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

Publication ,  Journal Article
Brunger, JM; Zutshi, A; Willard, VP; Gersbach, CA; Guilak, F
Published in: Arthritis & rheumatology (Hoboken, N.J.)
May 2017

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.

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Published In

Arthritis & rheumatology (Hoboken, N.J.)

DOI

EISSN

2326-5205

ISSN

2326-5191

Publication Date

May 2017

Volume

69

Issue

5

Start / End Page

1111 / 1121

Related Subject Headings

  • Tissue Engineering
  • Receptors, Interleukin-1
  • NF-kappa B
  • Mice
  • Interleukin-1
  • Inflammation
  • Induced Pluripotent Stem Cells
  • Gene Expression Regulation
  • Gene Expression
  • Gene Editing
 

Citation

APA
Chicago
ICMJE
MLA
NLM
Brunger, J. M., Zutshi, A., Willard, V. P., Gersbach, C. A., & Guilak, F. (2017). CRISPR/Cas9 Editing of Murine Induced Pluripotent Stem Cells for Engineering Inflammation-Resistant Tissues. Arthritis & Rheumatology (Hoboken, N.J.), 69(5), 1111–1121. https://doi.org/10.1002/art.39982
Brunger, Jonathan M., Ananya Zutshi, Vincent P. Willard, Charles A. Gersbach, and Farshid Guilak. “CRISPR/Cas9 Editing of Murine Induced Pluripotent Stem Cells for Engineering Inflammation-Resistant Tissues.Arthritis & Rheumatology (Hoboken, N.J.) 69, no. 5 (May 2017): 1111–21. https://doi.org/10.1002/art.39982.
Brunger JM, Zutshi A, Willard VP, Gersbach CA, Guilak F. CRISPR/Cas9 Editing of Murine Induced Pluripotent Stem Cells for Engineering Inflammation-Resistant Tissues. Arthritis & rheumatology (Hoboken, NJ). 2017 May;69(5):1111–21.
Brunger, Jonathan M., et al. “CRISPR/Cas9 Editing of Murine Induced Pluripotent Stem Cells for Engineering Inflammation-Resistant Tissues.Arthritis & Rheumatology (Hoboken, N.J.), vol. 69, no. 5, May 2017, pp. 1111–21. Epmc, doi:10.1002/art.39982.
Brunger JM, Zutshi A, Willard VP, Gersbach CA, Guilak F. CRISPR/Cas9 Editing of Murine Induced Pluripotent Stem Cells for Engineering Inflammation-Resistant Tissues. Arthritis & rheumatology (Hoboken, NJ). 2017 May;69(5):1111–1121.
Journal cover image

Published In

Arthritis & rheumatology (Hoboken, N.J.)

DOI

EISSN

2326-5205

ISSN

2326-5191

Publication Date

May 2017

Volume

69

Issue

5

Start / End Page

1111 / 1121

Related Subject Headings

  • Tissue Engineering
  • Receptors, Interleukin-1
  • NF-kappa B
  • Mice
  • Interleukin-1
  • Inflammation
  • Induced Pluripotent Stem Cells
  • Gene Expression Regulation
  • Gene Expression
  • Gene Editing