CN114075573A

CN114075573A - Engineered hepatocytes

Info

Publication number: CN114075573A
Application number: CN202010830851.4A
Authority: CN
Inventors: 丁秋蓉; 吴晓珊; 冯庄慧; 应浩
Original assignee: Shanghai Institute of Nutrition and Health of CAS
Current assignee: Shanghai Institute of Nutrition and Health of CAS
Priority date: 2020-08-18
Filing date: 2020-08-18
Publication date: 2022-02-22

Abstract

According to the invention, one or more of CDK1, CDK4, CCNB and CCND are overexpressed, the proliferation capacity of the human liver primary cells is induced, the cell amplification quantity and the passage frequency are obviously improved, and the efficient, rapid and long-term in-vitro human liver primary cell amplification is realized.

Description

Engineered hepatocytes

Technical Field

The invention belongs to the field of hepatocyte culture, and particularly relates to engineered hepatocytes, a culture method and application thereof.

Background

Whole liver, biopsy liver sections and primary hepatocytes are commonly used in liver studies. Whole liver, or biopsy liver sections, can retain cellular structure and liver-specific and hepatocyte-mesenchymal interactions in vivo, but their viability is limited (typically <48 hours) (March et al, 2015). Special cultures (e.g., in microfluidic devices) are required to prolong their hepatic activity but their hepatic functional activity is still not prolonged for weeks. In addition, cryopreservation of liver slices, due to donor deficiencies and donor-to-donor variability, not only does it not allow high throughput screening experiments, but also makes the overall experimental data difficult to interpret.

Primary hepatocytes are considered the "gold standard" for probing liver function because they are untransformed and relatively easy to use. Although human liver possesses a strong ability to regenerate in vivo, such as loss or increase in liver mass due to liver damage or pregnancy, liver cells will proliferate or die complementarily to restore the original liver/body weight ratio, and animal experiments as early as the last century have shown that after a partial liver resection (PHx) is performed, rats can survive after 70% of the rat liver weight is removed, and the remaining liver can regenerate to its original weight in a short time (Gaub and Iversen, 1984). However, once human primary hepatocytes are transplanted in vitro, the liver activity phenotype is rapidly lost (March et al, 2015). In conventional adherent culture, primary hepatocytes need to be seeded at very high density on collagen membranes for growth, and the function of mature hepatocytes declines rapidly within a few days, making primary hepatocytes cultured in vitro only suitable for short-term (<48 hours) experimental studies. That is, liver-related functions after hepatocytes have been transferred in vitro: including metabolism, detoxification, synthesis and secretion, are quickly lost, and in addition, the cell number is not multiplied any more, and passage expansion cannot be performed.

There remains a need in the art for efficient, rapid, long-term in vitro methods for human liver primary cell expansion.

Disclosure of Invention

In a first aspect, the invention provides a nucleic acid construct comprising coding sequences for any two, three or four of CDK1, CDK4, CCNB and CCND proteins.

In one or more embodiments, the nucleic acid construct comprises coding sequences for the following proteins: CDK1 and CDK4, CDK1 and CCNB, CDK1 and CCND, CDK4 and CCNB, CDK4 and CCND, CCNB and CCND, CDK1, CDK4 and CCNB, CDK1, CDK4 and CCND, CDK1, CCNB and CCND, CDK4, CCNB and CCND, or CDK1, CDK4, CCNB and CCND. Preferably, the nucleic acid construct comprises coding sequences for CDK1 and CCNB, CDK4 and CCND, or CDK1, CDK4, CCNB and CCND.

In one or more embodiments, CDK1 comprises SEQ ID No. 1 or a homologue or variant thereof having at least 90% sequence identity thereto and retaining CDK1 activity.

In one or more embodiments, CDK4 comprises SEQ ID No. 2 or a homologue or variant thereof having at least 90% sequence identity thereto and retaining CDK4 activity.

In one or more embodiments, the CCNB comprises SEQ ID No. 3 or a homolog or variant thereof that has at least 90% sequence identity thereto and retains CCNB activity.

In one or more embodiments, CCND comprises SEQ ID No. 4 or a homolog or variant thereof that has at least 90% sequence identity thereto and retains CCND activity.

In one or more embodiments, the nucleic acid construct is a recombinant vector or an expression vector.

The invention also provides lentiviruses comprising the nucleic acid constructs described herein.

In a second aspect, the invention provides a hepatocyte having upregulated expression or activity of any two, three or four of CDK1, CDK4, CCNB and CCND.

In one or more embodiments, the expression or activity of CDK1 and CCNB, CDK4 and CCND, or CDK1, CDK4, CCNB and CCND, is upregulated in the hepatocytes.

In one or more embodiments, the hepatocyte is a mammalian hepatocyte, preferably a human hepatocyte.

In one or more embodiments, the hepatocyte is an immature hepatocyte. The hepatocyte is a primary hepatocyte or a cell derived therefrom. The primary hepatocyte-derived cell is a hepatocyte of a primary hepatocyte cultured, for example, after 1-8 passages, preferably a hepatocyte cultured for 5 or 7 passages. The culturing comprises incubation with a medium comprising HM and/or HIM medium.

In one or more embodiments, the hepatocyte has the ability to mature in vivo or in vitro. Mature hepatocytes express or have increased expression of a hepatocyte signature marker. The hepatocyte characteristic marker is selected from one or more of ALB, CYP3A4, CYP2B6, CYP2E1 and Albumin.

In one or more embodiments, the hepatocyte is proliferative in vivo or in vitro.

In one or more embodiments, the hepatocyte is a non-cancerous hepatocyte.

In one or more embodiments, the hepatocytes may be passaged in vivo or in vitro for at least 10 passages, at least 15 passages, at least 17 passages, at least 20 passages, at least 23 passages, at least 30 passages, at least 40 passages.

In one or more embodiments, the number of cells per passage of the hepatocyte is increased at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold compared to a hepatocyte in which the expression or activity of the protein is not upregulated.

In one or more embodiments, the hepatocyte expresses Albumin.

In one or more embodiments, the hepatocyte has a nucleic acid construct as described herein in the first aspect.

The invention also provides a cell preparation comprising hepatocytes or an extract thereof as described herein. The extract is a protein or small molecule extract.

In one or more embodiments, the cell preparation is a cell culture, pharmaceutical composition, liver organoid, kit, device, medium, or system comprising a hepatocyte or an extract thereof described herein.

The present invention also provides a method for preparing a hepatocyte, enhancing the proliferative capacity of a hepatocyte, increasing the expansion number of a hepatocyte or increasing the number of passages of a hepatocyte or culturing a hepatocyte, which comprises (1) up-regulating the expression or activity of any two, three or four proteins of CDK1, CDK4, CCNB and CCND in a hepatocyte, and (2) optionally culturing the cell obtained in (1).

In one or more embodiments, (1) is overexpression of CDK1 and CCNB, CDK4 and CCND, or CDK1, CDK4, CCNB and CCND in hepatocytes.

In one or more embodiments, (1) is the introduction of a nucleic acid construct as described in the first aspect herein into a hepatocyte.

In one or more embodiments, the culturing of (2) comprises culturing in a medium comprising HIM medium and/or HM medium. Preferably, the culture is a 2-dimensional (2D) culture.

In one or more embodiments, CDK1 comprises SEQ ID No. 1 or a homologue or variant thereof having at least 90% sequence identity thereto and retaining CDK1 activity. In one or more embodiments, CDK4 comprises SEQ ID No. 2 or a homologue or variant thereof having at least 90% sequence identity thereto and retaining CDK4 activity. In one or more embodiments, the CCNB comprises SEQ ID No. 3 or a homolog or variant thereof that has at least 90% sequence identity thereto and retains CCNB activity. In one or more embodiments, CCND comprises SEQ ID No. 4 or a homolog or variant thereof that has at least 90% sequence identity thereto and retains CCND activity.

In one or more embodiments, the hepatocyte is an immature hepatocyte. The hepatocyte is a primary hepatocyte or a cell derived therefrom. In one or more embodiments, the primary hepatocyte is a mammalian primary hepatocyte, preferably a human primary hepatocyte. In one or more embodiments, the primary hepatocyte-derived cell is a hepatocyte of a primary hepatocyte that has been cultured for e.g. 1-8 passages, preferably a hepatocyte cultured for 5 or 7 passages; preferably, the culturing comprises incubation with a medium comprising HM and/or HIM medium.

In one or more embodiments, the hepatocyte is a non-cancerous hepatocyte.

In one or more embodiments, the hepatocyte has the ability to mature in vivo or in vitro. Mature hepatocytes express or have increased expression of a hepatocyte signature marker selected from one or more of ALB, CYP3a4, CYP2B6, CYP2E1, Albumin.

In one or more embodiments, the hepatocytes obtained in (1) may be passaged in vivo or in vitro for at least 10 passages, at least 15 passages, at least 17 passages, at least 20 passages, at least 23 passages, at least 30 passages, at least 40 passages.

In one or more embodiments, the number of cells per passage obtained in (1) is increased at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold compared to hepatocytes not overexpressing the protein.

In one or more embodiments, the hepatocyte expresses Albumin.

The invention also provides application of any two, three or four proteins or coding sequences thereof in enhancing the proliferation capacity of liver cells, increasing the amplification number of the liver cells, increasing the number of passages of the liver cells or culturing the liver cells, wherein the proteins or the coding sequences of any two, three or four proteins are CDK1, CDK4, CCNB and CCND.

In one or more embodiments, any two, three or four of the proteins CDK1, CDK4, CCNB and CCND are CDK1 and CCNB, CDK4 and CCND, or CDK1, CDK4, CCNB and CCND.

In one or more embodiments, the hepatocyte is up-regulated in expression or activity of CDK1 and CCNB, CDK4 and CCND, or CDK1, CDK4, CCNB and CCND. Preferably, the nucleic acid construct of the first aspect of the invention is comprised in said hepatocyte.

In one or more embodiments, the hepatocyte is an immature hepatocyte. The hepatocyte is a primary hepatocyte or a cell derived therefrom. The primary hepatocyte-derived cell is a hepatocyte of a primary hepatocyte cultured for e.g. 1-8 passages, preferably a hepatocyte cultured for 5 or 7 passages; preferably, the culturing comprises incubation with a medium comprising an HM and/or HIM medium.

In one or more embodiments, the hepatocyte is a non-cancerous hepatocyte.

In one or more embodiments, the hepatocyte has the ability to mature in vivo or in vitro. The hepatocyte has a maturation ability in vivo or in vitro. Mature hepatocytes express or have increased expression of a hepatocyte signature marker selected from one or more of ALB, CYP3a4, CYP2B6, CYP2E1, Albumin.

In one or more embodiments, the number of cells per passage of the hepatocyte is increased at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold compared to a hepatocyte that does not overexpress the protein.

In one or more embodiments, the hepatocyte expresses Albumin.

The invention also provides a method of screening for a drug comprising contacting a candidate drug with said hepatocytes and/or cell preparations and detecting a change in said hepatocytes and/or cell preparations.

The invention also provides the use of the hepatocytes and/or cell preparations described herein in drug screening, detecting drug clearance, detecting hepatotoxicity, or regenerative medicine.

The invention also provides the application of the liver cells and/or the cell products in the preparation of the cell products for preventing, treating and diagnosing liver diseases, liver transplantation and auxiliary treatment thereof and supporting liver auxiliary devices.

In one or more embodiments, the cell preparation is a cell culture, pharmaceutical composition, kit, device, medium or system, liver organoid comprising cells or extracts thereof as described herein.

The invention also provides a method of treating a liver disease comprising administering a cell or liver organoid as described herein to a patient in need thereof.

Drawings

FIG. 1, A: after overexpression of the combination of CDK1, CDK4, CCNB and CCND four factors (4F) in 293T cells, Western Blot detection was performed on Flag proteins overexpressing the factors. B: detecting mRNA expression levels of 7 th generation cells after 2F (CDK1-CCNB1), 4F (CDK1-CCNB1, CDK4-CCND1) and 2F (CDK4-CCND1) are respectively over-expressed by ProliHH; c: ProliHH was used to over-express 2F (CDK1-CCNB1), 4F (CDK1-CCNB1, CDK4-CCND1), and 2F (CDK4-CCND1) for each cell generation.

FIG. 2, A: brightfield plots at day 1 and day 6 after plating of 11 th and 16 th generation 2F cells after overexpression of four two factor combinations CDK4 and CCND (2F) in PoliHH cells. Scale bar: 200 mu m; b: brightfield plots at day 1 and day 6 after plating of 4F-th passage 4F cells after overexpression of the four factor combinations CDK1, CDK4, CCNB and CCND (4F) in PoliHH cells. Scale bar: 200 μm.

Fig. 3, a: growth curves of HH (P4-P7) and 2F (P11-14) cells in monolayer culture; b: growth curves of HH (P7-P8) and 4F (P21-22) cells in single layer culture.

Fig. 4, a: the mRNA expression levels of 2F human hepatocyte characteristic markers (ALB, CYP3a4, CYP2B6, CYP2E1) for 2D and 3D, P11 and P14, n-3, P <0.05, P < 0.01; b: mRNA expression levels of human hepatocyte characteristic markers for HH, 4F of 2D and 3D (ALB, CYP3a4, CYP2B6, CYP2E1), n-3, p <0.05, p < 0.01; c: the mRNA expression levels of human hepatocyte characteristic markers (ALB, CYP3a4, CYP2B6, CYP2E1) for HH passage 5, HH passage 8 and 4F passage 18 of 2D and 3D were examined by real-time quantitative PCR, and n was 3.

Fig. 5, a: taking an immunofluorescence staining image of HH and 2F organs cultured in 3D by a confocal microscope, wherein green fluorescence in the image is a mature hepatocyte marker Albumin; b: immunofluorescence staining images of 2D cultured HH and 4F cells and 3D cultured HH and 4F organs shot by a confocal microscope, wherein green fluorescence is a mature hepatocyte marker Albumin.

Fig. 6 shows that the expression levels of 9 genes (CYP3a4, CYP3a7, CYP1B1, CYP2E1, FBXO32, G6PD, RGCC, SULT1C2, TUBB2B) associated with human hepatotoxicity, n is 3, p is <0.05, and p is <0.01, in monolayer-cultured 4F cells after rifampicin treatment.

Fig. 7 shows that the expression levels of 9 genes (CYP3a4, CYP3a7, CYP1B1, CYP2E1, FBXO32, G6PD, RGCC, SULT1C2, TUBB2B) associated with human hepatotoxicity after treatment of 3D cultured 4F organoids with rifampicin were measured by real-time quantitative PCR, with n being 3, p being <0.05, and p being <0.01 relative to the 2D 4F solvent group. # p <0.05, # p <0.01, relative to the 3D 4F solvent group.

FIG. 8, genome-wide CNV (copy Number variants) assay 2D cultured HH cells of the fourth passage and 4F cells of the 20 th passage.

Detailed Description

It is to be understood that within the scope of the present invention, the above-described technical features of the present invention and the technical features described in detail below (e.g., the embodiments) may be combined with each other to constitute a preferred embodiment.

Studies have shown that a large number of expanded human hepatocytes ProliHH (proliferative HH) can be obtained by culturing human primary hepatocytes with a specific medium. These cells can be seeded into injured mouse liver at levels comparable to primary HH after transplantation and will mature after in vivo transplantation or in vitro differentiation. Proliferative HH can be used to study drug metabolism, liver viral infections and liver disease, and allows large scale expansion of transplantable HH protocols. Proliferative HH loses proliferation potency after 5-8 in vitro proliferation passages.

The inventor further induces the proliferation capacity of the human liver primary cells by up-regulating the expression or activity of CDK1, CDK4, CCNB or CCND, obviously improves the cell amplification quantity and the passage number, and realizes the efficient, rapid and long-term in-vitro amplification of the human liver primary cells. Hepatocytes with upregulated expression or activity may be passaged for at least 10 passages and the number of cells per passage increased at least 2-fold compared to hepatocytes with non-upregulated expression or activity of the protein.

As used herein, CDK1 (cyclin dependent kinase 1), CDK4 (cyclin dependent kinase 4), CCNB (cyclin B) or CCND (cyclin D) are defined as conventional in the art and may be a human protein or a homologous protein of any species, provided that it retains kinase or cyclin function in human hepatocytes. Illustratively, CDK1 comprises SEQ ID NO 1 or a homologue or variant thereof having at least 90% sequence identity thereto and retaining CDK1 activity; CDK4 comprises SEQ ID NO 2 or a homologue or variant thereof having at least 90% sequence identity thereto and retaining CDK4 activity; the CCNB comprises SEQ ID No. 3 or a homologue or variant thereof having at least 90% sequence identity thereto and retaining CCNB activity; CCND comprises SEQ ID NO. 4 or a homologue or variant thereof having at least 90% sequence identity thereto and retaining CCND activity.

In some embodiments, the upregulation of expression of the proteins is achieved by introducing a nucleic acid construct into the cell that expresses the proteins. The nucleic acid construct comprises a polynucleotide coding sequence for a protein described herein, and one or more control sequences operably linked to the sequences. The nucleic acid construct may be one nucleic acid construct comprising a plurality of proteins, or may be a plurality of nucleic acid constructs each expressing one or more proteins, for example, 4 nucleic acid constructs each expressing 4 proteins or 2 nucleic acid constructs each expressing 2 proteins. The polynucleotides of the invention may be manipulated in a variety of ways to ensure expression of the protein. The nucleic acid construct may be manipulated prior to insertion into the vector, depending on the type of expression vector or requirements. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

The control sequence may be an appropriate promoter sequence. The promoter sequence is typically operably linked to the coding sequence of the protein to be expressed. The promoter may be any nucleotide sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell. The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention. The control sequence may also be a suitable leader sequence, a nontranslated region of an mRNA which is important for translation by the host cell. The leader sequence is operably linked to the 5' terminus of the nucleotide sequence encoding the polypeptide. Any terminator which is functional in the host cell of choice may be used in the present invention.

In certain embodiments, the nucleic acid construct is a vector. The vector may be a cloning vector, an expression vector, or a homologous recombinant vector. The polynucleotides of the present invention can be cloned into many types of vectors, for example, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Cloning vectors may be used to provide coding sequences for therapeutic proteins and polypeptides of the invention, such as a nucleic acid molecule comprising coding sequences for therapeutic proteins and polypeptides. The expression vector may be provided to the cell in the form of a viral vector. Expression of a polynucleotide of the invention is typically achieved by operably linking the polynucleotide of the invention to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration into eukaryotic cells. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that may be used to regulate the expression of the desired nucleic acid sequence. Viral vector technology is well known in the art and is described, for example, in Sambrook et al (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and Molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. Homologous recombinant vectors are used to integrate the expression cassettes described herein into the host genome.

Generally, suitable vectors comprise an origin of replication functional in at least one organism, a promoter sequence, a convenient restriction enzyme site and one or more selectable markers. For example, in certain embodiments, the invention uses a pCDH-EF1-MCS vector containing a replication initiation site, a 3 'LTR, a 5' LTR, a polynucleotide described herein, and optionally a selectable marker.

Examples of suitable promoters are the EF1, TBG promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence operably linked thereto. Other constitutive promoter sequences may also be used, including, but not limited to, the simian virus 40(SV40) early promoter, mouse mammary cancer virus (MMTV), Human Immunodeficiency Virus (HIV) Long Terminal Repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, EB virus immediate early promoter, rous sarcoma virus promoter, and human gene promoters such as, but not limited to, actin promoter, myosin promoter, heme promoter, and creatine kinase promoter.

To assess the expression of a therapeutic protein, polypeptide, or portion thereof, the expression vector introduced into the cells may also contain either or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected by the viral vector. In other aspects, the selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in a host cell. Useful selectable markers include Flag, HA or V5. Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Expression of the reporter gene is measured at an appropriate time after the DNA has been introduced into the recipient cell. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein. Suitable expression systems are well known and can be prepared using known techniques or obtained commercially.

The polynucleotides described herein can generally be obtained by PCR amplification. Specifically, primers can be designed based on the nucleotide sequences disclosed herein, particularly open reading frame sequences, and the relevant sequences can be amplified using commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art as templates. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order. Alternatively, the nucleic acid molecules described herein can also be synthesized directly.

Methods for introducing and expressing genes into cells are known in the art. The vector may be readily introduced into a host cell by any method known in the art, for example, a mammalian, bacterial, yeast or insect cell. For example, the expression vector may be transferred into a host cell by physical, chemical or biological means.

Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.

Biological methods for introducing polynucleotides into host cells include the use of viral vectors, particularly retroviral vectors. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. Many virus-based systems have been developed for gene transfer into mammalian cells. The selected gene can be inserted into a vector and packaged into a retroviral particle using techniques known in the art. The recombinant virus can then be isolated and delivered to the subject cells in vivo or ex vivo. Many retroviral systems are known in the art. Lentiviruses are a genus of the family retroviridae. A lentiviral vector is a more complex retroviral vector. Reagents for lentiviral packaging are well known in the art, as are conventional lentiviral vector systems including pRsv-REV, pMDlg-pRRE, pMD2G and the desired interference plasmid. In one embodiment, a Lentiv lentiviral vector pCDH-EF1-MCS was used. Thus, in certain embodiments, the invention also provides a lentivirus for activating T cells, the virus comprising a retroviral vector as described herein and a corresponding packaging gene, such as gag, pol, vsvg and/or rev.

In addition to overexpression, the present invention also includes treating primary hepatocytes and the proliferative hepatocytes so obtained using methods or agents known in the art that upregulate CDK1, CDK4, CCNB, or CCND protein expression or activity. One of skill in the art is aware of methods or agents in the art that can up-regulate CDK1, CDK4, CCNB or CCND protein expression or activity.

Herein, the host cell contains and/or expresses the antibodies and/or fusion proteins described herein and optionally a therapeutic polypeptide. Herein, when a cell is referred to as containing or comprising, expressing a molecule, such as a polypeptide, "containing" means that the molecule is contained within or on the surface of the cell; by "expression" is meant that the cell produces the molecule. Host cells include both primary hepatocytes or cells derived therefrom, and various cells used in the production of hepatocytes, such as E.coli cells, e.g., to provide coding sequences for proteins of the invention or to provide vectors as described herein. In certain embodiments, provided herein is a primary hepatocyte that stably expresses a protein described herein.

Hepatocytes suitable for use in the present invention may be of various types of origin, including, but not limited to, hepatic progenitors, hepatic parenchymal cells, hepatic nonparenchymal cells (e.g., endothelial cells, stellate cells, macrophages, and lymphocytes of the liver), biliary cells, central paravenous hepatocytes, portal paravenous hepatocytes. In certain embodiments, the hepatocytes suitable for use in the present invention are primary hepatocytes or derived cells thereof obtained from liver tissue. In certain embodiments, after obtaining primary cells, derived cells may be obtained by culturing in HIM or HM medium. Herein, primary hepatocyte-derived cells cultured for 1-8 passages are also referred to as ProliHH.

Hepatocytes with up-regulated expression or activity of two or more of CDK1, CDK4, CCNB, and CCND herein are proliferative and have the ability to mature in vivo or in vitro. The hepatocytes may be passaged for at least 10 passages, at least 15 passages, at least 17 passages, at least 20 passages, at least 23 passages, at least 30 passages, at least 40 passages. The number of cells per passage of the hepatocyte is increased at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold compared to a hepatocyte in which the expression or activity of the protein is not upregulated. Herein, "XX passage" or "PXX" of cell passage refers to the passage number of cells under the current culture conditions. For example, 2D P8 refers to cells passaged 8 passages in the 2D culture.

Hepatocytes with upregulated expression or activity of CDK1, CDK4, CCNB, or CCND described herein express hepatocyte signature markers after maturation, with at least a 2-fold increase in expression compared to immature hepatocytes. For example, a hepatocyte having upregulated expression or activity of the protein has at least a 2-fold, at least a 4-fold, at least a 10000-fold increase in ALB expression after maturation as compared to an immature hepatocyte. The expression or activity of the protein is upregulated by at least 2-fold, at least 100-fold, at least 1000-fold increase in CYP3a4 expression after maturation as compared to immature hepatocytes. The expression or activity of the protein is up-regulated, and CYP2B6 expression in the mature hepatocytes is increased at least 2-fold, at least 50-fold, at least 600-fold compared to immature hepatocytes. The expression or activity of the protein is up-regulated in the mature CYP2E1 expression of the hepatocyte is increased by at least 2-fold, at least 30-fold, at least 200-fold, at least 10000-fold compared to the immature hepatocyte. The hepatocytes express Albumin. As used herein, "mature" of hepatocytes means that the proliferation function of hepatocytes is weakened, liver differentiation occurs, and highly functional cells are obtained. Mature hepatocytes have many more powerful functions than immature hepatocytes, such as synthesis and storage, secretion of bile, detoxification, defense, clearance of exogenous antigens or pathogens, hematopoiesis, and the like.

Hepatocytes herein expressing two or more of CDK1, CDK4, CCNB, and CCND can express hepatotoxicity-associated genes in response to a toxic stimulus (e.g., rifampin). Common hepatotoxicity-related genes are well known in the art and include, but are not limited to, CYP3a4, CYP3a7, CYP1B1, CYP2E1, FBXO32, G6PD, RGCC, SULT1C2, TUBB 2B. Methods for detecting expression of these genes are also known in the art, for example by detecting mRNA to analyze expression levels.

Also included herein are cell preparations comprising the hepatocytes or extracts thereof described herein. For example, the cell preparation can be a cell culture, pharmaceutical composition, kit, device, medium or system, e.g., a chip, etc., comprising the hepatocytes or extracts thereof described herein and a suitable culture medium. The medium may be a medium conventionally used in the art for culturing hepatocytes, such as HIM or HM medium. The cell culture may also be a three-dimensional (3D) cell culture, such as an organoid, a 3D printed tissue, and the like.

The cell preparation may also be a medicament or a pharmaceutical composition comprising the hepatocytes or an extract thereof described herein and a pharmaceutically acceptable excipient. Herein, pharmaceutically acceptable excipients refer to carriers, diluents and/or excipients that are pharmacologically and/or physiologically compatible with the subject and the active ingredient, including but not limited to: pH adjusting agents, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers and preservatives. More specifically, suitable pharmaceutically acceptable excipients may be those commonly used in the art for administration of hepatocytes.

Typically, the pharmaceutical composition comprises a therapeutically effective amount of hepatocytes or an extract thereof. A therapeutically effective amount refers to a dose that achieves treatment, prevention, alleviation, and/or amelioration of a disease or disorder in a subject. The therapeutically effective amount may be determined based on factors such as the age, sex, condition and severity of the condition, other physical conditions of the patient, etc. Herein, a subject or patient generally refers to a mammal, in particular a human.

The cell preparation can also be a kit containing the hepatocytes or extracts thereof described herein. The kit may also contain various reagents suitable for manipulating (e.g., culturing, transplanting, purifying, genetically engineering, maturing, etc.) the hepatocytes, and optionally instructions directing the operation of those skilled in the art.

The cell preparation can also be a device or system containing hepatocytes or extracts thereof as described herein, for use in, e.g., cell culture, prevention, treatment, or diagnosis of liver disease, transplantation of hepatocytes or tissue and adjunctive therapy thereof, fabrication of liver aid devices, drug screening, detection of clearance, detection of liver toxicity, and the like.

Also provided herein are methods of producing proliferative hepatocytes, methods of enhancing the proliferative capacity of hepatocytes, increasing the number of expanded hepatocytes, increasing the number of passages of hepatocytes, comprising culturing the hepatocytes as described herein. Specifically, the methods comprise (1) overexpressing any two, three, or four of the proteins CDK1, CDK4, CCNB, and CCND described herein in hepatocytes, and (2) optionally culturing the cells obtained in (1). Conditions for culturing hepatocytes such as medium, temperature, humidity, mode, etc. are well known in the art, for example, using HM or HIM medium. In certain embodiments, the culture is a 2-dimensional (2D) or 3-dimensional (3D) culture. The 2D culture described herein, also referred to as a monolayer cell culture, refers to the adherent spread of animal cells and the onset of mitosis, which gradually forms a dense monolayer of cells. The 3D culture refers to that carriers with different materials with three-dimensional structures and various cells of different types are cultured together in vitro, so that the cells can migrate and grow in the three-dimensional space structures of the carriers; or the cells are placed in a surface non-contact culture material which almost avoids cell adhesion, and cell-cell aggregation is generated through naturally secreted extracellular matrix (ECM) so as to support the construction of the 3D spheroids and organoids. 3D culture can better simulate the natural environment of cell growth in vivo.

Also provided herein are methods of culturing primary hepatocytes or cells derived therefrom, comprising (1) overexpressing any two, three, or four proteins of CDK1, CDK4, CCNB, and CCND in primary hepatocytes or cells derived therefrom, and (2) optionally culturing the cells obtained in (1). The primary hepatocyte-derived cell is a primary hepatocyte that has been cultured (e.g. using HM medium). The culture is a 2D or 3D culture. In one or more embodiments, the primary hepatocyte-derived cell is a cell in which the primary hepatocyte has been cultured in HM or HIM medium for 1-8 passages 2D, referred to as ProliHH. The method can culture primary hepatocytes or cells derived therefrom for at least 10 passages, at least 15 passages, at least 17 passages, at least 20 passages, at least 23 passages, at least 30 passages, at least 40 passages. Thus, also included herein is the use of any two, three or four proteins or their coding sequences of CDK1, CDK4, CCNB and CCND in enhancing hepatocyte proliferation potency, increasing the number of hepatocyte amplifications, increasing the number of hepatocyte passages or promoting hepatocyte maturation.

The invention also provides a cell therapy in which hepatocytes are genetically modified to express any two, three, or four of the proteins CDK1, CDK4, CCNB, and CCND described herein, and the hepatocytes are administered to a subject. The therapy can comprise administering to the subject a cell preparation described herein, e.g., a pharmaceutical composition. The dosage form, dosage, and administration route of the pharmaceutical composition can be adjusted according to the age, sex, disease state, desired amount of hepatocytes or extract, etc. of the patient. Such as gels, aerosols, tablets, capsules, powders, granules, syrups, solutions, suspensions, injections, powders, pills, controlled release formulations, infusion solutions, suspensions. The pharmaceutical composition typically contains a therapeutically effective amount of the hepatocytes. Routes of administration of the pharmaceutical compositions described herein may include, but are not limited to, subcutaneous injection, transdermal injection, implantation, topical administration, intramuscular injection, sustained release administration, oral administration, and the like.

The invention also provides methods of screening for a drug, detecting drug clearance, or detecting hepatotoxicity comprising contacting a candidate drug with said hepatocytes and/or cell preparations, and detecting a change in said hepatocytes and/or cell preparations. Accordingly, also provided herein is the use of the hepatocytes and/or cell preparations described herein in drug screening, detecting drug clearance, detecting hepatotoxicity, or regenerative medicine.

Thus, also included herein is the use of a hepatocyte according to any embodiment herein in the preparation of a cell preparation for the prevention, treatment or diagnosis of liver disease, liver transplantation and its adjuvant therapy, support of liver assist devices. The cell preparation is a cell culture, a medicament, a kit, a device, a medium or a system, a tissue, an organoid. The liver diseases comprise infectious diseases of liver, neoplastic diseases, vascular diseases, metabolic diseases, toxic diseases, autoimmune diseases, hereditary diseases, calculus of intrahepatic bile duct and the like. Such as liver injury, hepatitis, liver cirrhosis, fatty liver, liver echinococcosis, hepatocarcinoma, hepatic hemangioma, hepatic lipoma, and hepatic sarcoma.

Also included herein are organoid preparation methods comprising culturing hepatocytes as described herein, e.g., in 3D culture with HIM and/or HM media. As used herein, an "organoid" is a living liver entity that can be grown from hepatocytes. The cells and organoids of the invention may be non-human animals or humans. Typically, organoids have a capsular structure and a layer of cells containing buds and a central lumen. The organoids are generally elongate in shape and may include one or more bud structures. The liver organoids preferably comprise hepatocytes and cholangiocytes.

Embodiments of the present invention will be described in detail below with reference to embodiments. It will be appreciated by those skilled in the art that the following examples are illustrative only and should not be taken as limiting the scope of the invention. The examples are given without reference to the specific techniques or conditions, according to the techniques or conditions described in the literature of the art (for example, see J. SammBruk et al, molecular cloning, A laboratory Manual, third edition, scientific Press, translated by Huang Petang et al) or according to the instructions of the product. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Examples

Example 1

1. Cells and culture media

Human primary hepatocytes were purchased from bioremodeling IVT under accession numbers JFC, MRW.

HIM medium is referred to Zhang et al, 2018, Cell Stem Cell 23, 1-14, and the specific components are shown in the following table.

HM medium is referred to Zhang et al, 2018, Cell Stem Cell 23, 1-14. The HM medium is HLIM (human liver isolation medium) medium from which Rspo1, Noggin and forskolin were removed, and the specific composition is shown in the following table.

2. Vector construction

Primers for four genes, CDK1(NM _001786.5, 891bp, SEQ ID NO:1), CCNB (NM _031966.4, 1296bp, SEQ ID NO:2), CDK4(NM _000075.4, 906bp, SEQ ID NO:3) and CCND (NM _053056.3, 885bp, SEQ ID NO:4) were designed, the primer sequences included CDK 1-F: 5, CDK 1-T2A-R: 6, T2A-CCNB 1-F: 7, HA-CCNB 1-R: 8, V5-CDK 4-F: 9, CDK 4-P2A-R: 10, P2A-CCND 1-F: 11, CCND 1-R: 12 in SEQ ID NO.

Four genes were amplified by PCR (TOYOBO KOD-plus), and the target gene was amplified using primers for each of the four genes. And (3) recovering target bands by using the recovered target bands and pairwise combinations (CDK1-CCNB, CDK4-CCND) as templates, and amplifying CDK1-CCNB, CDK1-F, HA-CCNB1-R, CDK 5-CDK4-F and CCND1-R by using CDK1-F and HA-CCNB1-R as amplification templates and amplifying CDK 4-CCND. The PCR system was found in TOYOBO KOD-plus Standard system. Meanwhile, carrying out double-enzyme cutting on the vector pCDH-EF1-MCS by SalI and EcoRI, carrying out gel recovery on a target band, combining target genes pairwise, carrying out homologous recombination by adopting a Seamless Cloning Kit (D7010M), and inserting the target genes into the vector pCDH-EF 1-MCS. Then transforming Escherichia coli, picking single clone, extracting plasmid and sequencing.

3. Transfected cells

The above plasmids and PMDLg, REV and VSVG plasmids were packaged with PEI in 293T cells for lentivirus packaging, and lentivirus was collected for infection of ProliHH cells.

As shown in FIG. 1, after ProliHH was infected with 2F (CDK1-CCNB1), 4F (CDK1-CCNB1, CDK4-CCND1) and 2F (CDK4-CCND1) viruses, three groups of cells expressed more factors.

Example 2 proliferation potency and number of passages of Primary hepatocytes

Overexpression of a combination of two factors, cyclin-dependent kinase 4(CDK4) and cyclin D1(CCND), in human hepatocytes prolihh (hh) cells (2F) 2F cells were constructed. 2F cells at 2X 10⁴Per cm²Was inoculated in collagen I coated 6-well plates and cultured with HM media monolayer (2D). The cells were placed at 37 ℃ in hypoxia (5% O)₂、5％CO₂) Culturing in an incubator, replacing the culture medium every 2 days, and counting cells after culturing for 6 days. To promote hepatocyte maturation, 2F cells were plated at 1X 10⁵The density of individual cells/well was seeded in low adhesion 96-well plates (Kuraray CO. ltd), cultured in HIM medium for 3D, and placed at 37 ℃, 5% CO₂And (5) culturing for 9 days in an incubator. The cell bright field and growth curves are shown in FIGS. 2, A and 3, A, respectively. The results showed that the overexpression of CDK4 and CCND in PoliHH cells combined with the factor (2F) effectively induced the proliferation ability of ProLiHH cells, expanded the passable number of ProLiHH monolayer culture from 5-8 to 17 or more, and doubled the number of cells expanded per passage from 3-4 to about 10.

Overexpression of cyclin-dependent kinase 1(CDK1), cyclin-dependent kinase 4(CDK4), cyclin B1(CCNB) and cyclin D1(CCND) in human hepatocytes prolihh (hh) cells combined (4F) to construct 4F cells. 4F cells at 2X 10⁴Per cm²Was inoculated in collagen I coated 6-well plates and cultured with HM media monolayer (2D). The cells were incubated at 37 ℃ in low oxygen (5% O)₂、5％CO₂) Culturing in an incubator, replacing the culture medium every 2 days, and counting cells after culturing for 6 days. To promote hepatocyte maturation, 4F cells were plated at 1X 10⁵Density of individual cells/well was seeded in low adhesion 96-well plates (Kuraray CO. ltd), 3D cultured in HIM medium, and placed at 37 ℃, 5% CO₂And (5) culturing for 9 days in an incubator. The cell bright field and growth curves are shown in FIGS. 2, B and 3, B, respectively. The results showed that the proliferation capacity of ProLiHH cells was efficiently induced by overexpression of CDK1, CDK4, CCNB and CCND in PoliHH cells in combination (4F), passable numbers of ProLiHH monolayer culture were expanded from 5-8 to 23 or more, and the number of cells expanded per passage was doubled from 3-4 to about 10.

Example 3 significant changes in liver feature marker expression

Analyzing the gene expression level of the liver marker of the cells cultured in the monolayer and the organoids cultured in the 3D by adopting a real-time quantitative PCR method, and detecting the expression condition of the marker Albumin of the liver cells by adopting an immunofluorescence staining method. The results are shown in FIGS. 4 and 5.

According to fig. 4, a and 5, a, 2D expanded 2F human hepatocytes can restore the phenotype of mature hepatocytes in 3D organoid cultures. The mRNA expression level of human hepatocyte characteristic markers (albctyp 3a4, CYP2B6, CYP2E1) of the 3D cultures was significantly increased compared to the 2D cultured cells; immunofluorescent staining patterns show that the mature hepatocyte marker Albumin of the 3D culture is also more significantly expressed.

According to fig. 4, B, fig. 5, B, 2D expanded 4F human hepatocytes have both the characteristics of human hepatocytes and human hepatic progenitors, and the phenotype of mature hepatocytes can be restored in 3D organoid cultures. mRNA expression levels of human hepatocyte characteristic markers (ALB, CYP3a4, CYP2B6, CYP2E1) of the 3D cultures were significantly increased compared to the 2D cultured cells; immunofluorescent staining patterns show that the mature hepatocyte marker Albumin in 3D cultures is also more significantly expressed.

According to fig. 4, C, 3D 4F of the 8 th and 18 th generations did not have much difference in expression of the hepatocyte marker compared to 3D ProLiHH of the 5 th generation. The method shows that the cells after long-term passage are modified still have the capacity of restoring the mature phenotype of the liver cells.

Example 4 increased hepatotoxic Gene expression following cell administration treatment

4F cells at 2X 10⁴Per cm²In collagen I coated 12-well plates in monolayer culture. The cells were placed at 37 ℃ under hypoxia (5% O)₂、5％CO₂) Culturing in an incubator, replacing the culture medium every 2 days, culturing for 6 days, and then performing administration treatment for 48 hours by using rifampicin. The rifampicin concentration of the administration group is 10 mu M and 100 mu M (wherein the final DMSO concentration is less than or equal to 0.1%); the solvent control group was HM broth containing 0.1% DMSO.

The cultures after the above administration treatment were collected, and mRNA expression levels of hepatotoxicity-related genes (CYP3a4, CYP3a7, CYP1B1, CYP2E1, FBXO32, G6PD, RGCC, SULT1C2, TUBB2B) were analyzed by real-time quantitative PCR. Individual gene expression Using the GAPDH gene as an internal reference and the comparative Ct method (2)^-ΔΔCt) And (4) calculating.

As shown in fig. 6, 9 genes related to human hepatotoxicity were detected by qPCR, and the expression levels of 8 hepatotoxicity-related genes (CYP3a4, CYP3a7, CYP1B1, CYP2E1, FBXO32, G6PD, RGCC, SULT1C2) were significantly increased in 2D-cultured 4F cells 48 hours after administration of rifampicin as compared to the solvent group, and the expression levels of 5 genes (CYP3a4, CYP3a7, CYP1B1, FBXO32, G6PD, RGCC, SULT1C2) were increased as the administration concentration was increased. One of the main indicators for evaluating the function of drug metabolism of hepatocytes in vivo is the activity of hepatotoxicity-associated gene. This example demonstrates that the liver cells of the present invention have drug metabolism function on one hand, and can be used as a model for drug hepatotoxicity-related studies on the other hand.

Example 5 increase of hepatotoxic Gene expression following organoid drug treatment

In addition, 4F cells (1X 10) were used⁵One/well) were seeded on low-adhesion 96-well plates (Kuraray CO. ltd) and placed at 37 ℃ in 5% CO₂In the incubator, 3D culture was performed with HIM medium.After 9 days of culture, administration treatment with rifampicin was performed for 48 hours. The rifampicin concentration of the administration group is 10 mu M and 100 mu M (wherein the final DMSO concentration is less than or equal to 0.1%); the solvent control group was HIM medium containing 0.1% DMSO. The mRNA expression level was measured by quantitative PCR as described in example 3.

As shown in fig. 7, the organoids in the 3D 4F solvent group showed significantly higher levels of expression of 4 liver drug enzyme-related genes (CYP3a4, CYP3a7, CYP1B1, CYP2E1) than the cells in the 2D 4F solvent group; after 48 hours of administration of rifampicin, the expression levels of 6 hepatotoxicity-related genes (CYP3a4, CYP3a7, CYP1B1, CYP2E1, FBXO32, G6PD, SULT1C2) in the 3D administration group were significantly changed in response to rifampicin on average, and also significantly changed as compared to the 3D solvent group.

Example 6 genome-wide CNV copy number variation assay for genome stability

As shown in fig. 8, the whole genome CNV copy number variation assay results showed that no genomic instability was caused after 20 passages, except for 1 random variation.

To summarize: the experiment establishes a novel human primary liver cell long-term in vitro culture platform. The platform provides a method for efficiently, quickly and long-term in-vitro amplification of human liver primary cells. Based on ProliHH, the proliferous human liver cell line effectively induces the proliferation capacity of human liver primary cells after CDK4 and CCND (2F) or CDK1, CDK4, CCNB and CCND (4F) are over-expressed, and the cell amplification quantity and the passage number are remarkably improved. And the expanded liver cells can restore the phenotype of mature liver cells by a 3D organoid culture method.

In addition, preliminary experiments of in vitro administration for stimulating hepatotoxicity show that the hepatotoxicity characteristics caused by in vivo administration can be simulated by using the platform, so that the platform can be applied to in vitro screening of the hepatotoxicity of the medicament before clinical application. Based on the problems of the lack of in vitro liver donors, the limited in vitro culture viability of primary cells of human livers (generally less than 48 hours), the difficulty in reproducing a plurality of characteristics of human liver diseases by a mouse model and the like faced by the current clinical and scientific researches. The platform has the potential of opening up a new experimental approach for liver disease modeling, toxicology research, regenerative medicine and gene therapy, and also brings hope for clinical liver disease treatment problems.

Sequence listing

<110> Shanghai Nutrition and health institute of Chinese academy of sciences

<120> engineered hepatocytes

<130> 202350

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 891

<212> DNA

<213> Artificial Sequence

<400> 1

atggaagatt ataccaaaat agagaaaatt ggagaaggta cctatggagt tgtgtataag 60

ggtagacaca aaactacagg tcaagtggta gccatgaaaa aaatcagact agaaagtgaa 120

gaggaagggg ttcctagtac tgcaattcgg gaaatttctc tattaaagga acttcgtcat 180

ccaaatatag tcagtcttca ggatgtgctt atgcaggatt ccaggttata tctcatcttt 240

gagtttcttt ccatggatct gaagaaatac ttggattcta tccctcctgg tcagtacatg 300

gattcttcac ttgttaagag ttatttatac caaatcctac aggggattgt gttttgtcac 360

tctagaagag ttcttcacag agacttaaaa cctcaaaatc tcttgattga tgacaaagga 420

acaattaaac tggctgattt tggccttgcc agagcttttg gaatacctat cagagtatat 480

acacatgagg tagtaacact ctggtacaga tctccagaag tattgctggg gtcagctcgt 540

tactcaactc cagttgacat ttggagtata ggcaccatat ttgctgaact agcaactaag 600

aaaccacttt tccatgggga ttcagaaatt gatcaactct tcaggatttt cagagctttg 660

ggcactccca ataatgaagt gtggccagaa gtggaatctt tacaggacta taagaataca 720

tttcccaaat ggaaaccagg aagcctagca tcccatgtca aaaacttgga tgaaaatggc 780

ttggatttgc tctcgaaaat gttaatctat gatccagcca aacgaatttc tggcaaaatg 840

gcactgaatc atccatattt taatgatttg gacaatcaga ttaagaagat g 891

<210> 2

<211> 1296

<212> DNA

<213> Artificial Sequence

<400> 2

gcgctccgag tcaccaggaa ctcgaaaatt aatgctgaaa ataaggcgaa gatcaacatg 60

gcaggcgcaa agcgcgttcc tacggcccct gctgcaacct ccaagcccgg actgaggcca 120

agaacagctc ttggggacat tggtaacaaa gtcagtgaac aactgcaggc caaaatgcct 180

atgaagaagg aagcaaaacc ttcagctact ggaaaagtca ttgataaaaa actaccaaaa 240

cctcttgaaa aggtacctat gctggtgcca gtgccagtgt ctgagccagt gccagagcca 300

gaacctgagc cagaacctga gcctgttaaa gaagaaaaac tttcgcctga gcctattttg 360

gttgatactg cctctccaag cccaatggaa acatctggat gtgcccctgc agaagaagac 420

ctgtgtcagg ctttctctga tgtaattctt gcagtaaatg atgtggatgc agaagatgga 480

gctgatccaa acctttgtag tgaatatgtg aaagatattt atgcttatct gagacaactt 540

gaggaagagc aagcagtcag accaaaatac ctactgggtc gggaagtcac tggaaacatg 600

agagccatcc taattgactg gctagtacag gttcaaatga aattcaggtt gttgcaggag 660

accatgtaca tgactgtctc cattattgat cggttcatgc agaataattg tgtgcccaag 720

aagatgctgc agctggttgg tgtcactgcc atgtttattg caagcaaata tgaagaaatg 780

taccctccag aaattggtga ctttgctttt gtgactgaca acacttatac taagcaccaa 840

atcagacaga tggaaatgaa gattctaaga gctttaaact ttggtctggg tcggcctcta 900

cctttgcact tccttcggag agcatctaag attggagagg ttgatgtcga gcaacatact 960

ttggccaaat acctgatgga actaactatg ttggactatg acatggtgca ctttcctcct 1020

tctcaaattg cagcaggagc tttttgctta gcactgaaaa ttctggataa tggtgaatgg 1080

acaccaactc tacaacatta cctgtcatat actgaagaat ctcttcttcc agttatgcag 1140

cacctggcta agaatgtagt catggtaaat caaggactta caaagcacat gactgtcaag 1200

aacaagtatg ccacatcgaa gcatgctaag atcagcactc taccacagct gaattctgca 1260

ctagttcaag atttagccaa ggctgtggca aaggtg 1296

<210> 3

<211> 906

<212> DNA

<213> Artificial Sequence

<400> 3

gctacctctc gatatgagcc agtggctgaa attggtgtcg gtgcctatgg gacagtgtac 60

aaggcccgtg atccccacag tggccacttt gtggccctca agagtgtgag agtccccaat 120

ggaggaggag gtggaggagg ccttcccatc agcacagttc gtgaggtggc tttactgagg 180

cgactggagg cttttgagca tcccaatgtt gtccggctga tggacgtctg tgccacatcc 240

cgaactgacc gggagatcaa ggtaaccctg gtgtttgagc atgtagacca ggacctaagg 300

acatatctgg acaaggcacc cccaccaggc ttgccagccg aaacgatcaa ggatctgatg 360

cgccagtttc taagaggcct agatttcctt catgccaatt gcatcgttca ccgagatctg 420

aagccagaga acattctggt gacaagtggt ggaacagtca agctggctga ctttggcctg 480

gccagaatct acagctacca gatggcactt acacccgtgg ttgttacact ctggtaccga 540

gctcccgaag ttcttctgca gtccacatat gcaacacctg tggacatgtg gagtgttggc 600

tgtatctttg cagagatgtt tcgtcgaaag cctctcttct gtggaaactc tgaagccgac 660

cagttgggca aaatctttga cctgattggg ctgcctccag aggatgactg gcctcgagat 720

gtatccctgc cccgtggagc ctttcccccc agagggcccc gcccagtgca gtcggtggta 780

cctgagatgg aggagtcggg agcacagctg ctgctggaaa tgctgacttt taacccacac 840

aagcgaatct ctgcctttcg agctctgcag cactcttatc tacataagga tgaaggtaat 900

ccggag 906

<210> 4

<211> 885

<212> DNA

<213> Artificial Sequence

<400> 4

gaacaccagc tcctgtgctg cgaagtggaa accatccgcc gcgcgtaccc cgatgccaac 60

ctcctcaacg accgggtgct gcgggccatg ctgaaggcgg aggagacctg cgcgccctcg 120

gtgtcctact tcaaatgtgt gcagaaggag gtcctgccgt ccatgcggaa gatcgtcgcc 180

acctggatgc tggaggtctg cgaggaacag aagtgcgagg aggaggtctt cccgctggcc 240

atgaactacc tggaccgctt cctgtcgctg gagcccgtga aaaagagccg cctgcagctg 300

ctgggggcca cttgcatgtt cgtggcctct aagatgaagg agaccatccc cctgacggcc 360

gagaagctgt gcatctacac cgacaactcc atccggcccg aggagctgct gcaaatggag 420

ctgctcctgg tgaacaagct caagtggaac ctggccgcaa tgaccccgca cgatttcatt 480

gaacacttcc tctccaaaat gccagaggcg gaggagaaca aacagatcat ccgcaaacac 540

gcgcagacct tcgttgccct ctgtgccaca gatgtgaagt tcatttccaa tccgccctcc 600

atggtggcag cggggagcgt ggtggccgca gtgcaaggcc tgaacctgag gagccccaac 660

aacttcctgt cctactaccg cctcacacgc ttcctctcca gagtgatcaa gtgtgacccg 720

gactgcctcc gggcctgcca ggagcagatc gaagccctgc tggagtcaag cctgcgccag 780

gcccagcaga acatggaccc caaggccgcc gaggaggagg aagaggagga ggaggaggtg 840

gacctggctt gcacacccac cgacgtgcgg gacgtggaca tctaa 885

<210> 5

<211> 45

<212> DNA

<213> Artificial Sequence

<400> 5

gaccggcgcc tactctagac tagcgaattc atggaagatt atacc 45

<210> 6

<211> 36

<212> DNA

<213> Artificial Sequence

<400> 6

agggccggga ttctcctcca cgtcaccgca tgttag 36

<210> 7

<211> 42

<212> DNA

<213> Artificial Sequence

<400> 7

gtggaggaga atcccggccc tgcgctccga gtcaccagga ac 42

<210> 8

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 8

ccagaggttg attgtcgacc tagatttatg catagtccgg gacgtcatag ggata 55

<210> 9

<211> 55

<212> DNA

<213> Artificial Sequence

<400> 9

gaccggcgcc tactctagac tagcgaattc atgggtaagc ctatccctaa ccctc 55

<210> 10

<211> 64

<212> DNA

<213> Artificial Sequence

<400> 10

ccacgtctcc tgcttgcttt aacagagaga agttcgtggc ctccggatta ccttcatcct 60

tatg 64

<210> 11

<211> 63

<212> DNA

<213> Artificial Sequence

<400> 11

ctgttaaagc aagcaggaga cgtggaagaa aaccccggtc ctgaacacca gctcctgtgc 60

tgc 63

<210> 12

<211> 50

<212> DNA

<213> Artificial Sequence

<400> 12

ccagaggttg attgtcgacc tagatttaga tgtccacgtc ccgcacgtcg 50

Claims

1. A nucleic acid construct comprising coding sequences for any two, three or four proteins of CDK1, CDK4, CCNB and CCND,

preferably, the nucleic acid construct is a recombinant vector or an expression vector.

2. The nucleic acid construct of claim 1, wherein said nucleic acid construct comprises coding sequences for CDK1 and CCNB, CDK4 and CCND, or CDK1, CDK4, CCNB and CCND,

preferably, CDK1 comprises SEQ ID NO 1 or a homologue or variant thereof having at least 90% sequence identity thereto and retaining CDK1 activity;

preferably, CDK4 comprises SEQ ID NO 2 or a homologue or variant thereof having at least 90% sequence identity thereto and retaining CDK4 activity;

preferably, the CCNB comprises SEQ ID No. 3 or a homologue or variant thereof having at least 90% sequence identity thereto and retaining CCNB activity;

preferably, CCND comprises SEQ ID NO. 4 or a homolog or variant thereof that has at least 90% sequence identity thereto and retains CCND activity.

3. A lentivirus comprising the nucleic acid construct of claim 1 or 2.

A hepatocyte having upregulated expression or activity of any two, three or four of CDK1, CDK4, CCNB and CCND.

5. The hepatocyte according to claim 4, wherein the hepatocyte is a primary hepatocyte or a derived cell thereof,

preferably, the expression or activity of CDK1 and CCNB, CDK4 and CCND, or CDK1, CDK4, CCNB and CCND is up-regulated in the hepatocyte, more preferably the hepatocyte comprises the nucleic acid construct of claim 1 or 2.

6. A cell preparation comprising the hepatocyte or the extract thereof of claim 4 or 5,

preferably, the extract is a protein or small molecule extract;

preferably, the cell preparation is a cell culture, a pharmaceutical composition, a liver organoid, a kit, a device, a medium or a system comprising said hepatocytes or an extract thereof.

7. A method for preparing a hepatocyte, enhancing the proliferative capacity of a hepatocyte, increasing the expanded number of a hepatocyte or increasing the number of passages of a hepatocyte or culturing a hepatocyte, which comprises (1) up-regulating the expression or activity of any two, three or four proteins of CDK1, CDK4, CCNB and CCND in a hepatocyte, and (2) optionally culturing the cell obtained in (1),

preferably, (1) is overexpression of CDK1 and CCNB, CDK4 and CCND, or CDK1, CDK4, CCNB and CCND in hepatocytes, more preferably, (1) is introduction of the nucleic acid construct of claim 1 or 2 into hepatocytes.

Use of any two, three or four proteins or their coding sequences from CDK1, CDK4, CCNB and CCND to enhance hepatocyte proliferation potency, increase the number of hepatocyte amplifications, increase the number of hepatocyte passages or culture hepatocytes.

9. Use of the hepatocyte of claim 4 or 5 or the cell preparation of claim 6 in drug screening, detecting drug clearance, detecting hepatotoxicity or regenerative medicine.

10. Use of the liver cells of claim 4 or 5 or the cell preparation of claim 6 for the preparation of a cell preparation for the prevention, treatment, diagnosis of liver diseases, liver transplantation and adjuvant therapy thereof, supporting liver-assist devices.