Differentiation of human embryonic stem cells into hepatocytes in 2D and 3D culture systems in vitro
Open accessAbstract
Human embryonic stem cells (hESCs) have enormous potential as a source of cells for cell replacement therapies and as a model for early human development. In this study we examined the differentiating potential of hESCs into hepatocytes in two- and three-dimensional (2D and 3D) culture systems. Embryoid bodies (EBs) were inserted into a collagen scaffold 3D culture system or cultured on collagen-coated dishes and stimulated with exogenous growth factors to induce hepatic histogenesis. Immunofluorescence analysis revealed the expression of albumin (ALB) and cytokeratin-18 (CK-18). The differentiated cells in 2D and 3D culture system displayed several characteristics of hepatocytes, including expression of transthyretin, alpha-1-antitrypsin, cytokeratin 8, 18, 19, tryptophan-2,3-dioxygenase, tyrosine aminotransferase, glucose-6-phosphatase (G6P), cytochrome P450 subunits 7a1 and secretion of alpha-fetoprotein (AFP) and ALB and production of urea. In 3D culture, ALB and G6P were detected earlier and higher levels of urea and AFP were produced, when compared with 2D culture. Electron microscopy of differentiated hESCs showed hepatocyte-like ultrastructure, including glycogon granules, well-developed Golgi apparatuses, rough and smooth endoplasmic reticuli and intercellular canaliculi. The differentiation of hESCs into hepatocyte-like cells within 3D collagen scaffolds containing exogenous growth factors, gives rise to cells displaying morphological features, gene expression patterns and metabolic activities characteristic of hepatocytes and may provide a source of differentiated cells for treatment of liver diseases.
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Cells in a 3D culture environment differ in gene, protein, and cell receptor expression from 2D-cultured cells.
“In addition to the previously mentioned differences in physical and physiological properties, researchers have also found that cells in a 3D culture environment differ in gene, protein, and cell receptor expression from 2D-cultured cells. 8-10”
From Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors (2014) · cites this paper as [8]
The discussion (page not available in our copy) reports earlier ALB and G6P gene expression in 3D than in 2D culture. The immunocytochemical staining section (page not available) compares ALB- and CK-18-positive cells across the two systems, but the paper does not establish a general difference in protein expression, and the passages found do not address cell receptor expression. (LLM, conf=0.86)
Experimental — accuracy not yet measuredCells cultured in three-dimensional arrangements exhibit notable differences in morphology and physiology compared to cells grown in monolayers.
“cells cultured in three-dimensional arrangements exhibit notable differences in morphology and physiology compared to cells grown in monolayers [188,189,190].”
From Modeling Tumor Microenvironment Complexity In Vitro: Spheroids as Physiologically Relevant Tumor Models and Strategies for Their Analysis (2025) · cites this paper as [190]
Cells in the 3D culture environment differ morphologically and physiologically from cells in the 2D culture environment.
“In fact, research has found that cells in the 3D culture environment differ morphologically and physiologically from cells in the 2D culture environment. 8-10”
From Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors (2014) · cites this paper as [8]
Cells in a 3D culture differ morphologically and physiologically from their counterparts in a 2D setup.
“In fact, cells in a 3D culture differ morphologically and physiologically from their counterparts in a 2D setup (Baharvand et al., 2006; Edmondson et al., 2014).”
From In Vitro research Reproducibility: Keeping up High Standards (2019) · cites this paper as Baharvand et al., 2006
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- Authors
- Baharvand, H., Hashemi, S. M., Kazemi Ashtiani, S., & Farrokhi, A.
- Affiliations
- Royan Institute (IR)
- Corresponding author
- Baharvand, Hossein
- Venue
- The International Journal of Developmental Biology
- Publisher
- EHU Press
- Published
- 2006
- Volume
- 50
- Issue
- 7
- Pages
- 645-652
- ISSN
- 0214-6282 (ISSN-L)
- OpenAlex
- W2065122365
- Cited by
- 429
- References
- 29
- Citations by year
- 2026: 3 · 2025: 8 · 2024: 13 · 2023: 20 · 2022: 15 · 2021: 25 · 2020: 27 · 2019: 27 · 2018: 39 · 2017: 29 · 2016: 27 · 2015: 26 · 2014: 19 · 2013: 17 · 2012: 34
- FWCI
- 6.38
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- The International Journal of Developmental Biology (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Pluripotent Stem Cells Research
- Health Sciences › Medicine › Genetics › Mesenchymal stem cell research
- Health Sciences › Medicine › Surgery › Tissue Engineering and Regenerative Medicine
- Keywords
- human embryonic stem cells, hepatocyte differentiation, embryoid bodies, albumin, alpha-fetoprotein, urea synthesis, glucose-6-phosphatase, hepatocyte-like cells, cell replacement therapy
- MeSH terms
- Cell Differentiation, Cell Line, Humans, Immunohistochemistry, Molecular Sequence Data, Stem Cells, Cell Culture Techniques, Hepatocytes
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