Retroviral infection of murine embryonic stem cell derived embryoid body cells for analysis of hematopoietic differentiation

Emmanuel Bikorimana, Danica Lapid, Hyewon Choi, Richard Dahl

Research output: Contribution to journalArticle

3 Scopus citations

Abstract

Embryonic stem cells (ESCs) are an outstanding model for elucidating the molecular mechanisms of cellular differentiation. They are especially useful for investigating the development of early hematopoietic progenitor cells (HPCs). Gene expression in ESCs can be manipulated by several techniques that allow the role for individual molecules in development to be determined. One difficulty is that expression of specific genes often has different phenotypic effects dependent on their temporal expression. This problem can be circumvented by the generation of ESCs that inducibly express a gene of interest using technology such as the doxycycline-inducible transgene system. However, generation of these inducible cell lines is costly and time consuming. Described here is a method for disaggregating ESC-derived embryoid bodies (EBs) into single cell suspensions, retrovirally infecting the cell suspensions, and then reforming the EBs by hanging drop. Downstream differentiation is then evaluated by flow cytometry. Using this protocol, it was demonstrated that exogenous expression of a microRNA gene at the beginning of ESC differentiation blocks HPC generation. However, when expressed in EB derived cells after nascent mesoderm is produced, the microRNA gene enhances hematopoietic differentiation. This method is useful for investigating the role of genes after specific germ layer tissue is derived.

Original languageEnglish (US)
Article numbere52022
JournalJournal of Visualized Experiments
Issue number92
DOIs
StatePublished - Oct 20 2014

Keywords

  • Cellular biology
  • Embryoid body
  • Embryonic stem cell
  • Gene expression
  • Hematopoietic progenitor cells
  • Issue 92
  • Retrovirus
  • Temporal gene expression

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)
  • Medicine(all)

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