Next-level metabolic disease modeling with Cellartis® hiPSC-derived hepatocytes

Unlike any other hepatic cell model, complete kits, representing three hiPSC lineages, provide an inexhaustible supply of functional, mature hepatocytes to support long-term studies.

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Nov 22, 2017
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Challenges in metabolic disease modeling

The molecular mechanisms of metabolic disease progression in the liver are not well understood, and in vitro disease models are crucial to their discovery. Predictive in vitro models should exhibit metabolic characteristics linked to hepatic insulin resistance, lipid metabolism, and accumulation of free fatty acids. Currently, primary hepatocytes are the most common cell model, but their utility is fundamentally constrained by the substantial variation between donors and the finite number of cells that are harvestable from each donor.

Hepatocytes differentiated from human induced pluripotent stem (hiPS) cells, which can be derived noninvasively from a diseased or healthy person, eliminate these constraints and provide an unlimited supply of functional hepatocytes from each parental cell line. This model is particularly amenable to gene editing technology, which makes it possible to design cells with specific disease-relevant mutations—a boon for those studying the cause and progression of metabolic diseases of the liver. However, hiPS cell-derived hepatocytes are only useful as an in vitro model system and for gene editing applications if they recapitulate critical hepatocyte functions.

Until recently, the functionality of hiPS cell-derived hepatocytes was insufficient for modeling metabolic processes. To address this problem, we developed a robust hepatocyte differentiation protocol—which promotes a highly synchronized differentiation pattern across multiple hiPS cell lines [1]—and a novel maintenance medium, which enables the long-term culture of our hiPS cell-derived hepatocytes. Cellartis enhanced hiPS-HEP cells display adult characteristics and have substantial drug-metabolizing functionality that can be maintained for two weeks. To create these cells, we terminally differentiated three hiPS cell lines (ChiPSC12, ChiPSC18, and ChiPSC22; abbreviated C12, C18, and C22) from different donors into three lines of Cellartis enhanced hiPS-HEP cells; one of these lines is included in each complete kit. With up to three renewable, reliable sources of cells, functional studies reflecting interindividual variation can be performed with confidence due to low batch-to-batch variability.  

A new model for interrelated metabolic disorders

Metabolic disorders, including nonalcoholic fatty liver disease (NAFLD), metabolic syndrome, type 2 diabetes, and obesity have reached epidemic proportions. These diseases are all related to dysfunction of the liver, which performs over 500 critical functions including lipid metabolism and blood glucose regulation.

NAFLD occurs when fat is deposited (steatosis) in the liver due to metabolic dysfunction rather than alcohol use. NAFLD can progress to nonalcoholic steatohepatitis (NASH) and ultimately cirrhosis. Patients with NAFLD can often develop insulin resistance, while patients with diabetes can develop NASH, demonstrating a distinct role of the liver in mediating complex interactions between these interrelated metabolic diseases [2].

Metabolic syndrome is defined as abdominal obesity and elevated blood pressure, plasma glucose, serum triglycerides, or low high-density-lipoprotein levels and can lead to type 2 diabetes. Inherited metabolic disorders, like familial hypercholesterolemia, alpha-1-antitrypsin deficiency, and glycogen storage diseases, involve defective genes that cause metabolic enzyme deficiencies and liver dysfunction. Understanding these hereditary and interrelated disorders will require better in vitro liver models that capture important physiological features of disease progression.

Human primary hepatocytes and immortalized cell lines (e.g., HepaRG) are used to study these disorders, but due to these cells’ high variability, low availability, short assay window, or lack of physiological relevance, researchers studying metabolic diseases are searching for more robust and reliable models. Cellartis enhanced hiPS-HEP cells make progress towards addressing these challenges because they display key characteristics of mature hepatocytes and principal features of functional glucose and lipid regulation over at least a 14-day assay window. Because they are derived from Cellartis hiPS cells, they sidestep the problems inherent to other currently used in vitro models to provide consistency and stability. Moreover, the enhanced hiPS-HEP cells respond to steatosis-inducing conditions with an inflammatory response that mimics progressing NAFLD. Further describing these findings, the following information shows why these cells are ideal for metabolic disease studies.    

Mature hepatocyte markers

Hepatocyte nuclear factor 4α (HNF4α) is a transcription factor required for liver development and the control of expression of liver-specific genes, and it is associated with several critical metabolic pathways [3]. Quantification of HNF4α immunostainings showed that over 90% of hiPS cell-derived hepatocytes differentiated using our protocol express HNF4α [4]. Another liver-specific protein, Asialoglycoprotein receptor 1 (ASGPR1), is a surface marker for mature hepatocytes [5, 6].

In our experiments, both HNF4α and ASGPR1 proteins are well-expressed in the enhanced hiPS-HEP cells from all three hiPS cell lines. High mRNA expression levels of HNF4α and ASGPR1 are shown in the enhanced hiPS-HEP cells for an extended culture time and are comparable to levels in cryopreserved human primary hepatocytes (hphep cells) that have been grown for only a short time.  

Functional characteristics of mature hepatocytes

Of the many functions performed by mature hepatocytes in vivo, synthesis and secretion of both albumin and urea are commonly used to evaluate hepatocyte in vitro models. Albumin regulates the oncotic pressure of blood, and low serum albumin levels can indicate liver cirrhosis or chronic hepatitis. Urea is a normal byproduct of protein breakdown by the liver, and impaired urea synthesis can also indicate liver dysfunction. Another characteristic used to evaluate hepatocyte models is the presence of functional alpha-1-antitrypsin (α1AT), a protease inhibitor produced in the liver. α1AT deficiency leads to chronic tissue breakdown and liver damage.

Albumin (below, top row) and α1AT (below, bottom row) are well-expressed in the enhanced hiPS-HEP cells from all three hiPS cell lines, as shown by immunostaining (and mRNA expression data, not shown). Notably, only a subset of cells is strongly immunopositive for albumin in the enhanced hiPS-HEP cells and in hphep cultures, in agreement with the metabolic zonation [7] observed in liver slices (below).


Additionally, the enhanced hiPS-HEP cells express genes involved in the urea cycle within the same range as hphep cells, and the cells secrete albumin and urea. Over 20 days, albumin secretion in the enhanced hiPS-HEP occurs at comparable levels as in hphep cells cultured for a short time. Urea secretion in the enhanced hiPS-HEP cells is lower than in hphep cells, but it increases over time.  

Functional glucose regulation

Hepatocytes carry out energy metabolism by regulating glucose production. When blood glucose levels are high, hepatocytes respond to insulin by increasing glycogen storage, decreasing gluconeogenesis, and decreasing glycogenolysis. Conversely, when blood glucose levels are low, hepatocytes respond to glucagon and glucocorticoids by decreasing glycogen storage and producing glucose via gluconeogenesis and glycogenolysis. In NAFLD, metabolic syndrome, and type 2 diabetes, hepatocytes become insulin resistant, and glucose builds up in the blood. A relevant hepatocyte model for metabolic diseases should demonstrate normal insulin response and functional glucose regulation. The enhanced hiPS-HEP cells respond to insulin with phosphorylation of protein kinase B-α (Akt), even at low insulin concentrations, and the genes involved in glycogen metabolism, gluconeogenesis, and insulin signaling are expressed at similar levels as in hphep cells. Furthermore, the enhanced hiPS-HEP cells can store glycogen. Notably, both in hphep cells and the enhanced hiPS-HEP cultures, only a subset of hepatocytes is strongly stained for glycogen storage (shown by Periodic acid-Schiff staining, below)—again in agreement with the metabolic zonation observed in the liver lobe.

Functional lipid metabolism

In a healthy person, the liver transports fatty acids, sterols, and lipoproteins into and out of the liver in response to feeding and fasting. In various disease states, metabolic processes are dysfunctional, and the liver begins to accumulate these substances. Normally, the liver regulates the level of cholesterol, which is carried to and from tissues by lipoproteins in the blood, by taking up low-density lipoproteins (LDL) and secreting very-low-density lipoproteins (VLDL) and high-density lipoproteins (HDL). Proprotein convertase subtilisin/kexin type 9 (PCSK9) is an enzyme that regulates plasma cholesterol homeostasis through its interaction with the LDL receptor (LDLR). When an LDL particle (carrying cholesterol) binds to the LDLR, the particle is trafficked into the hepatocyte, and PCSK9 targets the receptor for lysosomal degradation. If PCSK9 is blocked, the LDL receptor is recycled back into the cell membrane and can remove additional LDL particles from the extracellular fluid. Therefore, PCSK9 is an excellent target for clinical inhibitors that lower blood LDL concentration and therefore cholesterol levels; in fact, two different drugs were approved for this purpose by the US Food and Drug Administration in 2015. The enhanced hiPS-HEP cells express PCSK9 and other genes that regulate blood cholesterol levels, as well as principal genes involved in uptake, synthesis, and beta-oxidation of fatty acids. The enhanced hiPS-HEP cells express high levels of the LDL receptor and take up fluorescently labeled LDL (red, below).

Modeling NAFLD  

NAFLD is an imbalance between the uptake and removal of lipids in hepatocytes, which leads to abnormal triglyceride accumulation, or steatosis. When inflammation occurs in late-stage NAFLD, it is diagnosed as NASH. One in vitro NASH model uses a steatosis-inducing medium [8] to induce expression of TNFα, a marker of inflammation and fibrosis—like the physiologic response to steatosis in progressing NAFLD.

Enhanced hiPS-HEP cells accumulate triglycerides in lipid droplets and show and elevated level of TNFα mRNA when exposed to steatosis-inducing media. Taken together, these results indicate the suitability of enhanced hiPS-HEP cells for modeling NAFLD.

To view the complete data set for the findings presented here, please read our detailed technical note.

More information

Human iPS cell-derived hepatocytes differentiated with our robust differentiation protocol and cultured using our novel maintenance medium provide an inexhaustible, consistent supply of functional hepatocytes that can be used to advance the understanding of diseases related to dysfunction in liver metabolism, including NAFLD/NASH, type 2 diabetes, and metabolic syndrome. This innovative tool represents an important step toward advancing the discovery of new treatments for metabolic and infectious disease, reducing the incidence of drug-induced liver injury, and developing new strategies for liver regeneration and transplantation.

Watch a webinar on how you can use enhanced hiPS-HEP cells in your long-term studies.

References

  • Ghosheh, N. et al. Highly Synchronized Expression of Lineage-Specific Genes during In Vitro Hepatic Differentiation of Human Pluripotent Stem Cell Lines. Stem Cells Int. 2016, 1–22 (2016).

  • Tilg, H., Moschen, A. R. & Roden, M. NAFLD and diabetes mellitus. Nat. Rev. Gastroenterol. Hepatol. 14, 32–42 (2016).

  • Gonzalez, F. J. Regulation of hepatocyte nuclear factor 4 alpha-mediated transcription. Drug Metab. Pharmacokinet. 23, 2–7 (2008).

  • Asplund, A. et al. One Standardized Differentiation Procedure Robustly Generates Homogenous Hepatocyte Cultures Displaying Metabolic Diversity from a Large Panel of Human Pluripotent Stem Cells. Stem Cell Rev. Reports 12, 90–104 (2016).

  • Takayama, K. et al. Prediction of interindividual differences in hepatic functions and drug sensitivity by using human iPS-derived hepatocytes. Proc. Natl. Acad. Sci. U. S. A. 111, 16772–7 (2014).

  • Peters, D. T. et al. Asialoglycoprotein receptor 1 is a specific cell-surface marker for isolating hepatocytes derived from human pluripotent stem cells. Development 143, 1475–81 (2016).

  • Jungermann, K. & Kietzmann, T. Oxygen: Modulator of metabolic zonation and disease of the liver. Hepatology 31, 255–260 (2000).

  • Sahai, A. et al. Roles of phosphatidylinositol 3-kinase and osteopontin in steatosis and aminotransferase release by hepatocytes treated with methionine-choline-deficient medium. Am. J. Physiol. Gastrointest. Liver Physiol. 291, G55–62 (2006).
Go to the profile of Takara Bio USA, Inc

Takara Bio USA, Inc

Takara Bio USA, Inc. provides kits, reagents, and services that help researchers explore questions about gene discovery, regulation, and function. Our mission is to develop high-quality innovative tools and services to accelerate discovery.

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