Sucessfull #transduction of #crypt cells and #organoids with #Magnetofection

HMGA1 amplifies Wnt signalling and expands the intestinal stem cell compartment and Paneth cell niche

Lingling Xian et al. Nature Communications, 2017.

Efficient transduction of crypt cells and mouse organoids from isolated crypts of the proximal small intestine with ViroMag R/L magnetic nanoparticles

Abstract

High-mobility group A1 (Hmga1) chromatin remodelling proteins are enriched in intestinal stem cells (ISCs), although their function in this setting was unknown. Prior studies showed that Hmga1 drives hyperproliferation, aberrant crypt formation and polyposis in transgenic mice. Here we demonstrate that Hmga1 amplifies Wnt/β-catenin signalling to enhance self-renewal and expand the ISC compartment. Hmga1 upregulates genes encoding both Wnt agonist receptors and downstream Wnt effectors. Hmga1 also helps to ‘build’ an ISC niche by expanding the Paneth cell compartment and directly inducing Sox9, which is required for Paneth cell differentiation. In human intestine, HMGA1 and SOX9 are positively correlated, and both become upregulated in colorectal cancer. Our results define a unique role for Hmga1 in intestinal homeostasis by maintaining the stem cell pool and fostering terminal differentiation to establish an epithelial stem cell niche. This work also suggests that deregulated Hmga1perturbs this equilibrium during intestinal carcinogenesis.

Lentivirus and transduction

For all lentiviral transductions, we modified an established protocol65 using magnetic nanoparticles (ViroMag R/L, OZ Bioscience, Inc.) and a magnetic plate (ViroMag R/L, OZ Bioscience, Inc., catalogue number: MF10000) to transduce crypt cells and organoids. Organoid fragments were seeded with 150 μl transduction medium into 48-well plates. Virus was added with viroMag R/L solution for 15 min at room temperature (2,500–3,000 virus particles per cells) to cells. The cell culture plate was placed on the magnetic plate for 30–60 min in a 37 °C tissue culture incubator. Cells were then incubated overnight at 37 °C. Organoid fragments and transduction media were then transferred to a 1.5 ml tube for centrifugation at 900 g for 5 min. The supernatant was discarded and tubes containing the pellet was placed on ice for 5 min. Next, 120 μl of matrigel was added and the pellet was resuspended by pipetting slowly up and down. Drops (30 μl) of basement matrix–cell mixtures were seeded into a new 48-well plate and incubated at 37 °C for 5–15 min until the basement matrix solidified. Media (ENRWntNic)6 was then added to each well and placed into a tissue culture incubator. Common ENR media6 was used and changed every 2–3 days beginning 4–6 days after the transduction.