Epithelial Cell Differentiation Research Articles

Activation of the mTOR pathway enhances PPARγ/SREBP-mediated lipid synthesis in human meibomian gland epithelial cells.

The involvement of the mechanistic targets of rapamycin (mTOR) pathway in lipid metabolism has been recently elucidated. However, its specific role in the Meibomian gland, where lipid metabolism is significant, remains not fully understood. We investigated the role of mTOR signaling system in the lipogenesis and differentiation of human meibomian gland epithelial cells (HMGECs). Treatment of HMGECs with rapamycin resulted in a reduction in lipid synthesis and the expression of PPARγ and SREBP-1, the major regulators of lipid synthesis. The phosphorylation of p70S6kinase and AKT, which are downstream signals of mTOR complexes 1 and 2, respectively, decreased following rapamycin treatment. In addition, when both mTOR complex 1 and 2 were suppressed using siRNA, there was a significant reduction in the expression of PPARγ and SREBP-1, along with a decrease in lipid synthesis in HMGECs. Our findings suggest that inhibiting the mTOR pathway diminishes the differentiation and adipogenesis of meibomian gland epithelial cells, and both mTOR complexes 1 and 2 appear to play a role in this activity.

Identification of feature genes in intestinal epithelial cell types.

The intestine, is responsible for food digestion, nutrient absorption, endocrine secretion, food residue excretion, and immune defense. These function performances are based on the intricate composition of intestinal epithelial cells, encompassing differentiated mature cells, rapidly proliferative cells, and intestinal stem cells. Although the characteristics of these cell types are well-documented, in-depth exploration of their representative markers and transcription factors is critical for comprehensive cell fate trajectory analysis. Here, we unveiled the feature genes in different cell types of the human and mouse gut through single-cell RNA sequencing analysis. Further, the locations of some specific transcription factors and membrane proteins were determined by immunofluorescence staining, and their role in regulating the proliferation and differentiation of intestinal epithelial cells were explored by CRISPR/Cas9 knockout. Therefore, this study not only reports new markers for various intestinal epithelial cell types but also elucidates the involvement of relevant genes in the determination of epithelial cell fate and maintenance of stem cell homeostasis, which facilitates the tracing and functional elucidation of intestinal epithelial cells.

Exploring the spatial distribution of interstitial cells in kidney tissue.

Interstitial cells are crucial to the development of kidney structure and function, although the mechanism underlying their role in it remains unclear to date. Our previous study identified cell clusters in human fetal kidney tissue, and we further analyzed the interstitial cell cluster within this context. We extracted the barcoded cDNA from tissue samples and prepared spatial transcriptome libraries. Sequencing data was quality-checked, normalized, and clusters were identified using Seurat. Single-cell and spatial data were integrated using MIA and cell types were deconvoluted. DEGs in interstitial cells were identified and functionally annotated using DAVID. CellPhoneDB was used to predict ligand-receptor interactions between cell types. The results of the present study revealed that this cluster of interstitial cells appeared to be scattered in the junction between the cortical and medullary regions. The subsequent KEGG pathway analysis revealed that the differentially expressed genes (DEGs) in this cluster of interstitial cells were involved in the WNT signaling pathway. The Gene Ontology (GO) analysis revealed that these DEGs were involved in multiple pathways associated with kidney development, with six of the genes (NKD2, TCF21, WNT5A, WNT4, MDK, and SFRP1) associated with kidney development exhibiting significant upregulation. Accordingly, it was inferred that these interstitial cells might be involved in regulating epithelial cell differentiation, ureteral bud development, and morphogenesis. The subsequent cell-cell communication analysis revealed that the cellular crosstalk was primarily regulated mainly by ligand-receptor pairs. Additionally, 17 genes reported to be associated with kidney disease were focused on, and these genes were found to be predominantly expressed in a single cell type. In summary, the present study revealed the characteristics of a previously identified cluster of interstitial cells in the kidney tissue, thereby providing fresh insights into the process of kidney development.

Molecular analysis of inherited disorders of cornification in polish patients show novel variants and functional data and provokes questions on the significance of secondary findings.

The Mendelian Disorders of Cornification (MeDOC) comprise a large number of disorders that present with either localised (palmoplantar keratoderma, PPK) or generalised (ichthyoses) signs. The MeDOC are highly heterogenic in terms of genetics and phenotype. Consequently, diagnostic process is challenging and before implementation of the next generation sequencing, was mostly symptomatic, not causal, which limited research on those diseases. The aim of the study was to genetically characterise a cohort of 265 Polish patients with MeDOC and to get insight into the skin lesions using transcriptome and lipid profile analyses. We detected causal variants in 85% (226/265) patients. In addition to the primary gene defect, a pathogenic variant in another gene involved in MeDOC pathology was identified in 23 cases. We found 150 distinct variants in 33 genes, including 32 novel and 16 recurrent (present in > 5 alleles). In 43 alleles large rearrangements were detected, including deletions in the STS, SPINK5, CERS3 and recurrent duplication of exons 10-14 in TGM1. The RNA analysis using samples collected from 18 MeDOC patients and 22 controls identified 1377 differentially expressed genes - DEG. The gene ontology analysis revealed that 114 biological processes were upregulated in the MeDOC group, including i.e. epithelial cell differentiation, lipid metabolic process; homeostasis; regulation of water loss via skin; peptide cross-linking. The DEG between TGM1 and ALOX12B patients, showed that RNA profile is highly similar, though fatty acid profile in epidermal scrapings of those patients showed differences e.g. for the very long chain fatty acids (VLCFAs; FAs ≥ C20), the very long-chain monounsaturated fatty acids (VLC-MUFAs, FAs ≥ C20:1) and the n6 polyunsaturated fatty acids (n6 PUFAs). Our results show that NGS-based analysis is an effective MeDOC diagnostic tool. The Polish MeDOC patients are heterogenic, however recurrent variants are present. The novel variants and high number of TGM1 and SPINK5 copy number variations give further insight into molecular pathology of MeDOC. We show that secondary variants in MeDOC-related genes are present in a significant group of patients, which should be further investigated in the context of phenotype modifiers. Finally, we provide novel RNA and lipid data that characterise molecularly MeDOC epidermis.

Targeting Gut microbiota as a therapeutic target in T2DM: a review of Multi-target interactions of probiotics, prebiotics, postbiotics, and synbiotics with the Intestinal Barrier

The global epidemic of type 2 diabetes mellitus (T2DM) imposes a substantial burden on public health and healthcare expenditures, thereby driving the pursuit of cost-effective preventive and therapeutic strategies. Emerging evidence suggests a potential association between dysbiosis of gut microbiota and its metabolites with T2DM, indicating that targeted interventions aimed at modulating gut microbiota may represent a promising therapeutic approach for the management of T2DM. In this review, we concentrated on the multifaceted interactions between the gut microbiota and the intestinal barrier in the context of T2DM. We systematically summarized that the imbalance of beneficial gut microbiota and its metabolites may constitute a viable therapeutic approach for the management of T2DM. Meanwhile, the mechanisms by which gut microbiota interventions, such as probiotics, prebiotics, postbiotics, and synbiotics, synergistically improve insulin resistance in T2DM are summarized. These mechanisms include the restoration of gut microbiota structure, upregulation of intestinal epithelial cell proliferation and differentiation, enhancement of tight junction protein expression, promotion of mucin secretion by goblet cells, and the immunosuppressive functions of regulatory T cells (Treg) and M2 macrophages. Collectively, these actions contribute to the amelioration of the body's metabolic inflammatory status. Our objective is to furnish evidence that supports the clinical application of probiotics, prebiotics, and postbiotics in the management of T2DM.

Ex Vivo Intestinal Organoid Models: Current State-of-the-Art and Challenges in Disease Modelling and Therapeutic Testing for Colorectal Cancer.

Despite improvements in participation in population-based screening programme, colorectal cancer remains a major cause of cancer-related mortality worldwide. Targeted interventions are desirable to reduce the health and economic burden of this disease. Two-dimensional monolayers of colorectal cancer cell lines represent the traditional in vitro models for disease and are often used for diverse purposes, including the delineation of molecular pathways associated with disease aetiology or the gauging of drug efficacy. The lack of complexity in such models, chiefly the limited epithelial cell diversity and differentiation, attenuated mucus production, lack of microbial interactions and mechanical stresses, has driven interest in the development of more holistic and physiologically relevant in vitro model systems. In particular, established ex vivo patient-derived explant and patient-derived tumour xenograft models have been supplemented by progress in organoid and microfluidic organ-on-a-chip cultures. Here, we discuss the applicability of advanced culturing technologies, such as organoid systems, as models for colorectal cancer and for testing chemotherapeutic drug sensitivity and efficacy. We highlight current challenges associated with organoid technologies and discuss their future for more accurate disease modelling and personalized medicine.

Candida tropicalis-derived vitamin B3 exerts protective effects against intestinal inflammation by promoting IL-17A/IL-22-dependent epithelial barrier function

ABSTRACT Candida tropicalis-a prevalent gut commensal fungus in healthy individuals – contributes to intestinal health and disease. However, how commensal C. tropicalis influences intestinal homeostasis and barrier function is poorly understood. Here, we demonstrated that the reference strain of C. tropicalis (MYA-3404) ameliorated intestinal inflammation in murine models of chemically induced colitis and bacterial infection. Intestinal colonization of C. tropicalis robustly upregulated the expression of IL-17A and IL-22 to increase barrier function and promote proliferation of intestinal epithelial cells in the mouse colon. Metabolomics analysis of fecal samples from mice colonized with C. tropicalis revealed alterations in vitamin B3 metabolism, promoting conversion of nicotinamide to nicotinic acid. Although nicotinamide worsened colitis, treatment with nicotinic acid alleviated disease symptoms and enhanced epithelial proliferation and Th17 cell differentiation. Oral gavage of C. tropicalis mitigated nicotinamide-induced intestinal dysfunction in experimental colitis. Blockade of nicotinic acid production with nicotinamidase inhibitors lowered the protective effects against colitis in mice treated with C. tropicalis. Notably, a clinical C. tropicalis strain isolated from patients with candidemia lacked the protective effects against murine colitis observed with the reference strain. Together, our results highlight a novel role for C. tropicalis in resolving intestinal inflammation through the modulation of vitamin B3 metabolism.

Assessment of equine intestinal epithelial junctional complexes and barrier permeability using a monolayer culture system.

Gastrointestinal disease is a leading cause of death in mature horses. A lack of in vitro modeling has impeded the development of novel therapeutics. The objectives of this study were to develop and further characterize a small intestinal monolayer cell culture derived from equine jejunum including establishing normal measurements of intestinal permeability and restitution. Three-dimensional enteroids, derived from postmortem sampling of equine jejunum, were utilized to develop confluent epithelial monolayers. The presence of differentiated intestinal epithelial cell types and tight junctions were confirmed using histology, reverse transcription PCR (RT-PCR), RNAscope, protein immunofluorescence and transmission electron microscopy. Transepithelial resistance (TER) and macromolecule flux were assessed as measurements of paracellular and transcellular permeability. Scratch assays were utilized to model and assess intestinal restitution. Monolayer cell cultures reached 100% confluency by ~5-7 days. Equine jejunum monolayers were confirmed as epithelial in origin, with identification of differentiated intestinal epithelial cell types and evidence of tight junction proteins. Function of the intestinal barrier was supported by acquisition of physiologically normal TER values (179.9 ± 33.7 ohms*cm2) and limited macromolecule flux (22 ± 8.8% at 60 min). Additionally, following a scratch wound, epithelial cell monolayers migrated to close gap defects within 24 h. In conclusion, this study describes the development of a novel intestinal epithelial monolayer cell culture for equine jejunum, and provides evidence of intestinal epithelial cell differentiation, formation of physiologically relevant barrier function and use as a model of intestinal restitution to test potential therapeutics for equine colic.

Detection of MUC1+/MUC2 and MUC5AC− Membrane-Associated Mucins in the Intraepithelial Surface Mucous Cells of the Developing Rabbit Esophagus

Introduction: Mucins are polydisperse molecules created to perform a variety of functions at the mucosal surface of the adult gastrointestinal tract. Two main groups of mucins could be identified: the membrane-associated mucins (MUC1, MUC4, MUC13, and MUC16), those bound to the apical plasma membrane of epithelial cells, and the secreted mucins (MUC2, MUC5AC, MUC5B, and MUC6), those secreted from the goblet cells. Little is known about the types and distribution patterns of mucins in prenatal life. Methods: We detected mucin-secreting cells in the developing rabbit esophagus though these cells are absent in the adult one. In order to identify the content and possible functions of these cells, we investigated the histochemical and immunohistochemical characteristics of their mucins. Results: Starting at 16th day of pregnancy, periodic acid Schiff (PAS), alcian blue (AB) pH (2.5), and PAS-AB combination intensely stained the mucous content, demonstrating both acidic and neutral mucopolysaccharides. Some blebs could be recognized on the free surface of the esophageal epithelium. Also, the mucous cells and some basal cells strongly immunoreacted with MUC1, but not MUC2, nor MUC5AC antibodies. Conclusion: Collectively, these data suggest that surface mucous cells are modified epithelial cells, not goblet cells, and may originate from the basal layer of the epithelial cells. A possible regulatory role for these MUC1-positive mucins in esophageal epithelial and mesenchymal cell differentiation and late organogenesis is suggested. However, future functional studies are recommended.

Autophagy-Mediated Cellular Remodeling during Terminal Differentiation of Keratinocytes in the Epidermis and Skin Appendages.

The epidermis of the skin and skin appendages, such as nails, hair and sebaceous glands, depend on a balance of cell proliferation and terminal differentiation in order to fulfill their functions at the interface of the body and the environment. The differentiation of epithelial cells of the skin, commonly referred to as keratinocytes, involves major remodeling processes that generate metabolically inactive cell remnants serving as building blocks of the epidermal stratum corneum, nail plates and hair shafts. Only sebaceous gland differentiation results in cell disintegration and holocrine secretion. A series of studies performed in the past decade have revealed that the lysosome-dependent intracellular degradation mechanism of autophagy is active during keratinocyte differentiation, and the blockade of autophagy significantly alters the properties of the differentiation products. Here, we present a model for the autophagy-mediated degradation of organelles and cytosolic proteins as an important contributor to cellular remodeling in keratinocyte differentiation. The roles of autophagy are discussed in comparison to alternative intracellular degradation mechanisms and in the context of programmed cell death as an integral end point of epithelial differentiation.

Vasoactive intestinal peptide promotes secretory differentiation and mitigates radiation-induced intestinal injury

BackgroundVasoactive intestinal peptide (VIP) is a neuronal peptide with prominent distribution along the enteric nervous system. While effects of VIP on intestinal motility, mucosal vasodilation, secretion, and mucosal immune cell function are well-studied, the direct impact of VIP on intestinal epithelial cell turnover and differentiation remains less understood. Intestinal stem and progenitor cells are essential for the maintenance of intestinal homeostasis and regeneration, and their functions can be modulated by factors of the stem cell niche, including neuronal mediators. Here, we investigated the role of VIP in regulating intestinal epithelial homeostasis and regeneration following irradiation-induced injury.MethodsJejunal organoids were derived from male and female C57Bl6/J, Lgr5-EGFP-IRES-CreERT2 or Lgr5-EGFP-IRES-CreERT2/R26R-LSL-TdTomato mice and treated with VIP prior to analysis. Injury conditions were induced by exposing organoids to 6 Gy of irradiation (IR). To investigate protective effects of VIP in vivo, mice received 12 Gy of abdominal IR followed by intraperitoneal injections of VIP.ResultsWe observed that VIP promotes epithelial differentiation towards a secretory phenotype predominantly via the p38 MAPK pathway. Moreover, VIP prominently modulated epithelial proliferation as well as the number and proliferative activity of Lgr5-EGFP+ progenitor cells under homeostatic conditions. In the context of acute irradiation injury in vitro, we observed that IR injury renders Lgr5-EGFP+ progenitor cells more susceptible to VIP-induced modulations, which coincided with the strong promotion of epithelial regeneration by VIP. Finally, the observed effects translate into an in vivo model of abdominal irradiation, where VIP showed to prominently mitigate radiation-induced injury.ConclusionsVIP prominently governs intestinal homeostasis by regulating epithelial progenitor cell proliferation and differentiation and promotes intestinal regeneration following acute irradiation injury.

Effects of Prebiotic Dietary Fibers on the Stimulation of the Mucin Secretion in Host Cells by In Vitro Gut Microbiome Consortia.

The gastrointestinal microbiota are important for human health. Dietary intake may modulate the composition and metabolic function of the gut microbiome. We examined how the breakdown of prebiotic dietary fibers by the gut microbiome affects mucin secretion by intestinal epithelial cells. Metagenomic analyses of in vitro gut microbiome consortia revealed taxonomic profiles and genetic diversity of carbohydrate-active enzymes that digest polysaccharides. Two independent consortia exhibited different abilities to produce acetic acid, propionic acid, and butyric acid via the fermentation of polysaccharides derived from dietary fibers of grains and mushrooms. Although acetic acid generally had the highest concentration, the ratios of butyric acid and propionic acid to acetic acid varied depending on the polysaccharide source. These short-chain fatty acids affected morphological differentiation and mucin secretion in HT-29 human intestinal epithelial cells. These results suggest that prebiotic dietary fibers can be digested and metabolized by the gut microbiome to short-chain fatty acids, which can affect gut epithelial cells both directly and indirectly via the modulation of the gut microbiota and their enzymes.

Role of Immunohistochemistry in the Diagnosis of Pilomatrical Tumors.

Pilomatrical skin tumors harbor mutations in CTNNB1, which encodes for β-catenin, a downstream effector of the Wnt signaling pathway responsible for the differentiation, proliferation, and adhesion of epithelial stem cells. Therefore, downstream molecules, such as CDX2, LEF-1, and SATB2, in the Wnt signaling pathway could be useful diagnostic markers. Here, we sought to investigate the potential of immunohistochemistry (IHC) to differentiate between pilomatricoma and pilomatrical carcinoma, as well as from other cutaneous adnexal tumors. We studied 88 cases of cutaneous tumors (14 pilomatrical carcinomas, 18 pilomatricomas, 13 basal cell carcinomas, 12 squamous cell carcinomas, 12 sebaceous carcinomas, 10 Merkel cell carcinomas, 7 trichoblastomas, and 2 hidradenocarcinomas) using a broad panel of IHC markers: β-catenin, SATB2, CDX2, LEF1, Ber-EP4, and PRAME. Pilomatricoma and pilomatrical carcinoma displayed >75% nuclear staining for β-catenin. CDX2 also strongly stained pilomatrical tumors; however, the staining distribution was limited in pilomatricoma and more widespread in pilomatrical carcinoma. But, overall, it was less than β-catenin. SATB2 and Ber-EP4 expressions were noted only in a subset of both pilomatrical carcinoma and pilomatricoma, whereas LEF-1 showed strong, diffuse nuclear positivity in both pilomatricoma and pilomatrical carcinoma. Among the IHC markers evaluated, none could distinguish between pilomatricoma and pilomatrical carcinoma. However, the combined use of β-catenin with CDX2 markers may assist in not only confirming the pilomatrical nature of the proliferation but also in differentiating benign from malignant cases when there is a significant presence of CDX2 staining. Despite these findings, the diagnosis should continue to primarily depend on a thorough histopathologic examination.

Rediscovering the human thymus through cutting-edge technologies.

Recent technological advances have transformed our understanding of the human thymus. Innovations such as high-resolution imaging, single-cell omics, and organoid cultures, including thymic epithelial cell (TEC) differentiation and culture, and improvements in biomaterials, have further elucidated the thymus architecture, cellular dynamics, and molecular mechanisms underlying T cell development, and have unraveled previously unrecognized levels of stromal cell heterogeneity. These advancements offer unprecedented insights into thymic biology and hold promise for the development of novel therapeutic strategies for immune-related disorders.

Loss of ARID3A perturbs intestinal epithelial proliferation-differentiation ratio and regeneration.

Intestinal stem cells at the crypt divide and give rise to progenitor cells that proliferate and differentiate into various mature cell types in the transit-amplifying (TA) zone. Here, we showed that the transcription factor ARID3A regulates intestinal epithelial cell proliferation and differentiation at the TA progenitors. ARID3A forms an expression gradient from the villus tip to the upper crypt mediated by TGF-β and WNT. Intestinal-specific deletion of Arid3a reduces crypt proliferation, predominantly in TA cells. Bulk and single-cell transcriptomic analysis shows increased enterocyte and reduced secretory differentiation in the Arid3a cKO intestine, accompanied by enriched upper-villus gene signatures of both cell lineages. We find that the enhanced epithelial differentiation in the Arid3a-deficient intestine is caused by increased binding and transcription of HNF1 and HNF4. Finally, we show that loss of Arid3a impairs irradiation-induced regeneration with sustained cell death and reprogramming. Our findings imply that Arid3a functions to fine-tune the proliferation-differentiation dynamics at the TA progenitors, which are essential for injury-induced regeneration.

7615 Genomic Consequences of GRHL2 Overexpression in ER+ Breast Cancer Cells

Abstract Disclosure: K.O. Allen: None. C. Zheng: None. N. Solodin: None. M.S. Ozers: Owner/Co-Owner; Self; Proteovista, LLC. C.L. Warren: None. P.E. Messa: None. D.T. Vogt: None. E.T. Alarid: None. Breast cancer progression is characterized by the presence and prevalence of metastatic disease. At a genomic level, metastasis can be facilitated via rearrangement of the chromatin landscape to expose oncogenic genes for transcription. Grainyhead like protein 2 (GRHL2) is a nuclear transcription factor implicated in epithelial cell differentiation. The GRHL2 motif has been found near activated estrogen receptor (ER) binding sites, and elevated levels of GRHL2 have been linked to worse prognosis in hormone receptor positive breast cancer patients. In its capacity to contribute to a more severe disease state when overexpressed, GRHL2 may function as either a transcription or pioneer factor to aid in the expression of metastasis-associated genes. Our lab previously showed that GRHL2 genomic binding precedes ER binding at co-regulated binding sites. Also, overexpression of GRHL2 in MCF7 cells for 24 hours did not appreciably alter gene expression by RNA-Seq. These data taken together indicate that GRHL2 may be exhibiting pioneer factor activity. However, the specific role of GRHL2 overexpression in hormone positive breast cancer progression remains unknown. In order to examine the mechanism of GRHL2 action, GRHL2 genomic binding was investigated using a tetracycline-inducible MCF7 model that allows controlled overexpression of GRHL2. ChIP studies were performed to determine GRHL2 binding intensity at specific and genome wide GRHL2 binding sites. Specific focus was given to established GRHL2 binding sites near oncogenic genes responsible for epithelial to mesenchymal transition (EMT), dormancy, and proto-oncogenic behavior. A similar analysis examined H3K27 acetylation to validate active gene transcription. An orthogonal technology, Specificity and Affinity for Protein (SNAP) microarrays, which characterizes direct GRHL2 binding to tiled genomic regions identified in ChIP-Seq analyses from multiple labs, was utilized to discriminate direct and indirect GRHL2 binding sites. The requirement for GRHL2 transactivation function in regulation of EMT related genes was further interrogated using transactivation GRHL2 dominant negative mutants. This work aims to distinguish the transcriptional and non-transcriptional activities of GRHL2 in breast cancer cells on a genome wide level and thereby, provide a deeper understanding of how overexpressed GRHL2 contributes to hormone positive breast cancer. Presentation: 6/3/2024

GZMA suppressed GPX4-mediated ferroptosis to improve intestinal mucosal barrier function in inflammatory bowel disease

BackgroundOur previous study has demonstrated a decreased colonic CD8+CD39+ T cells, enrichment of granzyme A (GZMA), was found in pediatric-onset colitis and inflammatory bowel disease (IBD) characterized by impaired intestinal barrier function. However, the influence of GZMA on intestinal barrier function remains unknown.MethodsWestern blotting(WB), real-time PCR (qPCR), immunofluorescence (IF) and in vitro permeability assay combined with intestinal organoid culture were used to detect the effect of GZMA on intestinal epithelial barrier function in vivo and in vitro. Luciferase, immunoprecipitation (IP) and subcellular fractionation isolation were performed to identify the mechanism through which GZMA modulated intestinal epithelial barrier function.ResultsHerein, we, for the first time, demonstrated that CD8+CD39+ T cells promoted intestinal epithelial barrier function through GZMA, leading to induce Occludin(OCLN) and Zonula Occludens-1(ZO-1) expression, which was attributed to enhanced CDX2-mediated cell differentiation caused by increased glutathione peroxidase 4(GPX4)-induced ferroptosis inhibition in vivo and in vitro. Mechanically, GZMA inhibited intestinal epithelial cellular PDE4B activation to trigger cAMP/PKA/CREB cascade signaling to increase CREB nuclear translocation, initiating GPX4 transactivity. In addition, endogenous PKA interacted with CREB, and this interaction was enhanced in response to GZMA. Most importantly, administration of GZMA could alleviate DSS-induced colitis in vivo.ConclusionThese findings extended the novel insight of GZMA contributed to intestinal epithelial cell differentiation to improve barrier function, and enhacement of GZMA could be a promising strategy to patients with IBD.

A-106 Determination of Urinary NGAL Reference Intervals from Specimens of Healthy Adult and Pediatric Individuals

Abstract Background Neutrophil gelatinase-associated lipocalin (NGAL) is a 25kDa protein implicated in multiple biological processes, including attenuation of apoptosis and differentiation of renal tubule epithelial cells and nephrons. NGAL is also produced and secreted by injured kidney tubule epithelial cells. As such, NGAL can serve as an early urinary biomarker for acute kidney injury (AKI). This multi-site, cross-sectional study aimed to establish reference intervals for urinary NGAL (uNGAL) in healthy pediatric and adult populations using a particle-enhanced turbidimetric assay. Methods Apparently healthy individuals, aged ≥ 3 months, were eligible for this study. Subjects with an active urinary tract infection (UTI) on the day of sample collection, acute kidney injury (AKI) or a history of AKI, stage 4 or 5 chronic kidney disease (CKD), known congenital anomalies of the kidney and urinary tract, known urothelial, urological, or kidney malignancies were excluded from the study. Subjects with uncorrected congenital heart disease, having undergone solid organ or bone marrow transplantation, receiving renal replacement therapy, and having undergone surgery (urologic, nephrectomy, or correction of congenital heart disease) were also excluded. A total of 688 subjects were screened, 677 were eligible, and 629 (91.4%) were considered evaluable per the protocol. The 59 excluded subjects were ineligible due to missing/incorrect information in the informed consent form, or not evaluable due to meeting exclusion criteria, mostly a positive UTI test. Urine samples were tested for NGAL on a Roche cobas® c501 analyzer in single determinations. Results Tables 1 and 2 describe the NGAL summary statistics and central 95% reference intervals by age and gender. Conclusions These data demonstrate the normal urine NGAL values in an apparently healthy pediatric and adult populations.

Mitochondrial-Derived Signaling Mediates Differentiation of Parietal Epithelial Cells into Podocytes.

Aims: Parietal epithelial cells (PECs) are potential stem cells within the glomerulus, migrating into site of podocyte loss to differentiate into podocytes. Little is known about the mechanism mediating differentiation of PECs into podocytes. Results: In vitro differentiation of PECs into podocytes led to upregulation of podocyte markers such as Wilms' tumor gene 1 (WT-1), Forkhead box C1 (FOXC1), synaptopodin and podocin, accompanied by increased mitochondrial abundance. Preincubation with a mitochondrial reactive oxygen species (ROS) inhibitor prevented all these events in PECs. In vivo, adriamycin (ADR)-treated mice exhibited albuminuria, decreased WT1 positive cells, and claudin-1 expressed in glomerular capillary tuft, as well as peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α) overproduction in PECs. Expression of the ROS-related molecule nuclear factor erythroid 2-related factor 2 (Nrf2) and its target protein Brahma-related gene 1 (Brg1) increased during differentiation of PECs into podocytes. Suppressing Nrf2 or Brg1 reduced the differentiation of PECs, whereas overexpression had the opposite effect. Brg1 directly regulated WT-1 transcription in PECs. Activation of Nrf2 with bardoxolone-methyl (CDDO-Me) resulted in less proteinuria and more WT1 positive cells in ADR mice. PECs conditional human Nrf2 knock-in mice showed increased WT1 cell numbers. Conclusion: It concluded that mitochondria-derived ROS mediated differentiation of PECs into podocytes via Nrf2 and Brg1 signaling.

Detection of Residual iPSCs Following Differentiation of iPSC-Derived Retinal Pigment Epithelial Cells.

Purpose: The goal of this study was to develop a lot release assay for iPSC residuals following directed differentiation of iPSCs to retinal pigment epithelial (RPE) cells. Methods: RNA Sequencing (RNA Seq) of iPSCs and RPE derived from them was used to identify pluripotency markers downregulated in RPE cells. Quantitative real time PCR (qPCR) was then applied to assess iPSC residuals in iPSC-derived RPE. The limit of detection (LOD) of the assay was determined by performing spike-in assays with known quantities of iPSCs serially diluted into an RPE suspension. Results: ZSCAN10 and LIN28A were among 8 pluripotency markers identified by RNA Seq as downregulated in RPE. Based on copy number and expression of pseudogenes and lncRNAs ZSCAN10 and LIN28A were chosen for use in qPCR assays for residual iPSCs. Reverse transcription PCR indicated generally uniform expression of ZSCAN10 and LIN28A in 21 clones derived from 8 iPSC donors with no expression of either in RPE cells derived from 5 donor lines. Based on qPCR, ZSCAN10, and LIN28A expression in iPSCs was generally uniform. The LOD for ZSCAN10 and LIN28A in qPCR assays was determined using spike in assays of RPE derived from 2 iPSC lines. Analysis of ΔΔCt found the limit of detection to be <0.01% of cells, equivalent to <1 iPSC/10,000 RPE cells in both iPSC lines. Conclusions: qPCR for ZSCAN10 and LIN28A detects <1 in 10,000 residual iPSCs in a population of iPSC-derived RPE providing an adequate LOD of iPSC residuals for lot release testing.