 CARTPI-0001-201 PECART-0031-2020 Red RANTECAR CAR-T 2019 00400200101918 PLEC2021-008094 PI-0014-2016 PEER-0286-2019, Spanish Government PLEC2021-008094 00123009/SNEO-20191072, Nicolas Monardes contracts from regional Ministry of Health 0006/2018 C2-0002-2019, German Research Foundation (DFG) FPU16/05467 FPU17/02268 FPU17/04327 MCI DIN2018-010180, Fundacion Andaluza Progreso y Salud, German Research Foundation (DFG) PEJ-2018-001760-A, Junta de Andalucia PE-0223-2018, Biomedicine Programme of the University of Granada (Spain)

BackgroundChimeric antigen receptor (CAR) T cells directed against CD19 have achieved impressive outcomes for the treatment of relapsed/refractory B lineage lymphoid neoplasms. However, CAR-T therapy still has important limitations due to severe side effects and the lack of efficiency in 40-50% of the patients. Most CARs-T products are generated using retroviral vectors with strong promoters. However, high CAR expression levels can lead to tonic signalling, premature exhaustion and over-stimulation of CAR-T cells, reducing efficacy and increasing side effects. TCR-like expression of the CAR through genome editing resulted in enhanced anti-tumour potency, reducing tonic signalling and improving CAR-T phenotype. In this manuscript, we searched for LVs that mimic the TCR expression pattern as a closer-to-clinic alternative for the generation of improved CAR-T cells.MethodsDifferent LVs containing viral and human promoters were analysed to select those that closely mimic a TCR-like kinetic profile upon T-cell activation. WAS gene proximal promoter-driven LVs (AW-LVs) were selected to express a second generation 4-1BB aCD19 CAR (ARI-0001) into T cells to generate AWARI CAR-T cells. TCR-like AWARI and EF1α-driven ARI CAR T cells were analysed for in vitro and in vivo killing efficiency using leukaemia and lymphoma cellular models. Tonic signalling, exhaustion markers and phenotype were determined by flow cytometry. Large-scale automated manufacturing of AWARI CAR-T cells was performed in a CliniMACs Prodigy bioreactor.ResultsOur data showed that LVs expressing the transgene through the WAS gene proximal promoter mimic very closely the TCR (CD3) expression pattern kinetic upon TCR stimulation or antigen encounter. Compared to EF1α-driven ARI CAR-T cells, AWARI CAR-T cells exhibited a higher proportion of naïve/stem cell memory T cells with less exhausted phenotype after efficient killing of CD19+ cells both in vitro and in vivo. AWARI CAR-T cells also showed lower tonic signalling and reduced secretion of pro-inflammatory cytokines and were efficiently manufactured in large-scale GMP-like conditions.ConclusionsWAS-gene-promoter driven LVs can be used to generate physiological 4-1BB-CAR-T cell products with lower tonic signalling, improved phenotype and a safer profile. We propose the use of TCR-like LVs as an alternative to strong-promoter driven LVs for the generation of CAR-T products.

Background aims Successful cell cryopreservation and banking remain a major challenge for the manufacture of cell therapy products, particularly in relation to providing a hermetic, sterile cryovial that ensures optimal viability and stability post-thaw while minimizing exposure to toxic cryoprotective agents, typically dimethyl sulfoxide (Me2SO). Methods In the present study, the authors evaluated the effectiveness and functionality of Limbo technology (Cellulis S.L., Santona, Spain). This system provides a hermetic vial with two compartments (one for adding cells with the cryoprotective agent solution and the other for the diluent solution) and an automated defrosting device. Limbo technology (Cellulis S.L.) allows reduction of the final amount of Me2SO, sidestepping washing and dilution steps and favoring standardization. The study was performed in several Good Manufacturing Practice laboratories manufacturing diverse cell therapy products (human mesenchymal stromal cells, hematopoietic progenitor cells, leukapheresis products, fibroblasts and induced pluripotent stem cells). Laboratories compared Limbo technology (Cellulis S.L.) with their standard cryopreservation procedure, analyzing cell recovery, viability, phenotype and functionality. Results Limbo technology (Cellulis S.L.) maintained the viability and functionality of most of the cell products and preserved sterility while reducing the final concentration of Me2SO. Conclusions Results showed that use of Limbo technology (Cellulis S.L.) offers an overall safe alternative for cell banking and direct infusion of cryopreserved cell products into patients.

Background & Aim Human platelet lysate (hPL) has long been used as a xeno-free culture supplement to expand different cell types. Its use is currently thriving given its potential therapeutic properties and its increasing importance in a clinical manufacturing context; however, to date, data regarding the stability of HPL solutions is limited. The general chapter "Raw materials for the production of cell-based and gene therapy medicinal products" (EP 5.2.12), gives examples of the critical quality attributes specific to each class of raw material. The aim of these studies has been to stablish the expiration time of our product in specific storage conditions. Methods, Results & Conclusion Five batches of our clinical grade hPL currently accredited by Spanish Medicine Agency, were selected for stability testing. Cells were culture (with 5%, 10% and 15% hPL) to analysed the stability of the supplemented medium with Hpl at 4 degrees. The two-year stability study has included the following controls: sterility, mycoplasma, adventitious viruses and endotoxin, activity test, pH, PDGF-AB, total proteins, albumin and IgGs. Sterility, mycoplasma, adventitious viruses and endotoxin level were performed after manufacturing (time 0) and after 24 months while activity test, pH level, PDGF-AB, total proteins, albumin and IgGs concentration were determined from each batch immediately after manufacturing (time zero) or after storage at -80 degrees for 6, 12 or 24 months. All the controls were performed according to European Pharmacopoeia if exist or according to approval procedures. No significant differences were observed regarding the proliferation capacity of fibroblasts cultured with 5%, 10% and 15% hPL for at least 40 days, maintaining the activity in culture. All microbiological controls met the acceptance criteria established to the validation at two years. No significant differences were observed in fibroblasts doubling time when we compared hPL solution and FBS at time 0 and after 24 months. PDGF-AB and pH remained stable at −80 degrees. Besides, total proteins, albumin and IgGs neither showed significant changes along 2 years. Here we show that our hPL solution remains stable in the supplemented medium for 40 days at 4 degrees without significant loss of activity and for 2 years at -80 degrees, maintaining its growth promotion activity in cells, pH and PDGF-AB levels, total proteins, albumin and IgGs concentrations, being an attractive alternative to commercial solutions for cell culture in a clinical context.

Background Effects of cell therapy on dilated cardiomyopathy (DCM) have been investigated in pre-clinical models using distinct cellular types in each study. A single study that compares the effectiveness of different cells is lacking. Methods We have compared the effects of intramyocardial injection (IMI) of bone marrow (BM)–derived mononuclear cells (MNCs), BM and adipose tissue (AT) mesenchymal stromal cells (BM-MSCs and AT-MSCs) on heart function, histological changes and myocardial ultrastructure in a rat model of DCM. Isogenic Wistar rats were used to isolate the different cell types and to induce DCM by autoimmune myocarditis. Animals were randomly assigned to receive BM-MNCs, BM-MSCs, AT-MSCs or placebo at day 42 by IMI. Serial echocardiography was used to assess cardiac function and hearts obtained after sacrifice at day 70, were used for histological and ultrastructural analysis. Serum levels of type B-natriuretic peptide (BNP) and vascular endothelial growth-factor (VEGF) were determined at different time points. Results BM-MSC treatment induced significant improvement in ejection fraction (EF), fractional shortening (FS), left ventricular systolic diameter (LVESD) and systolic volume (LVESV). In contrast, changes in echocardiographic parameters with respect to pre-treatment values in animals receiving placebo, AT-MSCs or BM-MNCs were not statistically significant. EF and FS in animals receiving AT-MSCs were superior to those receiving placebo. BM-MSC transplantation induced also improvement in cardiac fibers organization and capillary density, fibrotic tissue reduction, increase in final VEGF concentration and BNP decrease. Discussion IMI of BM or AT-MSCs improves LV function and induces more angiogenesis processes than BM-MNCs. In addition, BM-MSCs showed more anti-fibrotic effects and more ability to reorganize myocardial tissue compared with the other cell types.

Different studies have shown improvement in patients with idiopathic nonischemic dilated cardiomyopathy treated with cell-therapy. However, factors influencing responsiveness are not well known. This trial investigates functional changes and factors influencing the 6-month gain in ejection fraction in 27 patients with dilated cardiomiopathy treated with intracoronary cell-therapy.Patients received intracoronary infusion of autologous bone-marrow mononuclear cells (mean infused, 10.2 [2.9]×10(8)). Flow cytometry and functional analyses of the cells were also performed.The 6-month angiographic gain in ejection fraction ranged from -9% to 34% (mean, 9%). These changes were distinguished into 2 groups: 21 patients (78%) with a significant improvement at the 6-month evaluation (mean gain, 14 [7]%), and 6 patients who had no response (mean gain, -5 [3]%). The responders were younger as compared to the nonresponders (50 [12] years vs 62 [9] years; P.04). There was an inverse correlation (r=-0.41; P.003) between the gain in ejection fraction and the high density lipoprotein level, suggesting higher functional gain with low high density lipoprotein levels. The 24 h migratory capability of the infused cells was significantly reduced in the responders' group (5.4 [1.7]×10(8) vs 8.1 [2.3]×10(8); P.009 for vascular endothelial growth factor and 5.8 [1.7]×10(8) vs 8.4 [2.9]×10(8); P.002 for stromal cell-derived factor-1).Younger patients with dilated cardiomiopathy and lower plasma high density lipoprotein levels gain greater benefit from intracoronary cell-therapy. Functional improvement also seems to be enhanced by a lower migratory capacity of the infused cells.

Introduccion y objetivos Diferentes estudios han mostrado mejoria funcional en pacientes con miocardiopatia dilatada no isquemica tratada con terapia celular. Sin embargo, los factores que influyen en la respuesta no son bien conocidos. El presente estudio investiga los cambios funcionales y los factores que influyen en la mejora de la fraccion de eyeccion a los 6 meses en 27 pacientes con miocardiopatia dilatada tratados con terapia celular intracoronaria. Metodos Los pacientes recibieron una infusion intracoronaria de celulas mononucleares autologas de la medula osea (media de celulas infundidas, 10,2±2,9 × 108). En todos se efectuo analisis funcional y citometria de flujo de las celulas infundidas. Resultados La ganancia en fraccion de eyeccion observada a los 6 meses oscilo entre el ?9 y el 34% (media, 9%). Estos cambios formaron dos grupos de pacientes: 21 (78%) que mostraron una mejora significativa (ganancia media, 14±7%), frente a 6 (22%) que no mostraron respuesta (ganancia media, ?5±3%). Los respondedores eran mas jovenes (50±12 frente a 62±9 anos; p

Introduccion y objetivos. Diferentes estudios han demostrado que la infusion intracoronaria de celulas mononucleadas de la medula osea en pacientes con infarto agudo de miocardio mejora la funcion ventricular. Sin embargo, existe menos informacion sobre esta terapia en la fase cronica de un infarto. Este estudio analiza los cambios clinicos, ecocardiograficos y angiograficos observados en 19 pacientes con infarto anterior cronico revascularizado y funcion ventricular deprimida que fueron tratados con terapia celular. Metodos. Se estudio a los pacientes de forma seriada durante la fase del tratamiento, a los 6 meses y al ano. La medula osea autologa fue extraida mediante puncion aspirativa sobre cresta iliaca, y las celulas mononucleadas se aislaron por centrifugacion en gradiente de densidad. Se efectuo un estudio biologico in vitro de una muestra de las celulas infundidas, para citofluorometria, marcacion fenotipica y analisis de la capacidad quimiotactica de las celulas infundidas. Resultados. A los 6 meses y al ano de la terapia celular se observo una ligera mejoria clinica y de la funcion ventricular, mas acusada en un grupo de pacientes respondedores. Estos se caracterizaban por haberse sometido a revascularizacion de forma mas cercana a la terapia celular. Se observo una relacion inversa entre los parametros funcionales y los biologicos que traducen un estado de actividad proclive a la migracion. Conclusiones. La infusion intracoronaria de celulas mononucleares de la medula osea en pacientes con infarto anterior cronico parece producir una ligera mejoria clinica y funcional a los 6 meses que se mantiene al ano del tratamiento

It is not known whether high convective transport may have a role in modulating the chronic inflammation of hemodialysis (HD) patients. The aim of this study was to evaluate the effect of on-line hemodiafiltration (OL-HDF) on proinflammatory peripheral monocytes: Percentage of CD14+CD16+ cells and their telomere length and spontaneous or bacterial DNA-induced production of cytokines (TNF-alpha and IL-6). In a prospective, crossover study, 31 patients who were on high-flux HD (HF-HD) were evaluated. Patients underwent the following sequence of treatments (4 mo each): HF-HD (basal), OL-HDF (period 1), HF-HD (period 2), OL-HDF (period 3), and HF-HD (period 4). The dialysis characteristics were similar in the two modalities; the only difference was a higher convective transport in the OL-HDF than in the HF-HD. All patients who were on OL-HDF periods showed a significantly lower number of CD14+CD16+ cells than on HF-HD (18.5 +/- 2.3 basal versus 13.6 +/- 2.9 period 1 and 13.9 +/- 2.3 period 3; P = 0.001). By contrast, HF-HD restored the number of CD14+CD16+ cells to the basal values (19.2 +/- 2.8 and 18.6 +/- 1.4, periods 2 and 4, respectively; NS). During OL-HDF periods, the reduction of CD14+CD16+ was paralleled by a decreased number of short telomere cells. Spontaneous or bacterial DNA-induced production of cytokines (TNF-alpha and IL-6) was increased in HF-HD as compared with OL-HDF. In conclusion, these results demonstrate that as compared with HF-HD, OL-HDF markedly reduces the number of proinflammatory CD14+CD16+ cells and the production of TNF-alpha and IL-6. Future studies are needed to assess the possible therapeutic effect of convective transport on chronic inflammation that is associated with HD.

Introduction and objectives Studies have shown that intracoronary infusion of mononuclear bone marrow cells improves ventricular function in patients with acute myocardial infarction. However, less information is available about the use of this therapy during the chronic phase of a myocardial infarction. This study involved an analysis of the clinical, echocardiographic and angiographic changes observed in 19 patients with a revascularized chronic anterior myocardial infarction and depressed ventricular function who were treated by cell therapy. Methods A series of patients were monitored during treatment and 6 months and 1 year after treatment. Autologous bone marrow was obtained by needle aspiration of the iliac crest and mononuclear cells were isolated by density-gradient centrifugation. An in vitro biological study of a sample of the infused cells was performed using fluorocytometry, phenotype marking and an analysis of the chemotactic properties of the cells. Results Six months and 1 year after cell therapy, a modest improvement was observed in clinical status and ventricular function, which was most pronounced in the group of patients who responded. Characteristically, these patients were revascularized close to the time of cell therapy. There was an inverse relationship between functional recovery and biological parameters that reflected a state conducive to cell migration. Conclusions The intracoronary infusion of mononuclear bone marrow cells into patients with chronic anterior myocardial infarction appeared to result in a modest clinical and functional improvement after 6 months which was sustained up to 1 year after treatment.

Erythropoietin (EPO) prevents cell apoptosis induced by oxidative stress. Carbamylated EPO maintains the tissue-protective activities of the unmodified EPO but does not stimulate erythropoiesis. This study evaluates whether carbamylated erythropoietin is as effective as recombinant human erythropoietin in protecting endothelial progenitor cells (EPCs) from apoptosis without stimulating erythropoiesis. Experiments were performed in an erythroid cell line (UT-7) and in human EPCs. Cell signals regulating proliferation and apoptosis (Jak-2, Akt, Erk1/2, NFkappaB and Stat-5) were measured by Western blotting. In human EPCs, cell senescence, apoptosis and proliferation were assessed by acidic beta-gal and measurement of telomere length, TUNEL and PCNA labeling, respectively. Angiogenesis was evaluated using the endothelial tube formation assay. In UT-7, carbamylated erythropoietin (C-darbe) induced phosphorylation of the anti-apoptotic Jak-2/Akt signal and, as opposed to recombinant human erythropoietin (darbe), did not produce a significant activation of cell proliferating signals. Darbe increased the percent of proliferating EPCs and promoted angiogenesis. By contrast, C-darbe failed to stimulate proliferation of EPCs. Both C-darbe and darbe equally reduced apoptosis and senescence. Thus, C-darbe protects EPCs from apoptosis and does not increase erythropoiesis.

Chronic kidney disease (CKD) stage 4-5 patients have increased cardiovascular morbidity and mortality rates compared with the general population. Chronic inflammation has been proposed as a cardiovascular risk factor. We have previously demonstrated that the majority of CKD patients show a microinflammatory state with an increased percentage of CD14+/CD16+ monocytes in peripheral blood, even in patients who do not show clinical evidence of inflammatory disease. However, the role played by these microinflammatory cells on the endothelial damage that precede the development of cardiovascular disease has not been investigated.To study the effect of microinflammation on endothelial cell injury we have developed an experimental co-culture model in which isolated CD14+/CD16+ cells were seeded in 24-well tissue-culture plates. Human umbilical vein endothelial cells were placed on the top of the culture well in a insert that permitted intercellular soluble network communication. To stimulate the release of proinflammatory products, monocytes were activated with substimulating doses of bacterial DNA. Endothelial injury was characterized measuring intracellular reactive oxygen species activity and cell apoptosis.Only CD14+/CD16+ cells released proinflammatory cytokines when they were stimulated by bacterial DNA. In the culture wells in which inflammatory cytokines were detected, endothelial cells showed an increased reactive oxygen species activity and features of apoptosis.Our results support the hypothesis that independently of uremia, in CKD stage 4-5 patients microinflammation mediated by CD14+/CD16+ cells induces endothelial damage and thus may contribute to the increased risk of atherosclerosis and cardiovascular disease that has been reported in this population.

Background: Chronic kidney disease (CKD) stage 4-5 patients have increased cardiovascular morbidity and mortality rates compared with the general population. Chronic inflammation has been proposed a

The repair of the endothelium after inflammatory injury is essential to maintaining homeostasis. The link between inflammation-induced endothelial damage and repair has not been fully characterized in vivo. We have developed a rat model to evaluate the coupling of lipopolysaccharide (LPS)-induced endothelial injury and repair. Aortic endothelium injury was analyzed by both inmunohistochemistry and flow cytometry to quantify the number of endothelial cells and the percentage of apoptotic endothelial cells. We have also identified the percentage of circulating angiogenic cells capable of repairing the damaged endothelium. Erythropoietin was administered to inhibit LPS-induced endothelial apoptosis. Loss of the normal endothelial structure was observed in the aorta of the animals treated with LPS. Eight hours after LPS administration, the number of endothelial cells decreased by 40%, returning to normal after 24 h. There was a threefold increase in the percentage of circulating angiogenic cells, which did not return to normal levels until 48 h after LPS administration. Circulating angiogenic cell levels did not change when LPS-induced endothelial damage was prevented by erythropoietin. The endothelial injury caused by inflammation activates the mobilization of circulating angiogenic cells, thus completing endothelial repair. Inflammation without endothelial injury does not trigger the mobilization of circulating angiogenic cells.