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

Abstract

Objective 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. Methods 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. Results 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. Conclusion 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.

The potential of CRISPR/Cas9 genome editing for the study and treatment of intervertebral disc pathologies

“…In murine iPSCs subjected to chondrogenic differentiation, only complete homozygous CRISPR/Cas9 knockout of IL1R, but not heterozygous knockout, resulted in a cytokine‐resistant cartilage phenotype without chronic inflammation . Taking this approach further, an inflammation‐resistant cartilage with the ability to autonomously regulate its own inflammatory responses was recently engineered by CRISPR/Cas9 gene editing.…”

Designer Stem Cells: Genome Engineering and the Next Generation of Cell‐Based Therapies

“…Some of the first applications have been in the engineering of stem cells to allow for controlled attenuation of their detrimental responses to inflammatory cytokines, for example, IL‐1, tumor necrosis factor (TNF). For example, murine iPSCs have been engineered to harbor a functional deletion of the IL‐1 receptor I ( Il1r1 ) . These cells were capable of synthesizing a cartilaginous matrix that was protected against IL‐1‐mediated inflammation or tissue degradation, as measured by a decreased expression of proinflammatory genes and a reduced loss of proteoglycan content.…”

Genome Engineering for Personalized Arthritis Therapeutics

“…Proof-of-principle for this approach has been demonstrated by engineering murine pluripotent stem cells (PSCs) with functional deletion of the interleukin-1 receptor I ( Il1r1 ). [92] Knockout of Il1r1 resulted in iPSCs capable of producing articular cartilage resistant to IL-1α-mediated tissue degradation, measured from biochemical analysis of GAG content. [92] In other studies, epigenome editing – using dCas9-KRAB at loci encoding the cytokine receptors IL1R1 and TNFR1 – was used attenuate the catabolic response of human adipose stem cell (hASC)-derived chondrocyte-like cells to inflammatory environments.…”

“…[92] Knockout of Il1r1 resulted in iPSCs capable of producing articular cartilage resistant to IL-1α-mediated tissue degradation, measured from biochemical analysis of GAG content. [92] In other studies, epigenome editing – using dCas9-KRAB at loci encoding the cytokine receptors IL1R1 and TNFR1 – was used attenuate the catabolic response of human adipose stem cell (hASC)-derived chondrocyte-like cells to inflammatory environments. [93] Specifically, epigenetic silencing of IL1R1 and TNFR1 to their promoters (dCas9-KRAB targeting), mitigated the downstream activation of NF-κB, a transcription factor that initiates catabolic pathways; this protected cartilage matrix integrity in the presence of IL-1 or TNF.…”