Cell Culture Differentiation and Proliferation Conditions Influence the In Vitro Regeneration of the Human Airway Epithelium.

The human airway mucociliary epithelium can be recapitulated in vitro using primary cells cultured in an air-liquid interface (ALI), a reliable surrogate to perform pathophysiological studies. As tremendous variations exist among media used for ALI-cultured human airway epithelial cells, the aim of our study was to evaluate the impact of several media (BEGM, PneumaCult, Half & Half, and Clancy) on cell type distribution using single-cell RNA sequencing and imaging. Our work revealed the impact of these media on cell composition, gene expression profile, cell signaling, and epithelial morphology. We found higher proportions of multiciliated cells in PneumaCult-ALI and Half & Half, stronger EGF signaling from basal cells in BEGM-ALI, differential expression of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) entry factor ACE2, and distinct secretome transcripts depending on the media used. We also established that proliferation in PneumaCult-Ex Plus favored secretory cell fate, showing the key influence of proliferation media on late differentiation epithelial characteristics. Altogether, our data offer a comprehensive repertoire for evaluating the effects of culture conditions on airway epithelial differentiation and will aid in choosing the most relevant medium according to the processes to be investigated, such as cilia, mucus biology, or viral infection. We detail useful parameters that should be explored to document airway epithelial cell fate and morphology.

Novel dynamics of human mucociliary differentiation revealed by single-cell RNA sequencing of nasal epithelial cultures.

The upper airway epithelium, which is mainly composed of multiciliated, goblet, club and basal cells, ensures proper mucociliary function and can regenerate in response to assaults. In chronic airway diseases, defective repair leads to tissue remodeling. Delineating key drivers of differentiation dynamics can help understand how normal or pathological regeneration occurs. Using single-cell transcriptomics and lineage inference, we have unraveled trajectories from basal to luminal cells, providing novel markers for specific populations. We report that: (1) a precursor subgroup of multiciliated cells, which we have entitled deuterosomal cells, is defined by specific markers, such as DEUP1, FOXN4, YPEL1, HES6 and CDC20B; (2) goblet cells can be precursors of multiciliated cells, thus explaining the presence of hybrid cells that co-express markers of goblet and multiciliated cells; and (3) a repertoire of molecules involved in the regeneration process, such as keratins or components of the Notch, Wnt or BMP/TGFß pathways, can be identified. Confirmation of our results on fresh human and pig airway samples, and on mouse tracheal cells, extend and confirm our conclusions regarding the molecular and cellular choreography at work during mucociliary epithelial differentiation.

A Single-Cell Atlas of the Human Healthy Airways.

Rationale: The respiratory tract constitutes an elaborate line of defense that is based on a unique cellular ecosystem.Objectives: We aimed to investigate cell population distributions and transcriptional changes along the airways by using single-cell RNA profiling.Methods: We have explored the cellular heterogeneity of the human airway epithelium in 10 healthy living volunteers by single-cell RNA profiling. A total of 77,969 cells were collected at 35 distinct locations, from the nose to the 12th division of the airway tree.Measurements and Main Results: The resulting atlas is composed of a high percentage of epithelial cells (89.1%) but also immune (6.2%) and stromal (4.7%) cells with distinct cellular proportions in different regions of the airways. It reveals differential gene expression between identical cell types (suprabasal, secretory, and multiciliated cells) from the nose (MUC4, PI3, SIX3) and tracheobronchial (SCGB1A1, TFF3) airways. By contrast, cell-type-specific gene expression is stable across all tracheobronchial samples. Our atlas improves the description of ionocytes, pulmonary neuroendocrine cells, and brush cells and identifies a related population of NREP-positive cells. We also report the association of KRT13 with dividing cells that are reminiscent of previously described mouse "hillock" cells and with squamous cells expressing SCEL and SPRR1A/B.Conclusions: Robust characterization of a single-cell cohort in healthy airways establishes a valuable resource for future investigations. The precise description of the continuum existing from the nasal epithelium to successive divisions of the airways and the stable gene expression profile of these regions better defines conditions under which relevant tracheobronchial proxies of human respiratory diseases can be developed.

A cost effective 5΄ selective single cell transcriptome profiling approach with improved UMI design.

Single cell RNA sequencing approaches are instrumental in studies of cell-to-cell variability. 5΄ selective transcriptome profiling approaches allow simultaneous definition of the transcription start size and have advantages over 3΄ selective approaches which just provide internal sequences close to the 3΄ end. The only currently existing 5΄ selective approach requires costly and labor intensive fragmentation and cell barcoding after cDNA amplification. We developed an optimized 5΄ selective workflow where all the cell indexing is done prior to fragmentation. With our protocol, cell indexing can be performed in the Fluidigm C1 microfluidic device, resulting in a significant reduction of cost and labor. We also designed optimized unique molecular identifiers that show less sequence bias and vulnerability towards sequencing errors resulting in an improved accuracy of molecule counting. We provide comprehensive experimental workflows for Illumina and Ion Proton sequencers that allow single cell sequencing in a cost range comparable to qPCR assays.

The MIR34B/C genomic region contains multiple potential regulators of multiciliogenesis.

The MIR449 genomic locus encompasses several regulators of multiciliated cell (MCC) formation (multiciliogenesis). The miR-449 homologs miR-34b/c represent additional regulators of multiciliogenesis that are transcribed from another locus. Here, we characterized the expression of BTG4, LAYN, and HOATZ, located in the MIR34B/C locus using single-cell RNA-seq and super-resolution microscopy from human, mouse, or pig multiciliogenesis models. BTG4, LAYN, and HOATZ transcripts were expressed in both precursors and mature MCCs. The Layilin/LAYN protein was absent from primary cilia, but it was expressed in apical membrane regions or throughout motile cilia. LAYN silencing altered apical actin cap formation and multiciliogenesis. HOATZ protein was detected in primary cilia or throughout motile cilia. Altogether, our data suggest that the MIR34B/C locus may gather potential actors of multiciliogenesis.

CDC20B is required for deuterosome-mediated centriole production in multiciliated cells.

Multiciliated cells (MCCs) harbor dozens to hundreds of motile cilia, which generate hydrodynamic forces important in animal physiology. In vertebrates, MCC differentiation involves massive centriole production by poorly characterized structures called deuterosomes. Here, single-cell RNA sequencing reveals that human deuterosome stage MCCs are characterized by the expression of many cell cycle-related genes. We further investigated the uncharacterized vertebrate-specific cell division cycle 20B (CDC20B) gene, which hosts microRNA-449abc. We show that CDC20B protein associates to deuterosomes and is required for centriole release and subsequent cilia production in mouse and Xenopus MCCs. CDC20B interacts with PLK1, a kinase known to coordinate centriole disengagement with the protease Separase in mitotic cells. Strikingly, over-expression of Separase rescues centriole disengagement and cilia production in CDC20B-deficient MCCs. This work reveals the shaping of deuterosome-mediated centriole production in vertebrate MCCs, by adaptation of canonical and recently evolved cell cycle-related molecules.

[Chlamydia trachomatis infection in women using 2 family planning clinics]. / Infección por Chlamydia trachomatis en usuarias de dos clínicas de planificación familiar.

OBJECTIVE: To determine the prevalence of Chlamydia trachomatis in women attending two family planning clinics in Merida, Yucatan, Mexico. MATERIAL AND METHODS: From January to December 1998, a cross-sectional study was conducted in 1,100 sexually active women between 15 and 45 years of age. Study subjects had not received antibiotic therapy for at least one month prior to their visit to the clinic. Endocervical samples were taken for bacterial detection with the enzymatic Wellcozyme immunoassay method. Data were analyzed using the Epi-Info program. Statistical analysis was conducted using the chi-squared test and prevalence ratios. RESULTS: Seventy four women were positive to Chlamydia trachomatis (6.7%). Almost 50% of those women were asymptomatic. No statistically significant differences were found between the group with Chlamydia and the group without it who had vulvar-vaginal symptoms. Cervical changes were more frequent in infected women. More positive cases of Chlamydia infection were found in oral contraceptive users (8.3%) than among non users (5.4%). CONCLUSIONS: Study results confirm the high prevalence of asymptomatic infections and emphasize the importance of timely diagnosis to avoid infection sequelae. The English version of this paper is available at:http://www.insp.mx/salud/index.html.

Infección por Chlamydia trachomatis en usuarias de dos clínicas de planificación familiar / Chlamydia trachomatis infection in users of two family planning clinics

OBJETIVO: Determinar la prevalencia de infección por Chlamydia trachomatis en mujeres que acuden a dos clínicas de planificación familiar. MATERIAL Y MÉTODOS: Se llevó a cabo un estudio transversal en 1 100 mujeres sexualmente activas, aparentemente sanas, de Mérida, Yucatán, México, quienes acudieron a dos clínicas de planificación familiar en el periodo comprendido de enero a diciembre de 1998. Las mujeres incluidas en el estudio tuvieron entre 15 y 45 años de edad, y habían estado libres de tratamiento antibacteriano al menos durante el mes previo a su inclusión. Se obtuvieron muestras endocervicales y datos epidemiológicos. La detección de bacterias fue realizada por el método inmunoenzimático de marca Wellcozyme. Los datos fueron procesados en el programa EPI Info. Como métodos estadísticos se utilizaron la prueba de Z, ji cuadrada y como medida de asociación, la razón de prevalencias. RESULTADOS: Setenta y cuatro mujeres (6.7 por ciento) fueron positivas a infección por Chlamydia trachomatis. Cerca de 50 por ciento de las mujeres estuvo asintomática. No se encontró diferencia estadística entre la proporción de mujeres con y sin Chlamydia que tuvieron síntomas vulvovaginales; por el contrario, los cambios relacionados con cervicitis fueron más frecuentes en las pacientes infectadas. C trachomatis fue más frecuente en usuarias de anticonceptivos orales (8.3 por ciento) comparadas con mujeres que no tenían método anticonceptivo (5.4 por ciento). CONCLUSIONES: Los resultados de este estudio confirman la alta prevalencia de infecciones asintomáticas y pone énfasis en la importancia de un diagnóstico oportuno para evitar secuelas.