Hepatic differentiation of human pluripotent stem cells in miniaturized format suitable for high-throughput screen

Abstract

The establishment of protocols to differentiate human pluripotent stem cells (hPSCs) including embryonic (ESC) and induced pluripotent (iPSC) stem cells into functional hepatocyte-like cells (HLCs) creates new opportunities to study liver metabolism, genetic diseases and infection of hepatotropic viruses (hepatitis B and C viruses) in the context of specific genetic background. While supporting efficient differentiation to HLCs, the published protocols are limited in terms of differentiation into fully mature hepatocytes and in a smaller-well format. This limitation handicaps the application of these cells to high-throughput assays. Here we describe a protocol allowing efficient and consistent hepatic differentiation of hPSCs in 384-well plates into functional hepatocyte-like cells, which remain differentiated for more than 3 weeks. This protocol affords the unique opportunity to miniaturize the hPSCs-based differentiation technology and facilitates screening for molecules in modulating liver differentiation, metabolism, genetic network, and response to infection or other external stimuli.

Human stem cell-derived hepatocytes as a model for hepatitis B virus infection, spreading and virus-host interactions

“…The main challenges are to obtain HLCs exhibiting a mature and homogeneous level of differentiation and maintain their differentiation status in vitro . Here, we optimized our previous method to differentiate stem cells more efficiently into a homogeneous population of mature HLCs capable of retaining their phenotype for a prolonged period of time [8]. HLCs were obtained from hESCs or iPSCs (non-colony type monolayer-adapted) through a three-step differentiation protocol (Fig.…”

“…Non-colony monolayer type culture human embryonic stem cells (hESCs) H9 and iPSCs SC3D cell line have been described previously [7, 8] and were maintained on growth factor-reduced (GF(-)) Matrigel (0.4 mg/ml)(Corning, NY, USA) in mTeSR1 (StemCell Technologies, Vancouver, Canada). Cells were routinely passed 1 in 8 every 5–6 days with Accutase (Life Technologies, Frederick, USA) and reseeded with mTeSR1 medium in presence of 10 µM of Rock Inhibitor Y-27632 (Millipore, Billerica, USA) as described before [7, 8]. …”

Rhesus iPSC Safe Harbor Gene-Editing Platform for Stable Expression of Transgenes in Differentiated Cells of All Germ Layers

“…We adapted a recently published protocol that described the derivation of functional hepatocyte-like cells from human embryonic stem cells (ESCs) and iPSCs. 27,28 Although the kinetics of hepatic differentiation of RhiPSCs was similar to that of human iPSCs, we found that hypoxic culture (5% O 2 ) during definitive endoderm induction was necessary in RhiPSC differentiation ( Figure S7). After 4 days of differentiation, pluripotent genes were downregulated and RhiPSCs generated homogeneous definitive endoderm, which expressed SOX17 and FOXA2.…”

“…After 4 days, cells were transferred to a normoxic incubator and further differentiated to hepatoblasts in medium with 100 ng/ml hepatocyte growth factor and 1% DMSO for 8 days, as previously described. 27,28 Finally, cells were pushed toward hepatocyte-like cells with the addition of 10 À7 M dexamethasone for 3 additional days. At each time point, cells were analyzed by Taqman RT-qPCR and immunostaining as previously described.…”

Modeling Inborn Errors of Hepatic Metabolism Using Induced Pluripotent Stem Cells

“…Researchers’ ability to generate iPSCs from patients with inborn errors of hepatic metabolism and to use them to produce cells with hepatocyte characteristics in culture offers the possibility of using the cells for drug discovery 44 .…”