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INTESTINAL Publication-derived

Intestinal Organoids for Disease and Development

Full SOP with Academic or a plan

Source Beumer et al., 2020 · Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC Utrecht · 10.1016/j.cell.2020.04.036

Authors: Joep Beumer, Jens Puschhof, Julia Bauza-Martinez, Sarah A. Teichmann, Wei Wu, Hans Clevers Duration: 115 days 10 phases Cell source: Human iPSC

Abstract

A human organoid-based platform for studying enteroendocrine cells (EECs), which sense intestinal content and release hormones to regulate systemic metabolism and food intake. The protocol induces EEC formation in vitro by transient NEUROG3 expression, generates region-specific EEC subtypes from proximal small intestine, distal small intestine, and colon, and characterizes their transcriptomes and secreted products via single-cell RNA sequencing and mass spectrometry.

Cell source
Human iPSC
Application
Disease modeling; Secretome characterization; Enteroendocrine cell differentiation and functional studies

Protocol overview

45 steps across 10 phases

Phase 1 · Human Intestinal Organoid Establishment and Culture Days 1-7
  1. a Isolate and culture human intestinal organoids
  2. b Passage organoids
Phase 2 · NEUROG3-Induced Enteroendocrine Cell Differentiation Days 8-12
  1. a Induce NEUROG3 expression
  2. b Monitor EEC formation and hormone expression
  3. c Modulate signaling pathways to influence EEC subtype
  4. d Activate BMP signaling to generate villus-equivalent EECs
Phase 3 · Functional Validation and Hormone Secretion Testing Days 12-14
  1. a Test EEC maturity and secretory capacity with forskolin
  2. b Test EEC receptor function and inter-EEC communication
  3. c Assess olfactory receptor function with calcium imaging
Phase 4 · Generation of Hormone Reporter Organoids (EEC-TAG Biobank) Days 15-30
  1. a Clone targeting constructs for fluorescent hormone tagging
  2. b Electroporate CRISPR-Cas9 constructs into organoid cells
  3. c FACS sorting and clonal selection
  4. d Validate reporter organoids by immunofluorescence
Phase 5 · Single-Cell RNA Sequencing and Transcriptomic Analysis Days 31-45
  1. a Prepare single cells from EEC-enriched organoids
  2. b FACS sorting of EEC-enriched populations
  3. c Perform single-cell RNA sequencing (SORT-seq)
  4. d Cluster and analyze single-cell transcriptomes
  5. e Identify major EEC subtypes and cluster heterogeneity
  6. f Identify human-specific EEC markers and genes
Phase 6 · Bulk RNA Sequencing and Receptor/Transcription Factor Analysis Days 46-60
  1. a Sort and pool EEC populations for bulk RNA sequencing
  2. b Prepare cDNA libraries and perform deep RNA sequencing
  3. c Analyze EEC-subtype-specific receptors and sensory genes
  4. d Identify lineage-specifying transcription factors
Phase 7 · CRISPR-Cas9 Knockout Studies Days 61-75
  1. a Design and validate knockout constructs
  2. b Transfect organoid-derived cells with Cas9-gRNA plasmids
  3. c Screen clonal knockout organoids
  4. d Assess phenotypic consequences of transcription factor knockout
Phase 8 · Proteome Analysis of FACS-Sorted EEC Populations Days 76-85
  1. a FACS sort EEC populations for proteome analysis
  2. b Protein quantification and sample preparation
  3. c Trypsin digestion for bottom-up mass spectrometry
  4. d Perform liquid chromatography-mass spectrometry (LC-MS)
  5. e Process and analyze mass spectrometry data
Phase 9 · Secretome Analysis by Mass Spectrometry Days 86-100
  1. a Stimulate EEC organoids and collect conditioned media
  2. b Fractionate secreted proteins by molecular weight
  3. c Digest and prepare >10 kDa protein fraction for LC-MS
  4. d Prepare <10 kDa peptide fraction for direct LC-MS
  5. e Perform LC-MS/MS analysis of secretome
  6. f Analyze region-specific secretome differences
  7. g Characterize peptide processing events
Phase 10 · Validation Assays: qPCR, Immunofluorescence, and Functional Studies Days 101-115
  1. a Quantitative PCR validation of gene expression
  2. b Immunofluorescence staining of organoids
  3. c Immunofluorescence on primary human intestinal tissue
  4. d Fluorescence in situ hybridization (FISH) for transcript validation
  5. e Transmission electron microscopy (TEM) of EEC organoids
  6. f ELISA for quantification of secreted GLP-1 and other hormones

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Attribution

This SOP was written by Organthis based on the published method in Beumer et al., 2020. Once the originating laboratory has confirmed it, the SOP is marked Verified.

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