Author:
Breau Keith A.,Ok Meryem T.,Gomez-Martinez Ismael,Burclaff Joseph,Kohn Nathan P.,Magness Scott T.
Abstract
AbstractBackground & Aims2D monolayers of primary intestinal and colonic epithelial cells represent next-generation in vitro models of the gut. Efficient transgenesis and gene-editing in human intestinal stem cells (hISCs) would significantly improve utility of these models by enabling generation of reporter and loss/gain-of-function hISCs, but no published methods exist for transfecting 2D hISC monolayers. Electroporation has proven effective in other difficult-to-transfect cells; thus we applied this method to hISCs.MethodsTwenty-four electroporation parameters were tested, and the optimal condition for efficiency and viability was validated on hISCs from six anatomical regions along the small intestine and colon. PiggyBac™ transposase and Cas9 ribonucleoprotein (RNP) complexes were used for stable genomic integration of reporter genes. High-throughput methods for clone isolation, expansion, and screening were developed. An hISC OLFM4-emGFP reporter was generated and validated by qPCR, organoid assays, and hISC compartmentalization on a planar crypt-microarray (PCM) device.ResultsMaximum electroporation efficiency was 79.9% with a mean survival of 65%. Transfection of 105 hISCs produced ∼142 (0.14%) stable transposase-mediated clones. Transfection of OLFM4-targetting RNPs yielded ∼35% editing and 99/220 (45%) of antibiotic-resistant colonies analyzed expressed emGFP. OLFM4-emGFP hISCs applied to PCMs remained emGFP+ and proliferative in high-Wnt3a/R-spondin3/Noggin zones yet differentiated to emGFP-/KRT20+ cells outside engineered crypt zones. OLFM4-emGFP levels correlated with endogenous OLFM4. Olfm4-emGFPhigh cells were LGR5high/KRT20low, and demonstrated high organoid-forming potential.ConclusionsElectroporation of hISCs is highly efficient for stable transgenesis and transgenic lines can be generated in 3-4 weeks. Workflows mirror conventional culture methods, facilitating rapid integration into established tissue-culture operations. OLFM4high is a robust hISC marker with functional properties in culture.
Publisher
Cold Spring Harbor Laboratory