Generation of human colonic organoids from human pluripotent stem cells

Methods Cell Biol. 2020;159:201-227. doi: 10.1016/bs.mcb.2020.03.001. Epub 2020 Apr 8.


Advances in human pluripotent stem cell (hPSC) biology now allow the generation of organoids that resemble different regions of the gastrointestinal tract. Generation of region-specific organoids has been facilitated by developmental biology studies carried out in model organisms such as mouse, frog and chick. By mimicking embryonic development, hPSC-derived human colonic organoids (HCOs) can be generated through a stepwise differentiation, first into definitive endoderm (DE), then into mid/hindgut spheroids which are then patterned into posterior gut tissue which gives rise to HCOs following prolonged in vitro culture. HCOs undergo transitions similar to those observed in the developing colon of model organisms and human embryos. HCOs develop into tissue that resembles fetal colon on the basis of morphology, gene expression and presence of differentiated cell types. Generation of HCOs without the proper training or expertise can be a daunting task. Here, we describe a detailed protocol for differentiating hPSCs into HCOs, we include suggestions for troubleshooting these differentiations, and we discuss experimental design considerations. We have also highlighted the key advantages and limitations of the system.

Keywords: Definitive endoderm; Hindgut; Human colonic organoid; Human pluripotent stem cells.

MeSH terms

  • Cell Culture Techniques / methods*
  • Cell Differentiation
  • Colon / cytology*
  • Endoderm / cytology
  • Gene Expression Regulation
  • Human Embryonic Stem Cells / cytology
  • Humans
  • Organoids / cytology*
  • Organoids / metabolism
  • Pluripotent Stem Cells / cytology*
  • Pluripotent Stem Cells / metabolism
  • Spheroids, Cellular / cytology