Organoids are complex self-organized 3D cell culture models that are often derived from stem cells.1 Organoids have been generated from diverse tissues including brain,2 intestine,3 stomach,4 colon,5 liver,6 pancreas,6 lung,7 kidney8 and patient-derived tumors.9 Epithelial intestinal organoids, often referred to as enteroids or mini-guts, maintain the physiological characteristics of the gastrointestinal system and have been a useful cell culture tool to model intestinal development and disease including the study of colon cancer, celiac disease, inflammatory bowel diseases and host microbiome interactions.10 Traditional isolation techniques developed by Clevers et al.1 rely on lengthy primary tissue isolation, either from mouse or from difficult-to-source human tissue samples. Induced pluripotent stem cell-derived organoids would enable the rapid generation of patient-specific cell models from a wider range of human donors. We have generated a human iPSC-derived colon organoid system that provides highly characterized, assay-ready cryopreserved human colonic organoids and expansion media. Furthermore, our optimized serum-free media and reagents can be used to derive colonic organoids from any human iPS cell line using a simple three-step differentiation process.
Figure 1.Human colonic organoid differentiation workflow. Human colonic organoids can be generated from human iPS cells using a three-step differentiation protocol through definitive endoderm, hindgut endoderm, and colonic organoid expansion stages.
SCM302: Definitive Endoderm Induction Medium, SCM303: Hindgut Induction Medium, SCM304: 3dGRO™ Human Colon Organoid Expansion Medium
Note: Start with high-quality undifferentiated human ES/iPS cells (SCC271) that are ~70-80% confluent and contain <5% differentiated cells. The following protocol is for differentiation of one well of a six-well tissue-culture treated plate. Indicated volumes are for a single well. Adjust volumes as necessary.
Figure 2.Endoderm differentiation of human iPS cells. Flow cytometry analysis of human iPSC-derived definitive endoderm cells for endoderm markers demonstrates that cells are CXCR4+, c-Kit+, Sox-17+ , PDGFR- and FOXA2+ after four days of differentiation.
Figure 3.Differentiation of definitive endoderm cells into hindgut endoderm cells. Morphology of hindgut endoderm cells at A) day 2 B) day 3 and C) day 4 post hindgut endoderm induction.
Expansion of Colon Organoids
Figure 4.Human colon organoids. A) Colonic organoids encapsulated in Matrigel® matrix domes, two days post-thaw. B) By day 10-12 in culture, colon organoids occupy 85-90% of the dome, and are ready to be passaged.
Human colonic organoids can be cryopreserved using the 3dGRO™ Organoid Freezing Media (SCM301). This protocol is based on not using any dissociation reagents during passaging of human colonic organoids for cryopreservation. The recommended starting number of domes per vial is four per vial, with the assumption that the density of each dome is at 90%. If the density is less than 90%, freeze down more domes per vial.
Figure 5.Morphology of human iPS cell-derived colonic organoids. Mature human colon organoids have complex morphologies when cultured in three dimensions. A) 4X magnification B) 10X magnification.
Human Colon Organoid Characterization
Figure 6. Immunocytochemical (ICC) characterization of human colon organoids. Human iPS cell-derived colonic organoids are positive for CDX2, α-carbonic anhydrase-II, α-carbonic anhydrase-IV, mucin-5B, mucin-2 and E-cadherin.
Figure 7.Immunohistochemical (IHC) characterization of human colon organoids. A) Goblet cells identified with alcian blue staining B) proliferating cells identified using Ki67 antibody (red) C) nuclei and plasma protein identified using H&E staining.
We have developed a robust three-step differentiation protocol to generate human colonic organoids from human induced pluripotent stem cells (iPSCs). Colon organoids generated using this protocol express mature colon markers CDX2, α-carbonic anhydrase-II, α-carbonic anhydrase-IV, mucin 5B, mucin 2 and E-cadherin, and can be serially passaged over multiple passages without losing colon phenotypes. These organoids and serum-free media will provide researchers and drug discovery with highly validated new 3D cell models to study intestinal diseases.