Engineering fluorescent reporters in human pluripotent cells and strategies for live imaging human neurogenesis

Abstract

Investigation of cell behaviour and cell biological processes in human embryonic tissues is facilitated by creation of fluorescent reporters in human pluripotent stem cell lines, which can be differentiated into cell types of choice. Here we report use of a piggyBac transposon-mediated stable integration strategy to engineer human pluripotent stem cell reporter lines. These express a plasma membrane (pm) localised protein tagged with the fluorescent protein eGFP or mKate2, the photoconvertible nuclear marker H2B-mEos3.2, with or without pm-mKate2, and the cytoskeletal protein F-tractin tagged with mKate2. Focussing on neural development some of these lines were used to live image and quantify cell behaviours, including cell cycle progression and cell division orientation in spinal cord rosettes. Further, lipofection-mediated introduction of piggyBac constructs into human neural progenitors labelled single cells and small cell groups within rosettes, allowed monitoring of individual cell behaviours including neuronal delamination. Finally, using the F-tractin-mKate2 hiPSC line, actin dynamics were captured during proliferation of cortical neural rosettes. This study presents new tools and techniques with which to interrogate human cell behaviour and cell biology using live imaging approaches.