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

TY - JOUR

T1 - Engineering fluorescent reporters in human pluripotent stem cells and strategies for live imaging human neurogenesis

AU - Dady, Alwyn

AU - Davidson, Lindsay

AU - Loyer, Nicolas

AU - Rappich, Sophie

AU - Findlay, Greg

AU - Sanders, Timothy

AU - Januschke, Jens

AU - Storey, Kate G.

N1 - © 2025. Published by The Company of Biologists

PY - 2025/11

Y1 - 2025/11

N2 - Investigation of cell behaviour and cell biological processes underlying human development is facilitated by the creation of fluorescent reporters in human pluripotent stem cells, 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-localised protein tagged with the fluorescent proteins eGFP or mKate2, the photoconvertible nuclear marker H2B-mEos3.2, or the cytoskeletal protein F-Tractin tagged with mKate2. Focussing on neural development, 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, allowing individual cell behaviours including neuronal delamination to be monitored. Finally, using the F-Tractin-mKate2 hiPSC line, actin dynamics were captured during proliferation in cortical neural rosettes. This study presents and validates new tools and techniques with which to interrogate human cell behaviour and cell biology using live-imaging approaches.

AB - Investigation of cell behaviour and cell biological processes underlying human development is facilitated by the creation of fluorescent reporters in human pluripotent stem cells, 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-localised protein tagged with the fluorescent proteins eGFP or mKate2, the photoconvertible nuclear marker H2B-mEos3.2, or the cytoskeletal protein F-Tractin tagged with mKate2. Focussing on neural development, 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, allowing individual cell behaviours including neuronal delamination to be monitored. Finally, using the F-Tractin-mKate2 hiPSC line, actin dynamics were captured during proliferation in cortical neural rosettes. This study presents and validates new tools and techniques with which to interrogate human cell behaviour and cell biology using live-imaging approaches.

KW - human pluripotent stem cells

KW - human spinal cord development

KW - human cortical development

KW - live cell imaging

KW - piggyBac-mediated fluorescent reporters

KW - PiggyBac-mediated fluorescent reporters

KW - Live cell imaging

KW - Human spinal cord development

KW - Human pluripotent stem cells

KW - Human cortical development

UR - https://www.scopus.com/pages/publications/105020433810

U2 - 10.1242/dev.205082

DO - 10.1242/dev.205082

M3 - Article

C2 - 40748212

SN - 0950-1991

VL - 152

JO - Development

JF - Development

IS - 21

M1 - dev205082

ER -