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.

Different Nuclear Architecture in Human Sperm According to Their Morphology.

Human sperm parameters serve as a first step in diagnosing male infertility, but not in determining the potential for successful pregnancy during assisted reproductive technologies (ARTs) procedures. Here, we investigated the relationship between sperm head morphology at high magnification, based on strict morphologic criteria, and the nuclear architecture analyzed by fluorescence in situ hybridization (FISH). We included five men. Two of them had an elevated high-magnification morphology score of 6 points (Score 6) indicating high fertility potential, whereas three had a low score of 0 points (Score 0), indicating low fertility potential. We used FISH to study the inter-telomeric distance and the chromosomal territory area of chromosome 1 (Chr. 1). We then compared these two parameters between subjects with high and low scores. FISH data analysis showed that the inter-telomeric distance (ITD) and chromosomal territory area (CTA) of Chr. 1 were significantly higher in subjects with low scores (score 0) than high scores (score 6). Our results suggest that (i) there is a link between nuclear architecture and sperm head abnormalities, particularly vacuoles; and (ii) it is possible to select spermatozoa with normal nuclear architecture, which might indirectly explain the positive ART outcomes observed with this technique.

S100A alarmins and thymic stromal lymphopoietin (TSLP) regulation in severe asthma following bronchial thermoplasty.

RATIONALE: Severe asthma affects a small proportion of asthmatics but represents a significant healthcare challenge. Bronchial thermoplasty (BT) is an interventional treatment approach preconized for uncontrolled severe asthma after considering biologics therapy. It was showed that BT long-lastingly improves asthma control. These improvements seem to be related to the ability of BT to reduce airway smooth muscle remodeling, reduce the number of nerve fibers and to modulate bronchial epithelium integrity and behavior. Current evidence suggest that BT downregulates epithelial mucins expression, cytokine production and metabolic profile. Despite these observations, biological mechanisms explaining asthma control improvement post-BT are still not well understood. OBJECTIVES: To assess whether BT affects gene signatures in bronchial epithelial cells (BECs). METHODS: In this study we evaluated the transcriptome of cultured bronchial epithelial cells (BECs) of severe asthmatics obtained pre- and post-BT treatment using microarrays. We further validated gene and protein expressions in BECs and in bronchial biopsies with immunohistochemistry pre- and post-BT treatment. MEASUREMENTS AND MAIN RESULTS: Transcriptomics analysis revealed that a large portion of differentially expressed genes (DEG) was involved in anti-viral response, anti-microbial response and pathogen induced cytokine storm signaling pathway. S100A gene family stood out as five members of this family where consistently downregulated post-BT. Further validation revealed that S100A7, S100A8, S100A9 and their receptor (RAGE, TLR4, CD36) expressions were highly enriched in severe asthmatic BECs. Further, these S100A family members were downregulated at the gene and protein levels in BECs and in bronchial biopsies of severe asthmatics post-BT. TLR4 and CD36 protein expression were also reduced in BECs post-BT. Thymic stromal lymphopoietin (TSLP) and human ß-defensin 2 (hBD2) were significantly decreased while no significant change was observed in IL-25 and IL-33. CONCLUSIONS: These data suggest that BT might improve asthma control by downregulating epithelial derived S100A family expression and related downstream signaling pathways.

The Transcriptome Landscape of the In Vitro Human Airway Epithelium Response to SARS-CoV-2.

Airway-liquid interface cultures of primary epithelial cells and of induced pluripotent stem-cell-derived airway epithelial cells (ALI and iALI, respectively) are physiologically relevant models for respiratory virus infection studies because they can mimic the in vivo human bronchial epithelium. Here, we investigated gene expression profiles in human airway cultures (ALI and iALI models), infected or not with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), using our own and publicly available bulk and single-cell transcriptome datasets. SARS-CoV-2 infection significantly increased the expression of interferon-stimulated genes (IFI44, IFIT1, IFIT3, IFI35, IRF9, MX1, OAS1, OAS3 and ISG15) and inflammatory genes (NFKBIA, CSF1, FOSL1, IL32 and CXCL10) by day 4 post-infection, indicating activation of the interferon and immune responses to the virus. Extracellular matrix genes (ITGB6, ITGB1 and GJA1) were also altered in infected cells. Single-cell RNA sequencing data revealed that SARS-CoV-2 infection damaged the respiratory epithelium, particularly mature ciliated cells. The expression of genes encoding intercellular communication and adhesion proteins was also deregulated, suggesting a mechanism to promote shedding of infected epithelial cells. These data demonstrate that ALI/iALI models help to explain the airway epithelium response to SARS-CoV-2 infection and are a key tool for developing COVID-19 treatments.

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.

Platelets Purification Is a Crucial Step for Transcriptomic Analysis.

Platelets are small anucleate cells derived from the fragmentation of megakaryocytes and are involved in different biological processes especially hemostasis, thrombosis, and immune response. Despite their lack of nucleus, platelets contain a reservoir of megakaryocyte-derived RNAs and all the machinery useful for mRNA translation. Interestingly, platelet transcriptome was analyzed in health and diseases and led to the identification of disease-specific molecular signatures. Platelet contamination by leukocytes and erythrocytes during platelet purification is a major problem in transcriptomic analysis and the presence of few contaminants in platelet preparation could strongly alter transcriptome results. Since contaminant impacts on platelet transcriptome remains theoretical, we aimed to determine whether low leukocyte and erythrocyte contamination could cause great or only minor changes in platelet transcriptome. Using microarray technique, we compared the transcriptome of platelets from the same donor, purified by common centrifugation method or using magnetic microbeads to eliminate contaminating cells. We found that platelet transcriptome was greatly altered by contaminants, as the relative amount of 8274 transcripts was different between compared samples. We observed an increase of transcripts related to leukocytes and erythrocytes in platelet purified without microbeads, while platelet specific transcripts were falsely reduced. In conclusion, serious precautions should be taken during platelet purification process for transcriptomic analysis, in order to avoid platelets contamination and result alteration.

Using intracellular SCGB1A1-sorted, formalin-fixed club cells for successful transcriptomic analysis.

As opposed to surface marker staining, certain cell types can only be recognized by intracellular markers. Intracellular staining for use in cell sorting remains challenging. Fixation and permeabilization steps for intracellular staining and the presence of RNases notably affect preservation of high-quality mRNA. We report the work required for the optimization of a successful protocol for microarray analysis of intracellular target-sorted, formalin-fixed human bronchial club cells. Cells obtained from differentiated air-liquid interface cultures were stained with the most characteristic intracellular markers for club cell (SCGB1A1+) sorting. A benchmarked intracellular staining protocol was carried out before flow cytometry. The primary outcome was the extraction of RNA sufficient quality for microarray analysis as assessed by Bioanalyzer System. Fixation with 4% paraformaldehyde coupled with 0.1% Triton/0.1% saponin permeabilization obtained optimal results for SCGB1A1 staining. Addition of RNase inhibitors throughout the protocol and within the appropriate RNA extraction kit (Formalin-Fixed-Paraffin-Embedded) dramatically improved RNA quality, resulting in samples eligible for microarray analysis. The protocol resulted in successful cell sorting according to specific club cell intracellular marker without using cell surface marker. The protocol also preserved RNA of sufficient quality for subsequent microarray transcriptomic analysis, and we were able to generate transcriptomic signature of club cells.

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.

Roles of Mesenchymal Cells in the Lung: From Lung Development to Chronic Obstructive Pulmonary Disease.

Mesenchymal cells are an essential cell type because of their role in tissue support, their multilineage differentiation capacities and their potential clinical applications. They play a crucial role during lung development by interacting with airway epithelium, and also during lung regeneration and remodeling after injury. However, much less is known about their function in lung disease. In this review, we discuss the origins of mesenchymal cells during lung development, their crosstalk with the epithelium, and their role in lung diseases, particularly in chronic obstructive pulmonary disease.

NRG1/ErbB signalling controls the dialogue between macrophages and neural crest-derived cells during zebrafish fin regeneration.

Fish species, such as zebrafish (Danio rerio), can regenerate their appendages after amputation through the formation of a heterogeneous cellular structure named blastema. Here, by combining live imaging of triple transgenic zebrafish embryos and single-cell RNA sequencing we established a detailed cell atlas of the regenerating caudal fin in zebrafish larvae. We confirmed the presence of macrophage subsets that govern zebrafish fin regeneration, and identified a foxd3-positive cell population within the regenerating fin. Genetic depletion of these foxd3-positive neural crest-derived cells (NCdC) showed that they are involved in blastema formation and caudal fin regeneration. Finally, chemical inhibition and transcriptomic analysis demonstrated that these foxd3-positive cells regulate macrophage recruitment and polarization through the NRG1/ErbB pathway. Here, we show the diversity of the cells required for blastema formation, identify a discrete foxd3-positive NCdC population, and reveal the critical function of the NRG1/ErbB pathway in controlling the dialogue between macrophages and NCdC.

Molecular Profiling of Spermatozoa Reveals Correlations between Morphology and Gene Expression: A Novel Biomarker Panel for Male Infertility.

Choosing spermatozoa with an optimum fertilizing potential is one of the major challenges in assisted reproductive technologies (ART). This selection is mainly based on semen parameters, but the addition of molecular approaches could allow a more functional evaluation. To this aim, we used sixteen fresh sperm samples from patients undergoing ART for male infertility and classified them in the high- and poor-quality groups, on the basis of their morphology at high magnification. Then, using a DNA sequencing method, we analyzed the spermatozoa methylome to identify genes that were differentially methylated. By Gene Ontology and protein-protein interaction network analyses, we defined candidate genes mainly implicated in cell motility, calcium reabsorption, and signaling pathways as well as transmembrane transport. RT-qPCR of high- and poor-quality sperm samples allowed showing that the expression of some genes, such as AURKA, HDAC4, CFAP46, SPATA18, CACNA1C, CACNA1H, CARHSP1, CCDC60, DNAH2, and CDC88B, have different expression levels according to sperm morphology. In conclusion, the present study shows a strong correlation between morphology and gene expression in the spermatozoa and provides a biomarker panel for sperm analysis during ART and a new tool to explore male infertility.

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.

Transcriptome-Wide Analysis Reveals a Role for Extracellular Matrix and Integrin Receptor Genes in Otic Neurosensory Differentiation from Human iPSCs.

We analyzed transcriptomic data from otic sensory cells differentiated from human induced pluripotent stem cells (hiPSCs) by a previously described method to gain new insights into the early human otic neurosensory lineage. We identified genes and biological networks not previously described to occur in the human otic sensory developmental cell lineage. These analyses identified and ranked genes known to be part of the otic sensory lineage program (SIX1, EYA1, GATA3, etc.), in addition to a number of novel genes encoding extracellular matrix (ECM) (COL3A1, COL5A2, DCN, etc.) and integrin (ITG) receptors (ITGAV, ITGA4, ITGA) for ECM molecules. The results were confirmed by quantitative PCR analysis of a comprehensive panel of genes differentially expressed during the time course of hiPSC differentiation in vitro. Immunocytochemistry validated results for select otic and ECM/ITG gene markers in the in vivo human fetal inner ear. Our screen shows ECM and ITG gene expression changes coincident with hiPSC differentiation towards human otic neurosensory cells. Our findings suggest a critical role of ECM-ITG interactions with otic neurosensory lineage genes in early neurosensory development and cell fate determination in the human fetal inner ear.

Neuromedin B promotes chondrocyte differentiation of mesenchymal stromal cells via calcineurin and calcium signaling.

BACKGROUND: Articular cartilage is a complex tissue with poor healing capacities. Current approaches for cartilage repair based on mesenchymal stromal cells (MSCs) are often disappointing because of the lack of relevant differentiation factors that could drive MSC differentiation towards a stable mature chondrocyte phenotype. RESULTS: We used a large-scale transcriptomic approach to identify genes that are modulated at early stages of chondrogenic differentiation using the reference cartilage micropellet model. We identified several modulated genes and selected neuromedin B (NMB) as one of the early and transiently modulated genes. We found that the timely regulated increase of NMB was specific for chondrogenesis and not observed during osteogenesis or adipogenesis. Furthermore, NMB expression levels correlated with the differentiation capacity of MSCs and its inhibition resulted in impaired chondrogenic differentiation indicating that NMB is required for chondrogenesis. We further showed that NMB activated the calcineurin activity through a Ca2+-dependent signaling pathway. CONCLUSION: NMB is a newly described chondroinductive bioactive factor that upregulates the key chondrogenic transcription factor Sox9 through the modulation of Ca2+ signaling pathway and calcineurin activity.

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.

miR-155 Contributes to the Immunoregulatory Function of Human Mesenchymal Stem Cells.

Objectives: Mesenchymal stem/stromal cells (MSCs) are widely investigated in regenerative medicine thanks to their immunomodulatory properties. They exert their anti-inflammatory function thanks to the secretion of a number of mediators, including proteins and miRNAs, which can be released in the extracellular environment or in the cargo of extracellular vesicles (EVs). However, the role of miRNAs in the suppressive function of MSCs is controversial. The aim of the study was to identify miRNAs that contribute to the immunomodulatory function of human bone marrow-derived MSCs (BM-MSCs). Methods: Human BM-MSCs were primed by coculture with activated peripheral blood mononuclear cells (aPBMCs). High throughput miRNA transcriptomic analysis was performed using Human MicroRNA TaqMan® Array Cards. The immunosuppressive function of miRNAs was investigated in mixed lymphocyte reactions and the delayed type hypersensitivity (DTH) murine model. Results: Upon priming, 21 out of 377 tested miRNAs were significantly modulated in primed MSCs. We validated the up-regulation of miR-29a, miR-146a, miR-155 and the down-regulation of miR-149, miR-221 and miR-361 in additional samples of primed MSCs. We showed that miR-155 significantly reduced the proliferation of aPBMCs in vitro and inflammation in vivo, using the DTH model. Analysis of miRNA-mRNA interactions revealed miR-221 as a potential target gene that is down-regulated by miR-155 both in primed MSCs and in aPBMCs. Conclusion: Here, we present evidence that miR-155 participates to the immunosuppressive function of human BM-MSCs and down-regulates the expression of miR-221 as a possible inflammatory mediator.

Identification of a Novel Serum Proteomic Signature for Primary Sjögren's Syndrome.

Context: Primary Sjögren's syndrome (pSS) is a complex heterogeneous autoimmune disease (AID) which can mimic rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE). Our exploratory study investigated serum biomarkers that may discriminate pSS from RA and SLE. Methods: Serum concentrations of 63 biomarkers involved in immune cell trafficking, inflammatory response, cellular movement, and cell-to-cell signaling were measured in AID patients, included prospectively into the study at the Montpellier University Hospital. A multivariate analysis by multiple logistic regression was performed, and discriminative power assessed using logistic regression adjusted on significant demographic factors. Results: Among the 95 patients enrolled, 42 suffered from pSS, 28 from RA, and 25 from SLE. Statistical analysis showed that concentrations of BDNF (OR = 0.493 with 95% CI [0.273-0.891]; p = 0.0193) and I-TAC/CXCL11 (OR = 1.344 with 95% CI [1.027-1.76]; p = 0.0314) can significantly discriminate pSS from RA. Similarly, greater concentrations of sCD163 (OR = 0.803 with 95% CI [0.649-0.994]; p = 0.0436), Fractalkine/CX3CL1 (OR = 0.534 with 95% CI [0.287-0. 991]; p = 0.0466), MCP-1/CCL2 (OR = 0.839 with 95% CI [0.732-0.962]; p = 0.0121), and TNFa (OR = 0.479 with 95% CI [0.247-0.928]; p = 0.0292) were associated with SLE diagnosis compared to pSS. In addition, the combination of low concentrations of BDNF and Fractalkine/CX3CL1 was highly specific for pSS (specificity 96.2%; positive predictive value 80%) compared to RA and SLE, as well as the combination of high concentrations of I-TAC/CXCL11 and low concentrations of sCD163 (specificity 98.1%; positive predictive value 75%). Conclusion: Our study highlights biomarkers potentially involved in pSS, RA, and SLE pathophysiology that could be useful for developing a pSS-specific diagnostic tool.

Selective treatment pressure in colon cancer drives the molecular profile of resistant circulating tumor cell clones.

The characterization of circulating tumor cells (CTCs) holds promises for precision medicine because these cells are an important clinical indicator of treatment efficacy. We established the first and still only nine permanent colon CTC lines from peripheral blood samples of a patient with metastatic colon cancer collected at different time points during treatment and cancer progression. The study objectives were (i) to compare the gene expression profiles of these CTC lines, and (ii) to determine the main features acquired during treatment. The number of upregulated genes was higher in the CTC lines obtained after treatment, indicating that they acquired properties to escape treatment pressure. Among these upregulated genes, some are involved in the mTOR and PI3K/AKT signaling pathways. Moreover, cytidine deaminase expression was significantly increased in the CTC lines obtained after failure of the first- and second-line 5-fluorouracile-based treatments, suggesting that these CTCs can eliminate this specific drug and resist to therapy. Several enzymes involved in xenobiotic metabolism also were upregulated after treatment, suggesting the activation of detoxification mechanisms in response to chemotherapy. Finally, the significant higher expression of aldolase B in four of the six CTC lines obtained after treatment withdrawal and cancer progression indicated that these clones originated from liver metastases. In conclusion, these CTC lines generated at different time points during treatment of metastatic colon cancer in a single patient are characterized by the deregulation of different genes that promote (i) drug resistance, (ii) xenobiotic and energy metabolism, and (iii) stem cell properties and plasticity.

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.

Correction: Transcriptomic analysis of the developing and adult mouse cochlear sensory epithelia.

[This corrects the article DOI: 10.1371/journal.pone.0042987.].