Genetic Engineering of Human Embryonic Stem Cells for Precise Cell Fate Tracing during Human Lineage Development

Abstract

Summary: It is highly desirable to specify human developmental principles in an appropriate human model with advanced genetic tools. However, genetically engineering human cells with lineage-tracing systems has not been achieved. Here we introduce strategies to construct lineage-tracing systems in human embryonic stem cells (hESCs). The AAVS1 locus was suitable for the integration of the conditional reporter. The Cre-LoxP and Flp-FRT systems were highly sensitive, which may cause inaccurate lineage labeling in human cells. The recombination sensitivity and tracing fidelity could be finely tuned by modification of the LoxP recombination site. Moreover, tamoxifen-controllable CreERT2-LoxP and FlpERT2-FRT systems showed compelling advantages in tightly tracing human lineages temporally. In proof-of-principle experiments, we traced human PAX6+ neuroectoderm cells and revealed their full neural lineage differentiation potency both in vitro and in vivo. Devising and optimizing of lineage-tracing systems in hESCs will thus set up a solid foundation for human developmental studies. : In this article, Zhang and colleagues successfully constructed lineage-tracing systems in human pluripotent stem cells. Multiple strategies were designed in order to shaping the fidelity of the lineage-tracing system for studying human development both in vitro and in vivo. Tracing PAX6-expressing neuroectoderm identifies its full neural lineage potency in generating all spectrum of regional neural progenitors. Keywords: human embryonic stem cells, lineage-tracing, genetic engineering, development, PAX6, FOXA2