RESUMO
The potential impact of concrete mixtures containing steel slag (SS) as a partial replacement of natural aggregates (NA) on the terrestrial ecosystem was assessed using a battery of plant-based bioassays. Leaching tests were conducted on four concrete mixtures and one mixture containing only NA (reference concrete). Leachates were tested for phytotoxicity using seeds of Lepidium sativum, Cucumis sativus, and Allium cepa. Emerging seedlings of L. sativum and A. cepa were used to assess DNA damage (comet test). The genotoxicity of the leachates was also analyzed with bulbs of A. cepa using the comet and chromosome aberration tests. None of the samples caused phytotoxic effects. On the contrary, almost all the samples supported the seedlings; and two leachates, one from the SS-containing concrete and the other from the reference concrete, promoted the growth of C. sativus and A. cepa. The DNA damage of L. sativum and A. cepa seedlings was significantly increased only by the reference concrete sample. In contrast, the DNA damage in A. cepa bulbs was significantly enhanced by the reference concrete but also by that of a concrete sample with SS. Furthermore, all leachates caused an increase in chromosomal aberrations in A. cepa bulbs. Despite some genotoxic effects of the concrete on plant cells, the partial replacement of SS does not seem to make the concrete more hazardous than the reference concrete, suggesting the potential use of SS as a reliable recycled material. Environ Toxicol Chem 2023;42:2193-2200. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.
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Steel slags, the main waste product from the steel industry, may have several reuse possibilities. Among others, building applications represent a crucial field. However, the potential impact of harmful substances on the environment should be assessed. The aim of this study was to assess the phytotoxicity of steel slags (SS) and concrete mixtures cast with a partial replacement of SS (CSS). Leaching tests were carried out on four SS and four CSS according to EN 12457-2 and UNI EN 15863, respectively. Each leachate was assayed using root elongation tests on 30 seeds of Allium cepa, Cucumis sativus, and Lepidium sativum, respectively, and on 12 bulbs of A. cepa. The latter also allowed the analysis of other macroscopic parameters of toxicity (turgidity, consistency, colour change and root tip shape) and the evaluation of the mitotic index on 20,000 root tip cells per sample. None of the samples induced phytotoxic effects on the organisms tested: all samples supported seedlings emergence, verified by root elongation comparable to, or even greater than, that of the negative controls, and did not affect cell division, as evidenced by mitotic index values. The absence of phytotoxicity demonstrated by the leachates allows SS and SS-derived concrete to be considered as reliable materials suitable for use in civil constructions or in other engineering applications, with economic and environmental advantages, such as the reduction of the final disposal in landfills as well as the consumption of natural resources.
Assuntos
Resíduos Industriais , Aço , Resíduos Industriais/análise , Sementes/química , Materiais de Construção/toxicidadeRESUMO
TAK1 is a serine threonine kinase that mediates signal transduction induced by TGFß and bone morphogenetic proteins, and controls a variety of cell functions by modulating the downstream activation of NF-kkB, JNK, and p38. Heterozygous variants in the coding MAP3K7 gene cause the cardiospondylocarpofacial syndrome, characterized by various abnormalities. Skin fibroblasts derived from a patient carrying the MAP3K7 c.737-7A>G heterozygous variant were reprogrammed using Sendai viral vector system carrying the Yamanaka factors. The generated induced pluripotent stem cells (iPSC) line retained the original genotype, expressed pluripotency markers, and differentiated into cells of the three germ layers.
Assuntos
Anormalidades Múltiplas , Células-Tronco Pluripotentes Induzidas , Osteosclerose , Anormalidades Múltiplas/genética , Perda Auditiva Bilateral , Heterozigoto , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Insuficiência da Valva Mitral , Mutação , Osteosclerose/metabolismoRESUMO
We have developed Joubert syndrome (JS)-derived induced pluripotent stem cell (iPSC) lines from dermal fibroblasts biopsied from a female patient harbouring novel compound heterozygous mutations in CC2D2A gene. The newly established iPSC lines provide tremendous promises for development of JS-derived neuronal cell lines to uncover the molecular and cellular mechanisms underlying the pathogenesis of JS and to develop therapeutic interventions for treatment of JS.
Assuntos
Anormalidades Múltiplas , Anormalidades do Olho , Células-Tronco Pluripotentes Induzidas , Doenças Renais Císticas , Diferenciação Celular , Cerebelo/anormalidades , Anormalidades do Olho/genética , Feminino , Fibroblastos , Humanos , Mutação , Retina/anormalidadesRESUMO
Human platelet lysate (hPL) is considered a valid substitute to fetal bovine serum (FBS) in the expansion of mesenchymal stromal cells (MSC), and it is commonly produced starting from intermediate side products of whole blood donations. Through freeze-thaw cycles, hPL is highly enriched in chemokines, growth factors, and adhesion and immunologic molecules. Cell therapy protocols, using hPL instead of FBS for the expansion of cells, are approved by regulatory authorities without concerns, and its administration in patients is considered safe. However, published data are fairly difficult to compare, since the production of hPL is highly variable. This study proposes to optimize and standardize the hPL productive process by using instruments, technologies, and quality/safety standards required for blood bank activities and products. The quality and improved selection of the starting material (i.e., the whole blood), together with the improvement of the production process, guarantee a product characterized by higher content and quality of growth factors as well as a reduction in batch-to-batch variability. By increasing the number of freeze/thaw cycles from one (hPL1c) to four (hPL4c), we obtained a favorable effect on the release of growth factors from platelet α granules. Those changes have directly translated into biological effects leading to a decreasing doubling time (DT) of MSC expansion at 7 days (49.41 ± 2.62 vs. 40.61 ± 1.11 h, p < 0.001). Furthermore, mass spectrometry (MS)-based evaluation has shown that the proliferative effects of hPL4c are also combined with a lower batch-to-batch variability (10-15 vs. 21-31%) at the proteomic level. In conclusion, we have considered lot-to-lot hPL variability, and by the strict application of blood bank standards, we have obtained a standardized, reproducible, safe, cheap, and ready-to-use product.
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BACKGROUND: Cisplatin-based chemotherapy is the mainstay of pharmacological treatment of testicular germ cell tumors (TGCTs) that, together with early diagnosis, surgery, and/or radiotherapy, has dramatically improved the prognosis. However, under the pressure of such pharmacological therapy (both classical cytotoxic drugs and targeted therapy), cancer cells may develop resistance. Thus, combination therapy that may include cytotoxic drugs and targeted therapy could offer an advantage to curing cancers. Here, we investigated the in vitro and in vivo antitumor activity of cisplatin, as a single-agent or in combination with palbociclib. PATIENTS AND METHODS: The cell viability of Ntera-2/cl.D1 (NT2/D1) and 833K after exposure to palbociclib and/or cisplatin was evaluated by MTT dye reduction assay and by ATPLite Luminescence Assay. Gene and protein expression was evaluated by quantitative reverse transcription polymerase chain reaction and by western blot. Flow cytometric cell-cycle analysis was performed, as well. The in vivo experiments were conducted on NT2/D1 xenografts in AB zebrafish embryos exposed to the drugs. RESULTS: Palbociclib and cisplatin decreased TGCT cell viability both in vitro and in vivo. This effect was additive when cells were exposed to the drug combination. In the NT2/D1 cell lines, the drug combination also exerted a positive effect with regard to delaying cell recovery after the toxic insult. In the combination experiments, cisplatin-induced cell accumulation in G2/M was predominant compared with the palbociclib effect. CONCLUSIONS: These results could provide the rationale for developing further studies to improve the pharmacological treatment of TGCTs, but they must be demonstrated in a dedicated clinical trial.
Assuntos
Antineoplásicos , Neoplasias Testiculares , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Cisplatino/farmacologia , Cisplatino/uso terapêutico , Quinase 4 Dependente de Ciclina , Humanos , Masculino , Neoplasias Embrionárias de Células Germinativas , Piperazinas , Piridinas , Neoplasias Testiculares/tratamento farmacológico , Peixe-ZebraRESUMO
Peripheral blood mononuclear cells (PBMCs) derived from a healthy 40-year-old female were successfully transformed into induced pluripotent stem cells (iPSCs) by using the integration-free CytoTune-iPS Sendai Reprogramming method. The resulting iPSCs line exhibits a normal karyotype, expresses stemness markers and displays the differentiation capacity into the three germ layers. This human iPSCs line can be used as healthy control in disease modelling studies.
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Células-Tronco Pluripotentes Induzidas , Adulto , Diferenciação Celular , Reprogramação Celular , Feminino , Camadas Germinativas , Humanos , Leucócitos MononuclearesRESUMO
Steel slags (SS) are the major waste produced by iron and steel industry. Slags may be reused as recycled materials, instead of natural aggregates (NA), to reduce the final disposal in a landfill and the exploitation of raw materials. However, the reuse of SS may generate a potential release of toxic compounds for the environment and humans. The purpose of this study was to evaluate the toxicity and genotoxicity of SS, in comparison with NA, by using an integrated chemical-biological approach to enable their safe reuse in engineering applications. Leaching solutions from samples were obtained by using short-term leaching tests (CEN EN 12457-2, 2004) usually adopted for the evaluation of waste recovery and final disposal. Chemical analyses of leachates were performed according to the Italian legislation on waste recovery (Ministerial Decree 186/2006). The leaching solutions were assayed by using toxicity test on Daphnia magna. Moreover, mutagenicity/genotoxicity tests on Salmonella typhimurium, Allium cepa, and human leucocytes and fibroblasts were carried out. The releases of pollutants from all samples were within the limits of the Italian legislation for waste recovery. Despite the effects that SS and NA could have on different cells, in terms of toxicity and genotoxicity, globally, SS do not seem to be any more hazardous than NA. This ecotoxicological assessment, never studied before, is important for promoting further studies that may support the decision-making process regarding the use of such types of materials.
Assuntos
Poluentes Ambientais/toxicidade , Resíduos Perigosos/efeitos adversos , Aço/toxicidade , Linhagem Celular , Ecotoxicologia/métodos , Fibroblastos/efeitos dos fármacos , Humanos , Leucócitos/efeitos dos fármacos , Masculino , Testes de Mutagenicidade/métodos , Testes de Toxicidade/métodos , Instalações de Eliminação de ResíduosRESUMO
Novel and complementary experimental models are required for investigating the molecular mechanisms underlying the resistance to the available therapies of patients with major depression (Treatment-Resistant Depression, TRD) that occurs in at least one third of patients and need to be deeply investigated. Here, we have established a patient-specific disease model for TRD by reprogramming peripheral blood mononuclear cells (PBMCs) from two TRD patients into induced pluripotent stem cells (iPSCs), using non-integrating Sendai virus. These lines show the typical morphology of pluripotent cells, express pluripotency markers and displayed in vitro differentiation potential toward cells of the three embryonic germ layers.
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Células-Tronco Pluripotentes Induzidas , Diferenciação Celular , Reprogramação Celular , Depressão , Humanos , Leucócitos Mononucleares , Vírus Sendai/genéticaRESUMO
We have generated new disease-specific induced pluripotent stem cell (iPSC) lines from skin fibroblasts obtained from a female patient with Joubert syndrome (JS) caused by compound heterozygous mutations in C5orf42 gene. The generated iPSCs offer an unprecedented opportunity to obtain iPSC-derived neurons to investigate the pathogenesis of JS in vitro and to develop therapeutic strategies.
Assuntos
Anormalidades Múltiplas , Anormalidades do Olho , Células-Tronco Pluripotentes Induzidas , Doenças Renais Císticas , Diferenciação Celular , Cerebelo/anormalidades , Anormalidades do Olho/genética , Feminino , Humanos , Mutação , Retina/anormalidadesRESUMO
One of the main hurdles to improving scaffolds for regenerative medicine is the development of non-invasive methods to monitor cell proliferation within three-dimensional environments. Recently, an electrical impedance-based approach has been identified as promising for three-dimensional proliferation assays. A low-cost impedance-based solution, easily integrable with multi-well plates, is here presented. Sensors were developed using biocompatible carbon-based ink on foldable polyimide substrates by means of a novel aerosol jet printing technique. The setup was tested to monitor the proliferation of human mesenchymal stromal cells into previously validated gelatin-chitosan hybrid hydrogel scaffolds. Reliability of the methodology was assessed comparing variations of the electrical impedance parameters with the outcomes of enzymatic proliferation assay. Results obtained showed a magnitude increase and a phase angle decrease at 4 kHz (maximum of 2.5 kΩ and -9 degrees) and an exponential increase of the modeled resistance and capacitance components due to the cell proliferation (maximum of 1.5 kΩ and 200 nF). A statistically significant relationship with enzymatic assay outcomes could be detected for both phase angle and electric model parameters. Overall, these findings support the potentiality of this non-invasive approach for continuous monitoring of scaffold-based cultures, being also promising in the perspective of optimizing the scaffold-culture system.
RESUMO
Autosomal recessive osteopetrosis (ARO) is a rare inherited disorder leading to increased bone density with impairment in bone resorption. Among the genes responsible for ARO, the TCIRG1 gene, coding for the a3 subunit of the osteoclast proton pump, is mutated in more than 50% of the cases, increasing the importance of TCIRG1-iPSCs as disease model. We generated 3 iPSC clones derived from Peripheral Blood Mononuclear Cells (PBMCs) of a patient carrying the heterozygous mutations p.Y512X and c.2236 + 1G > A. A Sendai virus-based vector was used and the iPSCs were characterized for genetic identity to parental cells, genomic integrity, pluripotency, and differentiation ability.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Osteopetrose/genética , ATPases Vacuolares Próton-Translocadoras/genética , Humanos , Lactente , Masculino , MutaçãoRESUMO
AIMS: Atrial fibrillation (AF) is the most common type of cardiac arrhythmias, whose incidence is likely to increase with the aging of the population. It is considered a progressive condition, frequently observed as a complication of other cardiovascular disorders. However, recent genetic studies revealed the presence of several mutations and variants linked to AF, findings that define AF as a multifactorial disease. Due to the complex genetics and paucity of models, molecular mechanisms underlying the initiation of AF are still poorly understood. Here we investigate the pathophysiological mechanisms of a familial form of AF, with particular attention to the identification of putative triggering cellular mechanisms, using patient's derived cardiomyocytes (CMs) differentiated from induced pluripotent stem cells (iPSCs). METHODS AND RESULTS: Here we report the clinical case of three siblings with untreatable persistent AF whose whole-exome sequence analysis revealed several mutated genes. To understand the pathophysiology of this multifactorial form of AF we generated three iPSC clones from two of these patients and differentiated these cells towards the cardiac lineage. Electrophysiological characterization of patient-derived CMs (AF-CMs) revealed that they have higher beating rates compared to control (CTRL)-CMs. The analysis showed an increased contribution of the If and ICaL currents. No differences were observed in the repolarizing current IKr and in the sarcoplasmic reticulum calcium handling. Paced AF-CMs presented significantly prolonged action potentials and, under stressful conditions, generated both delayed after-depolarizations of bigger amplitude and more ectopic beats than CTRL cells. CONCLUSIONS: Our results demonstrate that the common genetic background of the patients induces functional alterations of If and ICaL currents leading to a cardiac substrate more prone to develop arrhythmias under demanding conditions. To our knowledge this is the first report that, using patient-derived CMs differentiated from iPSC, suggests a plausible cellular mechanism underlying this complex familial form of AF.
Assuntos
Potenciais de Ação/genética , Fibrilação Atrial/genética , Canais de Cálcio Tipo L/genética , Frequência Cardíaca/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação , Miócitos Cardíacos/metabolismo , Potenciais de Ação/efeitos dos fármacos , Antiarrítmicos/uso terapêutico , Fibrilação Atrial/tratamento farmacológico , Fibrilação Atrial/metabolismo , Fibrilação Atrial/fisiopatologia , Canais de Cálcio Tipo L/metabolismo , Estudos de Casos e Controles , Diferenciação Celular , Células Cultivadas , Resistência a Medicamentos/genética , Predisposição Genética para Doença , Frequência Cardíaca/efeitos dos fármacos , Humanos , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Pessoa de Meia-Idade , Irmãos , Sequenciamento do ExomaRESUMO
We report the generation of three isogenic iPSC clones (UNIBSi007-A, UNIBSi007-B, and UNIBSi007-C) obtained from fibroblasts of a patient with Aicardi Goutières Syndrome (AGS) carrying a homozygous mutation in RNaseH2B. Cells were transduced using a Sendai virus based system, delivering the human OCT4, SOX2, c-MYC and KLF4 transcription factors. The resulting transgene-free iPSC lines retained the disease-causing DNA mutation, showed normal karyotype, expressed pluripotent markers and could differentiate in vitro toward cells of the three embryonic germ layers.
Assuntos
Doenças Autoimunes do Sistema Nervoso/genética , Doenças Autoimunes do Sistema Nervoso/patologia , Técnicas de Cultura de Células/métodos , Linhagem Celular/patologia , Fibroblastos/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Mutação/genética , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/patologia , Ribonuclease H/genética , Sequência de Bases , Criança , Feminino , Humanos , Fator 4 Semelhante a Kruppel , Reprodutibilidade dos TestesRESUMO
Aicardi-Goutières syndrome (AGS) is an early-onset monogenic encephalopathy characterized by intracranial calcification, leukodystrophy and cerebrospinal fluid lymphocytosis. To date, seven genes have been related to AGS. Among these, IFIH1 encodes for MDA5, a cytosolic double-stranded RNA receptor, and is responsible for AGS type 7. We generated three isogenic iPSC clones, using a Sendai virus-based vector, starting from fibroblasts of a patient carrying a dominant mutation in IFIH1. All lines were characterized for genomic integrity, genetic uniqueness, pluripotency, and differentiation capability. Our clones might offer a good model to investigate AGS7 pathophysiological mechanism and to discover new biomarkers for this condition treatment.
Assuntos
Doenças Autoimunes do Sistema Nervoso/genética , Doenças Autoimunes do Sistema Nervoso/patologia , Técnicas de Cultura de Células/métodos , Linhagem Celular/patologia , Fibroblastos/patologia , Helicase IFIH1 Induzida por Interferon/genética , Mutação/genética , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/patologia , Adolescente , Sequência de Bases , Humanos , Células-Tronco Pluripotentes Induzidas , Masculino , Reprodutibilidade dos TestesRESUMO
Fibroblasts from a patient with Aicardi Goutières Syndrome (AGS) carrying a compound heterozygous mutation in TREX1, were reprogrammed into induced pluripotent stem cells (iPSCs) to establish isogenic clonal stem cell lines: UNIBSi006-A, UNIBSi006-B, and UNIBSi006-C. Cells were transduced using the episomal Sendai viral vectors, containing human OCT4, SOX2, c-MYC and KLF4 transcription factors. The transgene-free iPSC lines showed normal karyotype, expressed pluripotent markers and displayed in vitro differentiation potential toward cells of the three embryonic germ layers.
Assuntos
Doenças Autoimunes do Sistema Nervoso/genética , Doenças Autoimunes do Sistema Nervoso/patologia , Diferenciação Celular , Exodesoxirribonucleases/genética , Fibroblastos/patologia , Células-Tronco Pluripotentes Induzidas/patologia , Mutação , Malformações do Sistema Nervoso/genética , Malformações do Sistema Nervoso/patologia , Fosfoproteínas/genética , Células Cultivadas , Pré-Escolar , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , MasculinoRESUMO
Crohn's disease is a debilitating and incurable chronic inflammatory bowel disease, affecting millions of individuals worldwide, with an increasing frequency. Surgery must be applicable in half of the cases often with a disabling course, and pharmacological treatments may have adverse complications. We generated three isogenic clones of iPSCs from peripheral blood mononuclear cells (PBMCs) of a patient with Crohn's Disease under pharmacological treatment without adverse effects. Sendai virus based vector was used and the iPSCs were characterized for genetic uniqueness, genomic integrity, pluripotency, and differentiation ability. These iPSCs will be a powerful tool to develop tailored therapies.
Assuntos
Linhagem Celular/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Diferenciação Celular , Linhagem Celular/metabolismo , Células Cultivadas , Doença de Crohn/genética , Doença de Crohn/metabolismo , Doença de Crohn/fisiopatologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Leucócitos Mononucleares/citologia , Leucócitos Mononucleares/metabolismo , Masculino , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Adulto JovemRESUMO
The Cri du Chat Syndrome (CdCS) is a genetic disease resulting from variable size deletion occurring on the short arm of chromosome 5. The main clinical features are a high-pitched monochromatic cry, microcephaly, severe psychomotor and mental retardation with characteristics of autism spectrum disorders such as hand flapping, obsessive attachments to objects, twirling objects, repetitive movements, and rocking. We reprogrammed to pluripotency peripheral blood mononuclear cells derived from a patient carrying large deletion on the short arm of chromosome 5, using a commercially available non-integrating expression system. The iPSCs expressed pluripotency markers and differentiated in the three embryonic germ layers.
Assuntos
Técnicas de Reprogramação Celular , Síndrome de Cri-du-Chat , Células-Tronco Pluripotentes Induzidas , Leucócitos Mononucleares , Adulto , Síndrome de Cri-du-Chat/genética , Síndrome de Cri-du-Chat/metabolismo , Síndrome de Cri-du-Chat/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/patologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , MasculinoRESUMO
Physio-pathologic interrelationships between endothelial layer and graft-versus-host disease (GVHD) have been described leading to assess the entity "endothelial GVHD" as the early step for clinical manifestations of acute GVHD. The availability of the CellSearch system has allowed us to monitor Circulating Endothelial Cells (CEC) changes in allogeneic hematopoietic stem cell transplantation (allo-HSCT) as useful tool to help clinicians in GVHD diagnostic definition. We have compared CEC counts generated by an ad hoc designed polychromatic-flowcytometry (PFC) Lyotube with those of the CellSearch system. CEC were counted in parallel at 5 timepoints in 50 patients with malignant hematologic disorders undergoing allo-HSCT (ClinicalTrials.gov, NCT02064972). Spearman rank correlation showed significant association between CEC values at all time points (p = 0.0001). The limits of agreement was demonstrated by Bland Altman plot analysis, showing bias not significant at T1, T3, T4, while at T2 and T5 resulted not estimable. Moreover, Passing Bablok regression analysis showed not significant differences between BD Lyotube and CellSearch system. We show that CEC counts, generated with either the CellSearch system or the PFC-based panel, have a superimposable kinetic in allo-HSCT patients and that both counting procedures hold the potential to enter clinical routine as a suitable tool to assist clinicians in GVHD diagnosis.
Assuntos
Células Sanguíneas , Células Endoteliais/patologia , Citometria de Fluxo/métodos , Doença Enxerto-Hospedeiro/diagnóstico , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Transplante Homólogo/efeitos adversos , HumanosRESUMO
Circulating endothelial cells (CEC) represent a restricted peripheral blood (PB) cell subpopulation with high potential diagnostic value in many endothelium-involving diseases. However, whereas the interest in CEC studies has grown, the standardization level of their detection has not. Here, we undertook the task to align CEC phenotypes and counts, by standardizing a novel flow cytometry approach, within a network of six laboratories. CEC were identified as alive/nucleated/CD45negative/CD34bright/CD146positive events and enumerated in 269 healthy PB samples. Standardization was demonstrated by the achievement of low inter-laboratory Coefficients of Variation (CVL), calculated on the basis of Median Fluorescence Intensity values of the most stable antigens that allowed CEC identification and count (CVL of CD34bright on CEC ~ 30%; CVL of CD45 on Lymphocytes ~ 20%). By aggregating data acquired from all sites, CEC numbers in the healthy population were captured (medianfemale = 9.31 CEC/mL; medianmale = 11.55 CEC/mL). CEC count biological variability and method specificity were finally assessed. Results, obtained on a large population of donors, demonstrate that the established procedure might be adopted as standardized method for CEC analysis in clinical and in research settings, providing a CEC physiological baseline range, useful as starting point for their clinical monitoring in endothelial dysfunctions.