Sustained postconfluent culture of human mammary epithelial cells enriches for luminal and c-Kit+ subtypes.

BACKGROUND: A challenge in human mammary epithelial cell (HMEC) culture is sustaining the representation of competing luminal, myoepithelial, and progenitor lineages over time. As cells replicate in culture, myoepithelial cells come to dominate the composition of the culture with serial passaging. This drift in composition presents a challenge for studying luminal and progenitor cells, which are prospective cells of origin for most breast cancer subtypes. METHODS: We demonstrate the use of postconfluent culture on HMECs. Postconfluent culture entails culturing HMECs for 2-5 weeks without passaging but maintaining frequent feedings in low-stress M87A culture medium. In contrast, standard HMEC culture entails enzymatic subculturing every 3-5 days to maintain subconfluent density. RESULTS: When compared to standard HMEC culture, postconfluent culture yields increased proportions of luminal cells and c-Kit+ progenitor cells. Postconfluent cultures develop a distinct multilayered morphology with individual cells showing decreased physical deformability as compared to cells in standard culture. Gene expression analysis of postconfluent cells shows increased expression of lineage-specific markers and extracellular matrix components. CONCLUSIONS: Postconfluent culture is a novel, useful strategy for altering the lineage composition of HMECs, by increasing the proportional representation of luminal and progenitor cells. We speculate that postconfluent culture creates a microenvironment with cellular composition closer to the physiological state and eases the isolation of scarce cell subtypes. As such, postconfluent culture is a valuable tool for researchers using HMECs for breast cancer research.

A comparative study of in vitro air-liquid interface culture models of the human airway epithelium evaluating cellular heterogeneity and gene expression at single cell resolution.

BACKGROUND: The airway epithelium is composed of diverse cell types with specialized functions that mediate homeostasis and protect against respiratory pathogens. Human airway epithelial (HAE) cultures at air-liquid interface are a physiologically relevant in vitro model of this heterogeneous tissue and have enabled numerous studies of airway disease. HAE cultures are classically derived from primary epithelial cells, the relatively limited passage capacity of which can limit experimental methods and study designs. BCi-NS1.1, a previously described and widely used basal cell line engineered to express hTERT, exhibits extended passage lifespan while retaining the capacity for differentiation to HAE. However, gene expression and innate immune function in BCi-NS1.1-derived versus primary-derived HAE cultures have not been fully characterized. METHODS: BCi-NS1.1-derived HAE cultures (n = 3 independent differentiations) and primary-derived HAE cultures (n = 3 distinct donors) were characterized by immunofluorescence and single cell RNA-Seq (scRNA-Seq). Innate immune functions were evaluated in response to interferon stimulation and to infection with viral and bacterial respiratory pathogens. RESULTS: We confirm at high resolution that BCi-NS1.1- and primary-derived HAE cultures are largely similar in morphology, cell type composition, and overall gene expression patterns. While we observed cell-type specific expression differences of several interferon stimulated genes in BCi-NS1.1-derived HAE cultures, we did not observe significant differences in susceptibility to infection with influenza A virus and Staphylococcus aureus. CONCLUSIONS: Taken together, our results further support BCi-NS1.1-derived HAE cultures as a valuable tool for the study of airway infectious disease.

Serially passaged, conditionally reprogrammed nasal epithelial cells as a model to study epithelial functions and SARS-CoV-2 infection.

Primary airway epithelial cells (pAECs) cultivated at air-liquid interface (ALI) conditions are widely used as surrogates for human in vivo epithelia. To extend the proliferative capacity and to enable serially passaging of pAECs, conditional reprogramming (cr) has been employed in recent years. However, ALI epithelia derived from cr cells often display functional changes with increasing passages. This highlights the need for thorough validation of the ALI cultures for the respective application. In our study, we evaluated the use of serially passaged cr nasal epithelial cells (crNECs) as a model to study SARS-CoV-2 infection and effects on ion and water transport. NECs were obtained from healthy individuals and cultivated as ALI epithelia derived from passages 1, 2, 3, and 5. We compared epithelial differentiation, ion and water transport, and infection with SARS-CoV-2 between passages. Our results show that epithelia maintained major differentiation characteristics and physiological ion and water transport properties through all passages. However, the frequency of ciliated cells, short circuit currents reflecting epithelial Na+ channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) activity and expression of aquaporin 3 and 5 decreased gradually over passages. crNECs also expressed SARS-CoV-2 receptors angiotensin converting enzyme 2 (ACE2) and transmembrane serin2 protease 2 (TMPRSS2) across all passages and allowed SARS-CoV-2 replication in all passages. In summary, we provide evidence that passaged crNECs provide an appropriate model to study SARS-CoV-2 infection and also epithelial transport function when considering some limitations that we defined herein.

African-American Prostate Normal and Cancer Cells for Health Disparities Research.

Prostate cancer is the most frequently diagnosed solid malignancy in men. Epidemiological studies have shown African-American men to be at higher risk for developing prostate cancer and experience higher death as compared to other ethnic groups. Establishment of prostate cancer cell lines paired with normal cells derived from the same patient is a fundamental breakthrough in cell culture technology and provides a resource to improve our understanding of cancer development and pertinent molecular events. Previous studies have demonstrated that conditional reprogramming (CR) allows the establishment and propagation of patient-derived normal and tumor epithelial cell cultures from a variety of tissue types. Here, we report a new AA prostate cell model, paired normal and cancer epithelial cells from the same patient. "Tumor" cell culture AA-103A was derived from malignant prostate tissues, and "normal" cell culture AA-103B was derived from non-malignant prostate tissues from the prostatectomy specimen of an African-American male. These paired cell cultures have been propagated under CRC conditions to permit direct comparison of the molecular and genetic profiles of the normal epithelium and adenocarcinoma cells for comparison of biomarkers, enabling patient-specific pathological analysis, and molecular and cellular characterization. STR confirmed human origin albeit no karyotypic abnormalities in the two cell lines. Further quantitative PCR analyses demonstrated characteristic markers, including the high level of basal cell marker, the keratin 5 (KRT5) in normal cells and of luminal marker, the androgen receptor (AR) as well as the programmed death-ligand 1 (PD-L1) in tumor cells. Although 3-D sphere formation was observed, the AA-103A of tumor cells did not generate tumors in vivo. We report these paired primary epithelial cultures under CRC growth as a potentially useful tool for studies to understand molecular mechanisms underlying health disparities in prostate cancer.

Critical intestinal cells originate from the host in enteroid-derived tissue-engineered intestine.

BACKGROUND: Enteroid-derived tissue-engineered intestine (TEI) contains intestinal subepithelial myofibroblasts (ISEMFs) and smooth muscle cells (SMCs). However, these cell types are not present in the donor enteroids. We sought to determine the origin of these cell types and to quantify their importance in TEI development. MATERIALS AND METHODS: Crypts from pan-EGFP or LGR5-EGFP mice were used for enteroid culture and subsequent implantation for the production of TEI. TEI from pan-EGFP enteroids was labeled for smooth muscle alpha actin (SMA) to identify ISEMFs and SMCs and green fluorescent protein (GFP) to identify cells from pan-EGFP enteroids. Fluorescence in situ hybridization (FISH) for the Y chromosome was applied to TEI from male LGR5-EGFP enteroids implanted into female nonobese diabetic/severe combined immunodeficiency mice. To identify chemotactic effects of intestinal epithelium on ISEMFs, a Boyden chamber assay was performed. RESULTS: Immunofluorescence of TEI from pan-EGFP enteroids revealed GFP-positive epithelium with surrounding SMA positivity and no colocalization of the two. FISH of TEI from male LGR5-EGFP enteroids implanted into female nonobese diabetic/severe combined immunodeficiency mice revealed that only the epithelium was Y chromosome positive. Chemotactic assays demonstrated increased ISEMF migration in the presence of enteroids (983 ± 133) compared to that in the presence of either Matrigel alone (357 ± 36) or media alone (339 ± 24; P ≤ 0.05). CONCLUSIONS: Lack of GFP/SMA colocalization suggests that ISEMFs and SMCs are derived from host animals. This was confirmed by FISH which identified only epithelial cells as being male. All other cell types originated from host animals. The mechanism by which these cells are recruited is unknown; however, Boyden chamber assays indicate a direct chemotactic effect of intestinal epithelium on ISEMFs.

Production of tissue-engineered intestine from expanded enteroids.

BACKGROUND: Short bowel syndrome is a life-threatening condition with few solutions. Tissue-engineered intestine (TEI) is a potential treatment, but donor intestine is a limiting factor. Expanded epithelial surrogates termed enteroids may serve as a potential donor source. MATERIALS AND METHODS: To produce TEI from enteroids, crypts were harvested from mice and enteroid cultures established. Enteroids were seeded onto polymer scaffolds using Matrigel or culture medium and implanted in immunosuppressed mice for 4 wk. Histology was analyzed using Periodic acid-Schiff staining and immunofluorescence. Neomucosa was quantified using ImageJ software. To determine whether TEI could be produced from enteroids established from small intestinal biopsies, 2 × 2-mm pieces of jejunum were processed for enteroid culture, enteroids were expanded and seeded onto scaffolds, and scaffolds implanted for 4 wk. RESULTS: Enteroids in Matrigel produced TEI in 15 of 15 scaffolds, whereas enteroids in medium produced TEI in 9 of 15 scaffolds. Use of Matrigel led to more neomucosal surface area compared to media (10,520 ± 2905 µm versus 450 ± 127 µm, P < 0.05). Histologic examination confirmed the presence of crypts and blunted villi, normal intestinal epithelial lineages, intestinal subepithelial myofibroblasts, and smooth muscle cells. Crypts obtained from biopsies produced an average of 192 ± 71 enteroids. A single passage produced 685 ± 58 enteroids, which was adequate for scaffold seeding. TEI was produced in 8 of 9 scaffolds seeded with expanded enteroids. CONCLUSIONS: Enteroids can be obtained from minimal starting material, expanded ex vivo, and implanted to produce TEI. This method shows promise as a solution to the limited donor intestine available for TEI production in patients with short bowel syndrome.

Primary harbour seal (Phoca vitulina) airway epithelial cells show high susceptibility to infection by a seal-derived influenza A virus (H5N8).

Highly pathogenic avian influenza viruses of the H5N8 subtype have been circulating in Europe and Asia since 2016, causing huge economic losses to the poultry industry. A new wave of H5Nx infections has begun in 2020. The viruses mainly infect wild birds and waterfowl; from there they spread to poultry and cause diseases. Previous studies have shown that the H5N8 viruses have seldom spread to mammals; however, reports in early 2021 indicate that humans may be infected, and some incident reports indicate that H5Nx clade 2.3.4.4B virus may be transmitted to wild mammals, such as red foxes and seals. In order to get more information on how the H5N8 virus affects seals and other marine animals, here, we used primary cultures to analyze the cell tropism of the H5N8 virus, which was isolated from an infected grey seal (H5N8/Seal-2016). Primary tracheal epithelial cells were readily infected by H5N8/Seal -2016 virus; in contrast, the commonly used primary seal kidney cells required the presence of exogenous trypsin to initiate virus infection. When applied to an ex vivo precision-cut lung slice model, compared with recombinant human H3N2 virus or H9N2 LPAI virus, the H5N8/Seal-2016 virus replicated to a high titre and caused a strong detrimental effect; with these characteristics, the virus was superior to a human H3N2 virus and to an H9N2 LPAI virus. By using well-differentiated air-liquid interface (ALI) cultures, we have observed that ALI cultures of canines, ferrets, and harbour seals are more sensitive to H5N8/Seal-2016 virus than are human or porcine ALI cultures, which cannot be fully explained by sialic acid distribution. Our results indicate that the airway epithelium of carnivores may be the main target of H5N8 viruses. Consideration should be given to an increased monitoring of the distribution of highly pathogenic avian influenza viruses in wild animals.

Temporal Regulation of Notch Signaling and Its Influence on the Differentiation of Ex Vivo Cultured Limbal Epithelial Cells.

Purpose: Notch signaling plays a vital role in the differentiation and proliferation of corneal epithelial cells from limbal stem cells. The temporal regulation of Notch signaling during this differentiation remains unknown. Hence, we investigated the importance of temporal activation/blockage of Notch signaling during corneal differentiation.Methods: Human limbal epithelial cultures were established with and without Notch activators (rec-Human Jagged1 Fc chimera) and pharmacological blockers (LY-411575). The modulation of Notch signaling was done at different time points during cell differentiation, which were collected on Day 14 for further analysis of differentiation, proliferation, maturation and apoptosis using RT-qPCR and immunofluorescence staining.Results: The activation of Notch signaling at Day 8 resulted in the highest number of mature corneal epithelial cells (p = .008) and pro-apoptosis marker BAX (p = .0001) with no increase in the number of corneal progenitors, and proliferation marker Ki67 compared to untreated controls. Cultures grown in the presence of Notch signaling blockers showed a significantly higher number of corneal progenitors (p = .0001) and proliferation marker Ki67 (p = .02) but lower corneal epithelial marker CK3/CK12 (p = .0007) and no difference in the pro-apoptotic marker BAX compared to untreated controls.Conclusion: During the differentiation of limbal epithelial cells to the corneal epithelial cell type, Day 8 seems to be a crucial window to modulate Notch signaling for a customized outcome.

Preclinical Studies of a Rare CF-Causing Mutation in the Second Nucleotide Binding Domain (c.3700A>G) Show Robust Functional Rescue in Primary Nasal Cultures by Novel CFTR Modulators.

The combination therapies ORKAMBITM and TRIKAFTATM are approved for people who have the F508del mutation on at least one allele. In this study we examine the effects of potentiator and corrector combinations on the rare mutation c.3700A>G. This mutation produces a cryptic splice site that deletes six amino acids in NBD2 (I1234-R1239del). Like F508del it causes protein misprocessing and reduced chloride channel function. We show that a novel cystic fibrosis transmembrane conductance regulator CFTR modulator triple combination (AC1, corrector, AC2-2, co-potentiator and AP2, potentiator), rescued I1234-R1239del-CFTR activity to WT-CFTR level in HEK293 cells. Moreover, we show that although the response to ORKAMBI was modest in nasal epithelial cells from two individuals homozygous for I1234-R1239del-CFTR, a substantial functional rescue was achieved with the novel triple combination. Interestingly, while both the novel CFTR triple combination and TRIKAFTATM treatment showed functional rescue in gene-edited I1234-R1239del-CFTR-expressing HBE cells and in nasal cells from two CF patients heterozygous for I1234-R1239del/W1282X, nasal cells homozygous for I1234-R1239del-CFTR showed no significant response to the TRIKAFTATM combination. These data suggest a potential benefit of CFTR modulators on the functional rescue of I1234-R1239del -CFTR, which arises from the rare CF-causing mutation c.3700A>G, and highlight that patient tissues are crucial to our full understanding of functional rescue in rare CFTR mutations.

Epithelial monolayer culture system for real-time single-cell analyses.

Abstract Many epithelial cells form polarized monolayers under in vivo and in vitro conditions. Typically, epithelial cells are cultured for differentiation on insert systems where cells are plated on a porous filter membrane. Although the cultured monolayers have been a standard system to study epithelial physiology, there are some limits: The epithelial cells growing inside the commercial inserts are not optimal to visualize directly through lenses on inverted microscopes. The cell images are optically distorted and background fluorescence is bright due to the filter membrane positioned between the cells and the lens. In addition, the cells are not easily accessible by electrodes due to the presence of tall side walls. Here, we present the design, fabrication, and practical applications of an improved system for analysis of polarized epithelial monolayers. This new system allows (1) direct imaging of cells without an interfering filter membrane, (2) electrophysiological measurements, and (3) detection of apical secretion with minimal dilution. Therefore, our culture method is optimized to study differentiated epithelial cells at the single-cell and subcellular levels, and can be extended to other cell types with minor modifications.