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INTESTINAL Publication-derived

Niche-independent high-purity cultures of Lgr5+ intestinal stem cells and their progeny

Source Yin et al., 2014 · David H. Koch Institute for Integrative Cancer Research, MIT · 10.1038/nmeth.2737

👤 Xiaolei Yin, Henner F Farin, Johan H van Es, Hans Clevers, Robert Langer, Jeffrey M Karp ⏱ 11 days 📋 7 phases 🧫 Mouse Lgr5-EGFP intestinal stem cells

Abstract

This protocol describes the culture of Lgr5+ intestinal stem cells using CHIR99021 and valproic acid (VPA) to achieve nearly homogeneous stem cell cultures with ~100-fold higher colony-forming efficiency than conventional methods. The protocol maintains self-renewal while preserving multilineage differentiation capability into enterocytes, goblet cells, and Paneth cells.

Cell source
Mouse Lgr5-EGFP intestinal stem cells
Application
Maintenance and differentiation of intestinal stem cells

Protocol overview

30 steps across 7 phases

Crypt and Single-Cell Isolation Day 0
  1. 1 Tissue harvesting and preparation
  2. 2 Tissue fragmentation
  3. 3 EDTA incubation for crypt release
  4. 4 Crypt collection and purification
  5. 5 Single-cell isolation
  6. 6 FACS sorting of stem cells
Matrigel embedding and culture setup Day 0
  1. 1 Matrigel preparation
  2. 2 Cell embedding in Matrigel
  3. 3 Matrigel polymerization
  4. 4 Culture medium addition
Stem cell expansion (CV condition) Day 0–Day 6 and beyond
  1. 1 Medium change
  2. 2 Incubation
  3. 3 Cell passage as single cells (every 6 days)
  4. 4 Monitoring stem cell characteristics
Directed differentiation Day 6–Day 11
  1. 1 Preparation of homogeneous stem cell starting population
  2. 2 Transfer to differentiation condition – Enterocyte differentiation
  3. 3 Goblet cell differentiation
  4. 4 Paneth cell differentiation
  5. 5 Alternative – Spontaneous multilineage differentiation
Flow cytometry analysis As needed
  1. 1 Cell dissociation
  2. 2 Cell counting and preparation
  3. 3 Flow cytometry
Immunocytochemistry and microscopy As needed
  1. 1 Fixation
  2. 2 Matrigel disruption and cell isolation
  3. 3 Permeabilization
  4. 4 Antibody staining
  5. 5 EdU incorporation assay
  6. 6 Microscopy imaging
Molecular analysis – qPCR As needed
  1. 1 RNA extraction
  2. 2 Quantitative real-time PCR

Full SOP

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Attribution

This SOP was authored by Organthis based on the published method in Yin et al., 2014. The originating laboratory holds no rights in this SOP and has not endorsed it unless marked Verified.

This wording is awaiting legal review.

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