Determining cell division symmetry through the dissection of dividing cells using single-cell expression analysis

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• Cell division can be symmetric, producing identical sister cells, or asymmetric, producing distinct cells.
• Previous assessments of cell division symmetry relied on limited markers or functions.
• A comprehensive molecular comparison of sister cells has been lacking.

Purpose of the Study:

• To develop and validate a protocol for separating sister embryonic stem cells (ESCs).
• To enable single-cell, multiplexed gene expression analysis of sister ESCs.
• To provide a molecular basis for understanding cell division symmetry.

Main Methods:

• Micromanipulation to isolate dividing, paired sister ESCs.
• Single-cell lysis, reverse transcription, and gene-specific cDNA amplification.
• Multiplexed quantitative PCR (qPCR) for analyzing 48 ESC genes.

Main Results:

• A 10-day protocol for separating and analyzing sister ESCs was established.
• The protocol allows for detailed molecular comparison of sister cells at the gene expression level.
• The method is adaptable to other cell types in suspension culture.

Conclusions:

• This protocol offers a robust method for dissecting molecular differences between sister cells.
• It provides novel insights into the uniformity and variability of cells originating from the same division.
• The technique has broad applicability in stem cell research and developmental biology.