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Abstract
SummaryThe 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. Analyzing 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 behavior. Further, we use single-cell RNA sequencing (scRNA-seq) of iCOUNT-labeled NSPCs and their progenies from the developing mouse cortex and forebrain-regionalized human organoids to identify functionally relevant molecular pathways that are commonly regulated between mouse and human cells, depending on individual cell division histories. Thus, we developed a 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.
Highlights
Somatic stem cell proliferation does not end with embryogenesis, as many tissues such as skin, intestines, the blood system, and the central nervous system continue to rely on somatic stem cells for tissue homeostasis and repair (Barker, 2014; Bianconi et al, 2013; Brack and Rando, 2012; Donati and Watt, 2015; Gage, 2019; Morrison and Spradling, 2008)
We first tested the inducible cell division counter (iCOUNT) system in mouse embryonic stem cells. mESCs with H3.1-tagged iCOUNT expressed red histones, and 48 hours after co-transfection of a Cre and Cre-reporter plasmid, 81% of Cre-reporter expressing cells switched to green histones (Figure S1A)
Fluorescent time-lapse imaging of iCOUNT expressing mESCs over a period of 64 hours (Figure 1C; Video S1) revealed that the red fluorescence dropped by half on every cell division and was refilled by green fluorescence (Figures 1D and 1E, left panels, and S1F), confirming the theoretically expected values (Figures 1D and 1E, right panels)
Summary
Somatic stem cell proliferation does not end with embryogenesis, as many tissues such as skin, intestines, the blood system, and the central nervous system continue to rely on somatic stem cells for tissue homeostasis and repair (Barker, 2014; Bianconi et al, 2013; Brack and Rando, 2012; Donati and Watt, 2015; Gage, 2019; Morrison and Spradling, 2008). A number of tools have been developed recently with the aim of recording single-cell biographies based on a variety of potential experiences, such as the previous activity of multiple signaling pathways or even complete transcriptional profiles (Farzadfard and Lu, 2018; Frieda et al, 2017; Schmidt et al, 2018). These approaches have been successful in the context of cultured cells and bacteria using genetic approaches that allow turning back time and looking into the past of individual cells (Frieda et al, 2017; Schmidt et al, 2018; Tang and Liu, 2018). Overexpression of H2B-GFP in vivo was reported to be leaky in quiescent stem cells, to be diluted independent of cell division, to be loaded non-homogenously
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