Epithelial Cell Types Research Articles

Upregulation of FOXA2 in uterine luminal epithelium and vaginal basal epithelium of epiERα-/- (Esr1fl/flWnt7aCre/+) mice†.

Forkhead box protein A2 (FOXA2) is a pioneer transcription factor important for epithelial budding and morphogenesis in different organs. It has been used as a specific marker for uterine glandular epithelial cells (GE). FOXA2 has close interactions with estrogen receptor α (ERα). ERα binding to Foxa2 gene in the uterus indicates its regulation of Foxa2. The intimate interactions between ERα and FOXA2 and their essential roles in early pregnancy led us to investigate the expression of FOXA2 in the female reproductive tract of pre-implantation epiERα-/- (Esr1fl/flWnt7aCre/+) mice, in which ERα is conditionally deleted in the epithelium of reproductive tract. In the oviduct, FOXA2 is detected in the ciliated epithelial cells of ampulla but absent in the isthmus of day 3.5 post-coitum (D3.5) Esr1fl/fl control and epiERα-/- mice. In the uterus, FOXA2 expression in the GE appears to be comparable between Esr1fl/fl and epiERα-/- mice. However, FOXA2 is upregulated in the D0.5 and D3.5 but not PND25-28 epiERα-/- uterine luminal epithelial cells (LE). In the vagina, FOXA2 expression is low in the basal layer and increases toward the superficial layer of the D3.5 Esr1fl/fl vaginal epithelium, but FOXA2 is detected in the basal, intermediate, and superficial layers, with the strongest FOXA2 expression in the intermediate layers of the D3.5 epiERα-/- vaginal epithelium. This study demonstrates that loss of ERα in LE and vaginal basal layer upregulates FOXA2 expression in these epithelial cells during early pregnancy. The mechanisms for epithelial cell-type specific regulation of FOXA2 by ERα remain to be elucidated.

A novel 3D cell culture model to study the human small intestinal immune landscape.

Several subsets of mononuclear phagocytes and DCs (MDC) populate the small intestine (SI), and these cells reportedly exert specialized functions in anti-microbial immunity and tolerance. Given the specialized phenotype of these cells, differing from other MDC family members, including their putative circulating blood precursors, local intestinal factors play key instructive roles in their differentiation. We designed an SI cell culture model composed of three intestinal epithelial cell (IEC) types, including absorptive enterocytes (E cells), antigen delivering microfold (M) cells, and mucus-producing goblet (G) cells plus T lymphocytes and soluble B cell-derived factors. This model was used to study the differentiation fate of CD34+ hematopoietic progenitor cell-derived monocyte/DC precursors. Progeny cells can be analyzed after a 3-week co-culture period, mimicking the physiologic turn-over time of intestinal MDC. A dominant monocyte differentiation pathway was suppressed, in favor of partial differentiation along DC and macrophage pathways, with low percentages of cells acquired DC or macrophage markers. Moreover, E and G cells play opposing roles in CX3CR1+ vs CD103dim cell differentiation, indicating that both together might counter-balance M/DC differentiation. Thus, SI epithelial cells suppress M/DC differentiation, supporting a key role for exogenous factors in M/DC differentiation.

Isolation of Epithelial and Stromal Cells from Colon Tissues in Homeostasis and Under Inflammatory Conditions.

Inflammation of the gastrointestinal tract is a prevalent pathology in diseases such as inflammatory bowel disease (IBD). Currently, there are no therapies to prevent IBD, and available therapies to treat IBD are often sub-optimal. Thus, an unmet need exists to better understand the molecular mechanisms underlying intestinal tissue responses to damage and regeneration. The recent development of single-cell RNA (sc-RNA) sequencing-based techniques offers a unique opportunity to shed light on novel signaling pathways and cellular states that govern tissue adaptation or maladaptation across a broad spectrum of diseases. These approaches require the isolation of high-quality cells from tissues for downstream transcriptomic analyses. In the context of intestinal biology, there is a lack of protocols that ensure the isolation of epithelial and non-epithelial compartments simultaneously with high-quality yield. Here, we report two protocols for the isolation of epithelial and stromal cells from mouse and human colon tissues under inflammatory conditions. Specifically, we tested the feasibility of the protocols in a mouse model of dextran sodium sulfate (DSS)-induced colitis and in human biopsies from Crohn's patients. We performed sc-RNA sequencing analysis and demonstrated that the protocol preserves most of the epithelial and stromal cell types found in the colon. Moreover, the protocol is suitable for immunofluorescence staining of surface markers for epithelial, stromal, and immune cell lineages for flow cytometry analyses. This optimized protocol will provide a new resource for scientists to study complex tissues such as the colon in the context of tissue damage and regeneration. Key features • This protocol allows the isolation of epithelial and stromal cells from colon tissues. • The protocol has been optimized for tissues under inflammatory conditions with compromised cell viability. • This protocol is suitable for experimental mouse models of colon inflammation and human biopsies.

Abstract C044: A case and control genetic profile of tissue and serum in African American men

Abstract Prostate cancer is the most common non-cutaneous cancer among men. A man with 1,2 or 3 first degree relatives with prostate cancer, has a 2,5, and 11-fold increased risk of developing prostate cancer. The heritability rate of prostate cancer is 58%. African American men have the highest incidence and mortality rate of prostate cancer in American. Men of African descent, globally, are more likely to die from prostate cancer than any other ancestral groups. We performed Immunohistochemistry(IHC) and RNA seq analysis on African American tissue and serum samples, with a focus on HMGA2, which has been shown promote EMT, invasion, and metastasis in cancer. Acknowledgements: These studies were supported by NIH/NIMHD U54MD007590 and U54MD013376. Our sample set included 3 benign and 2 prostate cancer tumors from African Americans. IHC markers showed HMGA2 staining within epithelial cells of the prostate cancer tissue. This result validates studies that show that distinct subtypes of prostate cancer may arise from luminal and basal epithelial cell types. RNA seq results showed HMGA to be non-significantly down-regulated in tumor samples. The most differentially expressed (DE) gene, however, was SNORD116-18. Studies have shown SNORD116-18 expression to be associated with chronic lymphocytic leukemia in distinguishing prognostic groups. This finding may be a pathway of interest for further studies. RNA seq of serum gave homogeneous results, however, gene set enrichment analysis of the most differentially expressed genes within the serum showed overlap with inflammation pathways gene for upregulated DE genes and Methylation Pathway for downregulated DE genes. Further analysis on more samples from African Americans tissue and serum will be used to validate findings. Citation Format: Maxine S. Harlemon, Roni Bollag, Martha Terris, Ana Cecilia Millena, Nathan Bowen, Valerie Odero-Marah. A case and control genetic profile of tissue and serum in African American men [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C044.

Abstract PR004: The role of NOTCH1 in bronchial pre-malignant lesions

Abstract Continuous carcinogen exposure through smoking creates a field of injury along the airway and resulting in mutations which promote remodeling of the bronchial epithelium. Over time lesions can form and exhibit increasing abnormalities progressing from goblet cell hyperplasia (GCH) which is the earliest detectable premalignant state through hyperplasia and squamous metaplasia to incremental stages of dysplasia, carcinoma in situ (CIS) and eventually to frank carcinoma. Unfortunately, more than half of all lung cancer patients are diagnosed with metastatic disease with a dismal 5-year survival rate of only 5.2%. However, when diagnosed at an early stage, the 5-year survival rate is more than 10 times as high. Our group has identified NOTCH1 as the most frequently mutated gene in progressive lung premalignant lesions (PMLs). However, these mutations are significantly less common in advanced lung squamous cell carcinoma (LUSC), indicating a role for NOTCH1 in the pre-tumorigenic environment rather than contributing to the actual tumor mass. Transcriptomic profiling of NOTCH1 mutated lesions identified up regulation of peri-goblet cells (PGC), a newly identified bronchial epithelial cell type that is associated with smoking, and a down regulation of immune components. Using highly multiplex Imaging Mass Cytometry (IMC) to analyze the epithelium of NOTCH1 mutated PMLs and its interplay with the immune system in situ. We identified a shift in epithelial composition with an increase in un- and de-differentiated cells. Interestingly, PGC appear to be an intermediate cell type on a previously undescribed path to goblet cell differentiation. In a parallel approach we are modeling NOTCH1 driven dysplasia in vitro to gain insight into the molecular mechanisms of lesion progression. Primary human bronchial epithelial cells (hBECs) grown at the air-liquid interface (ALI) into mature epithelia are exposed to cigarette smoke condensate (CSC) or IL-13 to model the early stages of bronchial airway dysplasia. Continuous exposure of these differentiating cultures to either stimulus increases the abundance of goblet cells and PGCs in a dose dependent manner with IL-13 additionally leading to the loss of baso-apical organization of the epithelium. After successfully modeling the first stage of bronchial epithelial dysplasia we are now advancing our model by co-culturing primary hBECs with NOTCH1 mutated cells and to disentangle the pathway of basal-PGC-goblet cell differentiation. Understanding driving forces in early lesion progression will further our ability to discern progressive lesions from those that regress naturally and facilitate interventions that force lesions into regression. Citation Format: Roxana Michaela Pfefferkorn, Darren Jianjhih Chiu, Divya Lakshmi Venkatraman, Jennifer Ellen Beane, Sarah Anne Mazzilli, Joshua David Campbell. The role of NOTCH1 in bronchial pre-malignant lesions. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr PR004.

RASSF1A promotes radiosensitivity in nasopharyngeal carcinoma by promoting FoxO3a and inhibiting the Nrf2/TXNRD1 signaling pathway.

Radiotherapy is widely used as the first-line treatment for nasopharyngeal carcinoma (NPC). However, the resistance of some patients to treatment lowers its clinical effectiveness. Compared to typical epithelial cells, NPC markedly lowers the Ras-association domain family 1A (RASSF1A) protein expression. RASSF1A overexpression sensitizes NPC cells to radiotherapy. Mechanistically, RASSF1A promotes the expression of Forkhead box O3a (FoxO3a) in the nucleus and inhibits the Nuclear factor E2-related factor 2 (Nrf2) signaling pathway via binding to the Kelch-like ECH-associated protein 1 (Keap1) promoter. Through elevating intracellular ROS levels, RASSF1A overexpression inhibits the expression of thioredoxin reductase 1 (TXNRD1), a crucial Nrf2 target gene, and increases NPC sensitivity to radiation. Immunohistochemical staining of NPC tissue sections revealed that the expression of RASSF1A is negatively correlated with that of TXNRD1. The traditional Chinese medicine component andrographolide (AGP), which induces RASSF1A expression, increased the sensitivity of NPC cells to radiotherapy in vitro and in vivo. Our findings implied that RASSF1A increases the sensitivity of NPC to radiation by increasing FoxO3a expression in the nucleus, inhibiting the Nrf2/TXNRD1 signaling pathway, and elevating intracellular ROS levels. AGP targets RASSF1A and may be a promising adjuvant sensitizer for enhancing radiosensitivity in NPC.

Expansion of human amniotic epithelial cells using condition cell reprogramming technology

Human amniotic epithelial cells (hAECs) are non-immunogenic epithelial cells that can develop into cells of all three germline lineages. However, a refined clinically reliable method is required to optimize the preparation and banking procedures of hAECs for their successful translation into clinical studies. With the goal of establishing standardized clinically applicable hAECs cultured cells, we described the use of a powerful epithelial cell culture technique, termed Conditionally Reprogrammed Cells (CRC) for ex vivo expansion of hAECs. The well-established CRC culture method uses a Rho kinase inhibitor (Y-27632) and J2 mouse fibroblast feeder cells to drive the indefinite proliferation of all known epithelial cell types. In this study, we used an optimized CRC protocol to successfully culture hAECs in a CRC medium supplemented with xenogen-free human serum. We established that hAECs thrive under the CRC conditions for over 5 passages while still expressing pluripotent stem markers (OCT-4, SOX-2 and NANOG) and non-immunogenic markers (CD80, CD86 and HLA-G) suggesting that even late-passage hAECs retain their privileged phenotype. The hAECs-CRC cells were infected with a puromycin-selectable lentivirus expressing luciferase and GFP (green fluorescent protein) and stably selected with puromycin. The hAECs expressing GFP were injected subcutaneously into the flanks of Athymic and C57BL6 mice to check the tolerability and stability of cells against the immune system. Chemiluminescence imaging confirmed the presence and viability of cells at days 2, 5, and 42 without acute inflammation or any tumor formation. Collectively, these data indicate that the CRC approach offers a novel solution to expanding hAECs in humanized conditions for future clinical uses, while retaining their primary phenotype.Graphical abstract

Human Airway Epithelium Responses to Invasive Fungal Infections: A Critical Partner in Innate Immunity.

The lung epithelial lining serves as the primary barrier to inhaled environmental toxins, allergens, and invading pathogens. Pulmonary fungal infections are devastating and carry high mortality rates, particularly in those with compromised immune systems. While opportunistic fungi infect primarily immunocompromised individuals, endemic fungi cause disease in immune competent and compromised individuals. Unfortunately, in the case of inhaled fungal pathogens, the airway epithelial host response is vastly understudied. Furthering our lack of understanding, very few studies utilize primary human models displaying pseudostratified layers of various epithelial cell types at air-liquid interface. In this review, we focus on the diversity of the human airway epithelium and discuss the advantages and disadvantages of oncological cell lines, immortalized epithelial cells, and primary epithelial cell models. Additionally, the responses by human respiratory epithelial cells to invading fungal pathogens will be explored. Future investigations leveraging current human in vitro model systems will enable identification of the critical pathways that will inform the development of novel vaccines and therapeutics for pulmonary fungal infections.

Single-Cell RNA-Sequencing Reveals Epithelial Cell Signature of Multiple Subtypes in Chemically Induced Acute Lung Injury.

Alveolar epithelial cells (AECs) play a role in chemically induced acute lung injury (CALI). However, the mechanisms that induce alveolar epithelial type 2 cells (AEC2s) to proliferate, exit the cell cycle, and transdifferentiate into alveolar epithelial type 1 cells (AEC1s) are unclear. Here, we investigated the epithelial cell types and states in a phosgene-induced CALI rat model. Single-cell RNA-sequencing of bronchoalveolar lavage fluid (BALF) samples from phosgene-induced CALI rat models (Gas) and normal controls (NC) was performed. From the NC and Gas BALF samples, 37,245 and 29,853 high-quality cells were extracted, respectively. All cell types and states were identified and divided into 23 clusters; three cell types were identified: macrophages, epithelial cells, and macrophage proliferating cells. From NC and Gas samples, 1315 and 1756 epithelial cells were extracted, respectively, and divided into 11 clusters. The number of AEC1s decreased considerably following phosgene inhalation. A unique SOX9-positive AEC2 cell type that expanded considerably in the CALI state was identified. This progenitor cell type may develop into alveolar cells, indicating its stem cell differentiation potential. We present a single-cell genome-scale transcription map that can help uncover disease-associated cytologic signatures for understanding biological changes and regeneration of lung tissues during CALI.

ΔNp63 maintains the fidelity of the myoepithelial cell lineage and directs cell differentiation programs in the murine salivary gland

Salivary glands consist of several epithelial cell types of distinct lineages and functional characteristics that are established by directed differentiation programs of resident stem and progenitor cells. We have shown that ΔNp63, a crucial transcriptional regulator of stem/progenitor cells, is enriched in both the basal and myoepithelial cell (MEC) populations and that ΔNp63 positive cells maintain all the descendent epithelial cell lineages of the adult mouse salivary glands (mSGs). Although this pivotal role of ΔNp63 in driving the broader epithelial cell fate and identity in the mSG has been demonstrated, how ΔNp63 functions specifically in the commitment and differentiation of the MEC population is less understood. Using multiple genetic mouse models that allow for cell tracing, we show that ΔNp63 is critical in maintaining and renewing MECs, in part through the transcriptional regulation of Acta2 gene expression, a defining marker of this cell population. We demonstrate that during adult mSG homeostasis, ΔNp63 enriched MECs function as bipotent progenitor cells that maintain not only the MEC population, but also the distinctly different ductal cell lineages. The fidelity of this process is dependent on ΔNp63 expression, since MEC-specific ablation of ΔNp63 results in altered MEC differentiation and affects cellular plasticity resulting in aberrant differentiation of the intercalated ducts and acinar cells. In contrast, we find that the contribution of MECs to ductal and acinar cell regeneration following severe injury is independent of ΔNp63. Our observations offer new insights into cellular mechanisms driving MEC fate choices and differentiation programs in the context of salivary gland homeostasis and in response to injury and regeneration. Long term, these findings have implications for better treatment of salivary gland dysfunction through stem cell-based approaches.

STAGETOOL, a Novel Automated Approach for Mouse Testis Histological Analysis.

Spermatogenesis is a complex differentiation process that takes place in the seminiferous tubules. A specific organization of spermatogenic cells within the seminiferous epithelium enables a synchronous progress of germ cells at certain steps of differentiation on the spermatogenic pathway. This can be observed in testis cross-sections where seminiferous tubules can be classified into distinct stages of constant cellular composition (12 stages in the mouse). For a detailed analysis of spermatogenesis, these stages have to be individually observed from testis cross-sections. However, the recognition of stages requires special training and expertise. Furthermore, the manual scoring is laborious considering the high number of tubule cross-sections that have to be analyzed. To facilitate the analysis of spermatogenesis, we have developed a convolutional deep neural network-based approach named "STAGETOOL." STAGETOOL analyses histological images of 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI)-stained mouse testis cross-sections at ×400 magnification, and very accurately classifies tubule cross-sections into 5 stage classes and cells into 9 categories. STAGETOOL classification accuracy for stage classes of seminiferous tubules of a whole-testis cross-section is 99.1%. For cellular level analysis the F1 score for 9 seminiferous epithelial cell types ranges from 0.80 to 0.98. Furthermore, we show that STAGETOOL can be applied for the analysis of knockout mouse models with spermatogenic defects, as well as for automated profiling of protein expression patterns. STAGETOOL is the first fluorescent labeling-based automated method for mouse testis histological analysis that enables both stage and cell-type recognition. While STAGETOOL qualitatively parallels an experienced human histologist, it outperforms humans time-wise, therefore representing a major advancement in male reproductive biology research.

Do silver/hydroxyapatite and zinc oxide nano-coatings improve inflammation around titanium orthodontic mini-screws? In vitro study

Overcoming the failure percentage of orthodontic mini-screws (OMSs), which is about 30% of overall orthodontic cases, especially in malocclusion treatment that requires orthopaedic heavy forces, is a great challenge. Bacterial infections, soft tissue and bone inflammation, and weak connections between bones and the OMS surface are among the main causalities of this failure. The aim of the study is to evaluate in vitro the microbiological activities of the deposited nanomaterials (Silver/hydroxyapatite nanoparticles (Ag/HA NPs) and zinc oxide nanoparticles (ZnO NPs)) in terms of microbial inhibition. In addition, the in-vitro cytotoxicity and cytocompatibility of the synthesized nano-coatings prior to their in-vivo application in animal models were tested on four types of cells, namely, fibroblasts, osteocytes, osteoblasts, and oral epithelial cells. Ag/HA NPs and ZnO NPs were built up onto the surface of titanium OMSs by electrochemical deposition. This electrochemical deposition was performed on 50 orthodontic mini screws and the deposited materials were characterized with the aid of scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM/EDX) analysis, X-ray Diffraction (XRD) and nano-scratch test. In addition, the microbiological activities of the deposited nanomaterials were explored in vitro in terms of microbial inhibition. Furthermore, the cytotoxicity and cytocompatibility were tested on four types of cells, namely, fibroblasts, osteocytes, osteoblasts and oral epithelial cells. SEM images revealed spherical Ag NPs in the range of 40-70nm in diameter, rod-shaped HA NPs and porous scaly ZnO NPs on the surface of the OMSs. XRD analysis confirmed the crystal structures of AgNPs, HA NPs, and ZnO NPs. ZnO NPs coated OMS had the highest antimicrobial activity than Ag/HA coated OMS against Gram-positive, Gram-negative and fungal strains. Moreover, after incubation, the decrease in the number of bacterial colonies was significant with ZnO and Ag/HA nanoparticles (with the greatest decrease for the former), due to the potent antibacterial effect of nanoparticles against Escherichia coli and Enterococcus faecalis. Moreover, ZnO NPs-coated OMSs showed a better cytocompatibility with oral epithelium, bone cells, and fibroblasts compared to Ag/HA NPs. The suggested nanocoating is a promising strategy to overcome the development of an inflammatory zone around the fixed OMSs.

Abstract IA008: Mouse-INtraDuctal (MIND): An in vivo model for the discovery of epithelial/stromal cross talks that drive DCIS invasive and metastatic progression

Abstract A large fraction of human DCIS (>50%) may not need the multimodality treatment options currently offered to all patients. More importantly, while we may be overtreating many, we cannot identify those most at risk for invasion/metastasis. Revealing the cellular and molecular mechanisms by which some DCIS remain indolent while others advance to invasive and metastatic breast cancers is currently a clinical unmet need. To address this gap, we developed the Mouse-INtraDuctal (MIND) model, by which patient-derived (PDX) DCIS epithelial cells are injected intraductally and allowed to progress naturally in mice. Single cell RNA-sequencing (scRNA-seq) was utilized to profile the DCIS epithelial and stroma cells in progressors vs. non-progressors. To distinguish between stromal (diploid) cells and tumor (aneuploid) cells, we calculated CNA profiles from RNA using CopyKAT. Cell-type specific differential gene expression analysis of DCIS epithelial cells and microenvironment cell types in progressors and non-progressors was performed. We also predicted putative ligand/receptor interactions between the tumor cells and cell types in the microenvironment by CellPhoneDB. Among 37 PDX DCIS MIND models followed for a median of 9 months, 20 (54%) grafted into 95 glands, showed in vivo invasive progression (progressed) while 17 (46%), injected into 107 glands, remained non-invasive (non-progressed). ScRNA-seq was performed on 13 DCIS samples including 10 progressors and 3 non-progressors. The resulting data identified genes significantly upregulated (>log2 fold=1, FDR p<0.05) in the progressors. Notable genes included NEAT1, EIF4EBP1, SCGB2A2, TFF1 and TFF3 that were upregulated in the progressors. NEAT1, the core structural component of the paraspeckles, is frequently overexpressed in human cancers and its expression is correlated with worse survival in cancer patients. NEAT1 drives tumor progression by regulating genes involved in cellular growth, migration, invasion, metastasis, EMT, stemness, radio- and chemoresistance, supporting its role as a potential biomarker and therapeutic target. TFF1/TFF3 mRNAs show increased expression in metastatic breast cancers. EIF4EBP1 is located on chrom 8p11-p12 which is frequently amplified in breast cancer and is associated with poor clinical prognosis. CellPhoneDB identified expression of several receptor/ligand interactions including CD74/MIF involved in epithelial/stromal and stromal/stromal cross talks that may play a role in DCIS invasive progression. Conclusions: Future studies will validate our findings using patient DCIS samples with known long-term outcome and in vivo MIND models to further refine risk associated biomarkers for invasion/metastasis and to identify more effective preventive therapies. Citation Format: Fariba Behbod. Mouse-INtraDuctal (MIND): An in vivo model for the discovery of epithelial/stromal cross talks that drive DCIS invasive and metastatic progression [abstract]. In: Proceedings of the AACR Special Conference on Rethinking DCIS: An Opportunity for Prevention?; 2022 Sep 8-11; Philadelphia, PA. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_1): Abstract nr IA008.

Dental niche cells directly contribute to tooth reconstitution and morphogenesis.

Mammalian teeth develop from the inductive epithelial-mesenchymal interaction, an important mechanism shared by many organs. The cellular basis for such interaction remains elusive. Here, we generate a dual-fluorescence model to track and analyze dental cells from embryonic to postnatal stages, in which Pitx2+ epithelium and Msx1+ mesenchyme are sufficient for tooth reconstitution. Single-cell RNA sequencing and spatial mapping further revealed critical cellular dynamics during molar development, where tooth germs are organized by Msx1+Sdc1+ dental papilla and surrounding dental niche. Surprisingly, niche cells are more efficient in tooth reconstitution and can directly regenerate papilla cells through interaction with dental epithelium. Finally, from the dental niche, we identify a group of previously unappreciated migratory Msx1+ Sox9+ cells as the potential cell origin for dental papilla. Our results indicate that the dental niche cells directly contribute to tooth organogenesis and provide critical insights into the essential cell composition for tooth engineering.

Assessment of SARS-CoV-2 entry in gingival epithelial cells expressing CD147.

SARS-CoV-2, the causative agent of the debilitating COVID-19, is mainly transmitted by first infecting nose and lung epithelial cells. The mouth is also believed to be a viral portal site since certain types of oral epithelial cells were shown to express ACE2 receptor. However, it is unclear whether oral epithelial cells are directly infected by SARS-CoV-2. In this study, we addressed whether epithelial cells of the oral gingiva were susceptible to infection. Interestingly, we found that KRT5+ and KRT18+ gingival epithelial cells do not express ACE2 but highly express TMPRSS2 and Furin as well as CD147, which was proposed to be an alternative receptor for SARS-CoV-2. However, using SARS-CoV-2 pseudoviruses containing the spike protein, we observed that gingival epithelial cells were not susceptible to infection due to the lack of ACE2 expression and the inability of CD147 to mediate viral entry. These results strongly suggest that epithelial cells from the gingiva are not susceptible to SARS-CoV-2 and CD147 is not a receptor for the SARS-CoV-2 virus. The susceptibility of oral cells from other oral structures under healthy and pathological conditions still needs to be confirmed to better understand the role of the oral cavity in COVID-19 infection and transmission.

Human Cytomegalovirus Infection of Epithelial Cells Increases SARS-CoV-2 Superinfection by Upregulating the ACE2 Receptor.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), has caused widespread morbidity and mortality since its onset in late 2019. Here, we demonstrate that prior infection with human cytomegalovirus (HCMV) substantially increases infection with SARS-CoV-2 in vitro. HCMV is a common herpesvirus carried by 40%-100% of the population, which can reactivate in the lung under inflammatory conditions, such as those resulting from SARS-CoV-2 infection. We show in both endothelial and epithelial cell types that HCMV infection upregulates ACE2, the SARS-CoV-2 cell entry receptor. These observations suggest that HCMV reactivation events in the lung of healthy HCMV carriers could exacerbate SARS-CoV-2 infection and subsequent COVID-19 symptoms. This effect could contribute to the disparity of disease severity seen in ethnic minorities and those with lower socioeconomic status, due to their higher CMV seroprevalence. Our results warrant further clinical investigation as to whether HCMV infection influences the pathogenesis of SARS-CoV-2.

Full Remission of CAR-Deficient Tumors by DOTAP-Folate Liposome Encapsulation of Adenovirus.

Adenovirus (Ad)-based vectors have shown considerable promise for gene therapy. However, Ad requires the coxsackievirus and adenovirus receptor (CAR) to enter cells efficiently and low CAR expression is found in many human cancers, which hinder adenoviral gene therapies. Here, cationic 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-folate liposomes (Df) encapsulating replication-deficient Ad were synthesized, which showed improved transfection efficiency in various CAR-deficient cell lines, including epithelial and hematopoietic cell types. When encapsulating replication-competent oncolytic Ad (TAV255) in DOTAP-folate liposome (TAV255-Df), the adenoviral structural protein, hexon, was readily produced in CAR-deficient cells, and the tumor cell killing ability was 5× higher than that of the non-encapsulated Ad. In CAR-deficient CT26 colon carcinoma murine models, replication-competent TAV255-Df treatment of subcutaneous tumors by intratumoral injection resulted in 67% full tumor remission, prolonged survival, and anti-cancer immunity when mice were rechallenged with cancer cells with no further treatment. The preclinical data shows that DOTAP-folate liposomes could significantly enhance the transfection efficiency of Ad in CAR-deficient cells and, therefore, could be a feasible strategy for applications in cancer treatment.

Xenobiotic-sensing nuclear receptors as targets for phthalates-induced lung injury and antagonism of lycopene

Phthalates are extensively used in the production of plastics products and have been verified to induce lung injury. Lycopene (LYC) has proved an effective preventive and can be utilized to prevent phthalates-induced toxicity. However, the role of phthalate in pathogenesis of lung injury remain poorly researched, and little work has been devoted whether LYC could alleviate phthalate-induced lung toxicity via modulating nuclear xenobiotic receptors (NXRs) response. Here, di (2-ethylhexyl) phthalate (DEHP) is used as the representative of phthalates for further studies on toxicity of phthalates and the antagonistic role of LYC in phthalates-induced lung injury. We found that DEHP exposure caused alveoli destruction and alveolar epithelial cells type II damage. Mechanistically, DEHP exposure increased nuclear accumulation of aryl hydrocarbon receptor (AHR) and its downstream genes level, including cytochrome P450-dependent monooxygenase (CYP) 1A1 and CYP1B1. Constitutive androstane receptor (CAR) and their downstream gene level, including CYP2E1 are also increased after phthalates exposure. Significantly, LYC supplementation relieves lung injury from DEHP exposure by inhibiting the activation of NXRs. We confirm that NXRs plays a key role in phthalates-induced lung injury. Our study showed that LYC may have a positive role in alleviating the toxicity effects of phthalates, which provides an effective strategy for revising phthalates-induced injury.

Specific decellularized extracellular matrix promotes the plasticity of human ocular surface epithelial cells.

The ocular surface is composed of two phenotypically and functionally different epithelial cell types: corneal and the conjunctival epithelium. Upon injury or disease, ocular surface homeostasis is impaired resulting in migration of conjunctival epithelium on to the corneal surface. This can lead to incomplete transdifferentiation toward corneal epithelial-like cells in response to corneal basement membrane cues. We show that corneal extracellular matrix (ECM) proteins induce conjunctival epithelial cells to express corneal associated markers losing their conjunctival associated phenotype at both, mRNA and protein level. Corneal epithelial cells behave the same in the presence of conjunctival ECM proteins, expressing markers associated with conjunctival epithelium. This process of differentiation is accompanied by an intermediate step of cell de-differentiation as an up-regulation in the expression of epithelial stem cell markers is observed. In addition, analysis of ECM proteins by laminin screening assays showed that epithelial cell response is laminin-type dependent, and cells cultured on laminin-511 showed lower levels of lineage commitment. The phosphorylation and proteolysis levels of proteins mainly involved in cell growth and differentiation showed lower modifications in cells with lower lineage commitment. These observations showed that the ECM proteins may serve as tools to induce cell differentiation, which may have potential applications for the treatment of ocular surface injuries.

Characterization of alternative mRNA splicing in cultured cell populations representing progressive stages of human fetal kidney development

Nephrons are the functional units of the kidney. During kidney development, cells from the cap mesenchyme—a transient kidney-specific progenitor state—undergo a mesenchymal to epithelial transition (MET) and subsequently differentiate into the various epithelial cell types that create the tubular structures of the nephron. Faults in this transition can lead to a pediatric malignancy of the kidney called Wilms’ tumor that mimics normal kidney development. While human kidney development has been characterized at the gene expression level, a comprehensive characterization of alternative splicing is lacking. Therefore, in this study, we performed RNA sequencing on cell populations representing early, intermediate, and late developmental stages of the human fetal kidney, as well as three blastemal-predominant Wilms’ tumor patient-derived xenografts. Using this newly generated RNAseq data, we identified a set of transcripts that are alternatively spliced between the different developmental stages. Moreover, we found that cells from the earliest developmental stage have a mesenchymal splice-isoform profile that is similar to that of blastemal-predominant Wilms’ tumor xenografts. RNA binding motif enrichment analysis suggests that the mRNA binding proteins ESRP1, ESRP2, RBFOX2, and QKI regulate alternative mRNA splicing during human kidney development. These findings illuminate new molecular mechanisms involved in human kidney development and pediatric kidney cancer.