Single-cell RNA sequencing reveals metallothionein heterogeneity during hESC differentiation to definitive endoderm

Stem Cell Res. 2018 Apr;28:48-55. doi: 10.1016/j.scr.2018.01.015. Epub 2018 Jan 31.


Differentiation of human pluripotent stem cells towards definitive endoderm (DE) is the critical first step for generating cells comprising organs such as the gut, liver, pancreas and lung. This in-vitro differentiation process generates a heterogeneous population with a proportion of cells failing to differentiate properly and maintaining expression of pluripotency factors such as Oct4. RNA sequencing of single cells collected at four time points during a 4-day DE differentiation identified high expression of metallothionein genes in the residual Oct4-positive cells that failed to differentiate to DE. Using X-ray fluorescence microscopy and multi-isotope mass spectrometry, we discovered that high intracellular zinc level corresponds with persistent Oct4 expression and failure to differentiate. This study improves our understanding of the cellular heterogeneity during in-vitro directed differentiation and provides a valuable resource to improve DE differentiation efficiency.

Keywords: Definitive endoderm; Differentiation; Heterogeneity; RNA sequencing; Single cell; hPSC.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Cell Cycle Checkpoints
  • Cell Differentiation*
  • Endoderm / cytology*
  • Human Embryonic Stem Cells / cytology*
  • Human Embryonic Stem Cells / metabolism
  • Humans
  • Intracellular Space / metabolism
  • Metallothionein / metabolism*
  • Sequence Analysis, RNA / methods*
  • Single-Cell Analysis / methods*
  • Zinc / metabolism


  • Metallothionein
  • Zinc