Generation of In Vivo Traceable Hepatocyte-Like Cells from Human iPSCs.

Abstract

In this chapter, we describe a protocol for differentiation of human-induced pluripotent stem cells (iPSCs) into hepatocyte-like cells (HLCs) and their transduction with a lentivirus for gene transfer. Here, we engineer them to express the human sodium iodide symporter, which can be exploited as a radionuclide reporter gene, thereby enabling these cells to be tracked in vivo by single-photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging. Differentiation of HLCs from iPSCs involves three steps: induction of iPSCs to definitive endoderm, differentiation to a hepatic progenitor cell population, and maturation of immature HLCs. Once proliferation of hepatic progenitors has ceased and an immature HLC population is generated, lentiviral transduction can be performed. The immature hepatic gene expression profile/morphology at the stage of transduction will be compatible with further maturation following transgene expression either in vitro or in vivo, with expression of the transgene retained. We detail how transgenic cells can be imaged in vivo. While we provide a protocol for the NIS reporter gene, the cell engineering aspects of this protocol are transferable for use with other (reporter) genes if desired.