# Patent

## Title
Method for producing cells having characteristics of hepatocytes

## Abstract

The present invention relates to a method of producing cells having at least one characteristic of human hepatocytes as well as to cells produced by said method and uses of these cells.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application is a U.S. National Stage entry under 35 U.S.C. §371 of International Application No. PCT/EP2011/063871, filed on 12 Aug. 2011, designating the United States of America and published in English on 16 Feb. 2012, which in turn claims priority to European Patent Application No. 10172639.6, filed on 12 Aug. 2010, each of which is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
The present invention relates to a method of producing cells having at least one characteristic of human hepatocytes as well as to cells produced by said method and uses of these cells.
BACKGROUND OF THE INVENTION
The liver is an organ with a variety of functions such as glycogen storage, plasma protein synthesis, hormone production and detoxification and is thus necessary for survival. The cells of the main tissue of the liver which are responsible for most of the functions of the liver are called hepatocytes.
Human hepatocytes can be used for example for cell transplantation in acute and chronic liver failure. Furthermore, human hepatocytes are used by the pharmaceutical industry in vitro in pre-clinical drug testing and high throughput screening to determine whether a certain drug will exert deleterious effects on the human body. However, the use of primary hepatocytes is limited by the restricted availability of liver tissue, the early phenotypic changes occurring in cultured primary hepatocytes and their limited life span. Furthermore, primary hepatocytes exhibit large variability in CYP activities and in magnitude of induction after incubation with prototypical inducers. Hence, results obtained with primary hepatocytes may not be reproducible to the extent required for in vitro pre-clinical drug testing.
Efforts have thus been made to develop alternatives to the use of primary hepatocytes. One alternative is the use of immortalized cell lines such as HepG2 and HepaRG cells (Javitt (1990) The FASEB Journal 4: 161-168; Aninat et al. (2006) Drug Metabolism and Disposition 24: 75-83; Cerec et al. (2007) Hepatology 45: 957-967). However, many of these cell lines are of tumorigenic origin and do not retain the normal hepatocellular metabolizing capacities, so that their applicability in pharmacology and toxicology and even more so their applicability in vivo is greatly limited.
Other approaches use adult stem cells from bone marrow or induced pluripotent stem cells which are differentiated to hepatocytes (Fausto (2004) Hepatology 39(6): 1477-1487; Si-Tayeb et al. (2010) Hepatology 51(1): 297-305). However, these approaches require a complex isolation of the starting material and a complex differentiation protocol as well as a manipulation of the genome of these cells.
Finally, cells having characteristics of human hepatocytes, called “neo-hepatocytes”, have been obtained from peripheral monocytes by first treating the monocytes with a mixture of 5 ng/ml M-CSF and 0.4 ng/ml IL-3 for six days and subsequently treating the cells with 3 ng/ml FGF-4 for at least seven days (Ruhnke et a. (2005) Gastroenterology 128(7): 1774-1786; Ruhnke et al. (2005) Transplantation 79(9): 1097-1103; Ehnert et al. (2008) Drug Metab. Dispos. 36(9): 1922-1929). However, later it has been shown that these cells most closely resemble a population of serum-deactivated macrophages and it was concluded that although these cells exhibit some phenotypic characteristics of hepatocytes, these characteristics are acquired as part of a normal program of macrophage differentiation (Riquelme et al. (2009) Differentiation 77(3): 263-276). Furthermore, the differentiation protocol leading to “neo-hepatocytes” showed only a low reproducibility.
Hence, there is still a need for a method for producing cells having at least one characteristic of human hepatocytes with high reproducibility and a high cell yield and cells obtained by such a method.
OBJECT AND SUMMARY OF THE INVENTION
It is thus an object of the present invention to provide a method for producing cells having at least one characteristic of human hepatocytes which method is reproducible to a large extent and produces cells with a high yield.
It is a further object of the present invention to provide cells having at least one characteristic of human hepatocytes which cells are suitable for cell transplantation and in vitro drug testing.
These and further objects of the invention, as will become apparent from the description, are attained by the subject-matter of the independent claims.
Some of the preferred embodiments of the present invention form the subject-matter of the dependent claims.
The inventors of the present invention have found that the treatment of human peripheral monocytes with IL-3 and adenosine and subsequently with FGF-4 reproducibly produces cells having characteristics of human hepatocytes with a high cell yield. These cells are capable of undergoing apoptosis when treated with an apoptosis-inducing substance such as CD95 ligand. Furthermore, these cells show long-term expression of cytochrome enzymes and thus allow the determination of not only the acute toxicity, but also of the long-term toxicity of substances in an in vitro test. Furthermore, the inventors were able to show that the cells produced by the method of the present invention show differences in cytochrome expression depending on the gender of the donor of the peripheral monocytes, thus also facilitating investigation of gender-dependent differences in drug metabolism.
Accordingly, in one embodiment, the present invention provides a method for producing cells having at least one characteristic of human hepatocytes, said method comprising the steps of:
(a) seeding human monocytes in a culture vessel in a cell culture medium;
(b) culturing the monocytes in a cell culture medium comprising interleukin 3 (IL-3) and adenosine; and
(c) culturing the cells simultaneously with or subsequent to step (b) in a cell culture medium comprising fibroblast growth factor 4 (FGF-4).
In a preferred embodiment of the present invention the cell culture medium in step (b) further comprises macrophage colony-stimulating factor (M-CSF).
In a further preferred embodiment of the present invention the adenosine is present in the cell culture medium of step (b) in a concentration of 10-300 nM.
In still another preferred embodiment of the present invention, the cell culture medium in step (c) further comprises at least one compound selected from the group consisting of epidermal growth factor (EGF), glucagon, insulin and heparin, and, in an even more preferred embodiment of the present invention, the cell culture medium in step (c) comprises EGF, glucagon, insulin and heparin.
In a further preferred embodiment of the present invention, the cell culture medium in step (a), (b) and/or (c) is serum-free, and, in an even more preferred embodiment, the cell culture medium comprises albumin.
In a further embodiment of the present invention, the method further comprises the step of changing the cell culture medium at a time point less than one hour after seeding the human monocytes.
In still another embodiment of the present invention, the cell culture medium volume in step (b) and/or (c) is 40 to 90 μl/cm2 of the culture vessel.
In one embodiment of the present invention, the cells are cultured in step (b) of the method of the present invention for at least four days.
In still another embodiment of the present invention, the cells are cultured in step (c) of the method of the present invention for at least seven days.
The present invention further provides cells having at least one characteristic of human hepatocytes, which cells are obtainable by the method of the present invention and which can be kept in culture for at least 15 days.
Preferably, hepatocytes isolated from human liver are excluded.
More preferably, the cells are capable of undergoing apoptosis when incubated with a CD95-ligating antibody for 24 hours.
The present invention is further directed to the use of the cells of the present invention for in vitro toxicity studies or in cell therapy.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1: Influence of different culture conditions on protein content (a) and DPP-IV, γ-GT, ALT and LDH enzyme activities (b) after 14 days of culture of monocytes

    
    
        Lane 1: Cells established by the protocol described in Ruhnke et al. (2005) Gastroenterology 128(7): 1774-1786 called “neo-hepatocytes”
        Lane 2: Cells established according to the protocol of Ruhnke et al., but with reduced medium volume (about 70 μl/cm2), shorter adhesion time (about 20 minutes), medium without β-mercaptoethanol, 1 ng/ml IL-3 and 0.5 ng/ml M-CSF
        Lane 3: Conditions as in Lane 2, but with DMEM/Ham's F12 as basal medium, which further comprises 100 nM adenosine in the first culturing step
        Lane 4: Culture conditions as described in Lane 3, wherein the cell culture medium of the second culturing step additionally comprises 10 ng/ml recombinant human EGF, 0.28 IU recombinant human insulin, 5 nM recombinant human glucagon and 5 IU/ml heparin
    
    


FIG. 2: Influence of serum-free cultivation in 2% human albumin on different hepatocytic characteristics of the cells obtained by the method of the present invention after 14 days of culture of monocytes

    
    
        (a) Specific enzyme activities of DPP-IV, γ-GT, ALT and LDH in the cells of the present invention cultured under standard (std; i.e. 10% FCS) and serum-free conditions for the entire culture period, i.e. steps b) and c) of the method of the present invention (n=4 donors)
        (b) APAP toxicity in the cells of the present invention cultured under standard (i.e. 10% FCS) and serum-free conditions and incubated with different concentrations of APAP for 24 hours, as determined by release of LDH as a marker of cell damage (n=4 donors)
        (c) Induction of cytochrome CYP1A2 activity by treatment with 1 μM 3MC (3-methyl cholanthrene) for 48 hours in the cells of the present invention cultured under standard (std; i.e. 10% FCS) and serum-free (HA) conditions (n=3 donors)
        (d) Secretion of Factor VII in response to stimulation with different concentrations of menadione for 48 h in the cells of the present invention cultured under standard (i.e. 10% FCS) and serum-free conditions for different periods
        (e) Urea synthesis in the cells of the present invention cultured for 14 days under standard (i.e. 10% FCS) and serum-free conditions without (control) and with 100 μM NH4Cl for 24 hours
        (f) Morphology of the cells of the present invention cultured under standard (i.e. 10% FCS; upper picture) and serum-free conditions (lower picture)
    
    


FIG. 3: Induction of apoptosis in the cells of the present invention

    
    
        (a) Induction of apoptosis as measured by nuclear condensation visualized by HOECHST 33342 as marker of apoptosis in the cells of the present invention cultured for 21 days after incubation with different concentrations of the CD95 binding antibody APO for 24 hours
        (b) Caspase-3 activity in the cells of the present invention cultured for different periods and treated for 24 hours with different concentrations of the CD95 binding antibody APO.
    
    


FIG. 4: Comparison of hepatocyte-specific characteristics between the cells of the present invention (Metaheps) and primary human hepatocytes (pHH)

    
    
        (a) Urea synthesis normalized on cell protein (determined photometrically using the QuantiChromeTM Urea Assay Kit) under basal conditions and after treatment with 100 μM NH4Cl in primary human hepatocytes (culture day 4) and cells of the present invention (day 14) derived from the same donor
        (b) Induction of cytochrome CYP3A activity by treatment with 50 rifampicin for 24 hours in primary human hepatocytes (culture day 6) and cells of the present invention cultured for 14 days wherein the primary human hepatocytes and the cells of the present invention are derived from the same donor
        (c) Specific enzyme activities of DPP-IV, γ-GT (GGT) and ALT in primary human hepatocytes (culture day 4) and cells of the present invention cultured for 14 days, wherein the primary human hepatocytes and the cells of the present invention are derived from the same donor
    
    


FIG. 5: Comparison of the gene expression pattern between the cells of the present invention (Metaheps, MH) and primary human hepatocytes

    
    
        a) Correlation of gene expression patterns in Metaheps on culture days 3, 7 and 14 and primary human hepatocytes (culture day 4) of the same donor
        X- and Y-axis are log2 gene-expression normalized on beta-actin.
        b) Adaption of gene-expression in Metaheps to primary hepatocyte gene expression (on day 4 of culture) on culture days 3 (upper panel), day 7 (middle panel) and day 14 (lower panel). The darkest part shows the percentage of genes with very good adaptation, while the lightest part shows the percentage of genes which were not detectable.
        c) Comparison of gene groups coding for genes with specific physiological functions in Metaheps (culture day 14) and primary human hepatoctyes (culture day 4)
    
    


FIG. 6: Comparison of the cells of the present invention (culture day 14) with primary human hepatocytes (culture day 4) and/or neo-heaptocytes (neoheps) (culture day 14) established by the method described in Ruhnke et al. (2005) Gastroenterology 128(7): 1774-1786

    
    
        (a) Induction of apoptosis as measured by staining the cells with propidium iodide and subsequent FACS analysis in cells of the present invention (Metaheps) and NeoHeps derived from the same donor 500 ng/ml APO for 24 hours
        (b) Factor VIII synthesis in cells of the present invention (Metaheps), primary hepatocytes and NeoHeps (normalized to protein, presented as % of primary hepatocytes)
        (c) Factor VII synthesis in cells of the present invention (Metaheps), primary hepatocytes and NeoHeps (normalized to protein, presented as % of primary hepatocytes)
        (d) Inhibition of CYP1A2 activity in cells of the present invention (MH) cultured under standard (i.e. 10% FCS) and serum-free conditions and in NeoHeps (NH) by treatment of the cells with 500 μM caffeine as determined by the fluorescent substrate 3CEC
    
    


FIG. 7: Long term stability of CYP3A activities inducible by treatment with 10 μM phenyloin for 48 h in cells of the present invention from culture day 14 to 35 determined by using the luminescent P450 Glo assay (Promega).
FIG. 8: Long term toxicity in cells of the present invention cultured for 15 days in low concentrations of (a) diclofenac (50 μM) and (b) acetaminophen (500 μM) as measured by LDH activity
FIG. 9: Gender differences in activities of CYP1A2, 2C9 and 3A4 in cells of the present invention isolated from either male or female humans on culture day 14 determined by the luminescent method; “+” behind male or female means the activity after induction with 1 μM 3-methylcholanthrene for CYP1A2 and 50 μM rifampicin for CYP3A4 and CYP2C9



DETAILED DESCRIPTION OF THE INVENTION
Before describing in detail exemplary embodiments of the present invention, the following general definitions are provided.
The present invention as illustratively described in the following may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.
The present invention will be described with respect to particular embodiments but the invention is not limited thereto but only by the claims.
Where the term “comprising” is used in the present description and claims, it does not exclude other elements. For the purposes of the present invention, the term “consisting of” is considered to be a preferred embodiment of the term “comprising of”. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group which preferably consists only of these embodiments.
For the purposes of the present invention, the term “obtained” is considered to be a preferred embodiment of the term “obtainable”. If hereinafter e.g. a cell is defined to be obtainable by a specific method, this is also to be understood to disclose a cell which is obtained by this method.
Where an indefinite or definite article is used when referring to a singular noun, e.g. “a”, “an” or “the”, this includes a plural of that noun unless something else is specifically stated. The terms “about” or “approximately” in the context of the present invention denote an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. The term typically indicates deviation from the indicated numerical value of ±10%, and preferably of ±5%.
The term “human hepatocytes” means cells of the main tissue of the human liver which are involved in protein synthesis and storage, transformation of carbohydrates, synthesis of cholesterol, bile salts and phospholipids as well as detoxification of exogenous and endogenous substances. Primary human hepatocytes in culture usually display a hexagonal morphology and establish confluent cell layers including intercellular contacts made by gap junctions and an epithelial phenotype as determined by the expression of epithelial marker proteins such as cytokeratin 18 and pan-actin. Primary human hepatocytes, i.e. cells isolated from liver tissue without any transformation step, can be kept in culture only for a very limited time, i.e. 8 to 14 days, without losing their properties and viability.
Typical characteristics of human hepatocytes include, but are not limited to:

    
    
        activity of enzymes such as:
        
            dipeptidyl peptidase IV (DPP-IV) as measured, e.g., with the chromogenic substrate Gly-Pro-p-nitroanilide (Bauvois (1990) Eur. J. Immunol 20(3): 459-68);
            alanine aminotransferase (ALT) as measured, e.g., with the ALT-Kit purchased from Randox-Laboratories according to the manufacturer's instructions;
            gamma glutamyl transpeptidase (γ-GT), e.g. by determining the amount of p-nitroaniline resulting from the γ-GT catalyzed reaction of L-gamma-glutamyl-p-nitroanilide and glycylglycine to gamma-glutamyl-glycylglycide and p-nitroaniline may be determined (Szasz G: Gamma-glutamyl-transpeptidase, in: Methoden der Enzymatischen Analyse, 3rd ed., (Vol 1), edited by Bergmeyer H U, Weinheim/Bergstr., Verlag Chemie, 1974, pp 757-762);
            lactate dehydrogenase (LDH) as measured, e.g., using the cytotoxicity Kit obtained from Promega according to the manufacturer's instructions;
        
        
        secretion of glucose after glucagon stimulation as measured by photometric determination of NADPH using the Hexokinase/Glucose-6-phosphate Dehydrogenase-method (Bondar et al. (1974) Clin Chem 20/5: 586-590)
        ability to metabolize pharmacological substances such as acetaminophen (APAP) and diclofenac as measured for example by the release of the cytoplasmic enzyme LDH into the medium upon treatment with a selected pharmacological substance;
        expression of albumin, α-fetoprotein, asialoglycoprotein receptors 1 and 2, carbamoyl phosphate synthetase (1) and coagulation factors I and II as measured for example by Western Blot or RT-PCR analysis;
        expression of CYP 450 enzymes such as CYP1A2, CYP2A6, CYP2D6, CYP2P8-9, CYP2E1, CYP3A3-5, CYP2C9, CYP3A4 as measured for example by Western Blot or RT-PCR analysis;
        activity of CYP 450 enzymes such as CYP1A2, CYP2C9 and CYP3A4 as measured by luminescence (e.g. using P450-Glo-Assays (Promega) according to the manufacturer's instructions) or fluorescence (3-cyano-coumarine; Donato et al. (2004) Drug Metabolism and Disposition 32(7): 699-706);
        ability to synthesize urea after incubation with ammonium ions as measured for example by an enzymatic urea test (e.g. QuantiChromeTM Urea Assay Kit of Clonagen (Brussels, Belgium) according to the manufacturer's instructions);
        ability to produce factor VII after incubation with vitamin K as measured for example by direct ELISA for factor VII in the cell culture medium;
        ability to produce factor VIII as measured for example by sandwich-ELISA for factor VIII in the cell culture medium (Esposito et al. (2000) Kidney Int. 58:123-130; Ziyadeh et al. (1990) Am. J. Physiol. 259: F704-F714);
        secretion of albumin as measured for example by ELISA of the cell culture medium;
        expression of xenobiotic transporters such as proteins of the ABC-transporter family as measured for example by export of the fluorescent dyes rhodamine-123 or HOECHST 33342 (Brown C D (2008) Toxicol. Appl. Pharmacol. 233(3): 428-38); and
        gene expression patterns, e.g. of different CYP P450 genes and transcription factors such as E2F and of the RAR, RXR and PPAR families.
    
    


Details of suitable test methods for determining the above characteristics are described in the examples section below.
The cells produced by the method of the present invention show at least one, preferably two or three, more preferably four or five, even more preferably six, seven or eight and most preferably all of the above-listed characteristics.
In the process of the present invention, the above characteristics are preferably measured after the cells have been cultured for at least four days and not more than 42 days in the cell culture medium comprising FGF-4. More preferably, the characteristics are measured after the cells have been cultured for seven to 14 days in the cell culture medium comprising FGF-4 and most preferably the characteristics are measured after the cells have been cultured for 14 days in the cell culture medium comprising FGF-4.
The term “monocyte” refers to the leukocyte type which is characterized by high level expression of the cell surface antigen CD14.
These monocytes are usually obtained from whole blood treated with an anti-coagulant such as heparin, EDTA or citrate. The whole blood may be stored for up to 24 hours at 4° C. before the monocytes are isolated.
The whole blood can be separated into plasma and white and red blood cells preferably by density gradient centrifugation, wherein after the centrifugation the plasma is found in the supernatant below which there is a layer which contains the totality of the white blood cells, which layer is also referred to “buffy coat”. Below this “buffy coat” there is a phase containing the red blood cells. The “buffy coat” layer is then isolated and separated to obtain the monocytes, e.g. by centrifugation using a known process. For example, the “buffy coat” layer may be coated onto a lymphocyte separation medium such as Ficoll Hypaque and centrifuged. If the “buffy coat” is contaminated with erythrocytes, as apparent by the red staining of the “buffy coat”, the cells are incubated with an erythrocyte lysis buffer containing NH4Cl, KHCO3 and EDTA.
Alternatively, the monocytes can be obtained from whole blood by methods such as FACS, immunomagnetic bead sorting and magnetic-activated cell sorting on the basis of their expression of cell surface markers such as CD14.
The monocytes to be used in the method of the present invention can be obtained from any isolated human blood and the blood can also originate from organs such as the spleen, lymph nodes or bone marrow. In another embodiment of the present invention, the monocytes are isolated from ascites.
If the cells of the present invention, i.e. the cells having at least one characteristic of human hepatocytes, are intended to be used in cell therapy, the monocytes used as starting material in the method of the present invention are preferably autologous monocytes, i.e. monocytes originating from the blood of the patient who is subsequently to be treated with the cells of the present invention.
The term “seeding the cells” generally refers to introducing a cell mixture into an in vitro environment capable of promoting the survival and/or growth of the introduced cells or at least a certain population within the cell mixture such as the cells having at least one characteristic of human hepatocytes in the present case. The cells seeded in the method of the present invention are monocytes obtained from a suitable source such as peripheral blood or ascites. In a preferred embodiment the cells are seeded immediately after their isolation. However, it is also possible to introduce one or more additional steps between the isolation and the seeding, such as freezing or centrifuging the cells.
The cells are seeded at a density of 100,000-400,000 cells/cm2 of the culture vessel, preferably at a density of 150,000-350,000 cells/cm2 of the culture vessel, more preferably at a density of 200,000-300,000 cells/cm2 of the culture vessel and most preferably at a density of 250,000 cells/cm2 of the culture vessel.
The cells may be seeded in a cell culture medium comprising IL-3 and adenosine and optionally M-CSF. However, the cell culture medium into which the cells are seeded may also lack IL-3, adenosine and M-CSF, if the medium is to be changed within one hour, preferably within 20 minutes, after seeding the cells.
A “culture vessel” is any vessel which is suitable for growing cells in a culture medium or agar, either in fluid phase or adhered to an interior surface of the vessel. Types of such specialized vessels include roller bottles, spinner flasks, petri dishes and tissue flasks. Culture vessels are designed to be incubated in temperature, humidity and gas controlled environments to facilitate maximum cell or tissue growth. Generally, a layer of cell culture medium or agar covers the growing surface.
The portion of the vessel not utilized as a growing surface encloses the interior gaseous environment which surrounds the cell culture. Cells, tissues, microorganisms and the like are typically introduced into the interior of cell culture vessels through an opening in the vessel. After introduction of the cells the opening may be closed such that the cells are not in contact with the environment during the culturing of the cells.
In a preferred embodiment of the present invention, the culture vessel is treated for tissue culture, which means that the surface of the culture vessel is treated such that cells which usually grow in an adherent state, such as human hepatocytes, grow adherently, but cells which grow in suspension do not adhere or adhere only loosely. Such treatment for tissue culture may involve the irradiation of the vessel or the coating of the vessel, for example with a special plastic, polymer or nanostructure or with proteins of the extracellular matrix. Culture vessels treated for tissue culture can comprise tissue culture dishes and tissue culture flasks and are available from different suppliers such as Becton Dickinson, Greiner, Sigma and TPP.
The terms “cell culture” and “culturing of cells” refer to the maintenance and propagation of cells, preferably human cells, in vitro. Ideally the cultured cells do not differentiate spontaneously, but only when treated with a suitable stimulus such as FGF-4 used in the method of the present invention. Unless specified otherwise, when the present disclosure provides a time period for culturing the cells of the present invention this time period comprises both step (a) and step (b) of the method of the present invention.
“Cell culture medium” is used for the maintenance of cells in culture in vitro. For some cell types, the medium may also be sufficient to support the proliferation of the cells in culture. A medium according to the present invention provides nutrients such as energy sources, amino acids and anorganic ions. Additionally, it may contain a dye like phenol red, sodium pyruvate, several vitamins, free fatty acids, antibiotics, anti-oxidants and trace elements.
For culturing the cells of the present invention any standard medium such as Iscove's Modified Dulbecco's Media (IMDM), alpha-MEM, Dulbecco's Modified Eagle Media (DMEM), RPMI Media and McCoy's Medium is suitable. These media are well-known to a person skilled in the art and may be purchased from companies such as Cambrex (East Rutherford, USA), Invitrogen (San Diego, USA) and Sigma-Aldrich (Deisenhofen, Germany). Preferably, the medium is DMEM/Ham's F12 (1:1).
Preferably, the medium does not contain a sulfur compound such as 2-mercaptoethanol or dimethylsulfoxide (DMSO).
However, the medium to be used in the process of the present invention may contain other conventional cell culture medium supplements such as L-glutamine, penicillin and streptomycin.
In the method of the present invention, the monocytes are first cultured in a medium containing Interleukin 3 (IL-3) and adenosine (corresponding to step (b) of the method of the present invention).
IL-3 is a cytokine which stimulates the differentiation of multipotent hematopoietic stem cells into myeloid progenitor cells and which stimulates the proliferation of all cells in the myeloid lineage including monocytes. Preferably, human IL-3 is used and most preferably recombinant human IL-3 is used. Wild-type human IL-3 may be obtained for example from R&D Systems (Minneapolis, USA), GenWay (San Diego, USA) or Miltenyi Biotech (Bergisch Gladbach, Germany).
The term “IL-3” is intended also to comprise IL-3 analogs, i.e. variants having one or more amino acid substitutions, deletions and/or additions within the amino acid sequence of the IL-3 polypeptide which variants substantially maintain the receptor binding properties and the biological activity such as stimulating the proliferation of myeloid cells of wild-type IL-3. Suitable IL-3 analogs are described for example in Lopez et al. (1992) Proc. Natl. Acad. Sci. USA 89:11842-11846 and include IL-3 having an alanine on position 101, IL-3 having valine on position 116 and an IL-3 having alanine on position 101 and valine on position 116 of the polypeptide.
The IL-3 is present in the cell culture medium in step (b) and, optionally, step (c) of the method of the present invention in a concentration of 0.2 ng/ml to 2.5 ng/ml or 0.3 ng/ml to 2 ng/ml, preferably 0.4 ng/ml to 1.8 ng/ml or 0.5 ng/ml to 1.7 ng/ml, more preferably of 0.6 ng/ml to 1.6 ng/ml or 0.7 ng/ml to 1.5 ng/ml, even more preferably 0.8 ng/ml to 1.4 ng/ml or 0.9 ng/ml to 1.1 ng/ml and most preferably 1 ng/ml.
Adenosine is a nucleoside composed of a molecule of adenine attached to ribose sugar molecule moieties. It plays an important role in biochemical processes such as energy transfer and signal transduction. The term “adenosine” is intended also to comprise analogs of adenosine which bind to adenosine A3 receptor and potentiate the stimulatory effect of IL-3, such as N6-(3-iodobenzyl)adenosine-5′-N-methyluronamide (IB-MECA) (Hofer et al. (2009) Physiol. Res. 58: 247-252). The adenosine may be obtained from suitable suppliers such as Sigma-Aldrich, Deisenhofen, Germany.
The adenosine is present in the cell culture medium in step (b) and, optionally, step (c) of the method of the present invention in a concentration of 10-300 nM or 20-280 nM, preferably 30-250 nM or 40-220 nM, more preferably 50-200 nM or 60-180 nM, even more preferably 80-150 nM or 90-120 nM and most preferably 100 nM.
In a preferred embodiment of the present invention the cell culture medium comprises 0.2-2.5 ng/ml IL-3 and 10-300 nM adenosine, more preferably 0.6-1.6 ng/ml IL-3 and 50-200 nM adenosine, even more preferably 0.8-1.4 ng/ml IL-3 and 80-150 nM adenosine and most preferably 1 ng/ml IL-3 and 100 nM adenosine.
In a preferred embodiment, the medium used in step (b) and, optionally, step (c) of the method of the present invention further comprises macrophage colony-stimulating factor (M-CSF).
M-CSF (also referred to as CSF-1) is a cytokine produced by monocytes, fibroblasts and endothelial cells which induces the division of monocytes upon binding to its specific c-Fms receptor (also referred to as CSF-1R). Preferably, human M-CSF is used and most preferably recombinant human M-CSF is used. The M-CSF used in the cell culture medium may be obtained from suitable suppliers such as R&D Systems (Minneapolis, USA) or Sigma Aldrich (Deisenhofen, Germany).
The term “M-CSF” within the meaning of the present invention is intended also to comprise an analog of M-CSF which comprises one or more amino acid substitutions, deletions and/or additions with respect to the wild-type M-CSF amino acid sequence and which retains receptor binding and biological activity of M-CSF. Suitable M-CSF analogs include (Q17A, R21A) M-CSF and (E115A, N119A) M-CSF which are described for example in Taylor et al. (1994) J. Biol. Chem. 269(49): 31171-31177.
The M-CSF is present in the cell culture medium in step (b) and, optionally, step (c) of the method of the present invention in a concentration of 0.1 ng/ml to 7 ng/ml or 0.2 to 5 ng/ml, preferably 0.25 ng/ml to 3 ng/ml or 0.3 ng/ml to 2.5 ng/ml, more preferably 0.35 ng/ml to 1.0 ng/ml or 0.4 ng/ml to 0.8 ng/ml and most preferably 0.5 ng/ml.
In a preferred embodiment of the present invention the cell culture medium in step (b) comprises 0.2-2.5 ng/ml IL-3, 10-300 nM adenosine and 0.1 ng/ml-7 ng/ml M-CSF, more preferably 0.6-1.6 ng/ml IL-3, 50-200 nM adenosine and 0.3 ng/ml-2.5 ng/ml M-CSF, even more preferably 0.8-1.4 ng/ml IL-3, 80-150 nM adenosine and 0.4 ng/ml-0.8 ng/ml M-CSF and most preferably 1 ng/ml IL-3, 100 nM adenosine and 0.5 ng/ml M-CSF.
The cells are cultured in the cell culture medium comprising IL-3 and adenosine and optionally M-CSF, but not FGF-4, for at least 4 days, preferably at least 5 days, more preferably at least 6 days and most preferably for 7 days. The cells are cultured in the cell culture medium comprising IL-3 and adenosine and optionally M-CSF, but not FGF-4, for no more than ten days, preferably no more than nine days, more preferably no more than eight days and most preferably for seven days. Preferably, the cells are cultured in the cell culture medium comprising IL-3 and adenosine and optionally M-CSF, but not FGF-4, for at least 4 days and no more than ten days.
In a second culturing step of the method of the present invention (corresponding to step (c) of the method of the present invention) the cells are cultured in a cell culture medium comprising fibroblast growth factor 4 (FGF-4). In a preferred embodiment, the cell culture medium used in the first culturing step is removed and the cell culture medium comprising FGF-4 is added, i.e. the cells are cultured in the cell culture medium comprising FGF-4 subsequent to the culturing of the cells in the cell culture medium comprising IL-3 and adenosine and (optionally) M-CSF. The cell culture medium used in the second culturing step may be the same as or different from the cell culture medium used in the first culturing step. Hence, the medium may comprise IL-3, adenosine, (optionally) M-CSF and FGF-4 together or it may only comprise FGF-4. Preferably, the cell culture medium used in the second culturing step of the present invention does not comprise IL-3, M-CSF and adenosine. However, residual amounts, i.e. no more than 50% or 40%, preferably no more than 30% or 20%, more preferably no more than 15% or 10% and most preferably no more than 5% or 2% of the concentration in the first culturing step, of IL-3, adenosine and (optionally) M-CSF may be present in the second culturing step. In a further embodiment, the cells may be incubated in a medium comprising IL-3, adenosine, (optionally) M-CSF and FGF-4 immediately after seeding the cells,
Further, the basic medium used in the second culturing step may be the same as or different from the basic medium used in the first step, e.g if DMEM/Ham's F 12(1:1) is used in the first step, the medium used in the second step may also be DMEM/Ham's F12 (1:1) or it may be for example RPMI. Preferably, the medium used in the first and the second culturing step is the same and most preferably, the medium used in the first and the second culturing step is DMEM/Ham's F12 (1:1).
Fibroblast growth factors are a family of growth factors involved in angiogenesis, wound healing and embryonic development. They are heparin-binding proteins and interactions with cell surface associated heparan sulfate proteoglycans have been shown to be essential for FGF signal transduction. Preferably, human FGF-4 and most preferably recombinant human FGF-4 is used. Recombinant human FGF-4 may be obtained from suitable suppliers such as R&D Systems (Minneapolis, USA).
The concentration of FGF-4 in the cell culture medium in step (c) of the method of the present invention is 0.5-7 ng/ml, preferably 1-5 ng/ml, more preferably 2-4 ng/ml and most preferably 3 ng/ml.
In a preferred embodiment of the present invention, the cell culture medium used in the second culturing step of the present invention further comprises at least one compound selected from the group consisting of epidermal growth factor (EGF), glucagon, insulin and heparin, more preferably, the cell culture medium used in the second culturing step of the present invention further comprises EGF, glucagon and insulin and most preferably the cell culture medium used in the second culturing step of the present invention further comprises EGF, glucagon, heparin and insulin. EGF is a growth factor that plays an important role in the regulation of cell growth, proliferation, differentiation and survival. If present, the concentration of EGF in the cell culture medium in the second culturing step is 5-20 ng/ml, preferably 7-15 ng/ml, more preferably 8-12 ng/ml and most preferably 10 ng/ml. Preferably, human EGF and more preferably recombinant human EGF is used. The EGF used in the cell culture medium may be obtained from suitable suppliers such as Biochrom AG, Berlin, Germany.
Glucagon is a hormone which is secreted by the pancreas and which causes the liver to convert stored glycogen into glucose which is then released into the bloodstream. If present, the concentration of glucagon in the cell culture medium in the second culturing step of the method of the present invention is 2-10 nM, preferably 3-8 nM, more preferably 4-7 nM and most preferably 5 nM. Preferably, human glucagon and more preferably recombinant human glucagon is used. The glucagon used in the cell culture medium may be obtained from suitable suppliers such as Sigma-Aldrich, Deisenhofen, Germany.
Insulin is a peptide hormone which is produced by the islets of Langerhans in the pancreas which regulates the energy and glucose metabolism in the body. If present, the concentration of insulin in the cell culture medium in the second culturing step of the method of the present invention is 0.10-0.50 IU, preferably 0.15-0.40 IU, more preferably 0.17-0.35 IU, even more preferably 0.20-0.30 IU and most preferably 0.28 IU. Preferably, human insulin and more preferably recombinant human insulin is used. The insulin used in the cell culture medium may be obtained from suitable suppliers such as Biochrom AG, Berlin, Deutschland.
Heparin is a highly sulfated glycosaminoglycan which is widely used as an injectable anti-coagulant. If present, the concentration of heparin in the cell culture medium in the second culturing step of the method of the present invention is 2-10 IU/ml, preferably 3-8 IU/ml, more preferably 4-7 IU/ml and most preferably 5 IU/ml. The heparin used in the cell culture medium may be obtained from suitable suppliers such as Biochrom AG, Berlin, Germany.
Preferably, the cell culture medium in the second culturing step comprises 5-20 ng/ml EGF, 2-10 nM glucagon, 0.10-0.50 IU insulin, and 2-10 IU/ml heparin. More preferably, the cell culture medium in the second culturing step comprises 8-12 ng/ml EGF, 3-8 nM glucagon, 0.17-0.35 IU insulin, and 3-8 IU/ml heparin and most preferably the cell culture medium in the second culturing step comprises 10 ng/ml EGF, 5 nM glucagon, 0.28 IU insulin and 5 IU/ml heparin.
In an even more preferred embodiment of the present invention the cell culture medium in the second culturing step comprises 0.5-7 ng/ml FGF-4, 5-20 ng/ml EGF, 2-10 nM glucagon, 0.10-0.50 IU insulin and 2-10 IU/ml heparin, and even more preferably it comprises 1-5 ng/ml FGF-4, 8-12 ng/ml EGF, 3-8 nM glucagon, 0.17-0.35 IU insulin, and 3-8 IU/ml heparin. In a most preferred embodiment of the present invention, the cell culture medium in the second culturing step comprises 3 ng/ml FGF-4, 10 ng/ml EGF, 5 nM glucagon, 0.28 IU insulin and 5 IU/ml heparin.
In a preferred embodiment, the cell culture medium used in the second culturing step of the method of the present invention does not contain hepatocyte growth factor (HGF).
The cell culture medium used in the first and second culturing step may comprise further compounds such as bile acids or bile salts, for example taurocholic acid, glycocholic acid, cholic acid, chenodeoxycholic acid, deoxycholate and lithocholic acid; caffeine; and/or glucocorticoids such as hydrocortisone and dexamethasone. If deoxycholate is present in the medium, it is present in a concentration of 1 to 30 μM, preferably 2 to 25 μM or 3 to 20 μM, more preferably 4 to 18 μM or 5 to 15 μM and most preferably 10 μM. If caffeine is present in the medium, it is present in a concentration of 5 to 100 μM, preferably 10 to 80 μM or 20 to 70 μM, more preferably 30 to 65 μM or 40 to 60 μM and most preferably 50 μM.
The cells are cultured in the cell culture medium comprising FGF-4 and, optionally, one or more or all of EGF, glucagon, insulin and heparin for at least seven days, and preferably no longer than 50 days, more preferably no longer than 40 days, even more preferably no longer than 35 days and most preferably they are cultured for seven to 35 days. After at least four days, preferably after at least seven days and more preferably after at least 14 days of culture in a cell culture medium containing at least FGF-4 and, optionally, one or more of EGF, insulin, glucagon and heparin, the cells have acquired at least one characteristic of human hepatocytes as described above.
The term “medium change” is intended to mean the step of removing the medium which is in contact with the cells, e.g. by aspiration, and adding fresh medium, i.e. medium which has not been in contact with the cells before. Preferably, the medium is changed at a time-point less than 1 hr, more preferably less than 45 min, even more preferably less than 30 min, and most preferably 20 min or 10 min after seeding the monocytes.
A further step of medium change may occur 18-30 h, preferably 20-28 h, more preferably 22-26 h and most preferably 24 h after seeding the monocytes.
In another preferred embodiment of the present invention the cell culture medium used in the first and/or second culturing step is serum-free, i.e. it does not contain a serum component such as horse serum, human serum or calf serum (FCS). However, the serum-free medium may contain discrete proteins or bulk protein fractions. The use of serum-free medium allows the cells to be cultured with a designed set of conditions with as few variables as possible. Furthermore, if the cells produced by the method of the present invention are intended to be used for cell therapy, the use of serum in the medium is disadvantageous, as it also bears a contamination risk.
In an even more preferred embodiment, the serum-free medium comprises albumin such as human serum albumin or bovine serum albumin. Preferably, human serum albumin is present in the medium. The human serum albumin may be obtained from suitable suppliers such as Sigma (Deisenhofen, Germany) or Baxter (Unterschleiss-heim, Germany). If present, the albumin is present in the cell culture medium in a concentration of 0.5-10%, preferably 1-8%, more preferably 1.5-5% and most preferably 2%. Preferably, the albumin is present in the serum-free medium in the first and the second culturing step of the method of the present invention.
In a further preferred embodiment of the present invention, the volume of the cell culture medium is kept low in the first and/or the second culturing step, preferably the medium volume is kept low during the whole cultivation period, i.e. in both the first and the second culturing step. Preferably, the volume of the cell culture medium is 40 to 90 μl/cm2 or 50 to 85 μl/cm2, more preferably it is 60 to 80 μl/cm2 or 65 to 75 μl/cm2 and most preferably it is 70 μl/cm2 of the culture vessel. It has already been shown that the volume of the medium and therefore the height of the medium column above the cells influences the pericellular oxygen partial pressure (Gstraunthaler et al. (1999) Cell Physiol. Biochem. 9(3): 150-172; Wolff et al. (1993) Am. J. Physiol. 265(5): C1266-1270). It is thus assumed that the lower medium volume prevents the cells from becoming hypoxic.
After the second culturing step the cells having at least one characteristic of human hepatocytes may be harvested for further use, e.g. cell therapy. The term “harvesting the cells” is intended to mean that the cells are withdrawn from the cell culture and optionally processed for further use. The cells may e.g. be harvested by treatment with trypsin which detaches the adherent cells from the culture vessel. The cells may then be centrifuged to remove the cell culture medium. Furthermore, the cells may be washed one or more times, for example in PBS (phosphate buffered saline) or other buffered saline solutions. Alternatively, after harvesting the cells may be seeded again in a suitable medium in the same or a new culture vessel. Furthermore, for some applications such as in vitro toxicity tests the cells do not have to be harvested, but may also be used directly during or after the second culturing step.
The harvested cells may be used directly for cell therapy or in vitro toxicity tests or may be cryoconserved by freezing them at a temperature from about −196° C. to about −20° C., preferably at a temperature from about −196° C. and −80° C. Experiments have shown that the cells of the present invention maintain their characteristics after cryoconservation.
By the method of the present invention, at least 15−50×106, preferably at least 20-45×106 and more preferably 30−40×106 cells having at least one characteristic of human hepatocytes can be obtained from 100 ml peripheral blood within 10 to 14 days after seeding the cells.
The cells obtained by the method of the present invention, also called “cells of the present invention” or “Metaheps”, can be distinguished from primary human hepatocytes (phH) by their ability to be cultured for a longer period, i.e. at least 15 days, preferably at least 17 or 20 days, more preferably at least 23 or 25 days, even more preferably at least 28 or 32 days and most preferably at least 35 days without losing their metabolic properties and their viability. The cells of the present invention are cultured for no more than 56 days, preferably no more than 52 or 49 days, more preferably no more than 45 or 42 days and most preferably no more than 38 days. In this case, the culture period is calculated from the time-point at which the treatment with FGF-4 is started.
The term “metabolic properties” is intended to mean at least one characteristic of human hepatocytes as described above, i.e. activity of enzymes such as dipeptidyl peptidase IV (DPP-IV), alanine aminotransferase (ALT), gamma glutamyl transpeptidase (γ-GT) and lactate dehydrogenase (LDH); secretion of glucose after glucagon stimulation; ability to metabolize pharmacological substances such as acetaminophen (APAP) and diclofenac; expression of albumin, α-fetoprotein, asialoglycoprotein receptors 1 and 2, carbamoyl phosphate synthetase (1) and coagulation factors I and II; expression of CYP 450 enzymes such as CYP1A2, CYP2A6, CYP2D6, CYP2P8-9, CYP2E1, CYP3A3-5, CYP2C9, CYP3A4; inducible activity of CYP 450 enzymes such as CYP1A2, CYP2C9 and CYP3A4; ability to synthesize urea after incubation with ammonium ions; ability to produce factor VII after incubation with vitamin K; ability to produce factor VIII; secretion of albumin; and expression of xenobiotic transporters such as proteins from the ABC-transporter family. Within the meaning of the present invention the metabolic properties are not lost, if any of the above properties is at least 80%, preferably at least 82% or 85%, more preferably at least 87 or 90% and most preferably 92 or 95% of the properties of the cells of the present invention cultured for seven days in the medium containing FGF-4.
The viability of the cells of the present invention can be determined by methods known in the art, e.g. by staining with trypan blue wherein the dead cells take up the dye and stain blue and the living cells do not take up the dye. Within the meaning of the present invention, the cells retain their viability, if at a specific time-point less than 20%, preferably less than 18 or 15%, more preferably less than 12% or 10% and most preferably less than 8 or 5% of the cells are dead cells.
In contrast, primary human hepatocytes maintain hepatocyte-characteristics only for up to 14 days and only when cultured under special conditions, such as serum free collagen-sandwich (Tuschl et al. (2009) Chem Biol Interact. 181(1): 124-37; G. Elaut et al. (2006) Curr. Drug Metab. 7 (6): 629-660.)
Hence, in contrast to primary human hepatocytes the cells of the present invention are suitable for the study of the long-term toxicity of substances. The term “long-term toxicity” refers to the ability of cells of the present invention to respond to treatment with a pharmacological substance such as rapamycin or APAP with the induction of cytochrome P450 enzyme expression or activity or LDH release after at least 7 days, preferably at least 10 or 14 days, more preferably at least 20 or 25 days, even more preferably at least 28 or 30 days and most preferably at least 35 days in culture.
Further, the cells of the present invention can be distinguished from immortalized cell lines such as HepG2 by their ability to undergo apoptosis, for example after treatment with an antibody activating CD95 for 24 hours. Hence, the risk that the cells of the present invention develop to tumors after transplantation into a human is low. The ability to undergo apoptosis can be measured by staining the treated cells with HOECHST stain or by measuring caspase activity in the treated cells, as described in the examples below. Another difference between the cells of the present invention and HepG2 cells is the ability of the cells of the present invention to synthesize urea after treatment with ammonia and the induction of enzymes of the cytochrome P450 system.
Finally, in contrast to the cells called “neo-hepatocytes” as described for example in Ruhnke et al. (2005) Gastroenterology 128(7): 1774-1786, Ruhnke et al. (2005) Transplantation 79(9): 1097-1103 and Riquelme et al. (2009) Differentiation 77(3): 263-276, the cells of the present invention are capable of undergoing apoptosis when treated with 500 ng/ml of the CD95 activating antibody APO for 24 hours. The term “capable of undergoing apoptosis” means that after incubation of the cells of the present invention (culture day 14) with the CD95 activating antibody APO for 24 hours the number of dead cells (measured for example by staining the cells with propidium iodide and subsequent FACS analysis) increases at least by the factor 1.5, more preferably at least by the factor 1.8, even more preferably at least by the factor 2 and most preferably at least by the factor 2.5 compared to control cells not incubated with the antibody.
It was also found by others that the “neo-hepatocytes” do not react to fatal stimulation with the death-inducing cytokines TNF-α and CD95L, although they express the corresponding receptors (doctoral thesis of Ms. Isabelle Pochic, University of Konstanz: “The Use of NeoHepatocytes for Assessment of Metabolism-Dependent Human Acute Toxicity”, available at http://kops.ub.uni-konstanz.de/handle/urn:nbn:de:bsz:352-opus-57745). Hence, it was concluded that the “neo-hepatocytes” may not be useful in cell therapy.
The cells of the present invention also show higher levels of factor VII and VIII synthesis than the “neo-hepatocytes”. The synthesis of factor VII in the cells of the present invention is at least 5 times, preferably at least 10 times, more preferably at least 15 times and most preferably at least 20 times higher than in the “neo-hepatocytes”. The synthesis of factor VIII in the cells of the present invention is at least 3 times, preferably at least 5 times, more preferably at least 7 times and most preferably at least 10 times higher than in the “neo-hepatocytes”. If the cells of the present invention and the “neo-hepatocytes” are produced in a serum-free medium, the synthesis of factor VII in the cells of the present invention is at least 20 times, preferably at least 30 times, more preferably at least 40 times and most preferably at least 50 times higher than in the “neo-hepatocytes”. If the cells of the present invention and the “neo-hepatocytes” are produced in a serum-free medium, the synthesis of factor VIII in the cells of the present invention is at least 5 times, preferably at least 7 times, more preferably at least 10 times and most preferably at least 12 times higher than in the “neo-hepatocytes”.
Further, in contrast to the “neo-hepatocytes” the cells of the present invention show a cytochrome activity which cannot be inhibited by treating the cells with 500 μM caffeine.
The cells of the present invention may be used in vitro to study the toxicity of substances, for example in drug screening. For example, the cells may be starved in basal medium for 8-24 h and then incubated with the basal medium containing different amounts of the substances to be tested or the corresponding vehicle for controls. For incubations up to 72 h, the medium is not changed, for longer incubation periods, medium samples are acquired and the medium is changed 3 times a week. At the end of the incubation period, medium samples are drawn and cells are lysed in the corresponding lysis buffer (e.g. Triton X100/MOPS). After lysis, protein content and enzyme activities (DPP-IV, GGT, LDH, ALT) can be determined in the lysate.
As already outlined above, the cells of the present invention may be used to investigate the acute toxicity of test compounds, but also the long-term toxicity of the test compounds, for example when incubated with the test compound for at least 14 days, more preferably at least 21 days and even more preferably at least 28 days.
The cells of the present invention may also be used in an approach called “targeted metabolomics” to investigate the mechanism of action and the metabolism, in particular of newly developed substances. In this method, metabolites are examined qualitatively and quantitatively by automated mass spectrometrical high throughput methods and functionally annotated. Based on already existing metabolic profiles conclusions with respect to metabolic pathways involved can be drawn and target structures can be identified (see, e.g., Dudley et al. (2010) Adv. Protein Chem. Struct. Biol. 80: 45-83; Koal and Deigner (2010) Curr. Mol. Med. 10(2): 216-226).
Further, the cells of the present invention may be used in metabolic phenotyping to individually characterize the CYP-dependent metabolism for developing a patient-specific treatment regimen (see, e.g., Klein et al. (2010) Front. Pharmacol. 1:1-20).
Further, the cells of the present invention may be used in cell therapy by transplanting the cells into humans suffering from acute or chronic liver failure, e.g. in the course of diseases such as drug-induced or toxic liver failure, alcohol abuse, viral hepatitis e.g. B,B/D or C, iron or copper overload or non-alcoholic fatty liver disease, cholestatic or autoimmune liver diseases, genetically inherited liver diseases and liver diseases in children. The cells may also be used as an autologous vector for gene therapy, for example by transferring RNA, of monogenetic diseases such as phenylketonuria (see, e.g., Kormann et al. (2011) Nat. Biotech. 29: 154-157).
Preferably, the cell therapy uses autologous cells, i.e. cells produced from monocytes which have been isolated from the patient to be treated with the cells of the present invention.
For cell therapy, the cells are administered as suspension in sterile PBS, 0.9% NaCl or Ringer's solution by either intrasplenic, intraportal, intrahepatic or peripheral intravenous injection or a combination thereof.
A more complete understanding of the present invention can be obtained by reference to the following specific examples which are provided herein for the purposes of illustration only and are not intended to limit the scope of the invention.
EXAMPLES
1. Production of Cells Having Characteristics of Human Hepatocytes by the Method of the Present Invention
Whole blood from healthy donors was treated with heparin and separated by gradient centrifugation (400 g; room temperature; 30 minutes) using Ficoll in a 2:1 ratio of blood to Ficoll. The interphase was resuspended in ice-cold, sterile PBS in a 1:3 to 1:4 ratio of cell suspension to PBS and then centrifuged at 400 g for 5 minutes at 4° C. After the first wash, the supernatant was discarded and the cell pellet was resuspended in ice-cold, sterile PBS (for PBMCs from 50 ml whole blood, 20-30 ml of ice-cold, sterile PBS are used). If there was visible contamination by erythrocytes, this washing step is preceded by 10 min incubation in sterile hemolysis-buffer (8.29 g/l NH4Cl, 1 g/l KHCO3, 0.037 g/l EDTA) in the dark at room temperature. Again, after resuspension in PBS, cells were centrifuged for 5 minutes at 300 g (4° C.). Once again, the supernatant was discarded and cells were resuspended in PBS followed by a last centrifugation at 250 g for 5 minutes at 4° C.
After this final washing step, the PBS was discarded and cells were resuspended at 37° C. in a medium having the following composition:
DMEM/Ham's F12 (1:1)
10% FCS (standard conditions) or 2% human albumin (serum-free conditions)
2 mM glutamine
100 μg/ml streptomycin
1000 IE penicillin
1 ng/ml recombinant human IL-3 (R&D Systems)
100 nM adenosine (Sigma, Deisenhofen, Germany)
0.5 ng/ml recombinant human M-CSF (R&D Systems)
The cells were seeded in a density of 250.000 cells/cm2 in a Petri dish which had been pre-incubated with the culture medium 30-120 minutes before seeding of the cells. If cells were seeded in 96-well plates, the outer wells were left free and just filled with cell culture medium to omit differences in temperature and partial pressures of O2 and CO2.
After seeding, the cells were allowed to attach for 10-30 minutes. After attachment, the medium was replaced with 70 μl fresh medium per cm2 of the culture vessel. The cell culture medium was again replaced within the first 24 h of culture. After the first 24 hours of culture there was an adherent population of monocytes, characterized by small round cells with a bean-shaped nucleus. At this stage the cells showed little specific activities of DPP-IV, GGT and ALT, but there was a high expression of the monocyte marker CD14 and the pan-leukocyte-marker CD45.
After the first 24 hours, the medium was changed three times a week. Cells were cultured in the above medium for the first 4-10 days of culture.
After the cells were incubated with the medium comprising IL-3, M-CSF and adenosine, the medium was replaced with the same volume of a medium having the following composition:
DMEM/Ham's F12 (1:1)
10% FCS (standard conditions) or 2% human albumin (serum-free conditions)
2 mM glutamine
100 μg/ml streptomycin
1000 IE penicillin
3 ng/ml recombinant human FGF-4
10 ng/ml recombinant human EGF
5 nM recombinant human glucagon
0.28 IE recombinant human insulin
5 IE/ml heparin
The cells were incubated in this medium for at least seven days or for the time periods indicated in the figures and figure legends. During the second culturing step, there is again an alteration in morphology, resulting in a confluent or subconfluent layer of large, mono- or multinucleated, polygonal shaped cells with an organelle rich cytoplasm, the metaheps. This morphology is observed independent of the presence or absence of serum in the culture medium.
Before experiments were performed, cells were starved by incubation with DMEM/Ham's F12 1:1 containing only L-glutamine, penicilline and streptomycin (referred to as “basal medium”). The basal medium was also used for incubations to avoid interference of medium ingredients with the assays performed. Prior to lysis, cells were washed three times with ice-cold PBS.
2. Determination of Protein Content
Protein content was determined with the bicinchoninic acid (BCA) assay (Sigma, Deisenhofen, Germany): cells were grown on culture vessels, starved for 24 h in basal medium and incubated with experimental chemicals as indicated. After removal of culture media cells were lysed in Triton X-100/MOPS and 10 μl aliquots of the lysates were incubated for 30 min at 37° C. with 200 μl of the BCA-reagent (50 parts reagent+1 part 4% CuSO4). Absorbance was measured at 560 nm in a multilabel counter (Dynatech MR7000). Protein content was calculated using bovine serum albumin as a reference standard.
3. Determination of Enzyme Activities
a) LDH-Activity
LDH-activity was determined spectrophotometrically by measurement of β-NAD+ resulting from the LDH catalyzed reaction of pyruvate and β-NADH to lactate and β-NAD+, using a standard protocol (Bergmeyer H. U. (Hrsg.) “Methoden der enzymatischen Analyse”, 3. Aufl. 1974, Band 1, Verlag Chemie Weinheim/Bergstr.) or the Cyto Tox 96-Kit (Promega) according to the manufacturer's instructions.
b) ALT-Activity
The activity of ALT was measured by a two phase reaction consisting of the generation of pyruvate and L-glutamate from L-alanine and α-ketoglutarate by ALT and the subsequent quantification of pyruvate generation by LDH-dependent β-NAD+ formation (Schumann G et al. (2002) Clin Chem Lab Med 40(7):718-724).
Samples (cell lysates in Triton X-100/MOPS after centrifugation at 13.000 U/min at 4° C.) were incubated for 300 s at 37° C. with reaction mixture (100 mmol TRIS, 500 mmol L-alanine, 0.18 mmol β-NADH, 0.1 mmol pyridoxal-5′-phosphate, 1700 U/1 L-lactate-dehydrogenase, pH 7.15) in the absence of α-ketoglutarate. The reaction was started by addition of 15 mmol α-ketoglutarate and the decrease in β-NADH absorption was monitored for 180 s after a 90 s incubation period. Alternatively the ALT-Kit from Randox Chemicals was used according to the manufacturer's instructions.
c) Gamma-Glutamyl-Transpeptidase Activity
Gamma-glutamyl-transpeptidase activity was measured using the photometric determination of p-nitroaniline resulting from the γ-GT catalyzed reaction of L-γ-glutamyl-p-nitroanilide and glycylglycine to γ-glutamyl-glycylglycide and p-nitroaniline (Szasz G: Gamma-glutamyl-transpeptidase, in Methoden der Enzymatischen Analyse, 3rd ed., (Vol 1), edited by Bergmeyer H U, Weinheim/Bergstr., Verlag Chemie, 1974, pp 757-762).
Cells were rinsed three times with PBS, lysed with Triton-X 100, centrifuged (13.000 U/min, 10 min) and total γ-GT-activity was measured by incubating the cell lysates with 100 mM TRIS, 4 mM L-γ-glutamyl-p-nitroanilide and 50 mM glycylglycine. Absorption of p-nitroaniline was measured at 405 nm. ΔE/min was calculated from measurements at 5, 10, 15, 30 and 45 minutes.
d) DPP-IV-Activity
DPP-IV activity was measured using the photometric determination of p-nitroaniline resulting from the DPP-IV catalyzed cleavage of the chromogenic substrate Gly-L-Pro-p-nitroanilide.
Cells were lysed in Triton X-100/MOPS. After centrifugation of the lysates (13.000 U/min; 4° C.), the lysates were incubated with 0.2 mM Gly-L-Pro-pNA in 0.1 M TRIS (pH 8.0) at 37° C. and absorbance at 405 nm was read after 0, 5, 10, 15, 30 and 45 minutes to determine ΔE/min. DPP-IV activity was determined from a standard curve with p-nitroaniline and normalized to protein content as measured by the BCA-method.
4. Determination of P450 Cytochrome Activity
For CYP P450 activity measurement the P450 Glo assays (Promega) were used according to the manufacturer's instructions. Briefly, cells were starved in basal medium and incubated with different inducers (3-methylcholanthrene in concentrations from 0.1 to 10 μM, typically 1 μM for CYP1A2; 10 μM dexamethasone; 10 to 100 μM, typically 50 μM rifampicin; 5 to 10 μM, typically 10 μM phenyloin for CYP 3A4; and 50 μM rifampicin and 10 μM phenyloin for 2C9 for 24 h to 72 h). Typically, induction was performed using a 48 h incubation time. After induction, cells were rinsed three times with ice-cold PBS and incubated with basal medium containing the corresponding Luciferin-substrate according to the manufacturer's instruction and luminescence in the supernatant was measured after 30 min incubation with Luciferin detection reagent according to the manufacturer's instructions. Luminescence readings were normalized for protein content in the cell lysates as determined by the BCA-method.
To determine CYP1A2-activity using the fluorescent dye 3-cyano-7-ethoxycoumarine (Donato et al. (2004) Drug Metabolism and Disposition 32(7): 699-706), cells were treated with 3-methylcholanthrene for 48 hours and then rinsed three times with ice cold PBS, following incubation with 3-cyano-7-ethoxycoumarin (30 μM) in HEPES-buffered saline at 37° C. for 120 minutes. 75 μl of the supernatant were pipeted in an assay plate and incubated with 25 μl β-Glucuronidase/Arylsulfatase (1.5 μl/1 ml in Natriumacetat) at 37° C. for 2 h. After addition of 0.1 mM phosphate potassium buffer as quenching solution, fluorescence was measured at Excitation/Emission 408/455 nm in a plate fluorometer. Fluorescence readings were normalized to protein content as determined by the BCA-assay.
5. Determination of APAP Toxicity
Toxicity assays were performed after 24 h starvation of the cells with basal medium by incubation with different concentrations of APAP dissolved in basal medium for 24 h. LDH activity in media and lysates was determined as described above using the Cyto Tox 96 Kit from Promega and LDH release (%) was calculated by 100×(LDH-activity in media/(LDH-activity in media+LDH-activity in lysate).
6. Determination of Apoptosis
a) Apoptosis by HOECHST 33342 Staining
Cells were incubated with different concentrations of anti-APO (Alexis AXXORA Biochemicals, Lörrach, Germany)/Protein A (BioVision Research Products, Mountain View, USA) for 24-48 h. After the incubation period, cells were stained with HOECHST 33342 (10 μM) for 15 minutes and analysed after three washes with PBS using a fluorescence microscope.
b) Apoptosis by Caspase-3 Measurement
Caspase-3 activity in cell lysates was determined using the chromogenic caspase-3 substrate Ac-DEVD-pNA (final concentration 40 μM) as described in Benesic et al. (2006) Kidney Blood Press Res. 29(5): 280-293. The activity was assessed using a standard curve with p-nitroaniline and normalized to protein content as measured by the BCA method.
c) Assessment of Apoptotic Cells by FACS-Analysis
To evaluate apoptosis by DNA-fragmentation, FACS-analysis of cells stained with propidium-iodide was performed. Cells were starved for 24 h with basal medium and incubated with basal medium or basal medium containing Anti-APO (Alexis AXXORA Biochemicals, Lörrach, Germany) in the indicated concentrations. After 24 h incubation, cells were detached using Accutase, centrifuged at 250 g for 5 min (4° C.) and resuspended in ice-cold PBS containing 0.2% bovine serum albumin. Under continuous agitation pure ethanol was slowly added to a final concentration of 75% ethanol in PBS/BSA. Cells were frozen at −20° C. to allow fixation overnight. Afterwards, cells were centrifuged at 400 g for 5 min (4° C.). The supernatant was discarded and cells were stained with 50 μg/1 propidium iodide in PBS for at least 30 min in the dark, followed by FACS analysis. The apoptosis was assessed by evaluating the sub-G1 events.
7. Determination of Urea Synthesis
Urea synthesis was measured after 24 h starvation of the cells with basal medium, followed by 24 h incubation with different concentration of ammonium chloride (0-100 mM). The supernatants were collected and urea content was determined using the QuantiChromeTM Urea Assay Kit (Clonagen) according to the manufacturer's instructions. Urea content of the media was normalized to protein content in cell lysates as measured by the BCA method.
8. Determination of Factor VII Secretion
Factor VII secretion was measured by direct ELISA, in analogy to Esposito et al. (2000) Kidney Int. 58:123-130; Ziyadeh et al. (1990) Am. J. Physiol. 259: F704-F714). After starvation, cells were incubated for 48 h with different menadione concentrations as indicated. Media were collected and 100 μl aliquots were incubated in Immunosorb Plates (Nunc) with 200 μl Voller buffer (1.59 g Na2CO3+2.93 g NaHCO3 ad 100 ml aqua bidest) overnight at 4° C. The plate was rinsed three times with 200 μl PBS/0.05% Tween-20 and blocked with 200 μl 2% BSA in PBS/0.05% Tween-20 for 2 h. Afterwards the plate was emptied and incubated with 50 μl rabbit anti-factor VII antibody (Abcam, Cambridge, Great Britain) 1:1000 in 2%-BSA-PBS/0.05% Tween-20 for 1-2 h bei 4° C. Following three washes with 200 μl PBS/0.05% Tween-20, the plate was incubated for one hour with 50 μl anti-rabbit-peroxidase-Ak 1:5000 (Santacruz, Heidelberg, Germany) in 2%-BSA-PBS/0.05% Tween-20 at room temperature in the dark. After three rinses with 200 μl PBS/0.05% Tween-20 an incubation with 100 μl HRP-substrate (0.5 mg/ml o-phenylenediamine, 11.8 mg/ml Na2HPO4.2H2O, 7.3 mg/ml citric acid, 0.015% H2O2 in aqua bidest) in the dark for 15 and 45 minutes followed. Absorbance at 490 nm was read in a multiwell reader followed by wavelength correction (630 nm). The absorbance was normalized for cell lysate protein content as determined by the BCA method.
9. Determination of Factor VIII Secretion
Factor VIII secretion was measured by sandwich-ELISA, in analogy to Esposito et al. (2000) Kidney Int. 58:123-130; Ziyadeh et al. (1990) Am. J. Physiol. 259: F704-F714. After starvation, cells were incubated with basal medium for 24 h. Media were collected and 100 μl aliquots were incubated for 2 h in Immunosorb Plates (Nunc) precoated with 1 μg/ml goat Anti-human factor VIII (Santacruz, Heidelberg, Germany) at room temperature. The plate was rinsed three times with 200 μl PBS/0.05% Tween-20. Afterwards the plate was emptied and incubated with 50 μl rabbit anti-factor VIII (Santacruz, Heidelberg, Germany) 1:1000 in 1%-BSA-PBS/0.05% for 1 h at room temperature. Following three washes with 200 μl PBS/0.05% Tween-20, the plate was incubated for one hour with 50 μl anti-rabbit-peroxidase-Ak 1:5000 (Santacruz) in 1%-BSA-PBS at room temperature in the dark. After three rinses with 200 μl PBS/0.05%-Tween-20 an incubation with 100 μl HRP-substrate (0.5 mg/ml o-phenylenediamine, 11.8 mg/ml Na2HPO4.2H2O, 7.3 mg/ml citric acid, 0.015% H2O2 in aqua bidest) in the dark for 15 and 45 minutes followed.
Absorbance at 490 nm was read in a multiwell reader followed by wavelength correction (630 nm). The absorbance was normalized for cell lysate protein content as determined by the BCA method.
10) Gene Expression Analysis in Comparison to Primary Human Hepatocytes
Gene-expression of Metaheps and primary human hepatocytes (pHH) of the same donor was analyzed by a cRNA-Microarray (Kat. Nr. OHS 401, Human Toxicology and Drug Resistance, SA Biosciences). The genes which are analyzed with this array are shown in Table 1 below.
Monocytes and hepatocytes were acquired from patients undergoing partial hepatectomy who signed informed consent for hepatocyte donation and blood withdrawal. The patients were selected in cooperation with the Human Tissue and Cell Research Foundation (HTCR, c/o BioPark, Josef-Engert-Str. 9, 93053 Regensburg).
Hepatocytes were isolated and cultured as described in Weiss et al. (2003) J. Hepatol. 38(4): 476-482). After 4 days in culture mRNA was isolated from pHH using the Array Grade total RNA isolation Kit provided by SA Biosciences according to manufacturer's instructions.
The same was performed for Metaheps on days 3, 7 and 14 of culture in FGF-4.
According to the manufacturer's instructions the mRNA samples were transcribed into biotinylated cRNA and hybridized on the Array membrane. Afterwards a chemiluminescent detection was performed using the Chemiluminescence Detection Kit provided by SA Biosciences.
Arrays were analyzed by TIGR Spotfinder software and the data were analyzed using SPSS-software. Further, Cluster analysis was performed.
11) Long-Term Toxicity in the Cells of the Present Invention
To show the suitability of the cells of the present invention for long-term toxicity studies, the cells were produced as described above and incubated for 15 days with low concentrations of acetaminophen (APAP) and diclofenac (diclo). On incubation days 1, 3, 6, 8, 10, 13 and 15 medium samples were drawn and LDH activity in media was determined using the Cytotoxicity Kit from Promega. LDH release was calculated in % as 100×(LDH in media/LDH media+LDH lysate).
Low concentrations of diclofenac show long-term toxicity in Metaheps, whereas acetaminophen exerted acute toxicity in Metaheps
12) Discussion of the Results
The presented data show the hepatocyte-like characteristics of the cells of the invention. The important findings are summarized by expression and activity of CYP P450-enzymes, such as 1A2, 2C9 and 3A4, which are also inducible using prototypic substances (e.g. ligands for arylhydrocarbon receptor (AHR), pregnane-X-receptor (PXR), constitutive androstane-receptor (CAR) and glucocorticoid receptor (GR)). The most abundant CYP P450 enzyme in the liver, CYP 2E1 could be shown indirectly by APAP-toxicity and on the protein level. Furthermore, ammonia-dependent urea-synthesis, as well as synthesis of the coagulation factors VII and VIII by ELISA could be shown. Specific enzyme activities of DPP-IV, γ-GT, and LDH are present and comparable to primary hepatocytes or hepatocyte-derived cells (such as Huh-7 or HepG2). Thus, the cells of the present invention show hepatocyte-like metabolism, gene expression, synthesis function as well as enzyme activities. Furthermore, the cells of the present invention are capable of undergoing apoptosis induced by CD95-ligation, as shown by caspase-3 activity, HOECHST-stain and FACS-analysis. This is an important feature of cells which can potentially be used for therapeutic interventions.
The observed enzyme activities, metabolism and synthesis parameters have been compared to primary human hepatocytes in culture and the cells of the present invention exhibited at least 20% of hepatocyte factor VIII-synthesis, 30% of hepatocyte CYP3A-activity as well as 70% of hepatocyte-specific enzyme activities. Further, it has been shown that the cells of the present invention are useful for long-term toxicity studies.
Compared to cells cultivated by the method of Ruhnke et al. (2005) Gastroenterology 128(7): 1774-1786 (“neo-hepatocytes”), the cells of the present invention showed about 10 to 20-fold higher factor VIII release as well as a higher CYP1A2-activity measured by 3CEC-fluorescence. Moreover, the caffeine-insensitive metabolism of 3-CEC which is mostly mediated by CYP 2C9 is nearly absent in cells cultivated by the method of Ruhnke et al. Additionally, the latter cells did not show any measurable apoptosis after 24 h CD95-ligation, another marked difference to the cells of the invention.







TABLE 1







List of genes present in the cRNA microarray











Po-






si-






tion 
UniGene
GenBank
Symbol
Description














1
Hs.546292
NM_002954
RPS27A
Ribosomal protein 






S27a


2
Hs.546292
NM_002954
RPS27A
Ribosomal protein 






S27a


3
Hs.489033
NM_000927
ABCB1
ATP-binding cassette, 






sub-family B (MDR/






TAP), member 1


4
Hs.287827
NM_000443
ABCB4
ATP-binding cassette, 






sub-family B (MDR/






TAP), member 4


5
Hs.391464
NM_004996
ABCC1
ATP-binding cassette, 






sub-family C (CFTR/






MRP), member 1


6
Hs.368243
NM_000392
ABCC2
ATP-binding cassette, 






sub-family C (CFTR/






MRP), member 2


7
Hs.463421
NM_003786
ABCC3
ATP-binding cassette, 






sub-family C (CFTR/






MRP), member 3


8
Hs.368563
NM_005688
ABCC5
ATP-binding cassette, 






sub-family C (CFTR/






MRP), member 5


9
Hs.442182
NM_001171
ABCC6
ATP-binding cassette, 






sub-family C (CFTR/






MRP), member 6


10
Hs.544577
NM_002046
GAPDH
Glyceraldehyde-3-






phosphate 






dehydrogenase


11
Hs.544577
NM_002046
GAPDH
Glyceraldehyde-3-






phosphate 






dehydrogenase


12
Hs.544577
NM_002046
GAPDH
Glyceraldehyde-3-






phosphate 






dehydrogenase


13
Hs.546292
NM_002954
RPS27A
Ribosomal protein 






S27a


14
Hs.480218
NM_004827
ABCG2
ATP-binding cassette, 






sub-family G (WHITE), 






member 2


15
Hs.431048
NM_005157
ABL1
V-abl Abelson murine






leukemia viral oncogene






homolog 1


16
Hs.81934 
NM_001609
ACADSB
Acyl-Coenzyme A






dehydrogenase, short/






branched chain


17
Hs.232375
NM_000019
ACAT1
Acetyl-Coenzyme A






acetyltransferase 1






(acetoacetyl Coenzyme 






A thiolase)


18
Hs.171189 
NM_001621 
AHR
Aryl hydrocarbon 






receptor


19
Hs.525622
NM_005163 
AKT1
V-akt murine thymoma 






viral oncogene 






homolog 1


20
Hs.489365
NM_001283
AP1S1
Adaptor-related protein






complex 1, sigma 1 






subunit


21
Hs.158932 
NM_000038 
APC
Adenomatosis 






polyposis coli


22
Hs.496240
NM_000044 
AR
Androgen receptor






(dihydrotestosterone






receptor; testicular






feminization; spinal 






and bulbar muscular 






atrophy; Kennedy






disease)


23
Hs.131494 
NM_001668 
ARNT
Aryl hydrocarbon 






receptor nuclear 






translocator


24
Hs.435561 
NM_000051 
ATM
Ataxia telangiectasia 






mutated (includes






complementation groups






A, C and D)


25
Hs.377484 
NM_004323
BAG1
BCL2-associated 






athanogene


26
Hs.159428
NM_004324
BAX
BCL2-associated X 






protein


27
Hs.150749
NM_000633
BCL2
B-cell CLL/lymphoma 2


28
Hs.516966
NM_138578 
BCL2L1
BCL2-like 1


29
Hs.410026
NM_004050 
BCL2L2
BCL2-like 2


30
Hs.517461
NM_004327
BCR
Breakpoint Cluster 






Region


31
Hs.194143
NM_007294
BRCA1
Breast cancer 1, early 






onset


32
Hs.34012
NM_000059
BRCA2
Breast cancer 2, early 






onset


33
Hs.515162
NM_004343
CALR
Calreticulin


34
Hs.529890
NM_001746 
CANX
Calnexin


35
Hs.2490
NM_033292 
CASP1
Caspase 1, apoptosis-






related cysteine protease






(interleukin 1, beta, 






convertase)


36
Hs.5353
NM_001230
CASP10
Caspase 10, apoptosis-






related cysteine protease


37
Hs.369736 
NM_001228 
CASP8
Caspase 8, apoptosis-






related cysteine protease


38
Hs.502302
NM_001752 
CAT
Catalase


39
Hs.57907
NM_002989 
CCL21
Chemokine (C-C motif) 






ligand 21


40
Hs.514107 
NM_002983 
CCL3
Chemokine (C-C 






motif) ligand 3


41
Hs.75703
NM_002984 
CCL4
Chemokine (C-C 






motif) ligand 4


42
Hs.430646
NM_005190 
CCNC
Cyclin C


43
Hs.523852 
NM_053056 
CCND1
Cyclin D1 (PRAD1:






parathyroid 






adenomatosis 1)


44
Hs.244723
NM_001238 
CCNE1
Cyclin E1


45
Hs.79101
NM_004060
CCNG1
Cyclin G1


46
Hs.189772 
NM_006431
CCT2
Chaperonin containing 






TCP1, subunit 2 (beta)


47
Hs.491494
NM_005998 
CCT3
Chaperonin containing 






TCP1, subunit 3 






(gamma)


48
Hs.421509 
NM_006430
CCT4
Chaperonin containing 






TCP1, subunit 4 






(delta)


49
Hs.1600
NM_012073
CCT5
Chaperonin containing 






TCP1, subunit 5 






(epsilon)


50
Hs.368149
NM_006429
CCT7
Chaperonin containing 






TCP1, subunit 7 (eta)


51
Hs.125113
NM_006585
CCT8
Chaperonin containing 






TCP1, subunit 8 (theta)


52
Hs.19192
NM_001798
CDK2
Cyclin-dependent 






kinase 2


53
Hs.95577
NM_000075
CDK4
Cyclin-dependent 






kinase 4


54
Hs.370771
NM_000389
CDKN1A
Cyclin-dependent 






kinase inhibitor 1A 






(p21, Cipl)


55
Hs.238990
NM_004064
CDKN1B
Cyclin-dependent 






kinase inhibitor 1B






(p27, Kip1)


56
Hs.512599
NM_058195
CDKN2A
Cyclin-dependent 






kinase inhibitor 2A 






(melanoma, p16, 






inhibits CDK4)


57
Hs.435051
NM_001800
CDKN2D
Cyclin-dependent 






kinase inhibitor 2D 






(p19, inhibits CDK4)


58
Hs.535486 
NM_016280
CES4
Carboxylesterase 4-like


59
Hs.302002
NM_020985
CHAT
Choline 






acetyltransferase


60
Hs.291363
NM_007194
CHEK2
CHK2 checkpoint 






homolog (S. pombe)


61
Hs.104576 
NM_003654
CHST1
Carbohydrate 






(keratan sulfate Gal-6) 






sulfotransferase 1


62
Hs.516370
NM_004854
CHST10
Carbohydrate






sulfotransferase 10


63
Hs.8786
NM_004267
CHST2
Carbohydrate (N-






acetylglucosamine-






6-O) sulfotransferase 2


64
Hs.158304 
NM_004273
CHST3
Carbohydrate 






(chondroitin 6) 






sulfotransferase 3


65
Hs.251383 
NM_005769
CHST4
Carbohydrate (N-






acetylglucosamine 






6-O) sulfotransferase 4


66
Hs.156784
NM_012126
CHST5
Carbohydrate (N-






acetylglucosamine






6-O) sulfotransferase 5


67
Hs.157439
NM_021615
CHST6
Carbohydrate (N-






acetylglucosamine 






6-O) sulfotransferase 6


68
Hs.138155
NM_019886
CHST7 
Carbohydrate (N-






acetylglucosamine 






6-O) sulfotransferase 7


69
Hs.165724
NM_022467
CHST8 
Carbohydrate (N-






acetylgalactosamine 






4-O) sulfotransferase 8


70
Hs.436657
NM_001831
CLU
Clusterin (complement 






lysis inhibitor, 






SP-40,40, sulfated






glycoprotein 2, 






testosterone-repressed 






prostate


71
Hs.370408
NM_000754
COMT
Catechol-O-






methyltransferase


72
Hs.346950
NM_004378
CRABP1
Cellular retinoic acid 






binding protein 1


73
Hs.12068
NM_000755
Crat
Carnitine 






acetyltransferase


74
Hs.184085
NM_000394
CRYAA
Crystallin, alpha A


75
Hs.408767
NM_001885
CRYAB
Crystallin, alpha B


76
Hs.1349
NM_000758
CSF2
Colony stimulating 






factor 2 (granulocyte-






macrophage)


77
Hs.473087
NM_001905
CTPS
CTP synthase


78
Hs.413924
NM_001565
CXCL10
Chemokine (C—X—C 






motif) ligand 10


79
Hs.303980
NM_000781
CYP11A1 
Cytochrome P450, 






family 11, subfamily 






A, polypeptide 1


80
Hs.511880
NM_000498
CYP11B2 
Cytochrome P450, 






family 11, subfamily B, 






polypeptide 2


81
Hs.72912
NM_000499
CYP1A1
Cytochrome P450, 






family 1, subfamily A,






polypeptide 1


82
Hs.1361
NM_000761
CYP1A2
Cytochrome P450, 






family 1, subfamily A, 






polypeptide 2


83
Hs.154654
NM_000104
CYP1B1
Cytochrome P450, 






family 1, subfamily B, 






polypeptide 1


84
Hs.446065
NM_020674
CYP20A1 
Cytochrome P450, 






family 20, subfamily A, 






polypeptide 1


85
Hs.89663
NM_000782
CYP24A1
Cytochrome P450, 






family 24, subfamily A, 






polypeptide 1


86
Hs.91546
NM_019885
CYP26B1 
Cytochrome P450, 






family 26, subfamily B, 






polypep tide 1


87
Hs.439056
NM_000762
CYP2A6
Cytochrome P450, 






family 2, subfamily A, 






polypeptide 6


88
Hs.1360
NM_000767
CYP2B6
Cytochrome P450, 






family 2, subfamily B, 






polypeptide 6


89
Hs.282871
NM_000770
CYP2C8
Cytochrome P450, 






family 2, subfamily C, 






polypeptide 8


90
Hs.282624
NM_000771
CYP2C9
Cytochrome P450, 






family 2, subfamily C, 






polypeptide 9


91
Hs.534311
NM_000106
CYP2D6
Cytochrome P450, 






family 2, subfamily D, 






polypeptide 6


92
Hs.12907
NM_000773
CYP2E1
Cytochrome P450, 






family 2, subfamily E, 






polypeptide 1


93
Hs.363140
NM_000774
CYP2F1
Cytochrome P450, 






family 2, subfamily F, 






polypeptide 1


94
Hs.442527
NM_017460
CYP3A4
Cytochrome P450, 






family 3, subfamily A, 






polypeptide 4


95
Hs.150276
NM_000777
CYP3A5
Cytochrome P450, 






family 3, subfamily A, 






polypeptide 43


96
Hs.1645
NM_000778
CYP4A11
Cytochrome P450, 






family 4, subfamily A, 






polypeptide 11


97
Hs.436317
NM_000779
CYP4B1
Cytochrome P450, 






family 4, subfamily B, 






polypeptide 1


98
Hs.106242
NM_000896
CYP4F3
Cytochrome P450, 






family 4, subfamily F,






polypeptide 3


99
Hs.1644
NM_000780
CYP7A1
Cytochrome P450, 






family 7, subfamily A,






polypeptide 1


100
Hs.491869
NM_004820
CYP7B1
Cytochrome P450, 






family 7, subfamily B,






polypeptide 1


101
Hs.447793
NM_004391
CYP8B1
Cytochrome P450, 






family 8, subfamily B, 






polypeptide 1


102
Hs.505777
NM_004083
DDIT3
DNA-damage-inducible






transcript 3


103
Hs.83765
NM_000791
DHFR
Dihydrofolate reductase


104
Hs.517666
NM_007326
DIA1
Diaphorase (NADH)






(cytochrome b-5 






reductase)


105
Hs.335551
NM_001931
DLAT
Dihydrolipoamide S-






acetyltransferase 






(E2 component of 






pyruvate dehydrogenase 






complex)


106
Hs.445203
NM_001539
DNAJA1
DnaJ (Hsp40) homolog,






subfamily A, member 1


107
Hs.368078
NM_005880
DNAJA2
DnaJ (Hsp40) homolog,






subfamily A, member 2


108
Hs.459779
NM_005147
DNAJA3
DnaJ (Hsp40) homolog,






subfamily A, member 3


109
Hs.513053
NM_018602
DNAJA4
DnaJ (Hsp40) homolog,






subfamily A, member 4


110
Hs.515210
NM_006145
DNAJB1
DnaJ (Hsp40) homolog,






subfamily B, member 1


111
Hs.317192
NM_016306
DNAJB11
DnaJ (Hsp40) homolog,






subfamily B, member 11 


112
Hs.77768
NM_006736
DNAJB2
DnaJ (Hsp40) homolog, 






subfamily B, member 2


113
Hs.380282
NM_007034
DNAJB4
DnaJ (Hsp40) homolog,






subfamily B, member 4 


114
Hs.237506
NM_012266
DNAJB5
DnaJ (Hsp40) homolog,






subfamily B, member 5


115
Hs.6790
NM_012328
DNAJB9
DnaJ (Hsp40) homolog, 






subfamily B, member 9 


116
Hs.172847
NM_005528
DNAJC4
DnaJ (Hsp40) homolog,






subfamily C, member 4


117
Hs.164419
NM_025219
DNAJC5
DnaJ (Hsp40) homolog,






subfamily C, member 5


118
Hs.500156
NM_003315
DNAJC7
DnaJ (Hsp40) homolog,






subfamily C, member 7


119
Hs.433540
NM_014280
DNAJC8
DnaJ (Hsp40) homolog, 






subfamily C, member 8


120
Hs.335034
NM_000110
DPYD
Dihydropyrimidine






dehydrogenase


121
Hs.96055
NM_005225
E2F1
E2F transcription 






factor 1


122
Hs.488293
NM_005228
EGFR
Epidermal growth 






factor receptor






(erythroblastic






leukemia viral 






(v-erb-b) oncogene






homolog, avian)


123
Hs.326035
NM_001964
EGR1
Early growth response 1


124
Hs.181128
NM_005229
ELK1
ELK1, member of 






ETS oncogene family


125
Hs.89649
NM_000120
EPHX1
Hypothetical gene 






supported by AK124699


126
Hs.212088
NM_001979
EPHX2
Epoxide hydrolase 2,






cytoplasmic


127
Hs.446352
NM_004448
ERBB2
V-erb-b2 erythroblastic






leukemia viral oncogene






homolog 2, neuro/






glioblastoma derived 






oncogene homolog


128
Hs.118681
NM_001982
ERBB3
V-erb-b2 erythroblastic 






leukemia viral oncogene 






homolog 3 (avian)


129
Hs.390729
NM_005235
ERBB4
V-erb-a erythroblastic






leukemia viral oncogene






homolog 4 (avian)


130
Hs.435981
NM_001983
ERCC1
Excision repair cross-






complementing rodent 






repair deficiency, 






complementation group






1 (includes overlapping






antisense sequence)


131
Hs.469872
NM_000122
ERCC3
Excision repair cross-






complementing rodent 






repair deficiency, 






complementation






group 3 (xeroderma






pigmentosum group B






complementing)


132 
Hs.208124
NM_000125 
ESR1
Estrogen receptor 1


133 
Hs.443150
NM_001437
ESR2
Estrogen receptor 2 






(ER beta)


134 
Hs.284244
NM_002006
FGF2
Fibroblast growth 






factor 2 (basic)


135 
Hs.1424
NM_002021
FMO1
Flavin containing






monooxygenase 1


136 
Hs.386502
NM_002022 
FMO4
Flavin containing






monooxygenase 4


137 
Hs.303476
NM_001461
FMO5
Flavin containing






monooxygenase 5


138 
Hs.25647
NM_005252 
FOS
V-fos FBJ murine






osteosarcoma viral 






oncogene homolog


139 
Hs.80409
NM_001924 
GADD45A
Growth arrest and 






DNA-damage- 






inducible, alpha


140 
Hs.110571
NM_015675 
GADD45B
Growth arrest and 






DNA-damage-






inducible, beta


141 
Hs.17958
NM_004861 
GAL3ST1
Galactose-3-O-






sulfotransferase 1


142 
Hs.515258
NM_004864 
GDF15
Growth differentiation 






factor 15


143 
Hs.76686 
NM_000581
GPX1
Glutathione peroxidase 1


144 
Hs.2704
NM_002083
GPX2
Glutathione peroxixdase






2 (gastrointestinal)


145 
Hs.271510
NM_000637 
GSR
Glutathione reductase


146 
Hs.102484
NM_000847 
GSTA3
Glutathione S- 






transferase A3


147 
Hs.485557
NM_001512 
GSTA4
Glutathione S-






transferase A4


148 
Hs.301961
NM_000561 
GSTM1
Glutathione S- 






transferase M1


149 
Hs.279837
NM_000848 
GSTM2
Glutathione S- 






transferase M2 






(muscle)


150 
Hs.2006
NM_000849 
GSTM3
Glutathione S-






transferase M3 (brain)


151 
Hs.75652
NM_000851 
GSTM5
Glutathione S-






transferase M5


152 
Hs.190028
NM_004832 
GSTO1
Glutathione S-






transferase omega 1


153 
Hs.523836
NM_000852 
GSTP1
Glutathione S-






transferase pi


154 
Hs.268573
NM_000853
GSTT1
Glutathione S-






transferase theta 1


155 
Hs.1581
NM_000854 
GSTT2
Glutathione S-






transferase theta 2


156 
Hs.470611
NM_003642 
HAT1
Histone 






acetyltransferase 1


157 
Hs.509554
NM_001530 
HIF1A
Hypoxia-inducible 






factor 1, alpha subunit 






(basic helix-loop-helix 






transcription factor)


158 
Hs.517581
NM_002133
HMOX1
Heme oxygenase 






(decycling) 1


159
Hs.284279
NM_002134
HMOX2
Heme oxygenase 






(decycling) 2


160
Hs.42151
NM_006895
HNMT
Histamine N-






methyltransferase


161
Hs.121443
NM_139211
HOP
Homeodomain-only 






protein


162
Hs.530227
NM_005526
HSF1
Heat shock transcription 






factor 1


163
Hs.520028
NM_005345
HSPA1A
Heat shock 70 kDa 






protein 1A


164
Hs.80288
NM_005527
HSPA1L
Heat shock 70 kDa 






protein 1-like


165
Hs.432648
NM_021979
HSPA2
Heat shock 70 kDa 






protein 2


166
Hs.90093
NM_002154
HSPA4
Heat shock 70 kDa 






protein 4


167
Hs.3268
NM_002155
HSPA6
Heat shock 70 kDa 






protein 6 (HSP70B′)


168
Hs.180414
NM_006597
HSPA8
Heat shock 70 kDa 






protein 8


169
Hs.184233
NM_004134
HSPA9B
Heat shock 70 kDa 






protein 9B (mortalin-2)


170
Hs.520973
NM_001540
HSPB1
Heat shock 27 kDa 






protein 1


171
Hs.78846
NM_001541
HSPB2
Heat shock 27 kDa 






protein 2


172
Hs.41707
NM_006308
HSPB3
Heat shock 27 kDa 






protein 3


173
Hs.525600
NM_005348
HSPCA
Heat shock 90 kDa 






protein 1, alpha


174
Hs.509736
NM_007355
HSPCB
Heat shock 90 kDa 






protein 1, beta


175
Hs.471014
NM_002156
HSPD1
Heat shock 60 kDa 






protein 1 (chaperonin)


176
Hs.1197
NM_002157
HSPE1
Heat shock 10 kDa 






protein 1 (chaperonin 10)


177
Hs.36927
NM_006644
HSPH1
Heat shock 105 kDa/110






kDa protein 1


178
Hs.277704
NM_006389
HYOU1
Hypoxia up-regulated 1


179
Hs.20573
NM_000875
IGF1R
Insulin-like growth 






factor 1 receptor


180
Hs.487062
NM_000876
IGF2R
Insulin-like growth 






factor 2 receptor


181
Hs.274313
NM_002178
GFBP6
IInsulin-like growth 






factor binding protein 6


182
Hs.83077
NM_001562
IL18
Interleukin 18 






(interferon-gamma-






inducing factor)


183
Hs.1722
NM_000575
IL1A
Interleukin 1, alpha


184
Hs.126256
NM_000576
IL1B
Interleukin 1, beta


185
Hs.512234
NM_000600
IL6
Interleukin 6 






(interferon-beta 2)


186
Hs.36
NM_000595
LTA
Lymphotoxin alpha 






(TNF superfamily, 






member 1)


187
Hs.183109 
NM_000240
MAOA
Monoamine oxidase A


188
Hs.46732
NM_000898
MAOB
Monoamine oxidase B


189
Hs.369849 
NM_002392
MDM2
Mdm2, transformed 






3T3 cell double minute 






2, p53 binding protein






(mouse)


190
Hs.132966
NM_000245
MET
Met proto-oncogene






(hepatocyte growth 






factor receptor)


191
Hs.501522
NM_002412
MGMT
O-6-methylguanine-






DNA methyltransferase


192
Hs.389700
NM_020300
MGST1
Microsomal glutathione






S-transferase 1


193
Hs.81874
NM_002413
MGST2
Microsomal glutathione






S-transferase 2


194
Hs.191734 
NM_004528
MGST3
Microsomal glutathione






S-transferase 3


195
Hs.407995
NM_002415
MIF
Macrophage migration 






inhibitory factor






(glycosylation-






inhibiting factor)


196
Hs.195364 
NM_000249
MLH1
MutL homolog 1, colon






cancer, nonpolyposis 






type 2 (E. coli)


197
Hs.156519 
NM_000251
MSH2
MutS homolog 2, colon 






cancer, nonpolyposis






type 1 (E. coli)


198
Hs.374950 
NM_005952
MT1X
Metallothionein 1X


199
Hs.73133
NM_005954
MT3
Metallothionein 3 






(growth inhibitory 






factor (neurotrophic))


200
Hs.513488
NM_017458
MVP
Major vault protein


201
Hs.202453
NM_002467
MYC
V-myc 






myelocytomatosis






viral oncogene homolog






(avian)


202
Hs.21907
NM_007067
MYST2
MYST histone






acetyltransferase 2


203
Hs.35758
NM_012330
MYST4
MYST histone






acetyltransferase 






(monocytic leukemia) 4


204
Hs.155956
NM_000662
NAT1
N-acetyltransferase 1






(arylamine N-






acetyltransferase)


205
Hs.2
NM_000015
NAT2
N-acetyltransferase 2






(arylamine N-






acetyltransferase)


206
Hs.368783 
NM_016100
NAT5
N-acetyltransferase 5 






(ARD1 homolog, 






S. cerevisiae)


207 
Hs.14637
NM_003960 
NAT8
-acetyltransferase 8 






(camello like)


208 
Hs.431926
NM_003998 
NFKB1
Nuclear factor of kappa 






light polypeptide gene






enhancer in B-cells 1






(p105)


209 
Hs.73090
NM_002502 
NFKB2
Nuclear factor of kappa






light polypeptide gene






enhancer in B-cells 2 






(p49/p100)


210 
Hs.81328
NM_020529 
NFKBIA
Nuclear factor of kappa






light polypeptide gene 






enhancer in B-cells 






inhibitor, alpha


211 
Hs.9731
NM_002503 
NFKBIB
Nuclear factor of kappa 






light polypeptide gene






enhancer in B-cells






inhibitor, beta


212 
Hs.503911
NM_006169 
NNMT
Nicotinamide N-






methyltransferase


213 
Hs.462525
NM_153292
NOS2A
Nitric oxide synthase 






2A (inducible, 






hepatocytes)


214 
Hs.406515
NM_000903
NQO1
NAD(P)H 






dehydrogenase, 






quinone 1


215 
Hs.7303
NM_022002
NR1I2
Nuclear receptor 






subfamily 1, group I,






member 2


216 
Hs.349642
NM_005122
NR1I3
Nuclear receptor 






subfamily 1, group I, 






member 3


217 
Hs.534331
NM_002452
NUDT1
Nudix (nucleoside






diphosphate linked 






moiety X)-type motif 1


218
Hs.147433
NM_182649
PCNA
Proliferating cell 






nuclear antigen


219 
Hs.440967
NM_000940
PON3
Paraoxonase 3


220 
Hs.354056 
NM_000941
POR
P450 (cytochrome)






oxidoreductase


221 
Hs.485196
NM_006238 
PPARD
Peroxisome 






proliferative 






activated receptor, 






delta


222 
Hs.162646
NM_015869
PPARG
Peroxisome 






proliferative activated






receptor, gamma


223 
Hs.527078
NM_013261 
PPARGC1A
Peroxisome proliferative






activated receptor, 






gamma, coactivator 1


224 
Hs.180909
NM_002574
PRDX1
Peroxiredoxin 1


225 
Hs.432121
NM_005809
PRDX2
Peroxiredoxin 2


226 
Hs.201978
NM_000962 
PTGS1
Prostaglandin-






endoperoxide synthase 






1 (prostaglandin G/H 






synthase and






cyclooxygenase)


227 
Hs.196384
NM_000963 
PTGS2
Prostaglandin-






endoperoxide synthase






2 (prostaglandin






G/H synthase and






cyclooxygenase)


228 
Hs.440960
NM_005053
RAD23A
RAD23 homolog A 






(S. cerevisiae)


229 
Hs.242635
NM_005732
RAD50
RAD50 homolog 






(S. cerevisiae)


230 
Hs.446554
NM_002875
RAD51
RAD51 homolog 






(RecA homolog, 






E. coli) (S. cerevisiae)


231 
Hs.535499
NM_000964
RARA
Retinoic acid receptor, 






alpha


232 
Hs.436538
NM_000965
RARB
Retinoic acid receptor, 






beta


233 
Hs.1497
NM_000966
RARG
Retinoic acid receptor,






gamma


234 
Hs.408528
NM_000321
RB1
Retinoblastoma 1 






(including 






osteosarcoma)


235 
Hs.307905 
NM_006509
RELB
V-rel 






reticuloendotheliosis






viral oncogene 






homolog B, nuclear 






factor of kappa light 






polypeptidgene 






enhancer in B-cells






3 (avian)


236 
Hs.20084
NM_002957
RXRA
Retinoid X receptor, 






alpha


237 
Hs.388034
NM_021976
RXRB
Retinoid X receptor, 






beta


238 
Hs.26550
NM_006917
RXRG
Retinoid X receptor, 






gamma


239 
Hs.23978
NM_002967
SAFB
Scaffold attachment 






factor B


240 
Hs.241579
NM_001235
SERPINH1
Serine (or cysteine)






proteinase inhibitor, 






clade H (heat shock






protein 47), member 






1, (collagen binding






protein 1)


241 
Hs.443914
NM_000454
SOD1
Superoxide dismutase 






1, soluble (amyotrophic 






lateral sclerosis 1 






(adult))


242 
Hs.487046
NM_000636
SOD2
Superoxide dismutase 






2, mitochondrial


243 
Hs.458345
NM_000348
SRD5A2
Steroid-5-alpha-






reductase, alpha 






polypeptide 2 (3-oxo-5 






alpha-steroid delta4-






dehydrogenase alpha 2)


244 
Hs.546303
NM_003932
ST13
Suppression of






tumorigenicity 13 






(colon carcinoma) 






(Hsp70 interacting






protein)


245 
Hs.142
NM_001055
SULT1A
Sulfotransferase family, 






cytosolic, 1A, phenol-






preferring, member 1


246 
Hs.129742
NM_014465
SULT1B1 
Sulfotransferase family, 






cytosolic, 1B, member 1


247
Hs.436123
NM_001056
SULT1C1
Sulfotransferase family, 






cytosolic, 1C, member 1


248
Hs.312644
NM_006588
SULT1C2
Sulfotransferase family, 






cytosolic, 1C, member 2


249
Hs.479898
NM_005420
SULT1E1
Sulfotransferase family 






1E, estrogen-preferring, 






member 1


250
Hs.515835
NM_003167
SULT2A1
Sulfotransferase family, 






cytosolic, 2A, o






dehydrepiandrosterone






(DHEA) -preferring, 






member 1


251
Hs.369331
NM_004605
SULT2B1
Sulfotransferase family, 






cytosolic, 2B, member 1


252
Hs.189810
NM_014351
SULT4A1
Sulfotransferase family 






4A, member 1


253
Hs.509736 
NM_007355
HSPCB
Heat shock 90 kDa 






protein 1, beta


254
Hs.520757 
NM_001061
TBXAS1
Thromboxane A 






synthase 1 (platelet, 






cytochrome P450, 






family 5, subfamily A)


255
Hs.487054
NM_030752
TCP1
T-complex 1


256
Hs.241570 
NM_000594
TNF
Tumor necrosis factor 






(TNF superfamily, 






member 2)


257
Hs.204044
NM_003839
TNFRSF11A
Tumor necrosis factor






receptor superfamily,






member 11a, activator 






of NFKB


258
Hs.279594 
NM_001065
TNFRSF1A 
Tumor necrosis factor






receptor superfamily,






member 1A


259
Hs.478275 
NM_003810
TNFSF10
Tumor necrosis factor






(ligand) superfamily,






member 10


260
Hs.2007
NM_000639
FASLG
Fas ligand (TNF 






superfamily, member 6)


261
Hs.472737
NM_003286
TOP1
Topoisomerase (DNA) I


262
Hs.408312 
NM_000546
TP53
Tumor protein p53 (Li-






Fraumeni syndrome)


263
N/A
N/A
Pol1
Escherichia coli K12






MG1655 section 351 






of 400 of the complete 






genome


264
N/A
L08752
PUC18
PUC18 Plasmid DNA


265
Hs.534255 
NM_004048
B2M
Beta-2-microglobulin


266
Blank
Blank
Blank



267
Blank
Blank
Blank



268
Hs.444319
NM_000367
TPMT
Thiopurine S-






methyltransferase


269
Hs.421194
NM_003596
TPST1
Tyrosylprotein






sulfotransferase 1


270
Hs.438649
NM_003595
TPST2
Tyrosylprotein






sulfotransferase 2


271
Hs.192374
NM_003299
TRA1
Tumor rejection 






antigen (gp96) 1


272
N/A
X03205
18SrRNA
Human 18S ribosomal






RNA


273
N/A
N/A
AS1R3
Artificial Sequence 1 






Related 3 (70% identity)






(43/60)


274
N/A
N/A
AS1R2
Artificial Sequence 1 






Related 2 (80% identity)






(48/60)


275
N/A
N/A
AS1R1
Artificial Sequence 1 






Related 1 (90% identity)






(56/60)


276
N/A
N/A
AS1
Artificial Sequence 1


277
Hs.534255
NM_004048
B2M
Beta-2-microglobulin


278
Hs.534255
NM_004048
B2M
Beta-2-microglobulin


279
Hs.520640
NM_001101
ACTB
Actin, beta


280
Hs.460996
NM_003789
TRADD
TNFRSF1A-associated 






via death domain


281
Hs.369762 
NM_001071
TYMS
Thymidylate synthetase


282
Hs.250
NM_000379
XDH
Xanthine dehydrogenase


283
Hs.129727
NM_005431
XRCC2
-ray repair 






complementing






defective repair in 






Chinese hamster 






cells 2


284
N/A
N/A
BAS2C
Biotinylated Artificial






Sequence 2 






Complementary






sequence


285
N/A
N/A
BAS2C
Biotinylated Artificial






Sequence 2 






Complementary






sequence


286
N/A
N/A
BAS2C
Biotinylated Artificial






Sequence 2 






Complementary






sequence


287
N/A
N/A
BAS2C
Biotinylated Artificial






Sequence 2 






Complementary 






sequence


288
N/A
N/A
BAS2C
Biotinylated Artificial






Sequence 2 






Complementary 






sequence

## Claims

1. A method for producing cells having at least one characteristic of human hepatocytes, said method comprising the steps of:
(a) seeding human monocytes in a culture vessel in a cell culture medium;
(b) culturing the monocytes in a cell culture medium comprising interleukin 3 (IL-3) and adenosine; and
(c) culturing the cells subsequent to step (b) in a cell culture medium comprising fibroblast growth factor 4 (FGF-4), wherein the cell culture medium comprising FGF-4 does not comprise adenosine.

2. The method of claim 1, wherein the cell culture medium in step (b) further comprises macrophage-colony stimulating factor (M-CSF).

3. The method of claim 1, wherein the adenosine is present in the cell culture medium of step (b) in a concentration of 10 to 300 nM.

4. The method of claim 1, wherein the cell culture medium in step (c) further comprises at least one compound selected from the group consisting of epidermal growth factor (EGF), glucagon, insulin and heparin.

5. The method of claim 4, wherein the cell culture medium in step (c) comprises EGF, glucagon, insulin and heparin.

6. The method of claim 1, wherein the cell culture medium in steps (a), (b) and/or (c) is serum-free.

7. The method of claim 6, wherein the cell culture medium in steps (a), (b) and/or (c) comprises albumin.

8. The method of claim 1, further comprising a step of changing the cell culture medium at a time-point less than one hour after seeding the human monocytes.

9. The method of claim 1, wherein in step (b) and/or (c) the cell culture medium volume is 40 to 90 μl/cm2 of the culture vessel.

10. The method of claim 1, wherein in step (b) the cells are cultured for at least four days.

11. The method of claim 1, wherein in step (c) the cells are cultured for at least seven days.

12. Cells having at least one characteristic of human hepatocytes obtainable by the method of claim 1, wherein the cells can be kept in culture for at least 15 days.

13. Cells according to claim 12, wherein the cells are capable of undergoing apoptosis when incubated with a CD95-ligating antibody for 24 hours.

14. A method of using the cells of claim 12 in in vitro toxicity studies.