AI Article Synopsis
- Researchers have developed 2D cultures of human intestinal stem cells (hISCs) that offer alternatives to traditional 3D organoid systems.
- Electroporation has been successfully used on hISCs from various anatomical regions, achieving 80% transfection and allowing for gene editing and transgenesis.
- The study created a specific OLFM4-emGFP knock-in model and validated its utility in assessing hISC function, particularly highlighting that smaller hISCs with higher OLFM4 levels are the most effective at forming organoids.
Article Abstract
Two-dimensional (2D) cultures of intestinal and colonic epithelium can be generated using human intestinal stem cells (hISCs) derived from primary tissue sources. These 2D cultures are emerging as attractive and versatile alternatives to three-dimensional organoid cultures; however, transgenesis and gene-editing approaches have not been developed for hISCs grown as 2D monolayers. Using 2D cultured hISCs we show that electroporation achieves up to 80% transfection in hISCs from six anatomical regions with around 64% survival and produces 0.15% transgenesis by PiggyBac transposase and 35% gene edited indels by electroporation of Cas9-ribonucleoprotein complexes at the OLFM4 locus. We create OLFM4-emGFP knock-in hISCs, validate the reporter on engineered 2D crypt devices, and develop complete workflows for high-throughput cloning and expansion of transgenic lines in 3-4 weeks. New findings demonstrate small hISCs expressing the highest OLFM4 levels exhibit the most organoid forming potential and show utility of the 2D crypt device to evaluate hISC function.
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| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC9213823 | PMC |
| http://dx.doi.org/10.1016/j.stemcr.2022.04.005 | DOI Listing |
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