|Title||CRISPR/Cas9 editing of induced pluripotent stem cells for engineering inflammation-resistant tissues.|
|Publication Type||Journal Article|
|Year of Publication||2016|
|Authors||Brunger, JM, Zutshi, A, Willard, VP, Gersbach, CA, and Guilak, F|
|Journal||Arthritis and Rheumatology|
Pro-inflammatory cytokines such as interleukin 1 (IL-1) are elevated in diseased or injured tissues and promote rapid tissue degradation while preventing stem cell differentiation. The goals of this study were to engineer inflammation-resistant induced pluripotent stem cells (iPSCs) through deletion of the IL-1 signaling pathway and to demonstrate the utility of these cells for engineering replacements for diseased or damaged tissues.Targeted deletion of the interleukin 1 receptor 1 (Il1r1) gene in murine iPSCs was achieved using the RNA-guided, site-specific CRISPR/Cas9 genome engineering system. Clonal cell populations with homozygous and heterozygous deletions were isolated, and loss of receptor expression and cytokine signaling was confirmed by flow cytometry and transcriptional reporter assays, respectively. Cartilage was engineered from edited iPSCs and tested for its ability to resist IL-1-mediated degradation in gene expression, histological, and biomechanical assays after a three day treatment with 1 ng/ml IL-1α.Three of 41 clones isolated possessed the Il1r1(+/-) genotype. Four clones possessed the Il1r1(-/-) genotype, and flow cytometry confirmed loss of Il1r1 on the surface of these cells and led to an absence of NF-κB transcriptional activation after IL-1α treatment. Cartilage engineered from homozygous null clones was resistant to cytokine-mediated tissue degradation. By contrast, cartilage derived from wild-type and heterozygous clones exhibited significant degradative responses, highlighting the need for complete IL-1 blockade.This work demonstrates proof-of-concept of the ability to engineer custom-designed stem cells that are immune to pro-inflammatory cytokines (i.e., IL-1) as a potential cell source for cartilage tissue engineering. This article is protected by copyright. All rights reserved.
|Short Title||Arthritis and Rheumatology|