Chemicals and reagents were purchased from Thermo Fisher (Waltham, MA) or Sigma-Aldrich (St. Louis, MO) unless otherwise specified.
Small intestine enteroid culture and monolayer formation
Duodenal biopsies were obtained from two T1D pediatric patients and three pediatric HS (Table 1) undergoing endoscopy following approval by the Johns Hopkins University Institutional Review Board (IRB00044373). Informed consent was provided by the parents or legal guardians. All procedures and methods were performed under approved guidelines and regulations. Duodenal biopsies were collected from HS screened with endoscopy for gastrointestinal symptoms and who had histologically normal duodenum. Enteroids were established via the Hopkins Conte Basic and Translational Digestive Diseases Research Core Center (NIH/NIDDK P30) following an established protocol11,12,50. Briefly, human enteroids were maintained as cysts embedded in Matrigel (Corning #356231; Corning, NY) in 24-well plates and cultured in Wnt3A, Rspon-1, and Noggin containing growth medium. For differentiation of enteroids, growth medium was replaced with Wnt3A and Rspon free medium for 5–6 days11,12. Enteroid monolayers were generated by plating fragmented enteroids (in 100 µl growth medium) onto 0.4 μm pore polyester (PET) membrane 24-well cell culture inserts (Transwell; Corning, USA or Millipore, USA) pre-coated with human collagen IV (30 μg/ml; Sigma-Aldrich, USA) following our established protocols11,12. The formation of an enteroid monolayer and changes with differentiation were monitored by the measurement of TEER using an epithelial volt/ohm meter (EVOM; World Precision Instruments)12,51.
Brightfield imaging
Enteroids plated in Matrigel in 24 well plates were imaged on a Zeiss Axio Observer A1 inverted microscope (Zeiss, Oberkochen, Germany) with images captured on CellSense imaging software (Olympus, Tokyo, Japan). Images were viewed and processed using OlyVia (Olympus).
RNA isolation, reverse transcription, and real-time PCR
Enteroids were harvested from Matrigel using a Cultrex harvesting solution or from monolayers on Transwells11,51. Total RNA from harvested enteroids was extracted using the PureLink RNA Mini Kit (Life Technologies, Carlsbad, CA) according to the manufacturer’s protocol. Complementary DNA was synthesized from 1 to 2 μg of RNA using SuperScript VILO Master Mix (Life Technologies). Quantitative real-time PCR was performed using Power SYBR Green Master Mix (Life Technologies) on a QuantStudio 12 K Flex real-time PCR system (Applied Biosystems, Foster City, CA). Each sample was studied in triplicate, and 5 ng RNA-equivalent complementary DNA was used for each reaction. The relative fold changes in mRNA levels of genes were determined by using the 2-ΔΔCT method, with human 18S ribosomal RNA simultaneously studied and used as the internal control for normalization and shown as the fold change compared with relevant control. Commercially available primer pairs from OriGene Technologies (Rockville, MD) were used (Table 2).
Bulk RNA sequencing library preparation and sequencing
Total RNA extracted from undifferentiated and 6-day-differentiated 3D enteroids was used for RNAseq. The concentration and integrity of the extracted total RNA were estimated using the Quant-iT™ RiboGreen® RNA Assay Kit (Thermo Fisher Scientific) and the Tapestation (Agilent) using the RNA High Sense chip, respectively. Approximately 500 ng of total RNA was used for downstream RNA-seq Library preparation. Library preparation and sequencing were performed at Discovery Life Sciences, Huntsville, AL. Briefly, Polyadenylated RNAs were captured using NEBNext Magnetic Oligo d(T)25 Beads. The NEBNext mRNA Library Prep Reagent Set for Illumina (New England BioLabs Inc., Ipswich, MA, USA) was then used to prepare individually bar-coded libraries as per the manufacturer’s recommended protocol. Library concentration was assessed using the Quant-iT™ PicoGreen® dsDNA Reagent (Thermo Fisher Scientific), and the library quality was estimated by utilizing the Caliper LabChip GX DNA High Sense kit (PerkinElmer). Accurate quantification for sequencing applications was determined using the qPCR-based KAPA Biosystems Library Quantification Kit (Kapa Biosystems, Inc., Woburn, MA, USA). Paired-end sequencing (25 million, 100-bp, paired-end reads) was performed on Illumina NovaSeq 6000 sequencers (Illumina, Inc., San Diego, CA, USA) as per the manufacturer’s recommended protocol.
RNA-seq analysis
Raw sequence reads were checked for quality using the FASTQC package ver. 0.11.9; Illumina adapters and low-quality basepairs were trimmed using TrimGalore ver. 0.6.5 with default settings. Trimmed reads were aligned to human genome build GRCh38.98 using Gencode v40 and a count matrix was generated from the aligned reads using feature Counts. For alignment and counts, the Dragen Bio-IT Platform version 3.10 using specific RNA flags including duplicate marking was employed. Following count generation, we employed R version 4.1.0 and edgeR for normalization, testing procedures, PCA clustering, and differential expression analysis.
Principal component analysis
PCA plots were done with ggplot2 R package using the normalized TPM counts for the undifferentiated and differentiated clusters.
Heatmap DEG Volcano plot
Using the normalized counts from the DESeq2 analysis as the inputs, we created heatmaps for the top 50 differentially expressed genes (by p-adj values) using the pheatmap R package (R package version 1.0.12) and volcano plot depictions using the ggplot R package depicting the log2Foldchange on the x-axis and − log10(padj) on the y axis respectively.
GSEA on pathways enrichment
Pathway enrichment analysis was done using over-representation analysis (ORA) and the gene set enrichment analysis (GSEA) 52,53. The input data for these analyses were the output normalized counts from DESeq2. The Gene Ontology pathway compendium gene set compiled by the MsigDB database was used for the GSEA analysis54. The GSEA analysis was performed on rank files comprised of gene symbols and the corresponding log2 fold changes for all the expressed genes; enrichment was considered significant for adjusted p-value FDR < 0.25. Functional analyses on the differentially expressed genes were further performed using Over Representation Analysis (ORA) to determine whether known biological functions or processes are overrepresented. We used the hypergeometric distribution as implemented by MSigDB, with significance achieved for adjusted p-value FDR < 0.05.
Immunofluorescence staining and confocal imaging
Enteroid monolayers on Transwell inserts were washed with PBS and fixed in 4% paraformaldehyde for 30–45 min, incubated with 5% bovine serum albumin/0.1% saponin in phosphate-buffered saline for 1 h, and incubated with primary antibodies overnight at 4 °C. Primary antibodies used included: CHGA (rabbit polyclonal; 1:1000 dilution; Immunostar), DRA (SC-376187; mouse monoclonal, 1:500, Santa Cruz, Dallas, TX), NHE3 (NBP1-82,574; rabbit polyclonal; 1:1000, Novus, Littleton, CO), E-cadherin (Clone 36, rabbit polyclonal; 1:100, BD Biosciences) or SGLT-1 (rabbit polyclonal:1:250, Thermo Fisher Scientific). The next day, membranes were washed with PBS three times for 5 min each, and cells were then exposed to Alexa Fluor 488 goat anti-mouse and Alexa Fluor 594 goat anti-rabbit secondary antibodies (1:1000 dilution; Invitrogen, Waltham, MA, USA) or phalloidin (568 or 633) (Life Technologies) for 1 h. Nuclei were detected by incubating in DAPI for 15 min at room temperature. The membranes were washed with PBS three times for 5 min each and mounted on glass slides using ProLong Gold antifade mounting medium (Invitrogen, Waltham, MA, USA). Images were collected using × 40 oil (NA 1.25) immersion objective on an FV3000 confocal microscope (Olympus, Tokyo, Japan) with Olympus FV31S-SW and Fiji (ImageJ-2020) (NIH). For quantitative analysis, the same settings were used to image all samples.
Immunoblotting
Enteroids were rinsed 3 times and harvested in phosphate-buffered saline by scraping. Cell pellets were collected by centrifugation, solubilized in lysis buffer (60 mmol/L HEPES, 150 mmol/L NaCl, 3 mmol/L KCl, 5 mmol/L EDTA trisodium, 3 mmol/L ethylene glycol-bis(β-aminoethyl ether)—N,N,N′,N′—tetraacetic acid, 1 mmol/L Na 3 PO 4, and 1% Triton X-100, pH 7.4) containing a protease inhibitor cocktail, and homogenized by sonication. Protein concentration was measured using the bicinchoninic acid method. Proteins were incubated with sodium dodecyl sulfate buffer (5 mmol/L Tris–HCl, 1% sodium dodecyl sulfate, 10% glycerol, 1% 2-mercaptoethanol, pH 6.8) at 37 °C for 10 min, 50 µg total protein was loaded per well and separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on a 10% acrylamide gel, and transferred onto a nitrocellulose membrane. The blot was blocked with 5% nonfat milk, and probed with primary antibodies against DRA (SC-376187, 1:250), NHE3 (NBP1-82,574, 1:1000) or SGLT-1 (ab14686, rabbit, 1:1000, Abcam Waltham, MA), glyceraldehyde-3-phosphate dehydrogenase (G8795; mouse monoclonal, 1:5000, G8795; Sigma-Aldrich), overnight at 4 °C, followed by secondary antibody against mouse IgG (1:10,000) for 1 h at room temperature. All the primary antibodies used have been previously validated55,56,57. Protein bands were visualized and quantitated using an Odyssey system and Image Studio Lite Ver 4.0 (LI-COR Biosciences, Lincoln, NE, USA).
FITC-dextran permeability assay
Intestinal barrier integrity of 2D duodenal enteroid monolayers was evaluated using fluorescein isothio-cyanate (FITC)-dextran (4 kDa) flux assays. Human enteroids monolayers on Transwell inserts were maintained as an UD culture in Wnt3A, Rspon-1, and Noggin-containing growth medium and differentiated for six days by removing Wnt3A and Rspon from the medium. To measure changes in permeability in DF enteroids, the monolayers were treated with 5 mM EGTA on the apical side of the Transwells and incubated in a 37 °C incubator for 2 h. After 2 h, EGTA was removed and 1:100 dilution of 4 mg/ml of 4 kDa FITC-dextran prepared in DF media was added to the apical side of the Transwell and incubated in 5% CO2/95% air atmosphere at 37 °C incubator for 2 h. After 2 h, media was collected from the apical and basal sides and assayed using a fluorescent plate reader at an excitation/emission wavelength of 490/520 nm. The TEER was measured before EGTA treatment and at the end of the experiment. No EGTA-treated monolayers were used as the controls. Concentrations of FITC-dextran in the collected media were calculated using a standard curve of FITC-dextran with known concentrations prepared in DF media.
Organoid swelling assay
Fluid secretion was determined using the agonist-stimulated swelling assay in duodenal organoids25,26. Organoids were passaged weekly by mechanical disruption into single crypts that easily reseal and form new organoids. Approximately 50–200 organoids were plated in each well of a 24-well plate. On the day of the assay, the organoids were stained with calcein green, a fluorescent cell-permeable dye that is retained within living cells and facilitates live imaging by defining the plasma membrane. Then, forskolin (Fsk) 5 µM or ATP (5 µM) which causes an increase in intracellular adenylyl cyclase-cAMP or Ca2+ respectively were added to stimulate organoid swelling. Using live-cell microscopy, we observed a rapid expansion of both the lumen and total organoid surface area after the addition of the CFTR agonists19. DMSO-treated organoids showed no significant change in the surface area of organoids and hence were used as a control. Agonist-stimulated organoid swelling was calculated as described before using an organoid swelling analysis macro for ImageJ26. Briefly, to quantify swelling, total calcein-green surface areas were selected for each time point and expressed as a percentage increase of T = 0 min (set at 100%). The relative area increase was expressed per 10-min time interval, and measurements were generated for each condition (T = 0 to 60 min, baseline threshold set at 100%). The area under the curve (AUC) (T = 60 min, baseline 100%) was calculated using Prism.
Statistical analysis
Differentially expressed genes (DEGs) between groups were assessed using the DESeq2 ver. 1.42.0 package in R58. Raw count data were used as input for DESeq2 analysis. The cutoff criteria for assessing the DEGs was |log2FC|> 1.5 and false discovery rate (FDR)-adjusted p value < 0.05.
Statistical analyses were performed using GraphPad Prism (version 8.01, GraphPad Software, San Diego, CA, USA). Data points represent means of individual experiments performed on different enteroid lines. Data presented are mean ± s.e.m. of at least three-five independent experiments, with an error bar equaling one s.e.m. Statistical significance was determined using the Student’s t-test or one-way ANOVA and post-analysis correction. Differences were considered statistically significant at p value ≤ 0.05.
All authors had access to the study data and reviewed and approved the final manuscript.
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- Source: https://www.nature.com/articles/s41598-024-62282-x