Gene editing and reprogramming of human fibroblast cells (hFBs) to human pluripotent stem cells (hiPSCs).

Abstract

Predictive disease models play significant roles in advancing our knowledge of the pathology of human disease. In this field, animal models have been extensively employed and have provided crucial insights into the pathophysiological mechanisms of human disease. However, they often fail to fully capture many human phenotypes due to significant species differences in genomic responses. Human induced pluripotent stem cells (hiPSCs) are genetically reprogrammed cells that exhibit qualities remarkably similar to those of embryonic stem cells (ESC) and have emerged as a promising source for cell therapy and fundamental research in pathology. The ability to reprogram human fibroblast cells (hFBs) to iPSCs provides an opportunity to model human diseases. However, even hiPSCs from different persons have different genetic background, thus generation of isogenic unaffected control hiPSCs is necessary to study model human disease. Here, we describe methods to generate isogenic hFBs using CRISPR/Cas9 gene editing method, and subsequently reprogram them into iPSCs using commercially available Sendai virus vectors. Specifically, using the CRISPR/Cas9 system and Sendai virus vector, isogenic iPSC lines can be generated. This protocol provides a structured approach for obtaining multiple isogeneic hiPSC lines, which facilitate the modeling of various human diseases.