Morphologic and gene expression criteria for identifying human induced pluripotent stem cells

Area of Science:

• Stem Cell Biology
• Molecular Biology
• Cellular Morphology

Background:

• Induced pluripotent stem (iPS) cells are generated from somatic cells via forced expression of Oct3/4, Sox2, Klf4, and c-Myc.
• Currently, visual observation is used to identify iPS cells, lacking objective criteria for selection.
• This limitation hinders efficient and accurate iPS cell isolation and characterization.

Purpose of the Study:

• To establish objective criteria for identifying iPS cells.
• To analyze the morphology and gene expression of colonies generated during iPS cell induction.
• To identify reliable molecular markers for accurate iPS cell identification.

Main Methods:

• Analysis of 192-203 colonies from human fibroblasts transfected with four retroviral vectors (Oct3/4, Sox2, Klf4, c-Myc).
• Analysis of 192-199 colonies from human fibroblasts transfected with a single polycistronic retroviral vector encoding all four factors.
• Morphological categorization based on six parameters and gene expression analysis of emerged colonies.

Main Results:

• Morphologic features were categorized into seven subtypes (four vectors) and six subtypes (single vector), including iPS cell colonies.
• Essential iPS cell qualifications identified: single nucleolus, nucleus to nucleolus (N/Nls) ratio ~2.19, cell size ~43.5 µm², nucleus to cytoplasm (N/C) ratio ~0.87, cell density ~5900 cells/mm², and single cell layer.
• Endogenous Sox2 and Cdx2 were specifically expressed in iPS cells, unlike Oct3/4 and Nanog, which were found in other colonies.

Conclusions:

• Morphologic parameters provide objective criteria for classifying colonies during iPS cell induction.
• Endogenous Sox2 and Cdx2 expression are specific and reliable markers for accurate iPS cell identification.
• This study offers a robust method for distinguishing true iPS cells from other colony types.