CRISPR/Cas9 Editing of Murine Induced Pluripotent Stem Cells for Engineering Inflammation-Resistant Tissues.

Journal Article (Journal Article)


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.


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.


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.


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.

Full Text

Duke Authors

Cited Authors

  • Brunger, JM; Zutshi, A; Willard, VP; Gersbach, CA; Guilak, F

Published Date

  • May 2017

Published In

Volume / Issue

  • 69 / 5

Start / End Page

  • 1111 - 1121

PubMed ID

  • 27813286

Pubmed Central ID

  • PMC5406241

Electronic International Standard Serial Number (EISSN)

  • 2326-5205

International Standard Serial Number (ISSN)

  • 2326-5191

Digital Object Identifier (DOI)

  • 10.1002/art.39982


  • eng