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Quantitative Modeling of Metabolically Mature Na-Butyrate Induced Hepatocyte-like Cells from Embryonic Stem Cells

Nripen S. Sharma1, Marianthi Ierapetritou2, Rene S. Schloss3, and Martin L. Yarmush3. (1) Center for Engineering in Medicine, Shriner Burns Hospital, Massachussets General Hospital, Harvard Medical School, 51 Blossom Street, Boston, MA 02114, (2) Chemical & Biochemical Engineering, Rutgers University, 98 Brett Road, Piscataway, NJ 08854, (3) Biomedical Engineering, Rutgers University, 98, Brett Road, C-005,Engineering Building, Piscataway, NJ 08854

Embryonic stem (ES) cells serve as a promising technology to obtain specific cell types for a number of biomedical applications. Because traditional techniques, such as embryoid body formation result in a wide array of differentiated cells of hepatic, neuronal and cardiac lineages, strategies have been utilized which favor cell-specific differentiation to generate more uniformity. In the present study, we have investigated the use of sodium butyrate in a monolayer culture configuration to mediate hepatocyte-specific differentiation of murine embryonic stem cells. Several functional assays used to characterize hepatocyte function (viz. urea secretion, intracellular albumin content, cytokeratin 18 (CK18) staining and glycogen staining) were used to analyze the differentiating cell population, suggesting the presence of an enriched population of hepatocyte-like cells. Since mature hepatocytes mediate energy metabolism predominantly through oxidative means as opposed to hepatocyte precursors which are primarily glycolytic, we have performed a dynamic analysis of the glycolytic and functional capacity to characterize the differentiated cells. In conjunction with mitochondrial mass and activity measurements, we show that hepatocyte lineage cells mediate energy metabolism predominantly through glycolysis and thus represent an immature hepatocyte phenotype from an energetic standpoint (1). This metabolic and mitochondrial characterization can assist in evaluating hepatocyte differentiation and may prove useful in identifying key regulatory molecules in mediating further differentiation.

In order to mediate further differentiation, we have utilized key regulatory molecules for inducing mitochondrial development in the precursor populations. S-nitrosoacetyl penicillamine (SNAP), a nitric oxide donor, has been implicated in mitochondrial development in various cell lines (2). Since hepatocyte-like cells derived from embryonic stem cells are predominantly glycolytic as opposed to mature hepatocytes that are highly aerobic in nature, hepatocyte-like cells were exposed to 50,100,250 and 500 μM SNAP concentrations for 4 days. We have shown that 250 μM SNAP increased mitochondrial mass and activity, two components implicated in mitochondrial biogenesis on the 4th day of secondary culture. In addition, functional analysis revealed that hepatocyte functional characteristics, viz. urea secretion and intracellular albumin is maintained in the induced population. A morphological analysis showed that the metabolically mature cells were more granular and cuboidal as compared to the non-induced cells. This study is the only report that shows hepatic maturation from a metabolic standpoint in cells derived from embryonic stem cells.

In order to quantify the energetic and functional capacity of the differentiated cells, we have developed a reaction network model and utilized the tools of metabolic engineering to further analyze the system. This analysis investigates the development of an energetic model comprising of the glycolytic and oxidative phosphorylation pathway which (a) serves as a preliminary step in obtaining a comprehensive metabolic network for ES derived hepatocyte like cells and (b) provides a systematic means of assessing the effects of mitochondrial development inducers on intracellular metabolite fluxes. The energetic model consists of 21 intracellular metabolites and 25 fluxes. The assumptions for the simplified network are as follows: 1. No transport and biosynthetic fluxes are considered in the network. 2. Amongst the amino acids, only glutamine and glutamate uptake and metabolic reactions are considered for energy production and 3. Fatty acid oxidation pathway is not considered. In order to determine the intracellular fluxes under experimental conditions of mitochondrial induction, Metabolic Flux Analysis (MFA) (3) is utilized as a solution methodology. MFA consists of estimating intracellular fluxes under different environmental conditions based on extracellular metabolite measurements and mass balances. Using matrix notations, the model can be represented as a 20x25 matrix. Since in addition to 21 equations, we have 6 measured fluxes, viz. glucose, lactate, glutamine, glutamate, NH3 and urea; this leads to an over-determined system. The additional measurements are used for consistency testing and gross error detection.

As a comparison study, mature hepatocyte cultures are used for generating a metabolic network. This model reveals the numerical values of the metabolic fluxes that should be ideally present in the ES cell derived hepatocyte-like cells. Three different experimental conditions are considered viz. mature hepatocytes, Na-butyrate treated hepatocyte-like cells and Na-butyrate + SNAP induced hepatocyte-like cells. Thus, MFA can be utilized to examine the effects of different inducers on the intermediary metabolism of hepatocyte-like cells. This analysis decouples the effect of Na-butyrate and SNAP on metabolism and hence identify specific metabolic pathways altered by the reagents.

In addition to the metabolic network model, we have utilized mathematical programming techniques to optimize the biochemical environment of hepatocyte-like cell cultures towards the desired effect of increased albumin and urea synthesis. The relevance of the metabolic pathways have been investigated using previously established logic based programming techniques which determines which reactions are more important for maintaining hepatocyte function (4). This analysis helps in generating an energetic model for metabolically induced hepatocyte-like cells. Overall, the model is a preliminary step in quantifying the effects of SNAP on energetics and function of embryonic stem cell derived hepatocyte-like cells. This is the only report involving quantification of the effects of different inputs on the intermediary metabolism of hepatocyte-like cells derived from embryonic stem cells which can also be utilized for identifying potential targets for further inducing differentiated function.


1. Sharma NS, Shikhanovich R, Schloss R, Yarmush ML. Sodium butyrate treated embryonic stem cells yield hepatocyte-like cells expressing a glycolytic phenotype. Biotechnol Bioeng. 2006 Apr 7; [Epub ahead of print]. 2. Nisoli E, Clementi E, Paolucci C, Cozzi V, Tonello C, Sciorati C, Bracale R, Valerio A, Francolini M, Moncada S, Carruba MO. Mitochondrial biogenesis in mammals: the role of endogenous nitric oxide. Science. 2003 Feb 7; 299(5608):896-9. 3. Chan C, Berthiaume F, Lee K, Yarmush ML. Metabolic flux analysis of cultured hepatocytes exposed to plasma. Biotechnol Bioeng. 2003 Jan 5; 81(1):33-49. 4. Sharma NS, Ierapetritou MG, Yarmush ML. Novel quantitative tools for engineering analysis of hepatocyte cultures in bioartificial liver systems. Biotechnol Bioeng. 2005 Nov 5; 92(3):321-35.