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Background and Objectives: Children with multi-drug resistant idiopathic nephrotic syndrome (MDR-INS) usually progress to end-stage kidney disease with a consistent risk of disease recurrence after transplantation. New therapeutic options are needed for these patients. Mesenchymal stromal cells (MSCs) are multipotential non-hematopoietic cells with several immunomodulatory properties and growing clinical applications. Cord blood-derived MSC have peculiar anti-inflammatory and immunosuppressive properties. We aimed at assessing safety and efficacy of cord-blood-derived MSCs (CB-MSCs) in children with MDR-INS., Design, Setting, Participants: Prospective, open-label, single arm phase I-II pilot study. Pediatric patients with MDR-INS, resistant to at least two lines of therapy, were enrolled. Allogenic CB-MSCs were administered intravenously on days 0, 14, and 21 at a dose of 1.5 × 10 6 cells/kg. Patients were followed for at least 12 months. The primary outcomes were safety and toxicity. The secondary outcome was remission at 12 months evaluated by urinary protein/urinary creatinine ratio (uPr/uCr). Circulating regulatory T cells (Tregs) were monitored., Results: Eleven pediatric patients with MDR-INS (10 females, median age 13 years) resistant to a median of 3 previous lines of therapy were enrolled. All patients completed the CB-MSC infusion schedule. No patient experienced any infusion-related adverse event or toxicity. Nine patients were assessable for efficacy. At the 12 months follow-up after the treatment, the median uPr/uCr did not change significantly from baseline (8.13 vs. 9.07; p = 0.98), while 3 patients were in partial or complete remission. A lower baseline uPr/uCr was a predictor of remission (2.55 vs. 8.74; p = 0.0238). Tregs count was not associated with CB-MSCs therapy., Conclusions: CB-MSCs are safe and may have a role in the immunosuppressive therapy of pediatric patients with MDR-INS. This preliminary experience paves the way toward further phase II studies addressing MSC efficacy in immune-mediated kidney diseases., (© 2022. The Author(s).)

The majority of metastatic colorectal cancers (mCRC) are mismatch repair (MMR) proficient and unresponsive to immunotherapy, whereas MMR-deficient (MMRd) tumors often respond to immune-checkpoint blockade. We previously reported that the treatment of colorectal cancer preclinical models with temozolomide (TMZ) leads to MMR deficiency, increased tumor mutational burden (TMB), and sensitization to immunotherapy. To clinically translate these findings, we designed the ARETHUSA clinical trial whereby O6-methylguanine-DNA-methyltransferase (MGMT)-deficient, MMR-proficient, RAS-mutant mCRC patients received priming therapy with TMZ. Analysis of tissue biopsies and circulating tumor DNA (ctDNA) revealed the emergence of a distinct mutational signature and increased TMB after TMZ treatment. Multiple alterations in the nucleotide context favored by the TMZ signature emerged in MMR genes, and the p.T1219I MSH6 variant was detected in ctDNA and tissue of 94% (16/17) of the cases. A subset of patients whose tumors displayed the MSH6 mutation, the TMZ mutational signature, and increased TMB achieved disease stabilization upon pembrolizumab treatment., Significance: MMR-proficient mCRCs are unresponsive to immunotherapy. We provide the proof of concept that inactivation of MMR genes can be achieved pharmacologically with TMZ and molecularly monitored in the tissue and blood of patients with mCRC. This strategy deserves additional evaluation in mCRC patients whose tumors are no longer responsive to standard-of-care treatments. See related commentary by Willis and Overman, p. 1612. This article is highlighted in the In This Issue feature, p. 1599., (©2022 The Authors; Published by the American Association for Cancer Research.)

Sinusoidal Obstruction Syndrome (SOS) is a life threatening HSCT complication and it can rapidly evolve in Multiple Organ Dysfunction Syndrome, with a mortality exceeding 80%. Early treatment with defibrotide is the leading factor for efficacy. Its prophylactic use is recommended in the pediatric setting, but its value isn't validated for adults, although factors for individual risk assessment are debated. We here present a real-world experience of Defibrotide prophylaxis in adults at very high risk of SOS. We treated with prophylactic Defibrotide and Ursodeoxycholic Acid seven patients receiving allogeneic HSCT for high risk B-ALL, previously treated with single agent Inotuzomab-Ozogamicin. They all had other high risk factors for SOS such as previous hepatotoxicity, previous allo-HSCT, double alkylating conditioning. All patients received Treosulfan-Fludarabine conditioning, Thiotepa was added in 4 patients and 4GyTBI in 2 patients. GvHD prophylaxis included post-transplant cyclophosphamide, rapamycin and mycophenolate. Donor source was PBSC. Five patients received family MMRD transplant, 1 patient a MRD transplant and 1 patient a MUD transplant. Non-severe gastrointestinal bleeding occurred in two patients requiring defibrotide temporarily discontinuation. SOS occurred in 3/7 cases within 21 days after HSCT and no late-onset SOS were diagnosed. SOS caused death in all cases. All three patients were characterized by a common pattern of very high risk factors by prior HSCT, they all received a myeloablative conditioning with Treosulfan-Thiotepa and a MMRD transplant. Defibrotide prophylaxis apparently failed to protect against the development of SOS in those patients treated with a double alkylator-based conditioning regimen, while a possible efficacy for the other high-risk patients is debatable., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Giglio, Xue, Greco, Lazzari, Clerici, Lorentino, Mastaglio, Marktel, Lupo-Stanghellini, Marcatti, Corti, Bernardi, Piemontese, Ciceri and Peccatori.)

Background Aims: Advanced therapy medicinal products (ATMPs) are novel drugs based on genes, cells or tissues developed to treat many different diseases. Stability studies of each new ATMP need to be performed to define its shelf life and guarantee efficacy and safety upon infusion, and these are presently based on guidelines originally drafted for standard pharmaceutical drugs, which have properties and are stored in conditions quite different from cell products. The aim of this report is to provide evidence-based information for stability studies on ATMPs that will facilitate the interlaboratory harmonization of practices in this area., Methods: We have collected and analyzed the results of stability studies on 19 different cell-based experimental ATMPs, produced by five authorized cell factories forming the Lombardy "Plagencell network" for use in 36 approved phase I/II clinical trials; most were cryopreserved and stored in liquid nitrogen vapors for 1 to 13 years., Results: The cell attributes collected in stability studies included cell viability, immunophenotype and potency assays, in particular immunosuppression, cytotoxicity, cytokine release and proliferation/differentiation capacity. Microbiological attributes including sterility, endotoxin levels and mycoplasma contamination were also analyzed. All drug products (DPs), cryopreserved in various excipients containing 10% DMSO and in different primary containers, were very stable long term at <-150°C and did not show any tendency for diminished viability or efficacy for up to 13.5 years., Conclusions: Our data indicate that new guidelines for stability studies, specific for ATMPs and based on risk analyses, should be drafted to harmonize practices, significantly reduce the costs of stability studies without diminishing safety. Some specific suggestions are presented in the discussion., (Copyright © 2022 International Society for Cell & Gene Therapy. Published by Elsevier Inc. All rights reserved.)

Three-dimensional (3D) structured organoids are the most advanced in vitro models for studying human health effects, but their application to evaluate the biological effects associated with microplastic exposure was neglected until now. Fibers from synthetic clothes and fabrics are a major source of airborne microplastics, and their release from dryer machines is poorly understood. We quantified and characterized the microplastic fibers (MPFs) released in the exhaust filter of a household dryer and tested their effects on airway organoids (1, 10, and 50 µg mL -1 ) by optical microscopy, scanning electron microscopy (SEM), confocal microscopy and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). While the presence of MPFs did not inhibit organoid growth, we observed a significant reduction of SCGB1A1 gene expression related to club cell functionality and a polarized cell growth along the fibers. The MPFs did not cause relevant inflammation or oxidative stress but were coated with a cellular layer, resulting in the inclusion of fibers in the organoid. This effect could have long-term implications regarding lung epithelial cells undergoing repair. This exposure study using human airway organoids proved suitability of the model for studying the effects of airborne microplastic contamination on humans and could form the basis for further research regarding the toxicological assessment of emerging contaminants such as micro- or nanoplastics., (Copyright © 2022. Published by Elsevier Ltd.)

The complexes formed between Pb 2+ and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) were reinvestigated in aqueous solutions using a combination of pH potentiometry, UV-vis spectroscopy, and NMR spectroscopy. The thermodynamic data were supported by kinetics assays. Differently protonated complexes, i.e ., [PbH 3 L] + , [PbH 2 L], [PbHL] - , and [PbL] 2- , were detected, and the corresponding stability constants (log β ) at T = 298 K and I = 0.1 M NaCl were 33.1 ± 0.2, 32.00 ± 0.06, 29.28 ± 0.06, and 25.3 ± 0.1, respectively. Results differed significantly from those previously reported by Chaves et al. (Talanta1992, 39, 249) and Pippin et al. (Inorg. Chim. Acta1995, 239, 43) in both the speciation and the overall complex stability; the latter in particular was found to be remarkably higher. The work disclosed herein provides revised data on the Pb 2+ -DOTA complexes, which should be used as a new stability benchmark during the development of lead chelators., Competing Interests: The authors declare no competing financial interest., (© 2022 The Authors. Published by American Chemical Society.)

Aging is a physiological process that leads to a higher risk for the most devastating diseases. There are a number of theories of human aging proposed, and many of them are directly or indirectly linked to mitochondria. Here, we used mesenchymal stem cells (MSCs) from young and older donors to study age-related changes in mitochondrial metabolism. We have found that aging in MSCs is associated with a decrease in mitochondrial membrane potential and lower NADH levels in mitochondria. Mitochondrial DNA content is higher in aged MSCs, but the overall mitochondrial mass is decreased due to increased rates of mitophagy. Despite the higher level of ATP in aged cells, a higher rate of ATP consumption renders them more vulnerable to energy deprivation compared to younger cells. Changes in mitochondrial metabolism in aged MSCs activate the overproduction of reactive oxygen species in mitochondria which is compensated by a higher level of the endogenous antioxidant glutathione. Thus, energy metabolism and redox state are the drivers for the aging of MSCs/mesenchymal stromal cells., (© 2021 The Authors. Journal of Cellular Physiology published by Wiley Periodicals LLC.)

Background: Maternal cardiovascular risk factors have been associated with adverse maternal and fetal outcomes. Given the difficulty in establishing causal relationships using epidemiological data, we applied Mendelian randomization to explore the role of cardiovascular risk factors on risk of developing preeclampsia or eclampsia, and low fetal birthweight., Methods: Uncorrelated single-nucleotide polymorphisms associated systolic blood pressure (SBP), body mass index, type 2 diabetes, LDL (low-density lipoprotein) with cholesterol, smoking, urinary albumin-to-creatinine ratio, and estimated glomerular filtration rate at genome-wide significance in studies of 298 957 to 1 201 909 European ancestry participants were selected as instrumental variables. A 2-sample Mendelian randomization study was performed with primary outcome of preeclampsia or eclampsia (PET). Risk factors associated with PET were further investigated for their association with low birthweight., Results: Higher genetically predicted SBP was associated increased risk of PET (odds ratio [OR] per 1-SD SBP increase 1.90 [95% CI=1.45-2.49]; P =3.23×10 -6 ) and reduced birthweight (OR=0.83 [95% CI=0.79-0.86]; P =3.96×10 -18 ), and this was not mediated by PET. Body mass index and type 2 diabetes were also associated with PET (respectively, OR per 1-SD body mass index increase =1.67 [95% CI=1.44-1.94]; P =7.45×10 -12 ; and OR per logOR increase type 2 diabetes =1.11 [95% CI=1.04-1.19]; P =1.19×10 -3 ), but not with reduced birthweight., Conclusions: Our results provide evidence for causal effects of SBP, body mass index, and type 2 diabetes on PET and identify that SBP is associated with reduced birthweight independently of PET. The results provide insight into the pathophysiological basis of PET and identify hypertension as a potentially modifiable risk factor amenable to therapeutic intervention.

Human induced pluripotent stem cells (hiPSCs) must be manufactured as advanced therapy medicinal products (ATMPs) for innovative tissue replacement clinical applications. Yet, production of hiPSCs under current Good Manufacturing Practice (cGMP) presents many hurdles, such as the large-scale cell expansion needed to reach therapeutically-relevant hiPSC doses. For the monitoring of this phase, a fast and reliable cell counting method should be used. Conventional manual cell counting by the hemocytometer method is dependent on the operator's expertise and is time-consuming. Therefore, automation of sample preparation and analysis is needed to improve precision and rapidity of hiPSC cell counting. We investigated whether an automated cell counting method could be validated for use with hiPSCs, in comparison with a reference cell counting method included in the European Pharmacopeia, 10th edition. The proposed method was the fluorescence imaging-based NucleoCounter NC-100 system, whereas the reference method was manual cell counting using a Bürker hemocytometer. The validation strategy complied with EudraLex cGMP regulations for ATMP manufacturing and ICH Q2(R1) indications for validation of analytical methods. The use of the NucleoCounter NC-100 system for automated cell counting was validated, focusing on accuracy, specificity, intra- and inter-operator reproducibility, range and linearity, showing higher precision than the manual method. The automated method can be used more effectively than the manual one for hiPSC cell counting. Thus, this piece of work paves the way for all cGMP facilities that want to pursue hiPSC manufacturing for clinical use., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors. Published by Elsevier B.V.)

Background: A growing number of clinical trials have shown that regulatory T (T reg ) cell transfer may have a favorable effect on the maintenance of self-tolerance and immune homeostasis in different conditions such as graft-versus-host disease (GvHD), solid organ transplantation, type 1 diabetes, and others. In this context, the availability of a robust manufacturing protocol that is able to produce a sufficient number of functional T reg cells represents a fundamental prerequisite for the success of a cell therapy clinical protocol. However, extended workflow guidelines for nonprofit manufacturers are currently lacking. Despite the fact that different successful manufacturing procedures and cell products with excellent safety profiles have been reported from early clinical trials, the selection and expansion protocols for T reg cells vary a lot. The objective of this study was to validate a Good Manufacturing Practice (GMP)-compliant protocol for the production of T reg cells that approaches the whole process with a risk-management methodology, from process design to completion of final product development. High emphasis was given to the description of the quality control (QC) methodologies used for the in-process and release tests (sterility, endotoxin test, mycoplasma, and immunophenotype)., Results: The GMP-compliant protocol defined in this work allows at least 4.11 × 10 9 T reg cells to be obtained with an average purity of 95.75 ± 4.38% and can be used in different clinical settings to exploit T reg cell immunomodulatory function., Conclusions: These results could be of great use for facilities implementing GMP-compliant cell therapy protocols of these cells for different conditions aimed at restoring the T reg cell number and function, which may slow the progression of certain diseases., (© 2021. The Author(s).)

The purpose of this paper is to develop and validate a delta-radiomics score to predict the response of individual colorectal cancer liver metastases (lmCRC) to first-line FOLFOX chemotherapy. Three hundred one lmCRC were manually segmented on both CT performed at baseline and after the first cycle of first-line FOLFOX, and 107 radiomics features were computed by subtracting textural features of CT at baseline from those at timepoint 1 (TP1). LmCRC were classified as nonresponders (R-) if they showed progression of disease (PD), according to RECIST1.1, before 8 months, and as responders (R+), otherwise. After feature selection, we developed a decision tree statistical model trained using all lmCRC coming from one hospital. The final output was a delta-radiomics signature subsequently validated on an external dataset. Sensitivity, specificity, positive (PPV), and negative (NPV) predictive values in correctly classifying individual lesions were assessed on both datasets. Per-lesion sensitivity, specificity, PPV, and NPV were 99%, 94%, 95%, 99%, 85%, 92%, 90%, and 87%, respectively, in the training and validation datasets. The delta-radiomics signature was able to reliably predict R- lmCRC, which were wrongly classified by lesion RECIST as R+ at TP1, (93%, averaging training and validation set, versus 67% of RECIST). The delta-radiomics signature developed in this study can reliably predict the response of individual lmCRC to oxaliplatin-based chemotherapy. Lesions forecasted as poor or nonresponders by the signature could be further investigated, potentially paving the way to lesion-specific therapies.

In the last few years, there has been a considerable increase in the use of organoids, which is a new three-dimensional culture technology applied in scientific research. The main reasons for their extensive use are their plasticity and multiple applications, including in regenerative medicine and the screening of new drugs. The aim of this study was to better understand these structures by focusing on the choice of the best housekeeping gene (HKG) to perform accurate molecular analysis on such a heterogeneous system. This feature should not be underestimated because the inappropriate use of a HKG can lead to misleading data and incorrect results, especially when the subject of the study is innovative and not totally explored like organoids. We focused our attention on the newly described human pancreatic organoids (hPOs) and compared 12 well-known HKGs (ACTB, B2M, EF1α, GAPDH, GUSB, HPRT, PPIA, RNA18S, RPL13A TBP, UBC and YWHAZ). Four different statistical algorithms (NormFinder, geNorm, BestKeeper and ΔCt) were applied to estimate the expression stability of each HKG, and RefFinder was used to identify the most suitable genes for RT-qPCR data normalization. Our results showed that the intragroup and intergroup comparisons could influence the best choice of the HKG, making clear that the identification of a stable reference gene for accurate and reproducible RT-qPCR data normalization remains a critical issue. In summary, this is the first report on HKGs in human organoids, and this work provides a strong basis to pave the way for further gene analysis in hPOs., Competing Interests: The authors declare no competing interests.

Objective: The aim of this study is to evaluate the effect of bariatric surgery on long-term risk of VTEs in a large cohort of patients with obesity., Background: Obesity is a well-established risk factor for VTEs, such as pulmonary embolism and deep vein thrombosis. The rising prevalence of obesity and its associated co-morbidities, including VTE, represent a growing public health issue., Methods: A nested, retrospective matched cohort study was designed and conducted on prospectively collected national electronic healthcare records data from the Clinical Practice Research Datalink. Eight thousand, one hundred twelve patients were included in the study: the 4056 patients on the database who had undergone bariatric surgery, and equal numbers of age, sex, and body mass index matched controls. The primary endpoint was the occurrence of VTEs; secondary endpoints were the occurrence of deep vein thrombosis alone, pulmonary embolism alone., Results: Patients were followed up for a median of 10.7 years. The bariatric surgery cohort had a significantly lower occurrence of the primary outcome [hazard ratio (HR) 0.601; 95% confidence interval (CI) 0.430-0.841, P = 0.003]; mainly driven by a reduction in deep vein thrombosis (HR 0.523; 95% CI 0.349-0.783, P = 0·002) and not in pulmonary embolism (HR 0.882; 95% CI 0.511-1.521, P = 0.651)., Conclusions: The results of this nation-wide study set out to characterize the impact of bariatric surgery on long-term risk of thromboembolic events outline a significant reduction in thromboembolic events, driven by a reduction in deep vein thrombosis., Competing Interests: The authors report no conflicts of interest., (Copyright © 2019 Wolters Kluwer Health, Inc. All rights reserved.)

Human induced pluripotent stem cells (hiPSCs) are manufactured as advanced therapy medicinal products for tissue replacement applications. With this aim, the feasibility of hiPSC large-scale expansion in existing bioreactor systems under current good manufacturing practices (cGMP) has been tested. Yet, these attempts have lacked a paradigm shift in culture settings and technologies tailored to hiPSCs, which jeopardizes their clinical translation. The best approach for industrial scale-up of high-quality hiPSCs is to design their manufacturing process by following quality-by-design (QbD) principles: a scientific, risk-based framework for process design based on relating product and process attributes to product quality. In this review, we analyzed the hiPSC expansion manufacturing process implementing the QbD approach in the use of bioreactors, stressing the decisive role played by the cell quantity, quality and costs, drawing key QbD concepts directly from the guidelines of the International Council for Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use., (© 2021. The Author(s).)

We describe the case of a 45-year-old man affected by T-cell acute lymphoblastic leukaemia and diagnosed with COVID-19 early after an allogeneic haematopoietic stem cell transplant. The infectious disease was characterised by a severe and prolonged course, further complicated by a spontaneous pneumomediastinum and pneumopericardium. We successfully treated this patient with the antiviral drug remdesivir associated with two courses of COVID-19 convalescent plasma. This case report represents a good example of the typical clinical course of COVID-19 in severely immunosuppressed patients and gives evidence that in this population only a prompt treatment directed towards viral clearance can face the absence of a valid immune reactivity., Competing Interests: Competing interests: None declared., (© BMJ Publishing Group Limited 2021. No commercial re-use. See rights and permissions. Published by BMJ.)

Mesenchymal stromal cells (MSCs) are multipotent cells with anti-inflammatory properties. Here we tested the safety of MSCs in patients with progressive supranuclear palsy (PSP; ClinicalTrials.gov: NCT01824121; Eudract No. 2011-004051-39). Seven patients were treated. To improve the safety, protocol adjustments were made during the performance of the study. The objectives of our work were: (1) to assess the safety of MSCs and (2) to identify critical issues in cell therapies for neurodegenerative diseases. Autologous MSCs from the bone marrow of PSP patients were administered through the internal carotid arteries. 1-year survival and number of severe adverse events were considered as safety endpoints. Clinical rating scales, neuropsychological assessments, gait and posture analysis, single-photon emission computed tomography, positron emission tomography, and brain magnetic resonance (BMR) were performed at different follow-up times. Peripheral blood levels of inflammatory cytokines were measured before and after cell infusion. Six of the seven treated patients were living 1 year after cell infusion. Asymptomatic spotty lesions were observed at BMR after 24 h in six of the seven treated patients. The last patient in the preliminary cohort (Case 5) exhibited transiently symptomatic BMR ischemic alterations. No severe adverse events were recorded in the last two treated patients. Interleukin-8 serum concentrations decreased in three patients (Case 2, 3, and 4). An adaptive study design, appropriate and up-to-date efficacy measures, adequate sample size estimation, and, possibly, the use of a cellular and/or allogeneic cell sources may help in performing phase II trials in the field., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Giordano, Canesi, Isalberti, Marfia, Campanella, Vincenti, Cereda, Ranghetti, Palmisano, Isaias, Benti, Marotta, Lazzari, Montemurro, Viganò, Budelli, Montelatici, Lavazza, Rivera-Ordaz and Pezzoli.)

Nitrogen doping has been always regarded as one of the major factors responsible for the increased catalytic activity of Fe-N-C catalysts in the oxygen reduction reaction, and recently, sulfur has emerged as a co-doping element capable of increasing the catalytic activity even more because of electronic effects, which modify the d-band center of the Fe-N-C catalysts or because of its capability to increase the Fe-N x site density (SD). Herein, we investigate in detail the effect of sulfur doping of carbon support on the Fe-N x site formation and on the textural properties (micro- and mesopore surface area and volume) in the resulting Fe-N-C catalysts. The Fe-N-C catalysts were prepared from mesoporous carbon with tunable sulfur doping (0-16 wt %), which was achieved by the modulation of the relative amount of sucrose/dibenzothiophene precursors. The carbon with the highest sulfur content was also activated through steam treatment at 800 °C for different durations, which allowed us to modulate the carbon pore volume and surface area (1296-1726 m 2 g -1 ). The resulting catalysts were tested in O 2 -saturated 0.5 M H 2 SO 4 electrolyte, and the site density (SD) was determined using the NO-stripping technique. Here, we demonstrate that sulfur doping has a porogenic effect increasing the microporosity of the carbon support, and it also facilitates the nitrogen fixation on the carbon support as well as the formation of Fe-N x sites. It was found that the Fe-N-C catalytic activity [ E 1/2 ranges between 0.609 and 0.731 V vs reversible hydrogen electrode (RHE)] does not directly depend on sulfur content, but rather on the microporous surface and therefore any electronic effect appears not to be determinant as confirmed by X-ray photoemission spectroscopy (XPS). The graph reporting Fe-N x SD versus sulfur content assumes a volcano-like shape, where the maximum value is obtained for a sulfur/iron ratio close to 18, i.e., a too high or too low sulfur doping has a detrimental effect on Fe-N x formation. However, it was highlighted that the increase of Fe-N x SD is a necessary but not sufficient condition for increasing the catalytic activity of the material, unless the textural properties are also optimized, i.e., there must be an optimized hierarchical porosity that facilitates the mass transport to the active sites.

Introduction: Reducing toxicities while preserving efficacy in allogeneic stem cell transplant (allo-HCT) remains a particularly challenging problem. Different strategies to enhance the antitumor activity without increasing early and late adverse toxicities of the conditioning regimens have been investigated., Methods: The aim of "AlloTreo" prospective phase 2 clinical trial was to evaluate the efficacy and safety of a conditioning regimen based on Treosulfan (42 g/m 2 ) and fludarabine (https://clinicaltrials.gov/ct2/show/NCT00598624). We enrolled 108 patients with hematological diseases who received a first allo-HCT between June 2005 and January 2011, inside the frame of this trial at our center. Median age at allo-HCT was 49 (21-69) years. Disease Risk Index was low in 14 (13%) patients, intermediate in 73 (67.7%), high in 17 (15.7%), and very high in 4 (3.7%). Donors were human leukocyte antigen (HLA)-matched related in 50 cases, 10/10-matched unrelated in 36, and 9/10-mismatched unrelated in 22. Graft- versus -host disease (GvHD) prophylaxis consisted of cyclosporine-A and methotrexate. Anti-T-lymphocyte globulin (ATLG) was administered in patients receiving unrelated allo-HCT. Stem cell source was mainly peripheral blood stem cells (95%)., Results: Conditioning regimen was well tolerated. Full donor chimerism was documented for most patients (88%) at day +30. At 12 years, overall survival (OS) was 41.7% (32.2%-50.9%), progression-free survival (PFS) was 31.7% (23%-40.7%), GvHD-free/relapse-free survival was 20.9% (13.7%-29.1%), cumulative incidence (CI) of relapse was 44.5% (34.9%-53.6%), and transplant-related mortality (TRM) was 22.5% (15.1%-30.9%). CI of acute GvHD grades II-IV was 27.8% (19.7%-36.5%) at 100 days; 12-year CI of chronic GvHD was 40.7% (31.3%-49.9%). Relevant long-term adverse effects were 10 secondary malignancy, 3 fatal cardiovascular events, and 1 late-onset transplant-associated thrombotic microangiopathy. Ten successful pregnancies were reported after allo-HCT. In multivariate analysis, older age (≥60 years) at transplant [hazard ratio (HR), 2.157; p = 0.004] and a high/very high disease risk index (HR, 1.913; p = 0.026) were significantly associated with a lower OS., Conclusions: Overall, our data confirmed the myeloablative potential and safe toxicity profile of full dose Treo (42 g/m 2 ) especially for the younger population., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lazzari, Ruggeri, Lupo Stanghellini, Mastaglio, Messina, Giglio, Lorusso, Perini, Piemontese, Marcatti, Lorentino, Xue, Clerici, Corti, Bernardi, Assanelli, Greco, Ciceri and Peccatori.)

Post-transplantation cyclophosphamide (PTCy) has emerged as a promising graft-versus-host-disease (GVHD) prophylaxis in the setting of allogeneic hematopoietic stem cell transplantation (HSCT) from haploidentical donors and more recently in matched donor transplants. Herein, we describe our real-life experience on 249 adult patients undergoing allogeneic HSCT, from HLA-matched related (MRD), HLA-matched unrelated (MUD), or mismatched related donors (MMRD). Patients received unmanipulated peripheral blood stem cells (PBSCs), using a GVHD prophylaxis with PTCy and sirolimus. Mycophenolate mofetil was added in MUD or MMRD. In the HLA-matched donor group (MRD, n = 48, MUD, n = 50), the cumulative incidence of grades II-IV and III-IV acute GvHD was 23% and 9% at 100 days, respectively. The cumulative incidence of chronic GvHD was 25% at 2 years, severe only for 5% of the patients. The cumulative incidences of relapse and transplant-related mortality (TRM) were 31% and 9% at 2 years, respectively. The 2-year overall survival (OS) was 72% and progression-free survival (PFS) 60%; the composite endpoint of GvHD/relapse-free survival (GRFS) was 52% at 2 years. In the haploidentical donor group (n = 151), we documented a cumulative incidence of grades II-IV and III-IV acute GVHD of 35% and 20% at 100 days, respectively, and a cumulative incidence of chronic GvHD of 39% at 2 years. We observed severe chronic GVHD in 15% of the patients. The cumulative incidence of relapse and TRM was 32% and 25% at 2 years, respectively. The 2-year OS was 48%, whereas PFS was 43%; GRFS was 28% at 2 years. However, more patients in the haploidentical group presented high/very high disease risk index (DRI) and higher HCT-comorbidity index. In patients classified in the low-intermediate DRI, 2-year GRFS was 53% in MRD, 65% in MUD, and 46% in haploidentical HSCT (P = .33). Sirolimus-PTCy platform deserves further investigation as an alternative to calcineurin-inhibitor-based GVHD prophylaxis for all donor sources. In patients presenting a low-intermediate DRI, this strategy translates in relevant survival independently from the transplant source., (Copyright © 2021. Published by Elsevier Inc.)

Metastasis is facilitated by the formation of a "premetastatic niche," which is fostered by primary tumor-derived factors. Colorectal cancer (CRC) metastasizes mainly to the liver. We show that the premetastatic niche in the liver is induced by bacteria dissemination from primary CRC. We report that tumor-resident bacteria Escherichia coli disrupt the gut vascular barrier (GVB), an anatomical structure controlling bacterial dissemination along the gut-liver axis, depending on the virulence regulator VirF. Upon GVB impairment, bacteria disseminate to the liver, boost the formation of a premetastatic niche, and favor the recruitment of metastatic cells. In training and validation cohorts of CRC patients, we find that the increased levels of PV-1, a marker of impaired GVB, is associated with liver bacteria dissemination and metachronous distant metastases. Thus, PV-1 is a prognostic marker for CRC distant recurrence and vascular impairment, leading to liver metastases., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Bone marrow mesenchymal stem/stromal cells (BMSCs) show great promise for bone repair, however they are isolated by an invasive bone marrow harvest and their regenerative potential decreases with age. Conversely, cord blood can be collected non-invasively after birth and contains MSCs (CBMSCs) that can be stored for future use. However, whether CBMSCs can replace BMSCs targeting bone repair is unknown. This study evaluates the in vitro osteogenic potential of unprimed, osteogenically primed, or chondrogenically primed CBMSCs and BMSCs and their in vivo bone forming capacity following ectopic implantation on biphasic calcium phosphate ceramics in nude mice. In vitro, alkaline phosphatase (intracellular, extracellular, and gene expression), and secretion of osteogenic cytokines (osteoprotegerin and osteocalcin) was significantly higher in BMSCs compared with CBMSCs, while CBMSCs demonstrated superior chondrogenic differentiation and secretion of interleukins IL-6 and IL-8. BMSCs yielded significantly more cell engraftment and ectopic bone formation compared to CBMSCs. However, priming of CBMSCs with either chondrogenic or BMP-4 supplements led to bone formation by CBMSCs. This study is the first direct quantification of the bone forming abilities of BMSCs and CBMSCs in vivo and, while revealing the innate superiority of BMSCs for bone repair, it provides avenues to induce osteogenesis by CBMSCs.

The objective of this work was to characterize and study the behavior of the adsorption process of cellulose/biochar cryogels through isotherm models and adsorption kinetics. The cryogels were produced from a cellulose suspension obtained by mechanical fibrillation of 0.75 and 1.5% w/w unbleached long-fiber cellulose of the Pinus elliotti species. Into this suspension, 5, 10, and 20% w/w (relative to cellulose mass) biochar were added; then, the suspension was frozen and freeze-dried. After this, 2 mL of methyltrimethoxysilane (MTMS) was deposited on the cryogels. Characterization analyses were performed on the cryogels, including specific mass and porosity and sorption capacity, in addition to the study of adsorption kinetics and isotherms. The cryogels showed a porosity of above 90% and a specific gravity of less than 0.035 g cm -3 . The heterogeneous sorption capacity varied according to the concentration of cellulose used, and with the addition of 5% w/w biochar in the cellulose cryogel, the highest sorption capacity was obtained, 73 g g -1 of petroleum and 54 g g -1 of SAE20W50 oil. In the study of adsorption isotherms, the Freundlich model best fitted the process. Therefore, it was concluded that the process of petroleum adsorption by the cellulose cryogel occurs in multiple layers. In addition, the cellulose/biochar cryogel developed in the present work is suitable for use in the adsorption of organic liquids.

Mesenchymal stromal cells (MSC) are attractive candidates for the treatment of acute graft versus host disease (aGvHD) or autoimmune disorders. However, mechanisms of MSC recognition remain unclear and there are evidences that MSC are not totally immunoprivileged. Data suggest that MSC undergo apoptosis after infusion in presence of cytotoxic cells and their death could drive immunosuppression. In GvHD patients, that activity was associated with clinical response. It is mandatory to develop an in vitro potency testing predictor of the "in vivo" response to the therapy. We describe a flow cytometric assay based on differential immunostaining of target and effector cells where BM MSC are enumerated with fluorospheres to determine the loss of target cells after co-culture with PB MNC. 6/13 (46%) of BM MSC lots were lysed by PB MNC and the lysis was proportional to the E/T cell ratio. The method overcomes the problems linked to the use of dyes or radioactive, evidencing the limitations linked to the use of a single vital dye and proposing a precise gating strategy based on absolute cell counts where cells are left untouched. The assay is easy and could be used to predict the response of the patients to the therapy., Competing Interests: The authors declare no conflict of interest., (© 2021 The Author(s).)

The release of Extracellular Vesicles (EVs) into the bloodstream is positively associated with Particulate Matter (PM) exposure, which is involved in endothelial dysfunction and related to increased risk of cardiovascular disease. Obesity modifies the effects of PM exposure on heart rate variability and markers of inflammation, oxidative stress, and acute phase response. We isolated and characterized plasmatic EVs from six healthy donors and confirmed a positive association with PM exposure. We stratified for Body Mass Index (BMI) and observed an increased release of CD61+ (platelets) and CD105+ (endothelium) derived-EVs after high PM level exposure in Normal Weight subjects (NW) and no significant variations in Overweight subjects (OW). We then investigated the ability to activate endothelial primary cells by plasmatic EVs after both high and low PM exposure. NW-high-PM EVs showed an increased endothelial activation, measured as CD105+/CD62e+ (activated endothelium) EVs ratio. On the contrary, cells treated with OW-high-PM EVs showed reduced endothelial activation. These results suggest the ability of NW plasmatic EVs to communicate to endothelial cells and promote the crosstalk between activated endothelium and peripheral cells. However, this capacity was lost in OW subjects. Our findings contribute to elucidate the role of EVs in endothelial activation after PM exposure.

The distribution and isotopic composition (δ 13 C and δD) of lipids are proxies used to determinate paleoenvironmental conditions including precipitation regimes, vegetation changes and sources of organic matter, among others. This data article describes five datasets of distribution ( n -alkanes, fatty acids, n -alkanols and sterols) and isotopic composition ( n -alkanes and fatty acids) of lipids determined in 50 samples from a gravity core (GeoB16202-2) retrieved on the continental slope off northeastern Brazil. The core site is influenced by the Parnaiba freshwater discharge, the North Brazil Current and by the Intertropical Convergence Zone (ITCZ). Previous work focused on inorganic proxies in this core revealed important clues of climatic conditions during the Heinrich Stadial 1 (HS1). The baseline dataset of molecular and isotopic proxies of the organic matter provided here are additional and/or complimentary evidences to help elucidate past climate change during the Quaternary in the Equatorial Atlantic, where less information is available in comparison to other regions in this ocean., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article., (© 2020 The Authors.)

Huntington's disease (HD) is a neurodegenerative disorder characterized by motor disturbances, cognitive decline, and behaviour changes. A well-recognized feature of advanced HD is dysphagia, which leads to malnutrition and aspiration pneumonia, the latter being the primary cause of death in HD. Previous studies have underscored the importance of dysphagia in HD patients with moderate-to-advanced stage disease, but it is unclear whether dysphagia affects patients already at an early stage of disease and whether genetic or clinical factors can predict its severity. We performed fiberoptic endoscopic evaluation of swallowing (FEES) in 61 patients with various stages of HD. Dysphagia was found in 35% of early-stage, 94% of moderate-stage, and 100% of advanced-stage HD. Silent aspiration was found in 7.7% of early-stage, 11.8% of moderate-stage, and 27.8% of advanced-stage HD. A strong correlation was observed between disease progression and dysphagia severity: worse dysphagia was associated with worsening of motor symptoms. Dysphagia severity as assessed by FEES correlated with Huntington's Disease Dysphagia Scale scores (a self-report questionnaire specific for evaluating swallowing in HD). The present findings add to our understanding of dysphagia onset and progression in HD. A better understanding of dysphagia onset and progression in HD may inform guidelines for standard clinical care in dysphagia, its recognition, and management.

The COVID-19 epidemic represents an unprecedented global health emergency, further aggravated by the lack of effective therapies. For this reason, several clinical trials are testing different off-label drugs, already approved for other pathologies. Mesenchymal stem/stromal cells (MSCs) have been tested during the last two decades for the treatment of various pathologic conditions, including acute and chronic lung diseases, both in animal models and in patients. In particular, promising results have been obtained in the experimental therapy of acute respiratory distress syndrome, which represents the most threatening complication of COVID-19 infection. Furthermore, more recently, great interest has been devoted to the possible clinical applications of extracellular vesicles secreted by MSCs, nanoparticles that convey much of the biological effects and of the therapeutic efficacy of their cells of origin. This review summarizes the experimental evidence underlying the possible use of MSCs and of MSC-EVs in severe COVID-19 infection and underlines the need to evaluate the possible efficacy of these therapeutic approaches through controlled studies under the supervision of the Regulatory Authorities., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Melanoma progression is generally associated with increased transcriptional activity mediated by the Yes-associated protein (YAP). Mechanical signals from the extracellular matrix are sensed by YAP, which then activates the expression of proliferative genes, promoting melanoma progression and drug resistance. Which extracellular signals induce mechanotransduction, and how this is mediated, is not completely understood. Here, using secretome analyses, we reveal the extracellular accumulation of amyloidogenic proteins, i.e. premelanosome protein (PMEL), in metastatic melanoma, together with proteins that assist amyloid maturation into fibrils. We also confirm the accumulation of amyloid-like aggregates, similar to those detected in Alzheimer disease, in metastatic cell lines, as well as in human melanoma biopsies. Mechanistically, beta-secretase 2 (BACE2) regulates the maturation of these aggregates, which in turn induce YAP activity. We also demonstrate that recombinant PMEL fibrils are sufficient to induce mechanotransduction, triggering YAP signaling. Finally, we demonstrate that BACE inhibition affects cell proliferation and increases drug sensitivity, highlighting the importance of amyloids for melanoma survival, and the use of beta-secretase inhibitors as potential therapeutic approach for metastatic melanoma., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Background: Adult skin fibroblasts represent the most common starting cell type used to generate human induced pluripotent stem cells (F-hiPSC) for clinical studies. Yet, a foetal source would offer unique advantages, primarily the absence of accumulated somatic mutations. Herein, we generated hiPSC from cord blood multipotent mesenchymal stromal cells (MSC-hiPSC) and compared them with F-hiPSC. Assessment of the full activation of the pluripotency gene regulatory network (PGRN) focused on circular RNA (circRNA), recently proposed to participate in the control of pluripotency., Methods: Reprogramming was achieved by a footprint-free strategy. Self-renewal and pluripotency of cord blood MSC-hiPSC were investigated in vitro and in vivo, compared to parental MSC, to embryonic stem cells and to F-hiPSC. High-throughput array-based approaches and bioinformatics analyses were applied to address the PGRN., Findings: Cord blood MSC-hiPSC successfully acquired a complete pluripotent identity. Functional comparison with F-hiPSC showed no differences in terms of i) generation of mesenchymal-like derivatives, ii) their subsequent adipogenic, osteogenic and chondrogenic commitment, and iii) their hematopoietic support ability. At the transcriptional level, specific subsets of mRNA, miRNA and circRNA (n = 4,429) were evidenced, casting a further layer of complexity on the PGRN regulatory crosstalk., Interpretation: A circRNA map of transcripts associated to naïve and primed pluripotency is provided for hiPSC of clinical-grade foetal origin, offering insights on still unreported regulatory circuits of the PGRN to consider for the optimization and development of efficient differentiation protocols for clinical translation., Funding: This research was funded by Ricerca Corrente 2012-2018 by the Italian Ministry of Health., Competing Interests: Declaration of Competing Interest The method for isolation and reprogramming to pluripotency of long-living cord blood multipotent mesenchymal stromal cells is part of a technology developed by Dr. Lorenza Lazzari and Dr. Mario Barilani licensed by the Italian national patent agency Ufficio Italiano Brevettie Marchi (UIBM) with numbers 102017000141216 and 102017000141245 (international numbers WO2019/111197 and WO2019/111198)., (Copyright © 2020 The Author(s). Published by Elsevier B.V. All rights reserved.)

Purpose: Defects in the homologous recombination (HR) repair pathway are of clinical interest due to sensitivity of HR-deficient cells to PARP inhibitors. We were interested in defining PARP vulnerability in patients with metastatic colorectal cancer (mCRC) carrying KRAS and BRAF mutations who display poor prognosis, have limited therapeutic options, and represent an unmet clinical need., Experimental Design: We tested colorectal cancer cell lines, patient-derived organoids (PDO), and patient-derived xenografts (PDX) enriched for KRAS and BRAF mutations for sensitivity to the PARP inhibitor olaparib, and the chemotherapeutic agents oxaliplatin and 5-fluorouracil (5-FU). Genomic profiles and DNA repair proficiency of colorectal cancer models were compared with pharmacologic response., Results: Thirteen of 99 (around 13%) colorectal cancer cell lines were highly sensitive to clinically active concentrations of olaparib and displayed functional deficiency in HR. Response to PARP blockade was positively correlated with sensitivity to oxaliplatin in colorectal cancer cell lines as well as patient-derived organoids. Treatment of PDXs with olaparib impaired tumor growth and maintenance therapy with PARP blockade after initial oxaliplatin response delayed disease progression in mice., Conclusions: These results indicate that a colorectal cancer subset characterized by poor prognosis and limited therapeutic options is vulnerable to PARP inhibition and suggest that PDO-based drug-screening assays can be used to identify patients with colorectal cancer likely to benefit from olaparib. As patients with mCRC almost invariably receive therapies based on oxaliplatin, "maintenance" treatment with PARP inhibitors warrants further clinical investigation., (©2019 American Association for Cancer Research.)

The long-term efficacy of the Epidermal Growth Factor Receptor (EGFR)-targeted antibody cetuximab in advanced colorectal cancer (CRC) patients is limited by the emergence of drug-resistant (persister) cells. Recent studies in other cancer types have shown that cells surviving initial treatment with targeted agents are often vulnerable to alterations in cell metabolism including oxidative stress. Vitamin C (VitC) is an antioxidant agent which can paradoxically trigger oxidative stress at pharmacological dose. Here we tested the hypothesis that VitC in combination with cetuximab could restrain the emergence of secondary resistance to EGFR blockade in CRC RAS/BRAF wild-type models. We found that addition of VitC to cetuximab impairs the emergence of drug persisters, limits the growth of CRC organoids, and significantly delays acquired resistance in CRC patient-derived xenografts. Mechanistically, proteomic and metabolic flux analysis shows that cetuximab blunts carbohydrate metabolism by blocking glucose uptake and glycolysis, beyond promoting slow but progressive ROS production. In parallel, VitC disrupts iron homeostasis and further increases ROS levels ultimately leading to ferroptosis. Combination of VitC and cetuximab orchestrates a synthetic lethal metabolic cell death program triggered by ATP depletion and oxidative stress, which effectively limits the emergence of acquired resistance to anti-EGFR antibodies. Considering that high-dose VitC is known to be safe in cancer patients, our findings might have clinical impact on CRC patients treated with anti-EGFR therapies.

Background: Organoids are three-dimensional in vitro-grown cell clusters that recapitulate key features of native organs. In regenerative medicine, organoid technology represents a promising approach for the replacement of severely damaged organs, such as the pancreas in patients with type 1 diabetes. Isolation human pancreas organoids (hPOs) in chemically defined serum-free culture media would be a major milestone for this approach., Methods: Starting from discarded pancreatic tissues, we developed a large-scale process for obtaining clinically relevant quantities of undifferentiated organoids, obviating enzymatic digestion and operator-dependent pancreatic ducts picking steps. hPO identity was characterized by molecular and flow cytometry analysis., Results: This work demonstrates that it is possible to obtain a large-scale production of organoids. We introduced some innovations in the isolation, expansion, and freezing of hPOs from five donors. First of all, the choice of the starting material (islet-depleted pancreas) that allows obtaining a high quantity of hPOs at low passages. On the other hand, we introduced mechanical dissociation and we eliminated the picking step to exclude the operator-depending steps, without affecting the success of the culture (100% success rate). Another important improvement was to replace R-spondin-1 (Rspo1) conditioned medium with Rspo1 recombinant molecule to obtain a well-defined composition of the expansion medium. Finally, we implemented a GMP-compliant freezing protocol. hPOs showed exponential growth with diameter and area that increased three- and eight-fold in 7 days, respectively. Immunophenotypic profile and gene expression analysis revealed that hPOs were composed of ductal (82.33 ± 8.37%), acinar (2.80 ± 1.25%) cells, and pancreatic progenitors (5.81 ± 2.65%)., Conclusion: This work represents a milestone for a GMP-compliance hPO production and, ultimately, their clinical application as a type 1 diabetes therapy.

The environmental impacts of the urban expansion in Rio de Janeiro was evaluated based on the historical accumulation of black carbon (BC), aliphatic (AHs) and aromatic hydrocarbons (PAHs) and sterols in a sediment core retrieved from Botafogo Cove. BC related to oil combustion sources increased significantly since the 1990s. AHs were associated with petroleum inputs and revealed a high level of contamination. Multivariate statistical methods (a Principal Component Analysis associated with a linear multiple regression - PCA/LMR) applied to PAHs suggests changes in the sources in recent years. This can be ascribed to a reduction in pyrogenic emissions over the last four decades and to an increase in petrogenic inputs since the 1990s. The sterol dinosterol registered the increased eutrophication over the last three decades, but the sewage marker coprostanol was present at relatively low concentrations (0.40 to 1.16 μg g - 1 ) probably caused by enhanced bacterial activity in the sediment., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Invasive fungal diseases (IFDs) are a leading cause of infection-related-mortality after allogeneic hematopoietic stem cell transplantation (HSCT). In this prospective pilot study, we investigated the use of bedside lung ultrasound (US) in IFD management. Ten consecutive hematological patients, who developed pulmonary IFD after HSCT, were included in the study. Standard computed tomography scan and lung US were performed at IFD diagnosis and 10 days after antifungal treatment. The lung US demonstrated a high sensitivity in the detection of lung lesions at IFD diagnosis and in the follow-up examinations. It is of potential clinical relevance for IFD management in hematological patients., Competing Interests: No conflicts of interest, (Copyright © 2019 by The Korean Society of Infectious Diseases, Korean Society for Antimicrobial Therapy, and The Korean Society for AIDS.)

The therapeutic potential of mesenchymal stem cell (MSC) extracellular vesicles (EV) is currently under investigation in many pathological contexts. Both adult and perinatal MSC are being considered as sources of EV. Herein, we address antigen expression of cord blood and bone marrow MSC and released EV to define an identity and quality parameter of MSC EV as a medicinal product in the context of clinical applications. The research focuses on EV-shuttled neural/glial antigen 2 (NG2), which has previously been detected as a promising surface marker to distinguish perinatal versus adult MSC. Indeed, NG2 was significantly more abundant in cord blood than bone marrow MSC and MSC EV. Ultracentrifuge-isolated EV were then challenged for their pro-angiogenic properties on an xCELLigence system as quality control. NG2 + cord blood MSC EV, but not bone marrow MSC EV, promote bFGF and PDGF-AA proliferative effect on endothelial cells. Likewise, they successfully rescue angiostatin-induced endothelial cell growth arrest. In both cases, the effects are NG2-dependent. These results point at NG2 as an identity and quality parameter for cord blood MSC EV, paving the way for their clinical translation.

Metabolism and mitochondrial biology have gained a prominent role as determinants of stem cell fate and function. In the context of regenerative medicine, innovative parameters predictive of therapeutic efficacy could be drawn from the association of metabolic or mitochondrial parameters to different degrees of stemness and differentiation potentials. Herein, this possibility was addressed in human mesenchymal stromal/stem cells (hMSC) previously shown to differ in lifespan and telomere length. First, these hMSC were shown to possess significantly distinct proliferation rate, senescence status and differentiation capacity. More potential hMSC were associated to higher mitochondrial (mt) DNA copy number and lower mtDNA methylation. In addition, they showed higher expression levels of oxidative phosphorylation subunits. Consistently, they exhibited higher coupled oxygen consumption rate and lower transcription of glycolysis-related genes, glucose consumption and lactate production. All these data pointed at oxidative phosphorylation-based central metabolism as a feature of higher stemness-associated hMSC phenotypes. Consistently, reduction of mitochondrial activity by complex I and III inhibitors in higher stemness-associated hMSC triggered senescence. Finally, functionally higher stemness-associated hMSC showed metabolic plasticity when challenged by glucose or glutamine shortage, which mimic bioenergetics switches that hMSC must undergo after transplantation or during self-renewal and differentiation. Altogether, these results hint at metabolic and mitochondrial parameters that could be implemented to identify stem cells endowed with superior growth and differentiation potential.

Purpose: Patient-derived xenograft (PDX) models accurately recapitulate the tumor of origin in terms of histopathology, genomic landscape, and therapeutic response, but some limitations due to costs associated with their maintenance and restricted amenability for large-scale screenings still exist. To overcome these issues, we established a platform of 2D cell lines (xeno-cell lines, XL), derived from PDXs of colorectal cancer with matched patient germline gDNA available., Experimental Design: Whole-exome and transcriptome sequencing analyses were performed. Biomarkers of response and resistance to anti-HER therapy were annotated. Dependency on the WRN helicase gene was assessed in MSS, MSI-H, and MSI-like XLs using a reverse genetics functional approach., Results: XLs recapitulated the entire spectrum of colorectal cancer transcriptional subtypes. Exome and RNA-seq analyses delineated several molecular biomarkers of response and resistance to EGFR and HER2 blockade. Genotype-driven responses observed in vitro in XLs were confirmed in vivo in the matched PDXs. MSI-H models were dependent upon WRN gene expression, while loss of WRN did not affect MSS XLs growth. Interestingly, one MSS XL with transcriptional MSI-like traits was sensitive to WRN depletion., Conclusions: The XL platform represents a preclinical tool for functional gene validation and proof-of-concept studies to identify novel druggable vulnerabilities in colorectal cancer., (©2019 American Association for Cancer Research.)

Background: Although the outcome of allogeneic hematopoietic stem cell transplantation (allo-HSCT) has dramatically improved in the past decade, it is still compromised by transplant-related mortality (TRM), mainly caused by Graft-vs. -Host Disease (GvHD). Methods: We conducted a prospective observational study to ascertain the potential of serum interleukin-6 (IL6) levels, measured before conditioning and 7 days after allo-HSCT, in predicting acute GvHD, TRM and survival after allo-HSCT with Post-Transplant Cyclophosphamide (PT-Cy) based GvHD prophylaxis. Results: Between April 2014 and June 2017, we collected samples from 166 consecutive allo-HSCT patients. By ROC analysis, we identified a threshold of 2.5 pg/ml for pre-transplant IL6 and 16.5 pg/ml for post-transplant IL6. Both univariate and multivariate analyses confirmed the ability of high baseline IL6 levels to predict worse OS (HR 4.3; p < 0.01) and grade II-IV acute GvHD (HR 1.8; p = 0.04), and of high post-transplant IL6 to identify patients with worse OS (HR 3.3; p < 0.01) and higher risk of grade II-IV (HR 5; p < 0.01) and grade III-IV acute GvHD (HR 10.2; p < 0.01). In multivariate analysis, both baseline (HR 6.7; p < 0.01) and post-transplant high IL6 levels (HR 3.5; p = 0.02) predicted higher TRM. Conclusions: IL6 may contribute to the risk stratification of patients at major risk for aGvHD and TRM, potentially providing a window for additional prophylactic or preemptive strategies to improve the quality of life in the early post-transplant phase and the outcome of allo-HSCT., (Copyright © 2019 Greco, Lorentino, Nitti, Lupo Stanghellini, Giglio, Clerici, Xue, Lazzari, Piemontese, Mastaglio, Assanelli, Marktel, Corti, Bernardi, Ciceri and Peccatori.)

Surgical repair of large muscular defects requires the use of autologous graft transfer or prosthetic material. Naturally derived matrices are biocompatible materials obtained by tissue decellularization and are commonly used in clinical practice. Despite promising applications described in the literature, the use of acellular matrices to repair large defects has been only partially successful, highlighting the need for more efficient constructs. Scaffold recellularization by means of tissue engineering may improve not only the structure of the matrix, but also its ability to functionally interact with the host. The development of such a complex construct is challenging, due to the complexity of the native organ architecture and the difficulties in recreating the cellular niche with both proliferative and differentiating potential during growth or after damage. In this study, we tested a mouse decellularized diaphragmatic extracellular matrix (ECM) previously described by our group, for the generation of a cellular skeletal muscle construct with functional features. The decellularized matrix was stored using different conditions to mimic the off-the-shelf clinical need. Pediatric human muscle precursors were seeded into the decellularized scaffold, demonstrating proliferation and differentiation capability, giving rise to a functioning three-dimensional skeletal muscle structure. Furthermore, we exposed the engineered construct to cardiotoxin injury and demonstrated its ability to activate a regenerative response in vitro promoting cell self-renewal and a positive ECM remodeling. Functional reconstruction of an engineered skeletal muscle with maintenance of a stem cell pool makes this a promising tool toward future clinical applications in diaphragmatic regeneration. Stem Cells Translational Medicine 2019;8:858&869., (© 2019 The Authors. Stem Cells Translational Medicine published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

Stem cell identity and plasticity are controlled by master regulatory genes and complex circuits also involving non-coding RNAs. Circular RNAs (circRNAs) are a class of RNAs generated from protein-coding genes by backsplicing, resulting in stable RNA structures devoid of free 5' and 3' ends. Little is known of the mechanisms of action of circRNAs, let alone in stem cell biology. In this study, for the first time, we determined that a circRNA controls mesenchymal stem cell (MSC) identity and differentiation. High-throughput MSC expression profiling from different tissues revealed a large number of expressed circRNAs. Among those, circFOXP1 was enriched in MSCs compared to differentiated mesodermal derivatives. Silencing of circFOXP1 dramatically impaired MSC differentiation in culture and in vivo. Furthermore, we demonstrated a direct interaction between circFOXP1 and miR-17-3p/miR-127-5p, which results in the modulation of non-canonical Wnt and EGFR pathways. Finally, we addressed the interplay between canonical and non-canonical Wnt pathways. Reprogramming to pluripotency of MSCs reduced circFOXP1 and non-canonical Wnt, whereas canonical Wnt was boosted. The opposing effect was observed during generation of MSCs from human pluripotent stem cells. Our results provide unprecedented evidence for a regulatory role for circFOXP1 as a gatekeeper of pivotal stem cell molecular networks., (© The Author(s) 2019. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Electrocardiographic algorithms are particularly useful to differentiate, in the presence of a wide complex tachycardia, between supraventricular aberrancy and ventricular tachycardias (VT). There are numerous limitations to the sensitivity and specificity of these algorithms including the presence of accessory pathways, use of antiarrhythmic drugs, congenital heart diseases, electrolytes impairments, and artificial pacing. Once the diagnosis of VT has been reached, other algorithms can help in localizing the origin of the ventricular arrhythmia. These approaches are also limited by the anatomic structure of where the arrhythmia originates. This article illustrates the difficulties in applying common algorithms in many clinical circumstances., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Recently, we found a strict bone association between Fibroblast growth factor 23 (FGF23) and Fetuin-A, both involved in cardiovascular and mineral bone disorders. In this study, an uninvestigated bone marrow positivity for both was found. Though the role of exogenous FGF23 on mesenchymal cells (MSCs) was reported, no information is as yet available on the possible production of this hormone by MSCs. To further analyze these uninvestigated aspects, we studied human primary cells and mouse and human cell lines by means of immunostaining, qRT-PCR, enzyme linked immunosorbent assays, chromatin immunoprecipitation, transfection, and a streamlined approach for the FGF23⁻Fetuin-A interaction called Duolink proximity ligation assay. Mesenchymal cells produce but do not secrete FGF23 and its expression increases during osteo-differentiation. Fibroblast growth factor 23 is also involved in the regulation of Fetuin-A by binding directly to the Fetuin-A promoter and then activating its transcription. Both FGF23 overexpression and addition induced an upregulation of Fetuin-A in the absence of osteo-inducer factors. Fibroblast growth factor 23 and Fetuin-A promoter were increased by osteo-inducer factors with this effect being abolished after FGF23 silencing. In conclusion, both FGF23 and Fetuin-A are present and strictly linked to each other in MSCs with FGF23 driving Fetuin-A production. This mechanism suggests a role for these two proteins in the osteoblast differentiation.

The immunosuppressive properties of mesenchymal stromal cells (MSC) have been successfully tested to control clinical severe graft-versus host disease and improve survival. However, clinical studies have not yet provided conclusive evidence of their efficacy largely because of lack of patients' stratification criteria. The heterogeneity of MSC preparations is also a major contributing factor, as manufacturing of therapeutic MSC is performed according to different protocols among different centers. Understanding the variability of the manufacturing protocol would allow a better comparison of the results obtained in the clinical setting among different centers. In order to acquire information on MSC manufacturing we sent a questionnaire to the European Society for Blood and Marrow Transplantation centers registered as producing MSC. Data from 17 centers were obtained and analyzed by means of a 2-phase questionnaire specifically focused on product manufacturing. Gathered information included MSC tissue sources, MSC donor matching, medium additives for ex vivo expansion, and data on MSC product specification for clinical release. The majority of centers manufactured MSC from bone marrow (88%), whilst only 2 centers produced MSC from umbilical cord blood or cord tissue. One of the major changes in the manufacturing process has been the replacement of fetal bovine serum with human platelet lysate as medium supplement. 59% of centers used only third-party MSC, whilst only 1 center manufactured exclusively autologous MSC. The large majority of these facilities (71%) administered MSC exclusively from frozen batches. Aside from variations in the culture method, we found large heterogeneity also regarding product specification, particularly in the markers used for phenotypical characterization and their threshold of expression, use of potency assays to test MSC functionality, and karyotyping. The initial data collected from this survey highlight the variability in MSC manufacturing as clinical products and the need for harmonization. Until more informative potency assays become available, a more homogeneous approach to cell production may at least reduce variability in clinical trials and improve interpretation of results., (Copyright © 2018. Published by Elsevier Inc.)

Mesenchymal stromal cells (MSC) for cellular therapy in European Union are classified as advanced therapy medicinal products (ATMPs), and their production must fulfill the requirements of Good Manufacturing Practice (GMP) rules. Despite their classification as medicinal products is already well recognized, there is still a lack of information and indications to validate methods and to adapt the noncompendial and compendial methods to these peculiar biological products with intrinsic characteristics that differentiate them from classic synthetic or biologic drugs. In the present paper, we present the results of the validation studies performed in the context of MSC development as ATMPs for clinical experimental use. Specifically, we describe the validation policies followed for sterility testing, endotoxins, adventitious viruses, cell count, and immunophenotyping. Our work demonstrates that it is possible to fully validate analytical methods also for ATMPs and that a risk-based approach can fill the gap between the prescription of the available guidelines shaped on traditional medicinal products and the peculiar characteristics of these novel and extremely promising new drugs.

Targeting HER2 is effective in 24% of ERBB2 amplified metastatic colorectal cancer; however, secondary resistance occurs in most of the cases. We studied the evolution of individual metastases during treatment to discover spatially resolved determinants of resistance. Circulating tumor DNA (ctDNA) analysis identified alterations associated with resistance in the majority of refractory patients. ctDNA profiles and lesion-specific radiographic reports revealed organ- or metastasis-private evolutionary patterns. When radiologic assessments documented progressive disease in target lesions, response to HER2 blockade was retained in other metastases. Genomic and functional analyses on samples and cell models from eight metastases of a patient co-recruited to a postmortem study unveiled lesion-specific evolutionary trees and pharmacologic vulnerabilities. Lesion size and contribution of distinct metastases to plasma ctDNA were correlated., (Copyright © 2018 Elsevier Inc. All rights reserved.)