Protocol for directed differentiation of human pluripotent stem cells toward a hepatocyte fate

Cai, Jun; DeLaForest, Ann; Fisher, Joseph B.; Urick, Amanda; Wagner, Thomas E.; Twaroski, Kirk; Cayo, Max A.; Nagaoka, Masato; Duncan, Stephen A.

doi:10.3824/stembook.1.52.1

Cited by 28 publications

(28 citation statements)

References 30 publications

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“…109,110 Hepatocyte growth factor and oncostatin M are the most commonly used cytokines for hepatic maturation. 109,111,112 Although various modifications of cytokine concentration, application periods, and combinations were investigated, all of the protocols so far show that the hepatocyte-like cells (HLC) obtained still show an immature phenotype with reduced hepatic functionality when compared to PHH. 113,114 Approaches for the improvement of hepatic maturation.…”

Section: Human Pluripotent Stem Cellsmentioning

confidence: 99%

“…However, Matrigel, which is of biological origin with batch-to-batch variations, is still commonly used for the coating of culture vessels. 117,134,137 To enable better defined culture conditions, Matrigel was replaced in a number of studies by defined matrix components such as E-cadherin, 111,141 vitronectin, 142 or laminin. 107 A further possibility to increase reproducibility is to substitute recombinant growth factors with small molecules during the hepatic differentiation of pluripotent stem cells.…”

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confidence: 99%

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Cell sources forin vitrohuman liver cell culture models

Zeilinger

Freyer

Damm

et al. 2016

Exp Biol Med (Maywood)

178

160

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In vitro liver cell culture models are gaining increasing importance in pharmacological and toxicological research. The source of cells used is critical for the relevance and the predictive value of such models. Primary human hepatocytes (PHH) are currently considered to be the gold standard for hepatic in vitro culture models, since they directly reflect the specific metabolism and functionality of the human liver; however, the scarcity and difficult logistics of PHH have driven researchers to explore alternative cell sources, including liver cell lines and pluripotent stem cells. Liver cell lines generated from hepatomas or by genetic manipulation are widely used due to their good availability, but they are generally altered in certain metabolic functions. For the past few years, adult and pluripotent stem cells have been attracting increasing attention, due their ability to proliferate and to differentiate into hepatocyte-like cells in vitro. However, controlling the differentiation of these cells is still a challenge. This review gives an overview of the major human cell sources under investigation for in vitro liver cell culture models, including primary human liver cells, liver cell lines, and stem cells. The promises and challenges of different cell types are discussed with a focus on the complex 2D and 3D culture approaches under investigation for improving liver cell functionality in vitro. Finally, the specific application options of individual cell sources in pharmacological research or disease modeling are described.

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Section: Human Pluripotent Stem Cellsmentioning

confidence: 99%

mentioning

confidence: 99%

Cell sources forin vitrohuman liver cell culture models

Zeilinger

Freyer

Damm

et al. 2016

Exp Biol Med (Maywood)

178

160

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“…To confirm hepatic differentiation tendency of hPSC H9, KhES4, KhES3, and 201B7 maintained under KSR-based culture conditions, and 19-9-7T and its aberrant clone maintained under mTeSR1 culture conditions were differentiated toward hepatocyte-like cells according to previously reported differentiation protocol [31,60–62] with minor modifications. Briefly, dissociated hPSCs were cultured onto Matrigel Basement Membrane Matrix Growth Factor Reduced (Corning) in the MEF-conditioned medium for 3–4 days.…”

Section: Methodsmentioning

confidence: 99%

“…To confirm hepatic differentiation tendency of hPSC, H9, 253G1B1, and 201B7 after culturing in hESF-FX medium for more than five passages, the hPSCs were differentiated toward hepatocyte-like cells according to another previously reported differentiation protocol [31,60,61] with minor modifications. In brief, hPSCs dissociated with Accutase were seeded at a density of 600,000 cells per well in 24-well plates precoated with 300 μL/well Geltrex (Thermo Fisher Scientific) and cultured with mTeSR1 medium (Stemcell Technologies, Vancouver, Canada) in 4% O 2 /5% CO 2 .…”

Section: Methodsmentioning

confidence: 99%

Prediction of Differentiation Tendency Toward Hepatocytes from Gene Expression in Undifferentiated Human Pluripotent Stem Cells

Yanagihara

Liu

Kanie

et al. 2016

Stem Cells and Development

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Functional hepatocytes derived from human pluripotent stem cells (hPSCs) have potential as tools for predicting drug-induced hepatotoxicity in the early phases of drug development. However, the propensity of hPSC lines to differentiate into specific lineages is reported to differ. The ability to predict low propensity of hPSCs to differentiate into hepatocytes would facilitate the selection of useful hPSC clones and substantially accelerate development of hPSC-derived hepatocytes for pharmaceutical research. In this study, we compared the expression of genes associated with hepatic differentiation in five hPSC lines including human ES cell line, H9, which is known to differentiate into hepatocytes, and an hPSC line reported with a poor propensity for hepatic differentiation. Genes distinguishing between undifferentiated hPSCs, hPSC-derived hepatoblast-like differentiated cells, and primary human hepatocytes were drawn by two-way cluster analysis. The order of expression levels of genes in undifferentiated hPSCs was compared with that in hPSC-derived hepatoblast-like cells. Three genes were selected as predictors of low propensity for hepatic differentiation. Expression of these genes was investigated in 23 hPSC clones. Review of representative cells by induction of hepatic differentiation suggested that low prediction scores were linked with low hepatic differentiation. Thus, our model using gene expression ranking and bioinformatic analysis could reasonably predict poor differentiation propensity of hPSC lines.

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“…Although there remain many issues to be improved, some iPSC differentiation protocols are relatively straightforward, and have been successfully used by multiple groups to obtain mesoderm, [42][43][44] endoderm 45 and ectodermal [46][47][48] lineages. These protocols utilize available materials, the procedures are uncomplicated, the methods include simple cell purification steps such as sorting, and their reproducibility and usefulness have been demonstrated by other investigators.…”

Section: Practical Considerationsmentioning

confidence: 99%

A practical guide to induced pluripotent stem cell research using patient samples

Santostefano

Hamazaki

Biel

et al. 2015

Laboratory Investigation

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Approximately 3 years ago, we assessed how patient induced pluripotent stem cell (iPSC) research could potentially impact human pathobiology studies in the future. Since then, the field has grown considerably with numerous technical developments, and the idea of modeling diseases 'in a dish' is becoming increasingly popular in biomedical research. Likely, it is even acceptable to include patient iPSCs as one of the standard research tools for disease mechanism studies, just like knockout mice. However, as the field matures, we acknowledge there remain many practical limitations and obstacles for their genuine application to understand diseases, and accept that it has not been as straightforward to model disorders as initially proposed. A major practical challenge has been efficient direction of iPSC differentiation into desired lineages and preparation of the large numbers of specific cell types required for study. Another even larger obstacle is the limited value of in vitro outcomes, which often do not closely represent disease conditions. To overcome the latter issue, many new approaches are underway, including three-dimensional organoid cultures from iPSCs, xenotransplantation of human cells to animal models and in vitro interaction of multiple cell types derived from isogenic iPSCs. Here we summarize the areas where patient iPSC studies have provided truly valuable information beyond existing skepticism, discuss the desired technologies to overcome current limitations and include practical guidance for how to utilize the resources. Undoubtedly, these human patient cells are an asset for experimental pathology studies. The future rests on how wisely we use them.

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Protocol for directed differentiation of human pluripotent stem cells toward a hepatocyte fate

Cited by 28 publications

References 30 publications

Cell sources forin vitrohuman liver cell culture models

Cell sources forin vitrohuman liver cell culture models

Prediction of Differentiation Tendency Toward Hepatocytes from Gene Expression in Undifferentiated Human Pluripotent Stem Cells

A practical guide to induced pluripotent stem cell research using patient samples

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