CRISPR/Cas9 editing to generate a heterozygous COL2A1 p.G1170S human chondrodysplasia iPSC line, MCRIi019-A-2, in a control iPSC line, MCRIi019-A

Kung, Louise H. W.; Sampurno, Lisa; Yammine, Kathryn M.; Graham, Alison; McDonald, Penny; Bateman, John F.; Shoulders, Matthew D.; Lamandé, Shireen R.

doi:10.1016/j.scr.2020.101962

Cited by 10 publications

(10 citation statements)

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“…1 A , and Figs. S 1 and S 2 ) (Kung et al, 2020). These lines, along with the isogenic wild-type control, allow us to unambiguously assess the effect of the Gly1170Ser on collagen-II proteostasis – meaning its synthesis, folding, localization, and function – and how it affects the cells that express it and the matrix they deposit.…”

Section: Resultsmentioning

confidence: 99%

“…Generating the gene edited lines from ATCC fibroblast line 1502 is covered by the Human Research Ethics Committee of the Royal Children’s Hospital, Victoria, Australia (HREC #33118). 42 iPSC lines MCRIi019-A (wild-type control) and MCRIi019-A-2 ( COL2A1 p.Gly1170Ser heterozygote) and the homozygote variant iPSCs were grown in a feeder-free manner on Matrigel (Corning)-coated 6-well plates in Essential 8 (E8) medium (ThermoFisher Scientific). Media was changed daily.…”

Section: Methodsmentioning

confidence: 99%

“…S1 and S2). 42 These lines, along with the isogenic wild-type control, allow us to unambiguously assess the effect of the Gly1170Ser on collagen-II proteostasis -meaning its synthesis, folding, localization, and function -and how it affects the cells that express it and the matrix they deposit. We began by differentiating all three iPSC lines (wild-type, heterozygous Gly1170Ser, homozygous Gly1170Ser) to chondrocytes using the protocol recently described by Lamandé et al 35 Briefly, we expanded iPSCs in feeder-free monolayer culture, then guided them through differentiation to sclerotome by growth factor treatment over the course of 4 days.…”

Section: Cells Expressing Gly1170ser-substituted Collagen-ii Deposit ...mentioning

confidence: 99%

“…Recently, we reported the generation of iPSC lines harboring the heterozygote 42 or homozygote c.3508 GGT > AGT, p.Gly1170Ser mutation in COL2A1 , created via CRISPR/Cas9 gene editing of a wild-type, isogenic control line ( Fig. 1 , and Fig.S1 ).…”

Section: Introductionmentioning

confidence: 99%

“…The inherent ability of stem cells to proliferate makes these systems highly expandable and thus much more amenable for biochemistry and drug discovery than mouse models. Methods for producing cartilage in vitro in the correct developmental context, combined with gene editing technology to create genetically matched cell lines incorporating disease-causing mutations (Hosseini Far et al, 2019; Howden et al, 2019; Kung et al, 2020; Lilianty et al, 2021; Lilianty et al, 2020; Yammine et al, 2023), provide an exceedingly promising avenue for modeling, studying, and discovering potential therapies for genetic skeletal disorders, including the type-II collagenopathies.…”

Section: Introductionmentioning

confidence: 99%

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ER procollagen storage defect without coupled unfolded protein response drives precocious arthritis

Yammine,

Abularach,

Kim

et al. 2023

Preprint

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Collagenopathies are a group of clinically diverse disorders caused by defects in collagen folding and secretion. For example, mutations in the gene encoding collagen type-II (COL2A1), the primary collagen in cartilage, can lead to chondrodysplasias of various severities. One example is the Gly1170Ser substitution in procollagen-II, which causes precocious osteoarthritis and Legg-Calvé-Perthes disease. Here, we develop and characterize a novel induced pluripotent stem cell-based cartilage model of this disease, including both hetero- and homozygous genotypes. Biochemical characterization reveals that Gly1170Ser procollagen-II is notably slow to fold and secrete. Procollagen-II accumulates intracellularly, consistent with an endoplasmic reticulum (ER) storage disorder. Intriguingly, perhaps due to the pathologic substitution occurring within a triple-helical domain that lacks hydrophobic character, this ER protein accumulation is not recognized by cellular stress responses, such as the unfolded protein response. Interactome studies show that Gly1170Ser procollagen-II interacts to a greater extent with certain ER chaperones and modifying enzymes than wild-type, consistent with its slow folding. These findings provide mechanistic elucidation into the etiology of this disease. Moreover, the cartilage model developed here provides a valuable platform to rapidly screen and develop therapeutic strategies that can restore procollagen folding and secretion in this collagenopathy and others.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%