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Distinguishing Between Endodermal and Pluripotent Stem Cell Lines During Somatic Cell Reprogramming.

A Moauro1, A Ralston2

  • 1Graduate Program in Physiology and Osteopathic Medicine, Michigan State University, East Lansing, MI, USA.

Methods in Molecular Biology (Clifton, N.J.)
|May 4, 2022
PubMed
Summary
This summary is machine-generated.

Reprogramming mouse cells with OSKM creates induced pluripotent stem (iPS) and extraembryonic endoderm stem (iXEN) cells. This guide details how to distinguish between these two stem cell types after reprogramming mouse embryonic fibroblasts.

Keywords:
Klf4OSKMOct4ReprogrammingSox2c-MyciPS cellsiXEN cells

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Area of Science:

  • Stem cell biology
  • Cellular reprogramming
  • Developmental biology

Background:

  • Somatic cell reprogramming using Oct4, Sox2, Klf4, and c-Myc (OSKM) generates induced pluripotent stem (iPS) cells and induced extraembryonic endoderm stem (iXEN) cells.
  • Both iPS and iXEN cells frequently co-exist during reprogramming, necessitating clear differentiation methods.
  • Accurate distinction between iPS and iXEN cells is crucial for selecting and analyzing specific cell populations in research.

Purpose of the Study:

  • To provide a comprehensive protocol for reprogramming mouse embryonic fibroblasts (MEFs) into stem cells using retroviral delivery of OSKM.
  • To detail methods for distinguishing between iXEN and iPS cells at both the colony and single-cell levels.
  • To ensure researchers can reliably identify and isolate desired stem cell types post-reprogramming.

Main Methods:

  • Retroviral transduction of mouse embryonic fibroblasts (MEFs) with reprogramming factors (Oct4, Sox2, Klf4, c-Myc).
  • Culturing reprogrammed cells to form colonies.
  • Morphological and molecular analysis of colonies and single cells for differentiation markers.

Main Results:

  • Successful reprogramming of MEFs using the OSKM retroviral system.
  • Establishment of distinct criteria for differentiating iXEN and iPS cell colonies based on morphology.
  • Identification of specific markers and techniques for single-cell level discrimination between iXEN and iPS cells.

Conclusions:

  • The OSKM reprogramming method reliably generates both iPS and iXEN cells from MEFs.
  • Clear protocols for distinguishing iXEN and iPS cells are essential for experimental validity.
  • This chapter offers a practical guide for researchers working with OSKM-induced stem cells.