Differentiation of nonhuman primate embryonic stem cells into hepatocyte‐like cells

Kuai, Xiao Ling; Shao, Nan; Lu, Hong; Xiao, Shu Dong; Zheng, Qing Yin

doi:10.1111/1751-2980.12103

Cited by 8 publications

(6 citation statements)

References 28 publications

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“…A previous ultrastructural study of mESCs has shown that there is a clear increase in the cytoplasmic volume when ESCs are differentiated as EBs; in addition, there is an increase in protein synthesis (Sampath et al, 2008). In addition, many other investigations have studied ultrastructural morphology of EBs, which differentiated into various committed cell types, including cardiomyocytes (Taha et al, 2012), endothelial cells (Festag et al, 2007), hepatocytes (Kuai et al, 2014), skeletal muscle cells (Kawagoe et al, 2011), pancreatic exocrine enzyme-producing cells (Shirasawa et al, 2011), and renal cells (Kramer et al, 2006).…”

Section: Introductionmentioning

confidence: 99%

Ultrastructural Characteristics of Three Undifferentiated Mouse Embryonic Stem Cell Lines and Their Differentiated Three-Dimensional Derivatives: A Comparative Study

Alharbi

Elsafadi²,

Mobarak³

et al. 2014

Cellular Reprogramming

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The fine structures of mouse embryonic stem cells (mESCs) grown as colonies and differentiated in three-dimensional (3D) culture as embryoid bodies (EBs) were analyzed by transmission electron microscopy. Undifferentiated mESCs expressed markers that proved their pluripotency. Differentiated EBs expressed different differentiation marker proteins from the three germ layers. The ultrastructure of mESCs revealed the presence of microvilli on the cell surfaces, large and deep infolded nuclei, low cytoplasm-to-nuclear ratios, frequent lipid droplets, nonprominent Golgi apparatus, and smooth endoplasmic reticulum. In addition, we found prominent juvenile mitochondria and free ribosomes-rich cytoplasm in mESCs. Ultrastructure of the differentiated mESCs as EBs showed different cell arrangements, which indicate the different stages of EB development and differentiation. The morphologies of BALB/c and 129 W9.5 EBs were very similar at day 4, whereas C57BL/6 EBs were distinct from the others at day 4. This finding suggested that differentiation of EBs from different cell lines occurs in the same pattern but not at the same rate. Conversely, the ultrastructure results of BALB/c and 129 W9.5 ESCs revealed differentiating features, such as the dilated profile of a rough endoplasmic reticulum. In addition, we found low expression levels of undifferentiated markers on the outer cells of BALB/c and 129 W9.5 mESC colonies, which suggests a faster differentiation potential.

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Section: Introductionmentioning

confidence: 99%

Ultrastructural Characteristics of Three Undifferentiated Mouse Embryonic Stem Cell Lines and Their Differentiated Three-Dimensional Derivatives: A Comparative Study

Alharbi

Elsafadi²,

Mobarak³

et al. 2014

Cellular Reprogramming

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“…Standard cryopreservation (FBS, 10% DMSO) of LIV0APOLY cells had no effect on the doubling time (or viability) of the cells; in contrast, primary hepatocytes are reported to be poorly resistant to cryopreservation, which affects cell viability, morphology, and functionality ( 21 ). We found that the LIV0APOLY cell line expressed defined hepatocyte markers, such as albumin and CK18 ( 22 , 26 , 32 , 54 ). Expression of albumin in two-dimensionally cultured primary hepatocytes has been shown to decrease after approximately 1 wk, whereas cultured LIV0APOLY cells retained high expression of albumin over 45 days and for at least 14 days following differentiation.…”

Section: Discussionmentioning

confidence: 97%

Characterization of lipid metabolism in a novel immortalized human hepatocyte cell line

Green

Johnson

Amin

et al. 2015

American Journal of Physiology-Endocrinology and Metabolism

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The development of hepatocyte cell models that represent fatty acid partitioning within the human liver would be beneficial for the study of the development and progression of nonalcoholic fatty liver disease (NAFLD). We sought to develop and characterize a novel human liver cell line (LIV0APOLY) to establish a model of lipid accumulation using a physiological mixture of fatty acids under low- and high-glucose conditions. LIV0APOLY cells were compared with a well-established cell line (HepG2) and, where possible, primary human hepatocytes. LIV0APOLY cells were found to proliferate and express some mature liver markers and were wild type for the PNPLA3 (rs738409) gene, whereas HepG2 cells carried the Ile148Met variant that is positively associated with liver fat content. Intracellular triglyceride content was higher in HepG2 than in LIV0APOLY cells; exposure to high glucose and/or exogenous fatty acids increased intracellular triglyceride in both cell lines. Triglyceride concentrations in media were higher from LIV0APOLY compared with HepG2 cells. Culturing LIV0APOLY cells in high glucose increased a marker of endoplasmic reticulum stress and attenuated insulin-stimulated Akt phosphorylation whereas low glucose and exogenous fatty acids increased AMPK phosphorylation. Although LIV0APOLY cells and primary hepatocytes stored similar amounts of exogenous fatty acids as triglyceride, more exogenous fatty acids were partitioned toward oxidation in the LIV0APOLY cells than in primary hepatocytes. LIV0APOLY cells offer the potential to be a renewable cellular model for studying the effects of exogenous metabolic substrates on fatty acid partitioning; however, their usefulness as a model of lipoprotein metabolism needs to be further explored.

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“…Additionally, these HLCs displayed specific characteristics that define a typical "hepatocyte", as determined by both qualitative assays and qualitative analysis. 50 Significantly, our hepatic differentiation protocol is distinct from that of other reports of non-human primates, namely rhesus macaque ESCs 42,43 and cynomolgus ESCs. 48,49 We have used a modified three-step procedure that was previously developed in our laboratory to differentiate porcine iPS cells into HLCs.…”

Section: Dovepressmentioning

confidence: 97%

“…Aravalli et al ESCs from human, rodent, porcine, and monkey species have been differentiated into HLCs. [33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49] In this study, we have carried out hepatic differentiation of marmoset ESCs into HLCs and characterized them using a combination of immunohistochemistry, morphological analyses, RT-PCR, and biochemical assays. Activin A-treated marmoset ESCs and primary human hepatocytes (PHHs) were used as controls.…”

Section: Dovepressmentioning

confidence: 99%

<p>Hepatic Differentiation of Marmoset Embryonic Stem Cells and Functional Characterization of ESC-Derived Hepatocyte-Like Cells</p>

Aravalli¹,

Collins²,

Hapke³

et al. 2020

HMER

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Background: Primary human hepatocytes (PHHs) are the ideal candidates for studying critical liver functions such as drug metabolism and toxicity. However, as they are isolated from discarded livers that are unsuitable for transplantation, they possess limited expansion ability in vitro and their enzymatic functions deteriorate rapidly because they are often of poor quality. Therefore, there is a compelling reason to find reliable alternative sources of hepatocytes. Methods: In this study, we report on efficient and robust differentiation of embryonic stem cells (ESC) from the common marmoset Callithrix jacchus into functional hepatocyte-like cells (HLC) using a simple, and reproducible three-step procedure. ESC-derived HLCs were examined by morphological analysis and tested for their expression of hepatocyte-specific markers using a combination of immunohistochemistry, RT-PCR, and biochemical assays. Primary human hepatocytes were used as controls. Results: ESC-derived HLCs expressed each of the hepatocyte-specific markers tested, including albumin; α-fetoprotein; asialoglycoprotein receptor 1; α-1 antitrypsin; hepatocyte nuclear factors 1α and 4; cytokeratin 18; hepatocyte growth factor receptor; transferrin; tyrosine aminotransferase; alkaline phosphatase; c-reactive protein; cytochrome P450 enzymes CYP1A2, CYP2E1 and CYP3A4; and coagulation factors FVII and FIX. They were functionally competent as demonstrated by biochemical assays in addition to producing urea. Conclusion: Our data strongly suggest that marmoset HLCs possess characteristics similar to those of PHHs. They could, therefore, be invaluable for studies on drug metabolism and cell transplantation therapy for a variety of liver disorders. Because of the similarities in the anatomical and physiological features of the common marmoset to that of humans, Callithrix jacchus is an appropriate animal model to study human disease conditions and cellular functions.

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Differentiation of nonhuman primate embryonic stem cells into hepatocyte‐like cells

Abstract: With cytokine induction, rhesus monkey ESC differentiated into cells displaying morphological features, gene expression patterns and metabolic activities characteristic of hepatocytes.

Cited by 8 publications

References 28 publications

Ultrastructural Characteristics of Three Undifferentiated Mouse Embryonic Stem Cell Lines and Their Differentiated Three-Dimensional Derivatives: A Comparative Study

Ultrastructural Characteristics of Three Undifferentiated Mouse Embryonic Stem Cell Lines and Their Differentiated Three-Dimensional Derivatives: A Comparative Study

Characterization of lipid metabolism in a novel immortalized human hepatocyte cell line

<p>Hepatic Differentiation of Marmoset Embryonic Stem Cells and Functional Characterization of ESC-Derived Hepatocyte-Like Cells</p>

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