318. Targeted Genome Editing of Human Induced Pluripotent Stem Cells Using CRISPR/CAS9 to Generate a Knock-in Type II Collagen Reporter for the Purification of Chondrogenic Cells

Abstract

INTRODUCTION: Differentiation of human induced pluripotent stem cells (hiPSCs) to articular chondrocytes may provide a strategy for engineering cartilage tissue for therapy and functional characterization of patient-specific cartilage. Development of robust protocols will be particularly important for therapeutic applications. However, the difficulty of generating a pure population of chondrogenic precursors has hindered development of such protocols. We sought to isolate a chondrogenic cell population by engineering a novel hiPSC line to express GFP downstream of the COL2A1 gene using CRISPR/Cas9. METHODS: Reporter hiPSCs lines were generated via transfection of guide RNA/Cas9-T2A-GFP expression vector and COL2A1-P2A-GFP targeting vector (Fig 1AFig 1A). Targeted clones were identified by junction PCR. Reporter validation was performed with lentiviral SOX9 overexpression and recombinant TGF-β3 treatment to activate COL2A1 expression. Cells were imaged with fluorescence microscopy at Days 4, 7, and 10. GFP+ and GFP− cells were sorted at Day 14 using fluorescence activated cell sorting (FACS). Gene expression from cell populations was analyzed with qRT-PCR. Reporter lines were differentiated to chondrogenic precursors via ectomesenchymal specification and TGF-β3 treatment. RESULTS: COL2A1-P2A-GFP reporter hiPSC lines were generated with CRISPR/Cas9 (Fig 1A/BFig 1A/B). Imaging after COL2A1 activation demonstrated the presence of GFP signal only in treated cells (Fig 1CFig 1C). Clonally derived cell lines displayed varied expression of GFP as measured by FACS (Fig 1DFig 1D). GFP signal was highly correlated with COL2A1 gene expression (r2=0.95) (Fig 1EFig 1E). GFP+ cells were enriched for chondrogenic markers such as COL2A1 and ACAN (p<0.05) (Fig 2AFig 2A). After differentiation to chondrogenic precursors, 4.5% of cells were GFP+ (Fig 2BFig 2B). This GFP+ population was enriched for COL2A1 transcript (Fig 2CFig 2C), suggesting isolation of a chondrogenic population. DISCUSSION: We show the characterization of a knock-in COL2A1 hiPSC reporter cell line using CRISPR/Cas9. The reporter functions specifically during COL2A1 upregulation and may be useful as a surrogate metric with wide dynamic range and high specificity for determining COL2A1 expression. The GFP+ population is enriched for chondrocyte-specific markers, namely COL2A1 and ACAN. The reporter enriches for COL2A1 expression after multi-staged differentiation, suggesting its utility for tissue engineering applications.View Large Image | Download PowerPoint SlideView Large Image | Download PowerPoint Slide