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CRISPR/Cas9 Editing of Murine Induced Pluripotent Stem Cells for Engineering Inflammation‐Resistant Tissues
Author(s) -
Brunger Jonathan M.,
Zutshi Ananya,
Willard Vincent P.,
Gersbach Charles A.,
Guilak Farshid
Publication year - 2017
Publication title -
arthritis and rheumatology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 4.106
H-Index - 314
eISSN - 2326-5205
pISSN - 2326-5191
DOI - 10.1002/art.39982
Subject(s) - induced pluripotent stem cell , biology , crispr , flow cytometry , proinflammatory cytokine , cytokine , stem cell , cas9 , microbiology and biotechnology , inflammation , gene , immunology , embryonic stem cell , genetics
Objective Proinflammatory cytokines such as interleukin‐1 (IL‐1) are found in elevated levels in diseased or injured tissues and promote rapid tissue degradation while preventing stem cell differentiation. This study was undertaken to engineer inflammation‐resistant murine 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. Methods Targeted deletion of the IL‐1 receptor type I (IL‐1RI) gene in murine iPSCs was achieved using the RNA‐guided, site‐specific clustered regularly interspaced short palindromic repeat (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, histologic, and biomechanical assays after a 3‐day treatment with 1 ng/ml of IL‐1α. Results Three of 41 clones isolated possessed the IL‐1RI +/− genotype. Four clones possessed the IL‐1RI −/− genotype, and flow cytometry confirmed loss of IL‐1RI on the surface of these cells, which led to an absence of NF‐κB transcription activation after IL‐1α treatment. Cartilage engineered from homozygous null clones was resistant to cytokine‐mediated tissue degradation. In contrast, cartilage derived from wild‐type and heterozygous clones exhibited significant degradative responses, highlighting the need for complete IL‐1 blockade. Conclusion This work demonstrates proof‐of‐concept of the ability to engineer custom‐designed stem cells that are immune to proinflammatory cytokines (i.e., IL‐1) as a potential cell source for cartilage tissue engineering.