Generation of a healthy heavy smoker patient-derived induced pluripotent stem cell line UHOMi007-A from peripheral blood mononuclear cells.

Human pluripotent stem cells (hiPSC) represent a unique opportunity to model lung development and chronic bronchial diseases. We generated a hiPSC line from a highly characterized healthy heavy smoker male donor free from emphysema or tobacco related disease. Peripheral blood mononuclear cells (PBMCs) were reprogrammed using integration-free Sendai virus. The cell line had normal karyotype, expressed pluripotency hallmarks, and differentiated into the three primary germ layers. The reported UHOMi007-A iPSC line may be used as a control to model lung development, study human chronic bronchial diseases and drug testing.

Intimate partner violence as a risk factor for venous thromboembolism in women on combined oral contraceptives: An international matched case-control study.

BACKGROUND: Intimate partner violence (IPV) targeting women is probably underestimated during a woman's lifetime. Venous thromboembolism (VTE) is a multifactorial disease associated with haemostasis-activating conditions. Minor injuries can trigger VTE. OBJECTIVES: We aimed to look for an association between VTE and IPV in women taking combined oral contraceptives (COCs) METHODS: We performed a multicentric, international, matched case-control study. Patients were women with a first VTE associated with COC intake. Controls were women taking COCs undergoing regular gynaecological check-ups. Patients and Controls were matched for country, age, length of COC intake and type (997 pairs). IPV was evaluated using the WAST self-administrated questionnaire. RESULTS: IPV, defined as a WAST score value at least 5, was diagnosed in 33 Controls (3.3 %) and 109 patients (10.9 %), conditional odds ratio (OR): 3.586, 95 % confidence interval (2.404-5.549), p < 0.0001. After multivariate analysis, the adjusted OR was 3.720 (2.438-5.677), p < 0.0001. Sensitivity analysis using increasing WAST score thresholds confirmed the association. CONCLUSIONS: A first VTE in women taking COCs is associated with IPV. This association can have strong human consequences but also raises significant medical issues, for instance on the haemorrhagic risk of anticoagulant treatments in abused women. Pathophysiological studies are warranted.

New erythrocyte parameters derived from the Coulter principle relate with red blood cell properties-A pilot study in diabetes mellitus.

In routine hematological instruments, blood cells are counted and sized by monitoring the impedance signals induced during their passage through a Coulter orifice. However, only signals associated with centered paths in the aperture are considered for analysis, while the rejected measurements, caused by near-wall trajectories, can provide additional information on red blood cells (RBC), as recent publications suggest. To assess usefulness of two new parameters in describing alterations in RBC properties, we performed a pilot study to compare blood samples from patients with diabetes mellitus (DM), frequent pathological condition associated with impairment in RBC deformability, versus controls. A total of 345 blood samples were analyzed: 225 in the DM group and 120 in the control group. A diagram of [Formula: see text] and [Formula: see text], the two new parameters derived from the analysis of impedancemetry pulses, was used to compare distribution of RBC subpopulations between groups. To discriminate RBC from DM and control individuals, based on our multiparametric analysis, we built a score from variables derived from [Formula: see text] matrix which showed good performances: area under the receiving operating characteristic curve 0.948 (0.920-0.970), p<0.0001; best discriminating value: negative predictive value 94.7%, positive predictive value was 78.4%. These results seem promising to approach RBC alterations in routine laboratory practice. The related potential clinically relevant outcomes remain to be investigated.

Clonal haematopoiesis of indeterminate potential in patients with venous thromboembolism.

Over the last decade, the concept of Clonal haematopoiesis of undetermined potential (CHIP) has emerged. Low frequency somatic mutations in hematopoietic cells can occur with age and might allow formation of clones in individuals with no characterized haematological pathology. These CHIP mutations are associated with an increased risk of cancer or atherothrombosis, and their prevalence are more and more studied in pathologies with an inflammatory component. In our study, we analysed, by next generation sequencing, the prevalence of CHIP mutation in 94 patients with deep venous thrombosis (DVT), distinguishing two clinical phenotypes: provoked distal and non-provoked proximal DVTs. We show that there is no difference in CHIP prevalence between these two groups, nor with a matched-aged control group. The number of mutation per patients and the affected genes remain also the same between the three groups. Consequently and despite the relative small number of patients in each cohort, it seems that CHIP is not a strong concern in venous thromboembolism.

Antiphospholipid Antibodies in Pregnancy: Maternal and Neonatal Implications.

Antiphospholipid antibodies (aPL Abs) have long been associated with the occurrence of certain specific pregnancy morbidities, affecting both mother and fetus. Antithrombotic-based prophylactic regimens are the standard of care. Their intensity is modulated by the thrombotic history and has greatly improved the prognosis related to spontaneous morbidity. Observational studies show that this treatment is still associated with the persistence of excess of late-pregnancy placental diseases, calling for new or complementary developments, yet to be validated. Rigorous prospective multicentric validation of clinical and laboratory parameters capable of identifying those women and fetuses at a risk of pejorative evolution, thus early prognosis, is a priority issue. These will make it possible to develop customized treatments and test them. Furthermore, there are still concerns, particularly neurodevelopmental ones, about children born to aPL Ab-positive mothers, and clarification based on regular, more systematic evaluations is required. Even after pregnancy, women with a pure obstetrical antiphospholipid syndrome are at a greater risk of venous and arterial thrombosis over time, and prevention needs to be improved. These women also appear to develop more psychiatric and mood disorders. Central nervous system imaging using high-resolution techniques has shown subtle impairments in the white matter, associated with the most pathogenic aPL Abs and the clinical significance of this is under investigation. These mothers also seem to develop an excess of cancers. The systemic impact of aPL Abs is gradually being suspected, although this requires further evidence, and prevention should be envisaged.

Combined oral contraceptive-associated venous thromboembolism revealing an antiphospholipid syndrome: International retrospective study of outcomes.

INTRODUCTION: Limitations in the data used to define thromboprophylaxis for patients with antiphospholipid antibodies (aPLAbs) and thrombosis include uncertainties after an initial provoked venous thromboembolic event (VTE). We aimed to study such cases associated with combined oral contraceptive (COC) intake. METHODS: We retrospectively analysed thrombotic outcomes after a first COC-associated VTE and positive aPLAbs, with a low risk HERDOO2 score, on low-dose aspirin (LDA) secondary thromboprophylaxis, seen from 2010 to 2021 in 3 tertiary referral centres, one in France and 2 in Russia. Data from 264 patients (distal deep vein thrombosis DVT: 62.9 %), cumulating in 1327.7 patient-years of observation, were collected. RESULTS: There were 22 cases of thrombosis: 16 distal DVTs, 3 proximal, 1 pulmonary embolism (PE) and 2 transient ischemic attacks. Recurrence rate was 1.66 per 100 patient-years (p-y; 95 % CI: 0.96-2.33). No major bleeding occurred. Risk factors affecting recurrence-free survival were the time between first COC intake and VTE (p < 0.0001; the shortest, the lower), proximal DVT (p = 0.021), active smoking (p = 0.039), an associated systemic disease (p = 0.043) and circulating monocyte counts (p = 0.001). CONCLUSIONS: We observed a low risk of recurrence which was modulated by classical risk factors for VTE. These observational data may provide clues for future randomized controlled trials.

Differentiation of Human Induced Pluripotent Stem Cells from Patients with Severe COPD into Functional Airway Epithelium.

Background: Chronic Obstructive Pulmonary Disease (COPD), a major cause of mortality and disability, is a complex disease with heterogeneous and ill-understood biological mechanisms. Human induced pluripotent stem cells (hiPSCs) are a promising tool to model human disease, including the impact of genetic susceptibility. Methods: We developed a simple and reliable method for reprogramming peripheral blood mononuclear cells into hiPSCs and to differentiate them into air−liquid interface bronchial epithelium within 45 days. Importantly, this method does not involve any cell sorting step. We reprogrammed blood cells from one healthy control and three patients with very severe COPD. Results: The mean cell purity at the definitive endoderm and ventral anterior foregut endoderm (vAFE) stages was >80%, assessed by quantifying C-X-C Motif Chemokine Receptor 4/SRY-Box Transcription Factor 17 (CXCR4/SOX17) and NK2 Homeobox 1 (NKX2.1) expression, respectively. vAFE cells from all four hiPSC lines differentiated into bronchial epithelium in air−liquid interface conditions, with large zones covered by beating ciliated, basal, goblets, club cells and neuroendocrine cells, as found in vivo. The hiPSC-derived airway epithelium (iALI) from patients with very severe COPD and from the healthy control were undistinguishable. Conclusions: iALI bronchial epithelium is ready for better understanding lung disease pathogenesis and accelerating drug discovery.

The Risk of Thrombosis Around Pregnancy: Where Do We Stand?

Pregnancy and puerperium increase the relative risk of venous thromboembolism (VTE) and the absolute risk remains low, around 1 per 1,000, with induced mortality of around 1 per 100,000. Analysis of large databases has helped specify the modes of presentation and risk factors (RF) whose impact is greater after than before childbirth, since VTE during pregnancy and post-partum obey different RFs. The evolution of the population concerned (mostly women over 35, obese, of multi-ethnicity undergoing medically assisted reproduction) affects the frequency of these RFs. Pulmonary embolism (PE) is over-represented after childbirth, but 30% of PE in pregnancy occurs without any RFs. Recommendations for prevention, mainly from expert groups, are heterogeneous and often discordant. Low molecular weight heparins (LMWH) are the mainstay of pharmacological thromboprophylaxis, in a field where randomized controlled studies are definitely lacking. VTE risk assessment in pregnancy must be systematic and repetitive. Risk assessment methods and scores are beginning to emerge to guide thromboprophylaxis and should be used more systematically. In the future, analyzing observational data from huge, nationwide registries and prospective cluster clinical trials may bring to light clinically relevant outcomes likely to feed comprehensive guidelines.

Pregnancy after Combined Oral Contraceptive-Associated Venous Thromboembolism: An International Retrospective Study of Outcomes.

BACKGROUND: Few data are available on thrombotic outcomes during pregnancy and puerperium occurring after an initial provoked venous thromboembolic (VTE) event. OBJECTIVES: To describe thrombotic outcomes during pregnancy after a first combined oral contraceptive (COC)-associated VTE and the factors associated with recurrence. METHODS: This was an international multicentric retrospective study on patients referred for thrombophilia screening from January 1, 2010 to January 1, 2021 following a first COC-associated VTE, including women with neither inherited thrombophilia nor antiphospholipid antibodies and focusing on those who had a subsequent pregnancy under the same thromboprophylaxis treatment. Thrombotic recurrences during pregnancy and puerperium as well as risk factors for recurrence were analyzed. RESULTS: We included 2,145 pregnant women. A total of 88 thrombotic events, 58 antenatal and 29 postnatal, occurred, mostly during the first trimester of pregnancy and the first 2 weeks of puerperium. Incidence rates were 49.6 (37-62) per 1,000 patient-years during pregnancy and 118.7 (78-159) per 1,000 patient-years during puerperium. Focusing on pulmonary embolism, incidence rates were 1.68 (1-4) per 1,000 patient-years during pregnancy and 65.5 (35-97) per 1,000 patient-years during puerperium.Risk factors for antenatal recurrences were maternal hypercholesterolemia and birth of a very small-for-gestational-age neonate. A risk factor for postnatal recurrence was the incidence of preeclampsia. CONCLUSION: Our multicentric retrospective data show significant rates of VTE recurrence during pregnancy and puerperium in women with a previous VTE event associated with COC, despite a unique low-molecular-weight heparin-based thromboprophylaxis. These results may provide benchmarks and valuable information for designing future randomized controlled trials.

Reference values of coagulation assays performed for thrombophilia screening after a first venous thrombosis and their intra-patient associations.

INTRODUCTION: No reference values are currently available for coagulation assays performed for thrombophilia screening prescribed according to guidelines, after a first venous thromboembolic (VTE) event, and we have no idea of the intra-patient associations between results. METHODS: We performed a retrospective study of consecutive prescriptions fulfilling guidelines in a French university hospital from 2010 to 2019 (n = 3842) from the Glims® laboratory information system. We collected results of 12 parameters: aPTT, PT, fibrinogen (Fg), one-stage clotting methods for factors VIII, IX, XI and II (FVIII, FIX, FXI, FII), antithrombin (using an amidolytic assay: AT), protein C and S (using clotting assays: PC and PS) and mixing tests of a lupus-anticoagulant sensitive aPTT and of DRVVT. RESULTS: We show the results of the 12 parameters from 3603 individual files with less than 6 missing values, then describe these distributions and correlations between results from 2930 files with no missing value. We give the frequency of results described as indicating a risk of first VTE or of VTE recurrence. We propose 2 quantitative scores linking the 12 parameters at the individual level and reflecting their degree of dispersion with respect to their mean, describe the values of these scores and their associations with thrombophilic results. CONCLUSIONS: These normal values should help laboratory workers to validate process results and to assess their degree of originality. Our 2 scores should help to determine the intra-patient plausibility of associations of results. The usefulness of these laboratory scores for predicting clinically-relevant outcomes deserves to be investigated.

CRISPR/Cas9-mediated gene knockout and interallelic gene conversion in human induced pluripotent stem cells using non-integrative bacteriophage-chimeric retrovirus-like particles.

BACKGROUND: The application of CRISPR/Cas9 technology in human induced pluripotent stem cells (hiPSC) holds tremendous potential for basic research and cell-based gene therapy. However, the fulfillment of these promises relies on the capacity to efficiently deliver exogenous nucleic acids and harness the repair mechanisms induced by the nuclease activity in order to knock-out or repair targeted genes. Moreover, transient delivery should be preferred to avoid persistent nuclease activity and to decrease the risk of off-target events. We recently developed bacteriophage-chimeric retrovirus-like particles that exploit the properties of bacteriophage coat proteins to package exogenous RNA, and the benefits of lentiviral transduction to achieve highly efficient, non-integrative RNA delivery in human cells. Here, we investigated the potential of bacteriophage-chimeric retrovirus-like particles for the non-integrative delivery of RNA molecules in hiPSC for CRISPR/Cas9 applications. RESULTS: We found that these particles efficiently convey RNA molecules for transient expression in hiPSC, with minimal toxicity and without affecting the cell pluripotency and subsequent differentiation. We then used this system to transiently deliver in a single step the CRISPR-Cas9 components (Cas9 mRNA and sgRNA) to generate gene knockout with high indel rate (up to 85%) at multiple loci. Strikingly, when using an allele-specific sgRNA at a locus harboring compound heterozygous mutations, the targeted allele was not altered by NHEJ/MMEJ, but was repaired at high frequency using the homologous wild type allele, i.e., by interallelic gene conversion. CONCLUSIONS: Our results highlight the potential of bacteriophage-chimeric retrovirus-like particles to efficiently and safely deliver RNA molecules in hiPSC, and describe for the first time genome engineering by gene conversion in hiPSC. Harnessing this DNA repair mechanism could facilitate the therapeutic correction of human genetic disorders in hiPSC.

Generation of four severe early-onset chronic obstructive pulmonary disease (COPD) patient-derived induced pluripotent stem cell lines from peripheral blood mononuclear cells.

Evidence highlights the concept of multiple trajectories leading to COPD. Early-life events (i.e., in utero lung development) may influence the maximally attained lung function and increase the risk to develop COPD. Human pluripotent stem cells (hiPSC) represent a unique opportunity to model lung development. We generated hiPSC lines from four highly characterized COPD patients with early onset and severe phenotype. Peripheral blood mononuclear cells (PBMCs) were reprogrammed using integration-free Sendai Virus. The cell lines had normal karyotype, expressed pluripotency hallmarks, and differentiated into the three primary germ layers. These lines offer a tool to study early-life origins of COPD.

Targeted therapy in eosinophilic chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is a common and preventable airway disease causing significant worldwide mortality and morbidity. Lifetime exposure to tobacco smoking and environmental particles are the two major risk factors. Over recent decades, COPD has become a growing public health problem with an increase in incidence. COPD is defined by airflow limitation due to airway inflammation and small airway remodelling coupled to parenchymal lung destruction. Most patients exhibit neutrophil-predominant airway inflammation combined with an increase in macrophages and CD8+ T-cells. Asthma is a heterogeneous chronic inflammatory airway disease. The most studied subtype is type 2 (T2) high eosinophilic asthma, for which there are an increasing number of biologic agents developed. However, both asthma and COPD are complex and share common pathophysiological mechanisms. They are known as overlapping syndromes as approximately 40% of patients with COPD present an eosinophilic airway inflammation. Several studies suggest a putative role of eosinophilia in lung function decline and COPD exacerbation. Recently, pharmacological agents targeting eosinophilic traits in uncontrolled eosinophilic asthma, especially monoclonal antibodies directed against interleukins (IL-5, IL-4, IL-13) or their receptors, have shown promising results. This review examines data on the rationale for such biological agents and assesses efficacy in T2-endotype COPD patients.

Generation of the induced pluripotent stem cell line UHOMi002-A from peripheral blood mononuclear cells of a healthy male donor.

Human pluripotent stem cells (hiPSC) are highly valuable tools to model lung development and chronic bronchial diseases. We generated a hiPSC line from a highly characterized 40-year-old healthy male nonsmoking donor. Peripheral blood mononuclear cells (PBMCs) were reprogrammed using integration-free Sendai Virus. The cell line had normal karyotype, expressed pluripotency hallmarks, and differentiated into the three primary germ layers. The reported UHOMi002-A iPSC line may be used as a control to model lung development, study human chronic bronchial diseases and drug testing.

Pipeline for the Generation and Characterization of Transgenic Human Pluripotent Stem Cells Using the CRISPR/Cas9 Technology.

Recent advances in genome engineering based on the CRISPR/Cas9 technology have revolutionized our ability to manipulate genomic DNA. Its use in human pluripotent stem cells (hPSCs) has allowed a wide range of mutant cell lines to be obtained at an unprecedented rate. The combination of these two groundbreaking technologies has tremendous potential, from disease modeling to stem cell-based therapies. However, the generation, screening and molecular characterization of these cell lines remain a cumbersome and multi-step endeavor. Here, we propose a pipeline of strategies to efficiently generate, sub-clone, and characterize CRISPR/Cas9-edited hPSC lines in the function of the introduced mutation (indels, point mutations, insertion of large constructs, deletions).

[Lung organoids]. / Les organoïdes pulmonaires.

As burden of chronic respiratory diseases is constantly increasing, improving in vitro lung models is essential in order to reproduce as closely as possible the complex pulmonary architecture, responsible for oxygen uptake and carbon dioxide clearance. The study of diseases that affect the respiratory system has benefited from in vitro reconstructions of the respiratory epithelium with inserts in air/liquid interface (2D) or in organoids able to mimic up to the arborescence of the respiratory tree (3D). Recent development in the fields of pluripotent stem cells-derived organoids and genome editing technologies has provided new insights to better understand pulmonary diseases and to find new therapeutic perspectives.

TITLE: Les organoïdes pulmonaires. ABSTRACT: L'impact en santé publique des pathologies respiratoires chroniques ne cesse de croître. Dans ce contexte, il paraît indispensable d'améliorer les modèles d'études du poumon afin de reproduire au plus proche l'architecture pulmonaire complexe, garante des fonctions d'oxygénation et d'épuration du gaz carbonique. Les connaissances actuelles en physiopathologie respiratoire résultent en partie des études de modèles de reconstitution d'épithélium bronchique in vitro à partir de cellules primaires, en deux dimensions sur des inserts, ou en trois dimensions, en organoïdes mimant jusqu'à l'arborescence pulmonaire. Le développement de ces modèles in vitro a connu un nouvel essor grâce aux organoïdes pulmonaires issus de cellules souches pluripotentes et la démocratisation des outils d'édition du génome. Ces apports technologiques récents offrent de nouvelles perspectives en matière de thérapeutiques ou de compréhension physiopathologique et pourraient, dans le futur, ouvrir les portes de la médecine régénératrice pulmonaire.

Recurrent Genetic Abnormalities in Human Pluripotent Stem Cells: Definition and Routine Detection in Culture Supernatant by Targeted Droplet Digital PCR.

Genomic integrity of human pluripotent stem cells (hPSCs) is essential for research and clinical applications. However, genetic abnormalities can accumulate during hPSC generation and routine culture and following gene editing. Their occurrence should be regularly monitored, but the current assays to assess hPSC genomic integrity are not fully suitable for such regular screening. To address this issue, we first carried out a large meta-analysis of all hPSC genetic abnormalities reported in more than 100 publications and identified 738 recurrent genetic abnormalities (i.e., overlapping abnormalities found in at least five distinct scientific publications). We then developed a test based on the droplet digital PCR technology that can potentially detect more than 90% of these hPSC recurrent genetic abnormalities in DNA extracted from culture supernatant samples. This test can be used to routinely screen genomic integrity in hPSCs.

Generation of the induced pluripotent stem cell line UHOMi001-A from a patient with mutations in CCDC40 gene causing Primary Ciliary Dyskinesia (PCD).

Primary Ciliary Dyskinesia (PCD) is a rare heterogeneous genetic disorder affecting motile cilia structure and function leading to lung disease. We generated induced pluripotent stem cells (iPSCs) from dermal fibroblasts of a female PCD patient carrying disease-causing variants in the CCDC40 gene. Reprogramming was performed with the human OSKM transcription factors using the Sendai-virus delivery system. The resulting transgene free iPSCs had normal karyotype, expressed pluripotency markers, could differentiate into the three germ layers in vivo and retained the disease-causing CCDC40 mutations. This iPSC line could be useful to model PCD disease and test gene therapy strategies. Resource Table.

Induced Pluripotent Stem Cells for Primary Ciliary Dyskinesia Modeling and Personalized Medicine.

Primary ciliary dyskinesia (PCD) is a rare and heterogeneous genetic disorder that affects the structure and function of motile cilia. In the airway epithelium, impaired ciliary motion results in reduced or absent mucociliary clearance that leads to the appearance of chronic airway infection, sinusitis, and bronchiectasis. Currently, there is no effective treatment for PCD, and research is limited by the lack of convenient models to study this disease and investigate innovative therapies. Furthermore, the high heterogeneity of PCD genotypes is likely to hinder the development of a single therapy for all patients. The generation of patient-derived, induced pluripotent stem cells, and their differentiation into airway epithelium, as well as genome editing technologies, could represent major tools for in vitro PCD modeling and for developing personalized therapies. Here, we review PCD pathogenesis and then discuss how human induced pluripotent stem cells could be used to model this disease for the development of innovative, patient-specific biotherapies.