Protocol

Method for Single-Cell Sorting and Expansion of Genetically Modified Human Embryonic Stem Cells

Institute for Stem Cell Biology and Regenerative Medicine, Department of Obstetrics and Gynecology, Stanford University, Palo Alto, CA 94304-5542, USA

¹Corresponding author (reneer{at}stanford.edu)

INTRODUCTION

The potential for human embryonic stem cells (hESCs) to differentiate into all three embryonic germ layers, and the germline, can be used as a tool to understand the mechanisms of cell lineage-specific differentiation and development in vitro and in vivo. Therefore, it is important to develop technologies for the manipulation of hESCs and their effective utilization in research. One such manipulation is genetic modification. Reliable methods for genetic modification of hESCs with ubiquitous and tissue-specific reporters will assist in the identification and isolation of specific cell types following hESC differentiation. This protocol describes a method for hESC transduction with a ubiquitous eGFP reporter construct using self-inactivating lentivirus, followed by single-cell isolation and expansion of homogenous genetically modified hESCs using fluorescence-activated cell sorting (FACS).