Generation of three isogenic gene-edited Huntington's disease human embryonic stem cell lines with DOX-inducible NGN2 expression cassette in the AAVS1 safe locus

Luisana Duque Villegas; Abinaya Chandrasekaran; Sofie Amalie Flintholm Andersen; Anne Nørremølle; Benjamin Schmid; Mahmoud A Pouladi; Kristine Freude

doi:10.1016/j.scr.2024.103408

Generation of three isogenic gene-edited Huntington's disease human embryonic stem cell lines with DOX-inducible NGN2 expression cassette in the AAVS1 safe locus

Stem Cell Res. 2024 Mar 28:77:103408. doi: 10.1016/j.scr.2024.103408. Online ahead of print.

Authors

Luisana Duque Villegas¹, Abinaya Chandrasekaran², Sofie Amalie Flintholm Andersen², Anne Nørremølle¹, Benjamin Schmid³, Mahmoud A Pouladi⁴, Kristine Freude⁵

Affiliations

¹ Dept. of Cellular and Molecular Medicine, The Panum Institute, The Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen 2200N, Denmark.
² Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, 1870 Frederiksberg, Denmark.
³ Bioneer A/S, Kogle Alle 2, 2970 Hørsholm, Denmark.
⁴ Department of Medical Genetics, Centre for Molecular Medicine and Therapeutics, Djavad Mowafaghian Centre for Brain Health, British Columbia Children's Hospital Research Institute, University of British Columbia, Vancouver, BC, Canada.
⁵ Department of Veterinary and Animal Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, 1870 Frederiksberg, Denmark. Electronic address: kkf@sund.ku.dk.

PMID: 38569398
DOI: 10.1016/j.scr.2024.103408

Abstract

Neurogenin 2 (NGN2), a neuronal transcription factor, can expedite differentiation of stem cells into mature glutamatergic neurons. We have utilized an allelic series of previously published and characterized isogenic Huntington's disease (IsoHD) human embryonic stem cell lines (Ooi et al., 2019), carrying different CAG repeat lengths in the first exon of the huntingtin gene. These IsoHDs were modified using CRISPR/Cas9 to insert NGN2 under the TET-ON doxycycline inducible promoter. The resulting IsoHD-NGN2 cell lines retained pluripotency in the absence of doxycycline (DOX), and via addition of DOX to the culturing media differentiation to neurons was achieved within 14 days.