piggyBac Transposon Mediated Reprogramming and Flow Cytometry Analysis of CD44 and ICAM1 Cell-Surface Marker Changes

Abstract

Generation of iPSCs is inefficient and the molecular mechanisms underlying reprogramming are not well understood. While several studies have demonstrated that reprogramming is not entirely a random process and contains predictable stepwise changes, varying degrees of cellular heterogeneity that arise in different reprogramming systems can obscure the process. Among several reprogramming systems available, delivery of polycistronic reprogramming factor expression cassettes with piggyBac transposon into mouse embryonic fibroblasts (MEFs) is one of the simplest and most robust reprogramming approaches that provide a low background of partially reprogrammed cells. Using two novel cell surface markers, ICAM1 and CD44, clear cell population changes undergoing reprogramming can be observed over a time course upon induction of the reprogramming factors. Consequently, this technique allows for easy identification of factors that enhance or delay reprogramming, and can be a useful strategy in elucidating key mechanisms for efficient generation of iPSCs.

Keywords: CD44; Flow cytometry; ICAM1 (CD54); Induced pluripotent stem cells (iPSCs); Mouse embryonic fibroblasts (MEF); Nanog-GFP; Reprogramming; piggyBac transposon.