Derivation of enteric neuron lineages from human pluripotent stem cells

Nat Protoc. 2019 Apr;14(4):1261-1279. doi: 10.1038/s41596-019-0141-y. Epub 2019 Mar 25.

Abstract

The enteric nervous system (ENS) represents a vast network of neuronal and glial cell types that develops entirely from migratory neural crest (NC) progenitor cells. Considerable improvements in the understanding of the molecular mechanisms underlying NC induction and regional specification have recently led to the development of a robust method to re-create the process in vitro using human pluripotent stem cells (hPSCs). Directing the fate of hPSCs toward the enteric NC (ENC) results in an accessible and scalable in vitro model of ENS development. The application of hPSC-derived enteric neural lineages provides a powerful platform for ENS-related disease modeling and drug discovery. Here we present a detailed protocol for the induction of a regionally specific NC intermediate that occurs over the course of a 15-d interval and is an effective source for the in vitro derivation of functional enteric neurons (ENs) from hPSCs. Additionally, we introduce a new and improved protocol that we have developed to optimize the protocol for future applications in regenerative medicine, in which components of undefined activity have been replaced with fully defined culture conditions. This protocol provides access to a broad range of human ENS lineages within a 30-d period.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Antigens, CD / genetics
  • Antigens, CD / metabolism
  • Biomarkers / metabolism
  • Cell Culture Techniques*
  • Cell Differentiation
  • Cell Lineage / physiology
  • Enteric Nervous System / cytology*
  • Enteric Nervous System / metabolism
  • Gene Expression
  • Genes, Reporter
  • Homeodomain Proteins / genetics
  • Homeodomain Proteins / metabolism
  • Humans
  • Intercellular Signaling Peptides and Proteins / genetics
  • Intercellular Signaling Peptides and Proteins / metabolism
  • Intestine, Small / cytology*
  • Intestine, Small / metabolism
  • Neural Crest / cytology*
  • Neural Crest / metabolism
  • Neurons / cytology*
  • Neurons / metabolism
  • PAX3 Transcription Factor / genetics
  • PAX3 Transcription Factor / metabolism
  • Pluripotent Stem Cells / cytology*
  • Pluripotent Stem Cells / metabolism
  • Regenerative Medicine / methods

Substances

  • Antigens, CD
  • Biomarkers
  • HOXB5 protein, human
  • Homeodomain Proteins
  • HoxB3 protein, human
  • Intercellular Signaling Peptides and Proteins
  • PAX3 Transcription Factor
  • PAX3 protein, human