Visualization of individual cell division history in complex tissues

The division potential of individual stem cells and the molecular consequences of successive rounds of proliferation remain largely unknown. Here, we developed an inducible cell division counter (iCOUNT) that reports cell division events in human and mouse tissues in vitro and in vivo. Analysing cell division histories of neural stem/progenitor cells (NSPCs) in the developing and adult brain, we show that iCOUNT can provide novel insights into stem cell behaviour. Further, we used single cell RNA-sequencing (scRNA-seq) of iCOUNT-labelled NSPCs and their progenies from the developing mouse cortex and forebrain-regionalized human organoids to identify molecular pathways that are commonly regulated between mouse and human cells, depending on individual cell division histories. Thus, we developed a novel tool to characterize the molecular consequences of repeated cell divisions of stem cells that allows an analysis of the cellular principles underlying tissue formation, homeostasis, and repair. RNA sequencing of i) single cells isolated from human iCount forebrain organoids, ii) single nuclei isolated from embryonic cortices of iCount mice and iii) bulk WT and iCount mouse ESCs. For the human single cell dataset, human forebrain organoids were injected with Cre (1μg/μl) and electroporated using the AMAXA Nucleofector device. 4 and 7 days after electroporation (39 days in culture), cells were dissociated. Single cells were isolated by flow cytometry and prepared for scRNA-seq according to the Smart-Seq2 protocol. For the mouse single nuclei dataset, tamoxifen (180mg/kg) was injected into pregnant H3.1-iCOUNT x ROSA26:CreERT2 mice at E13.5. 38h later, embryonic cortices were dissected and the nuclei were dissociated. Single nuclei were isolated by flow cytometry and prepared for snRNA-seq according to the Smart-Seq2 protocol. For the mouse bulk RNA-seq dataset, three independent biological replicates of WT and iCount mESCs were collected using TrypLE washed and resuspended in DPBS containing RNAse inhibitor. RNA extraction was performed using the PureLink RNA Mini Kit according to the manufacturer’s protocol. PolyA selection was performed and libraries were prepared with the TruSeq RNA library prep kit (Illumina).