By micro-dissecting dividing cells and combining multiplexed qPCR analyses, the researchers were able to examine the similarity between murine embryonic stem cells (mESCs) for the first time.
So far, the team has processed over 500 single sister cells and conducted multiplexed gene expression analyses using 48 ESC genes.
While single cells have been isolated previously by conventional sorting methods, the new technique is the only way to know whether two single cells were derived from a common parental cell.
Their study, published in Nature Protocols today, explains how they separated paired embryonic stem cells, which was covered in a news story in September.
Their method used Nocodazole to enrich dividing sister cells, a phase contrast microscope to identify viable pairs and capillary-based micro-dissection to isolate living single cells from cell culture.
Their method leaves the isolated sister ESCs viable, allowing for various functional analyses to be performed. The cells can be processed to conduct gene expression analyses or other single cell assay methodologies such as analysing DNA methylation.
The method is also applicable for isolating other types of suspension cells, which could help determine the similarity between sister haematopoietic precursor cells at the molecular level.
Dr Tomoyuki Sawado, leader of the Stem Cells and Chromatin Team at The Institute of Cancer Research, said: “Our multiplexed assay system provides a novel experimental platform to evaluate cell division symmetry at single cell levels. The system will help to identify mechanisms that cause epigenetic diversity, which emerges as an important problem in stem cell biology and also for effective cancer treatment.”
- Jasnos L, Sawado T, (2014) Determining cell division symmetry through the dissection of dividing cells using single cell expression analysis,Nat. Protoc, doi:10.1038/nprot.2014.032