Optimization of adenoviral gene transfer in human pluripotent stem cells

Biochem Biophys Res Commun. 2021 Feb 19;541:78-83. doi: 10.1016/j.bbrc.2021.01.009. Epub 2021 Jan 19.

Abstract

Human pluripotent stem cells, such as embryonic stem cells and induced pluripotent stem cells, have the potential to differentiate into a wide variety of cells in vitro and have applications in basic developmental biology research and regenerative medicine. To understand the process of differentiation from pluripotent stem cells to functional cells, it is necessary to efficiently and safely transfer and express exogenous genes. We attempted to optimize the efficient transfer of genes into pluripotent stem cells using adenoviral vectors. Comparative study of the activities of three representative ubiquitously active promoters revealed that only the CA promoter allowed robust transgene expression in human pluripotent stem cells. In addition, we established a protocol that allowed us to efficiently introduce target genes and ensure their expression even in small numbers of cells. Adenoviral vector infection of pluripotent stem cells in single-cell suspension culture yielded high gene transfer efficiency with low cytotoxicity, without losing the undifferentiated state of the pluripotent stem cells. This optimized system will facilitate developmental biology research and regenerative medicine using pluripotent stem cells.

Keywords: Adenoviral vector; Gene transfer; Human ES cells; Human iPS cells; Human pluripotent stem cell.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenoviridae / genetics*
  • Adenoviridae / physiology
  • Cell Culture Techniques
  • Cells, Cultured
  • Gene Transfer Techniques / standards*
  • Genes, Reporter / genetics
  • Genetic Vectors / genetics*
  • Green Fluorescent Proteins / genetics
  • Humans
  • Pluripotent Stem Cells / cytology
  • Pluripotent Stem Cells / metabolism*
  • Promoter Regions, Genetic / genetics

Substances

  • enhanced green fluorescent protein
  • Green Fluorescent Proteins