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. 2017:1639:151-160.
doi: 10.1007/978-1-4939-7163-3_15.

Protocol for Directed Differentiation of Human Induced Pluripotent Stem Cells (iPSCs) to a Hepatic Lineage

Joseph E Kaserman  1 , Andrew A Wilson  2
Affiliations

Protocol for Directed Differentiation of Human Induced Pluripotent Stem Cells (iPSCs) to a Hepatic Lineage

Joseph E Kaserman et al. Methods Mol Biol. 2017.

Abstract

Directed differentiation is a powerful cell culture technique where developmental pathways are applied to a pluripotent progenitor in order to generate specific terminally differentiated cell populations. Here, we describe a serum-free protocol using growth factors in defined concentrations to derive iPSC-hepatic cells starting from both feeder and feeder-free conditions. The generated iPSC-hepatic cells are developmentally similar to fetal stage hepatocytes, and when generated from patients with genetic mutations such as alpha-1 antitrypsin deficiency recapitulate pathologic changes associated with clinical disease, such as protein misfolding, intracellular retention of misfolded proteins, and elevated levels of ER stress.

Keywords: Definitive endoderm; Directed differentiation; Induced pluripotent stem cell (iPSC); iPSC-hepatic cell.

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Figures

Fig. 1
Fig. 1
Intracellular AAT Accumulation in PiZZ Alpha-1 Antitrypsin Deficiency iPSC-Hepatic Cells. Flow cytometry of fixed, permeabilized iPSC-hepatic cells using anti-AAT and anti-FOXA1 antibodies demonstrates a significantly increased level of intracellular AAT protein in PiZZ iPSC-hepatic cells compared to wild-type PiMM cells. This is quantified by the main fluorescence intensity (MFI) which approximates the amount of stained intracellular AAT protein
Fig. 2
Fig. 2
Morphological Changes at Key Time Points during iPSC-hepatic Cell Directed Differentiation. The addition of the modal analogue Activin A to undifferentiated cells helps drive them to definitive endoderm. By day 5, cells no longer form tight distinct colonies but instead have informed a confluent monolayer. Following passaging and treatment with high dose BMP4, HGF, and dexamethasone, the cells begin to assume features typical of hepatocytes, and with further maturation by day 25 have the classic polygonal morphology
Fig. 3
Fig. 3
Day 5 Definitive Endoderm Staining. Flow cytometry of day 5 iPSCs using anti-ckit and anti-CXCR4 antibodies demonstrates highly efficient induction of definitive endoderm
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