JP2004187679A - Method for detecting or isolating liver stem cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for detecting or separating liver stem cells from a mass of cells in a fetal liver, an adult liver under regeneration, etc. <P>SOLUTION: A gene of an Itm2A protein (Integral membrane protein 2A) is specifically and highly expressed in the liver stem cells in the fetal liver and the adult liver under regeneration. Further, it is confirmed that the gene comprises a type 2 membrane protein. Therefore, the liver stem cells are detectable or separable by using the Itm2A protein or a mRNA encoding the protein as an indicator. Thus, the method for detecting or separating the liver stem cells comprises detecting or separating the liver stem cells in a sample separated from a living body to the outside by using the Itm2A protein or the mRNA encoding the protein as the indicator. <P>COPYRIGHT: (C)2004,JPO&NCIPI

Description

  The present invention relates to a method for detecting or separating hepatic stem cells from a cell population in fetal liver or regenerating adult liver. INDUSTRIAL APPLICABILITY The present invention is useful for liver regeneration medicine, development of artificial liver, and the like.

  The only basic treatment for severe liver disease (fulminant hepatitis, cirrhosis, liver cancer, etc.) is to use a liver transplant, but there are problems such as a shortage of donors and the need to perform immunosuppressive therapy for a lifetime. The development of a law is desired. One of them is cell transplantation therapy using hepatic stem cells. Liver stem cells are defined as hepatic stem cells that have the ability to differentiate into both hepatocytes and bile duct epithelial cells, and have a high proliferative capacity with the ability to self-renew, and are extremely close to fetal liver and adult liver. It is said that there are a few. In recent years, many studies have been conducted to induce hepatic stem cells or hepatocytes from embryonic stem cells (ES cells), blood stem cells, and other stem cells in bone marrow. Here, one of the important problems is a method of identifying hepatic stem cells from a miscellaneous cell population and separating them with high purity. If hepatic stem cells can be specifically recognized, and they can be separated and recovered with high purity, the proliferation and differentiation induction of hepatic stem cells (undifferentiated hepatocytes) outside the body can be used for hybrid artificial liver and cell transplantation therapy. It is extremely useful for clinical applications, and can be a model experimental system in vitro for drug metabolism and infection / proliferation of hepatitis virus. In order to identify hepatic stem cells and to separate them with high efficiency, it is considered that a method using an antibody against a cell surface antigen is most appropriate, and this method is frequently used for separating hematopoietic stem cells. Almost no cell surface antigens of hepatic stem cells have been identified so far, but recently, a method of enriching hepatic stem cells from mouse fetal liver using the expression of known 5 to 6 types of cell surface antigens as an index (Non-patented) Reference 1) was reported. However, since this method uses a plurality of antibodies, the operation is complicated and hepatic stem cells cannot be separated from the adult liver. The group of the present inventors has developed a mouse embryo 14.5 by using a signal trap method (Non-patent Document 2) for specifically cloning a molecule having a signal sequence, that is, a gene encoding a cell surface antigen or a secretory protein. The present inventors have identified dlk, a gene highly expressed in the liver of the day, and found that hepatic stem cells can be purified to a high degree in one step from fetal liver using an anti-dlk monoclonal antibody (Patent Document 1). However, there is still very little information on cell surface antigens of hepatic stem cells, and it is important to identify as many hepatic stem cell surface antigens as possible.

Suzuki, A. et al. (2000) Hepatology 32: 1230-1239 Kojima, T. and Kitamura, T. (1999) Nat.Biotechnol. 17: 487-490 Kirchner, J. et al. J. Exp. Med. 190: 217-228 (1999) WO 02 / 103033A1

  It is an object of the present invention to provide a method for detecting or separating hepatic stem cells from a cell population such as in fetal liver or regenerating adult liver.

  The present inventors have conducted intensive studies and found that, in hepatic stem cells in the fetal liver and in the regenerating adult liver, the gene for the Itm2A (Integral membrane protein 2A) protein is specifically highly expressed. The present inventors have confirmed that it is a type 2 membrane protein, have reached the conclusion that it is possible to detect or separate hepatic stem cells using the Itm2A protein or mRNA encoding it as an index, and completed the present invention.

  That is, the present invention provides a method for detecting or separating hepatic stem cells, which detects or separates hepatic stem cells in a sample separated in vitro using the Itm2A protein or mRNA encoding the same as an index. The present invention also provides a monoclonal antibody for detecting or separating hepatic stem cells, which reacts with the extracellular region of Itm2A as an antigen-antibody. Further, the present invention provides a nucleic acid for measuring Itm2A mRNA or cDNA, which hybridizes with the mRNA or cDNA of Itm2A.

  The present invention has provided a method for efficiently detecting or separating hepatic stem cells from a cell population such as a fetal liver or a regenerating adult liver. According to the method of the present invention, hepatic stem cells can be identified and separated and recovered with high purity. Liver stem cells isolated in this way can be used for hepatocyte transplantation, which is expected as a next-generation regenerative medicine replacing organ transplantation, hybrid artificial liver, drug metabolism experiments in vitro, etc. The present invention is expected to make a significant contribution in these fields.

  As specifically described in the Examples below, the present inventors have compared the gene expression of mouse embryonic 12.5-day-old immature hepatocytes and embryonic 17.5-day-old prenatal hepatocytes by cDNA chip analysis and found that the membrane protein Integral Membrane protein 2A (Itm2A: Genbank accession No. NM_008409) was found to be highly expressed in immature hepatocytes in mouse embryos. As a result of Northern blot, immunostaining, and Itm2A mRNA expression analysis by whole-mount in situ hybridization, it was highly expressed in fetal hepatocytes from mouse embryos 10.5 to 12.5 days, and then rapidly attenuated. It was found that it was not expressed at all. The fetal liver functions as a hematopoietic organ and is mainly composed of immature hepatocytes and blood cells, but Itm2A was not expressed in blood cells at all, but was expressed in immature hepatocytes. The immature hepatocytes at this stage are also called hepatoblasts, and are considered to be hepatic stem cells having the ability to differentiate into two types of cells in the future, hepatocytes and bile duct epithelial cells. In addition, the expression of mRNA of human Itm2A (Genbank accession No. NM_004867) was examined using a total RNA sample of human fetal liver, and expression was observed in human fetal liver from 6 weeks to 12 weeks of embryo. In other words, Itm2A was found to be a membrane protein expressed in fetal immature hepatocytes not only in mice but also in humans.

  Next, 70% partial hepatectomy was performed on rats in which 2-acetylaminofluorene (2-AAF) was pre-administered to suppress the proliferation ability of mature hepatocytes, and oval cells around the portal vein ( oval cells) appear (Evarts, RP., et al (1989) Cancer Research, 49: 1541-1547). Oval cells are cells with high proliferative potential, and are considered to be hepatic stem cells in adult liver because they have both properties of immature hepatocytes and bile duct epithelial cells. These oval cells are considered to be responsible for liver regeneration instead of hepatocytes in a state where the division and proliferation of hepatocytes are suppressed due to severe liver disease or the like. The origin of this oval cell is largely unknown, but data has recently been reported that it arises from transdifferentiation of hematopoietic stem cells in bone marrow (Matsusaka, S. et al. (1999) Hepatology. 29 (3) : 670-6; Petersen, BE., Et al. (1999) Science 284: 1168-1170; Theise, ND., Et al. (2000) Hepatology. 31: 235-240). When the expression of Itm2A in the liver was examined in a model in which oval cells appeared in the rat, the expression was observed from 4 days after partial hepatectomy, and the expression was observed until day 13 while gradually attenuating. Then disappeared. This expression pattern is not expressed in mature hepatocytes and is similar to the expression pattern of alpha fetoprotein expressed in oval cells (Pertersen, BE. Et al. (1989) Hepatology. 27: 1030 -1038), suggesting that Itm2A is expressed in oval cells.

  From the above results, Itm2A is a membrane protein expressed in both hepatic stem cells in fetal liver and hepatic stem cells in adult liver, and it is possible to identify hepatic stem cells using the expression of Itm2A gene or protein as an index It is. In addition, since Itm2A is a cell membrane protein, using a monoclonal antibody that recognizes the extracellular region of Itm2A, using a cell separation device such as FACS (Fluorescein activated cell sorting) or MACS (Magnetic beads cell sorting), hepatic stem cell Can be separated. The hepatic stem cells isolated in this manner can be used for hepatocyte transplantation, which is expected as a next-generation regenerative medicine replacing organ transplantation, hybrid artificial liver, drug metabolism experiments in vitro, and the like.

  As described above, the Itm2A protein and the cDNA sequence encoding it are already known, and the mouse Itm2A cDNA sequence and the amino acid sequence encoded by it are described in Genbank accession No.NM_008409, and the human Itm2A cDNA. The sequence and the amino acid sequence it encodes have been described in Genbank accession No. XM_084285. These sequences are shown in SEQ ID NOs: 1 and 3 in the sequence listing, respectively.

  Since the Itm2A gene is expressed in hepatic stem cells, hepatic stem cells can be detected by examining the expression of the Itm2A gene. The expression of the Itm2A gene can be examined by measuring (detecting, semi-quantifying or quantifying) the Itm2A protein or mRNA encoding the same in cells. The measurement of mRNA in cells can be performed by a conventional method. That is, for example, as described in the Examples below, it can be performed by Northern blotting, or reverse transcription PCR (RT-PCR) can be performed, and the PCR product can be electrophoresed. Can be performed. Alternatively, by performing RT-PCR by a real-time detection PCR (RTD-PCR) method, it is possible to accurately quantify the amount of cDNA used as a template, and thus the amount of mRNA. Alternatively, mRNA can be directly amplified by the NASBA method or the like and measured by electrophoresis, and then Northern blot after electrophoresis. These methods are all conventional methods, and the necessary reagent kits and devices are commercially available. In addition, since the cDNA sequence of Itm2A is known (described in SEQ ID NOs: 1 and 3), the probes and primers required for these methods can be easily designed. Examples are given. Therefore, measurement of mRNA encoding Itm2A protein can be easily performed by those skilled in the art. The probe or primer used for the detection or amplification of the mRNA of Itm2A (or cDNA obtained using the mRNA as a template) preferably has a sequence complementary to any strand of the mRNA or cDNA of Itm2A, It is also possible to use those having a base mismatch of 10% or less, preferably 5% or less of the size of the probe or primer. By using a primer having such a mismatch, a desired restriction enzyme site can be provided to the amplification product. Such restriction sites may be convenient when integrating the amplification product into a vector. The size of the probe or primer is not particularly limited, but is 15 bases or more, preferably 20 bases or more, and there is no particular upper limit on the size, as in a conventional method. In the case of a primer, the size is usually 50 bases or less. The length is preferably 40 bases or less, and in the case of a probe, the total length is suitable. The present invention also provides nucleic acids for measuring Itm2A mRNA or cDNA, such as these probes and primers, which hybridize with the mRNA or cDNA of Itm2A.

  As specifically described in the Examples below, itm2A protein was confirmed to be a type 2 membrane protein. The type 2 membrane protein is a type of membrane protein in which the N-terminal side is present in the cell and the C-terminal side is outside the cell. Therefore, by preparing a monoclonal antibody having the C-terminal region of the Itm2A protein as an epitope and contacting the monoclonal antibody with a cell, the Itm2A protein present on the cell surface and the monoclonal antibody are bound by an antigen-antibody reaction. Thus, hepatic stem cells can be detected and separated using this. A method for detecting or separating cells that react with an antigen-antibody with the monoclonal antibody by utilizing an antigen-antibody reaction between the antigen on the cell surface and the monoclonal antibody against the antigen is well known in the art. If a monoclonal antibody that reacts with the above Itm2A as an antigen-antibody can be obtained, it can be easily performed. For example, it can be detected or separated by a known method such as a method using immunohistochemistry, FACS (fluorescence activated cell sorting), MACS (magnetic cell sorting), or the like. In the detection method and separation method of the present invention, it is preferable to detect the expression of the Itm2A gene by detecting the Itm2A protein. Non-invasive detection of Itm2A protein expression without fixing or lysing cells by detecting Itm2A expression using antibodies or ligands that can bind to the extracellular region of the Itm2A protein expressed on the cell surface It is possible to do. In this case, cells can be detected or separated efficiently by using a cell sorter such as FACS or MACS. FACS and MACS are well known in this field, and devices such as a cell sorter are commercially available. Therefore, if a monoclonal antibody having the extracellular region (C-terminal region) of Itm2A protein as an epitope can be obtained, hepatic stem cells Can be easily detected and separated.

  Since the nucleotide sequence of the Itm2A cDNA is known (SEQ ID NOS: 1 and 3), the extracellular region (C-terminal region) of the Itm2A protein can be easily prepared by a conventional method. In the mouse Itm2A cDNA shown in SEQ ID NO: 1, a region downstream of about 376 nt is an extracellular region, and in the human Itm2A cDNA shown in SEQ ID NO: 3, a region downstream of about 368 nt is an extracellular region. Therefore, a peptide having an amino acid sequence (the number of amino acid residues is 5 or more, preferably 10 or more) encoded by the whole region or a part of the extracellular region is prepared, and is used as an antigen. By preparing a monoclonal antibody by a conventional method, a monoclonal antibody having the extracellular region of the Itm2A protein as an epitope can be prepared. If the peptide used as the immunogen is short, it can be synthesized by chemical synthesis, and if it is long, it can be prepared by genetic engineering. As specifically described in the Examples below, cDNA for Itm2A can be prepared in large quantities by PCR from hepatic stem cells in fetal liver or regenerating adult liver. Therefore, the extracellular region or the partial region in the cDNA can be amplified by PCR, inserted into an appropriate vector, and introduced into a host cell to prepare a large amount of the desired peptide. Peptides used as immunogens can be easily prepared using a commercially available peptide synthesizer in the case of chemical synthesis, or can be easily prepared in the usual manner by the method outlined above when prepared by genetic engineering techniques. . When a peptide used as an immunogen is relatively short (for example, 10 amino acid residues or less), the peptide is transferred to another protein carrier (for example, keyhole limpet hemocyanin (KLH)) in order to efficiently elicit an immune response. ) Can also be used as an immunogen. This is also a common law. The present invention also provides such a monoclonal antibody for detecting or separating hepatic stem cells, which has an antigen-antibody reaction with the extracellular region of Itm2A.

  As specifically described in the Examples below, it was confirmed that hepatic stem cells were present in fetal liver and regenerating adult liver. Therefore, target tissues to which the method of the present invention is applied include fetal liver and regenerating adult liver. With respect to fetal liver, the Itm2A gene is highly expressed in fetal liver at 10.5 to 12.5 days of fetal life in mice and in fetal liver at 6 to 12 weeks of gestation in humans. For the regenerating adult liver, a partial hepatectomy is performed in a state in which the proliferation ability of mature hepatocytes is suppressed by pre-administration of a liver toxin such as 2-acetylaminofluorene (2-AAF). Small cells called oval cells appear around, but since the hepatic stem cells are included in this population of oval cells, the method of the present invention can be performed on oval cell groups in the regenerating adult liver. it can. Furthermore, since hepatic stem cells may also be present in tissues other than the liver such as bone marrow and embryonic stem cells (ES cells), these tissues or cell populations can also be subjected to the method of the present invention. .

  The applicant of the present application has previously found that a cell membrane protein called dlk (delta-like) is highly expressed in fetal hepatocytes, and the fetal hepatocytes can be efficiently separated by MACS using an anti-dlk monoclonal antibody. How to develop. dlk itself is known, and its amino acid sequence and cDNA sequence are shown in, for example, human dlk in Genbank accession numbers U15979 and NM_003836. Rat dlk is indicated in Genbank accession numbers AB046763 and D84336 and the like. Bovine dlk is shown in Genbank accession number AB009278. In addition, a monoclonal antibody having an extracellular region of dlk as an epitope is also known (Kaneta, M. et al. (2000) J. Immunol. 164: 256-264), and embryos are produced by MACS using this known monoclonal antibody. Hepatocytes can be separated (Patent Document 1). Since hepatic stem cells that highly express Itm2A are contained in such dlk-positive hepatocytes, first, anti-dlk antibody-positive cells are first selected from a sample, and the method of the present invention is applied to the selected cell population. Then, the hepatic stem cells can be efficiently detected or separated. The following embodiment employs this method.

  As specifically described in the Examples below, Itm2A was highly expressed in both mouse and human hepatic stem cells. As can be seen from SEQ ID NOs: 1 and 3, the amino acid sequences of the mouse and human Itm2A proteins have high homology. Although both mice and humans belong to the class Mammalia, they differ at the eye level and are very different organisms in mammals. Nevertheless, itm2A is also highly expressed in hepatic stem cells of other animals other than mice and humans, particularly other mammals, since highly homologous Itm2A is highly expressed in hepatic stem cells. , Is thought to be a universal protein. Therefore, the method of the present invention can be applied to animals other than mice and humans.

  As described above, the liver stem cells isolated by the method of the present invention are used for hepatocyte transplantation, which is expected as a next-generation regenerative medicine instead of organ transplantation, hybrid artificial liver, drug metabolism experiments in vitro, and the like. It is possible. In particular, the finding that hepatic stem cells can be separated from regenerating adult liver is important. Isolation of hepatic stem cells from the patient's own liver allows the transplantation of the hepatic stem cells into the patient, and in this case, rejection of the transplant occurs because the cells to be transplanted are the patient's own. Absent.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

1. Materials and methods
Preparation of mouse embryonic hepatocytes C57 / BL6, slc mice (Japan SLC) were used in all experiments. Livers were isolated from 200 fetal mice at 12.5 days of embryo and 50 fetal mice at 17.5 days of embryo, respectively, and the liver was digested by collagenase treatment to prepare a cell suspension. Next, the cell suspension was sequentially reacted with a hamster anti-dlk monoclonal antibody (Kaneta, M. et al. (2000) J. Immunol. 164: 256-264), a biotinylated anti-hamster antibody, and streptavidin beads. Were used to prepare hepatic stem cells and prenatal fetal hepatocytes. The purity of dlk-positive cells prepared by this method was 95% or more in embryonic day 12.5 cells and 85% or more in embryonic day 17.5 cells.

Rat oval cell induction model 8-week-old male F344 rats were orally administered with 2-acetylaminofluorene (Wako Pure Chemical Industries) at a dose of 1.5 mg / kg (body weight) once a day for 4 days, and 70% A partial hepatectomy was performed. The day when the partial hepatectomy was performed was designated as day 0, and the same amount of 2-acetylaminofluorene was orally administered for 5 days from the next day. Livers were collected on Day 0, 4, 7, 9, 11, 13, and 20 and used for gene expression analysis. Untreated liver was used as a control. Further, on Day 7, parenchymal cells and non-parenchymal cells were prepared from liver tissue by combining the two-step collagenase digestion method and low-speed centrifugation (500 rpm, 1 min), and used for gene expression analysis (described later). The preparation of parenchymal cells and non-parenchymal cells from liver tissue was specifically performed as follows. After laparotomy of the rat under Nembutal anesthesia, ligating the inferior vena cava in the lower liver, inserting a catheter into the portal vein and starting to flow the preperfusion solution (Ca2 +, Mg2 + free Hanks solution) for 10 minutes at the same flow rate of 20 mL / min After perfusion, the liver was similarly perfused with a 0.05% collagenase solution (collagenase type IA, Sigma) for 10 minutes to digest the liver. The liver was collected in a 50 ml centrifuge tube, pre-perfused liquid was added thereto, and the mixture was suspended well, followed by centrifugation at 500 rpm for 1 min. The supernatant was collected as a non-parenchymal cell fraction and the precipitate was collected as a parenchymal cell fraction. After washing 4 to 5 times, RNA was prepared.

DNA chip analysis Trizol reagent (trade name, manufactured by Nippon Gene Co., Ltd.), Oligotex dT30 (trade name, Daiichi Kagaku (Manufactured by KK). The prepared mRNA was subjected to DNA chip analysis using a mouse GEM2 microarray manufactured by Incyte Genomics (USA) using a DNA chip carrying 9514 clones.

Gene expression analysis RNA was extracted from the collected liver or cells using Trizol reagent (Nippon Gene). In gene expression analysis by Northern blot, 10 micrograms of total RNA per lane was electrophoresed on a formaldehyde denaturing gel, transferred to a nylon membrane, and then hybridized using a DIG-labeled cDNA probe (described later). Was. Probe detection was performed by chemiluminescence using CDP-star as a substrate. Gene expression analysis by RT-PCR method, cDNA was synthesized from the extracted RNA using a First-strand cDNA synthesis kit (trade name, manufactured by Amersham Pharmacia Biotech), and the expression of Itm2A was analyzed using PCR. . The PCR primers are 5'-att tac cat ggt gag atg tg-3 'for the forward primer and 5'-aag tgt cta atc ttc cag ca-3' for the reverse primer, and a 500 bp PCR product using these primers. Was amplified. Also, using this PCR product as a template, a DIG-labeled cDNA probe for mouse Itm2A was prepared. A DIG-labeled cDNA probe for human Itm2A was prepared using a template obtained by cutting out a band with EcoRI / HindIII from a pCRII vector into which the following human full-length Itm2A cDNA was incorporated.

Isolation of mouse and human Itm2A full-length cDNA and construction of expression vector PCR primers were designed based on the gene sequence information of mouse Itm2A (Genbank accetion No. NM_008409) and human Itm2A (Genbank accetion No. XM_084285). For insertion into an expression vector maintaining the orientation, a mouse was designed with a forward primer to which a restriction enzyme digestion sequence by EcoRI was added at the 5 ′ end and a reverse primer to which a restriction enzyme digestion sequence by XhoI was added. For humans, a reverse primer was designed to which a restriction enzyme digestion sequence with Hind III was added. In the case of mouse, cDNA prepared from liver of 12.5 days of embryonic embryo was used as a template, and in the case of human, cDNA synthesized from total RNA prepared from commercially available human fetus was used as a template. Synthesized (includes up to 45 bp of sequence before the termination codon and has no termination codon). Primer sequences are as follows: mouse forward: 5'-gga att cca gcc caa gat act gat tc-3 ', reverse: 5'-ccg ctc gag cgg gtg tct aat ctt cca gca tt-3', human forward: 5 '-cga tct cct ctt gca gtc tgc-3', reverse: 5'-aag ctt ctc ttg aca gat ctt ggt c-3 '. After amplification by PCR, developing by agarose gel electrophoresis, extracting and extracting the target band, the mouse Itm2A PCR product was digested with EcoRI and XhoI, and the expression vector (pcDNA4 / Myc-His: Invitrogen) ) At the EcoRI / XhoI site. Since this expression vector has a Myc tag and a His tag on the C-terminal side, the expressed protein is a fusion protein of Itm2A, Myc tag, and His tag. The human Itm2A PCR product was T / A cloned into a pCRII vector (Invitrogen).

Gene transfer into cultured cells An expression vector (pcDNA4 / Myc-His) incorporating the full-length mouse Itm2A cDNA was introduced into the mouse fibroblast cell line NIH-3T3 cells by lipofection. This operation was specifically performed as follows. A mixture of 8 μg of DNA and 16 μL of LIPOFECTAMINE PLUS reagent (Invitrogen) was added to 500 μL of OPTI-MEM medium (solution A), and left at room temperature for 15 minutes. Then, a solution (solution B) was prepared by mixing 24 μL of LIPOFECTAMINE reagent (Invitorogen) and 500 μL of OPTI-MEM medium, and the solution A and the solution B were mixed and left at room temperature for 15 minutes. Next, 4 mL of OPTI-MEM medium was added to make a total volume of 5 mL (solution C). The NIH-3T3 cells seeded on a 10 cm diameter culture dish the day before were washed once with OPTI-MEM medium, and then C solution was added thereto and placed in a CO ₂ incubator (37 ° C., 5% CO ₂ , 95% Air ) For 3 hours, 5 mL of DMEM / 20% FBS medium was added, and the cells were further cultured. The next day, the medium was replaced with a DMEM / 10% FBS medium. 48 hours after gene introduction, the cells were detached from the culture dish by trypsin treatment to prepare a cell suspension, and expression analysis by FACS was performed (described later).

FACS analysis A cell suspension of NIH3T3 cells transfected with an expression vector (pcDNA4 / Myc-His) incorporating the full-length mouse Itm2A cDNA was reacted with an anti-Myc monoclonal antibody or an anti-His monoclonal antibody (4 ° C., 15 minutes). ). After washing with PBS, it was reacted with biotinylated anti-mouse IgG (4 ° C., 15 minutes), washed with PBS, and reacted with streptavidin-FITC or streptavidin-APC (4 ° C., 15 minutes). The expression of Itm2A was analyzed by FACS Calibur (BECTON DICKINSON) using the expression of Myc tag or His tag as an index.

Preparation of Anti-Itm2A Polyclonal Antibody The intracellular region of mouse Itm2A cDNA was amplified by PCR, and the amplified product was inserted into the EcoRI / SalI site of a GST fusion protein expression vector (pGEX-5X-1: Amersham Pharmacia Biotech). The resulting expression vector was introduced into Escherichia coli, a fusion protein of GST and Itm2A was induced by adding IPTG, and then affinity purification was performed using a glutathione column. Using this as an antigen, rabbits were immunized to prepare anti-mouse Itm2A antibody. The PCR primers used for cloning the intracellular region of Itm2A were 5'-gga att cca gcc caa gat act gat tc-3 'on the forward side and 5'-gtc gac ccc aga gag cca tct ttc t-3' on the reverse side. there were.

7 μm frozen sections were prepared using an immunohistochemical cryostat and fixed with 4% paraformaldehyde. The immunostaining for Itm2A was carried out by sequentially reacting an anti-Itm2A antibody (rabbit IgG) and a biotinylated anti-rabbit IgG, and staining with DAB as a substrate. As counterstaining, nuclear staining with hematoxylin was performed.

Whole-mount in situ hybridization Whole embryos of embryos at embryonic days 10.5 and 11.5 were hybridized using a DIG-labeled Itm2A RNA probe. The detection of the probe was performed by reacting with an alkaline phosphatase-labeled anti-DIG antibody and then performing chemical coloring using NBT / BCIP as a substrate.

Preparation of Itm2A monoclonal antibody
In order to prepare a monoclonal antibody having the C-terminal region of the Itm2A protein as an epitope, the method described in "Materials and Methods" in Example 1, "Isolation of full-length mouse and human Itm2A cDNA and construction of expression vector". A cell suspension of COS-7 cells transfected with an expression vector (pcDNA4 / Myc-His) incorporating the full-length mouse Itm2A cDNA is mixed 1: 1 with an immunostimulant (Complete Freund's adjuvant: Wako Pure Chemical Industries). The resulting emulsion was injected into both footpads of 6-week-old Wistar rats (female) at 1 × 10 ⁷ cells each for immunization. Booster immunization was performed 3 days and 10 days after the initial immunization, and on the day after the final immunization, the lymph nodes of both feet were collected from the rats under Nembutal anesthesia to prepare lymphocytes. Subsequently, cell fusion was performed with a mouse myeloma cell line (P3X) by the polyethylene glycol method. In a 96-well plate, culturing was performed in a RMPI1640 / 10% FBS medium containing HAT (aminopterin, hypoxanthine, thymidine) in a CO ₂ incubator (37 ° C., 5% CO ₂ , 95% air).

Screening of anti-Itm2A monoclonal antibody-producing hybridomas After about 9 days from the start of culture, HEK293 cells expressing the mouse Itm2A gene (Example 1) were used for culture supernatants of about 150 hybridomas that had proliferated and formed colonies. Insert the expression vector (pcDNA4 / Myc-His) incorporating the mouse Itm2A full-length cDNA described in the section “Isolation of mouse and human Itm2A full-length cDNA and construction of expression vector” in “Materials and Methods” in “ Screening was performed by FACS analysis using a cell suspension transfected into HEK293 cells by the method described in “Gene Transfer”. The cell suspension was reacted with the culture supernatant (4 ° C., 15 minutes). After washing with PBS, it was reacted with biotinylated anti-rat IgG (4 ° C., 15 minutes), washed with PBS, and reacted with streptavidin-PE (4 ° C., 15 minutes). Analysis was performed using a FACS Calibur (BECTON DICKINSON). The culture supernatant that was positive in the screening by FACS was then screened by immunostaining using fetal mouse hepatocytes. This operation was specifically performed as follows. All cells including hepatocytes were prepared from fetal mice at 12.5 days of embryonic age by the method described in the section “Preparation of mouse embryonic hepatocytes” in the above “Materials and Methods”. The cells were spread on a slide glass using a cell collection centrifuge (Cyto Spin 3; SHANDON) at a cell number of 1 × 10 ⁵ cells (room temperature, 800 rpm, 3 minutes). The smear was fixed with 4% paraformaldehyde, and then treated with PBS containing 1.5% normal goat serum (room temperature, 30 minutes). Next, the culture supernatant was reacted sequentially with a biotinylated anti-rat antibody, and staining was performed using DAB as a substrate. As counterstaining, nuclear staining with hematoxylin was performed. As a result, two types of hybridoma cell lines recognizing Itm2A were established (clone 3A2, 8B1).

Cell separation using magnetic beads All cells including hepatocytes were prepared from embryonic 13.5 day-old fetal mice by the method described in the section "Preparation of mouse embryonic hepatocytes" in the above "Materials and Methods". . The cells were treated with a cell permeation reagent for FACS (BECTON DICKINSON) (room temperature, 5 minutes). After centrifugation (1000 rpm, 5 minutes), the cells were sequentially reacted with the culture supernatant containing the above-described anti-Itm2A monoclonal antibody, biotinylated anti-rat antibody, and streptavidin beads (each reaction condition was 4 ° C., 15 ° C., respectively). Minute). Next, Itm2A-positive cells were separated using autoMACS. This operation was specifically performed as follows. After completion of the above series of reactions, the cell suspension was centrifuged (1000 rpm, 5 minutes), and then suspended in 3 mL of a flow cytometry sheath solution (BIOSURE) containing 0.5% BSA. The cell suspension was set in autoMACS (MILTENYI BIOTEC), and Itm2A-positive cells were separated by positive selection using the separation program POSSELD2 of the same apparatus.
result
1. cDNA chip analysis As described above, dlk (delta-like) is a cell membrane protein highly expressed in fetal liver cells, and by combining anti-dlk monoclonal antibody and MACS (Magnetic beads cell sorting), from fetal liver, Only hepatocytes can be recovered with high purity (Patent Document 1). Comparison of gene expression between dlk-expressing cells of embryonic liver of day 12.5 and embryonic dlk-expressing cells of day 17.5 was performed by DNA chip analysis. The DNA chip used for the analysis was a mouse GEM2 microarray manufactured by Incyte Genomics (USA) and a DNA chip on which 9514 clones were mounted. Embryonic hepatocytes at day 12.5 of embryo (dlk-expressing cells) are called hepatoblasts and have the ability to differentiate into hepatocytes and biliary epithelial cells in both directions. It is considered that hepatic stem cells that also have self-renewal ability are included in these. On the other hand, embryonic hepatocytes on day 17.5 of embryonic life are just before birth, most of which are fate-determined in hepatocytes, and it is time to start expressing genes for liver-specific metabolic enzymes such as glucose 6 phosphatase and tyrosine aminotransferase. . Therefore, by comparing the expression of these two genes, it can be expected to identify hepatoblast-specific genes, and thus hepatic stem cell-specific genes, and genes whose expression is induced with development. As a result of the analysis, out of all 9514 clones, the expression of 218 clones was enhanced, and the expression of 133 clones was decreased. For the purpose of identifying genes specifically expressed in hepatic stem cells, we focused on 133 clones whose expression decreased during development. As a result, the decrease rate was large (more than 2 times). Attention was focused on Integral membrane protein 2A (Itm2A: Genbank accetion No. NM_008409), which is expected to be a membrane protein.

2. Gene expression analysis of Itm2A in fetal liver
In the DNA chip analysis, itm2A was highly expressed on embryonic day 12.5, and the expression intensity was less than half on embryonic day 17.5. Thus, the expression of Itm2A in the developing liver was actually confirmed by Northern blot. As a result, high expression was observed in the liver at 12.5 days of fetal life, and thereafter, the expression was rapidly attenuated. Although some expression was observed on 16.5 days of fetal life, no expression was detected in adult liver. The liver at day 12.5 of the embryo functions as a hematopoietic organ and contains a large number of blood cells. To determine whether Itm2A is expressed in embryonic hepatocytes or blood cells, fractionate dlk-positive and negative cells from the 12.5-day embryonic liver using autoMACS and separate each fraction. When the expression of Itm2A in the cells was examined by RT-PCR, Itm2A was specific to dlk-positive cells. In embryonic liver, the expression of dlk was specific to embryonic hepatocytes and not to blood cells, suggesting that Itm2A was also specifically expressed in embryonic hepatocytes. In order to trace the timing of the expression, fetal whole-mount in situ hybridization was performed on the embryos at 10.5 days and 11.5 days. As a result, the expression was observed in both liver primordia. In mice, around 9.5 days of embryogenesis, liver primordium was formed from a part of the intestinal tract, but it was found that Imm2A was already expressed immediately thereafter. The Itm2A gene has very high homology not only in mice but also in humans (Genbank accetion No. XM_084285). Whether or not expression was observed in the human fetal liver as well as in the mouse was examined by Northern blot using a commercially available human fetal liver total RNA sample (TAKARA). , Expression of Itm2A was observed.

3. Gene expression analysis by immunohistochemistry Northern blot and RT-PCR in embryonic mouse liver suggested that Itm2A was expressed in embryonic hepatocytes. However, for further confirmation, anti-Itm2A antibody (rabbit IgG) was prepared. Was used to immunostain liver sections at 12.5 days of embryonic age. As a result, Itm2A was stained in embryonic hepatocytes and not in blood cells, like alpha-fetoprotein and dlk expressed in embryonic hepatocytes. In addition, double staining of dlk and Itm2A was performed using cytospin specimens of cell suspensions obtained by treating embryonic liver with collagenase. As a result, the staining of dlk and Itm2A completely matched.

Four. FACS analysis
From its primary structure, Itm2A is predicted to be a single-transmembrane type 2 membrane protein, but no paper has proven that it actually appears on the cell membrane. In order to prepare a monoclonal antibody against the extracellular region of Itm2A and to isolate Itm2A-expressing hepatic stem cells, it is necessary to first confirm that Itm2A is really a type 2 membrane protein. The type 2 membrane protein is a type of membrane protein in which the N-terminal side is present in the cell and the C-terminal side is outside the cell. Mouse Itm2A cDNA cloned by PCR is cloned into an expression vector (pcDNA4 / Myc-His: Invitrogen) so that it becomes a fusion protein of Myc tag and His tag at the C-terminus. If it is a type 2 membrane protein, the Myc tag and His tag added to the C-terminal side will be extracellular when expressed in cells, so FACS analysis using anti-Myc antibody and anti-His antibody It is possible. When this Itm2A-Myc-His fusion protein was forcibly expressed in NIH3T3 cells and analyzed by FACS with anti-Myc antibody and anti-His antibody, 4.3% of anti-Myc antibody and 16.5% of anti-His antibody showed a shift. Was. That is, it was confirmed that Itm2A was a type 2 membrane protein. The difference between the two values is considered to be the difference in gene transfer efficiency between experiments.

5. Analysis of gene expression of Itm2A in rat oval cells (hepatic stem cells in adult liver) The liver is a very regenerative organ. Occurs and returns to the original liver weight in about a week. The liver regeneration in this case is such that the mature hepatocytes proliferate, but the hepatotoxin such as 2-acetylaminofluorene (2-AAF) is pre-administered to suppress the proliferative ability of the mature hepatocytes. When the resection is performed, small cells called oval cells appear around the portal vein. Oval cells are cells with high proliferative potential, and are considered to be hepatic stem cells in adult liver because they have both properties of immature hepatocytes and bile duct epithelial cells. Thus, it was examined whether Itm2A, which was suggested to be expressed in fetal liver stem cells during development, is also expressed in oval cells, that is, hepatic stem cells in the adult liver that emerges during liver regeneration. At normal 70% partial hepatectomy, no expression of Itm2A was detected at all, but gene expression of Itm2A was only induced when 2-AAF administration was combined with 70% partial hepatectomy to induce the appearance of oval cells. It was induced 4 days after partial hepatectomy and continued to develop on day 13 with gradual attenuation, and disappeared after 20 days. This expression pattern was very similar to the expression pattern of the oval cell marker alpha-fetoprotein when it appeared in oval cells, suggesting that Itm2A is expressed in oval cells.

6. FACS analysis of fetal hepatocytes using anti-Itm2A monoclonal antibody (clone 3A2)
Itm2A is presumed to be a type 2 membrane protein from the amino acid sequence, and mouse or human Itm2A gene was exogenously transfected into animal cells as shown in “4. FACS analysis” in “Results”. In this case, it was experimentally confirmed that it was expressed on the cell surface as a type 2 membrane protein. Using the prepared anti-Itm2A monoclonal antibody and performing FACS analysis on liver cells of mouse embryonic day 12.5 according to a conventional method, it was not possible to detect the expression of Itm2A. Kirchner et al. Found that In two cell lines derived from T cells, Itm2A is usually expressed on the cell surface, but most of it is expressed on the membrane of intracellular organelles, and the cell surface is activated by T cell activation. (Non-Patent Document 3). Therefore, fetal liver cells were treated with a cell membrane infiltration reagent (BECTON DICKINSON), then reacted sequentially with anti-Itm2A monoclonal antibody, biotinylated anti-rat IgG, and streptavidin PE, and subjected to FACS. It was confirmed that the expression was strong. This result suggests that In fetal liver cells, Itm2A is usually localized in intracellular organelles. Therefore, in the following experiments, cells were treated with a cell membrane permeating reagent, and then FACS or cell separation using magnetic beads was performed.

7. Comparison of expression between Itm2A and dlk by FACS analysis
dlk (delta-like) is a cell membrane protein highly expressed in fetal liver cells.By combining anti-dlk monoclonal antibody and MACS (Magnetic beads cell sorting), only hepatocytes can be recovered from fetal liver with high purity. (Patent Document 1). From the pattern of gene expression in the liver, the Itm2A gene is more likely to be a more immature cell marker because its expression is lost earlier than the dlk gene. When two-dimensional analysis was performed by FACS using an anti-dlk monoclonal antibody and an anti-Itm2A monoclonal antibody, 94% and 89% of the dlk-positive cells were Itm2A-positive among the embryos at 12.5 days and 13.5 days of the embryo, respectively. On the other hand, 45% of the dlk-positive cells were Itm2A-positive in the embryonic day 15.5 on which the liver developed. This result indicates that Itm2A is a marker expressed on immature hepatic stem cells.

8. ITM2A monoclonal antibody hepatic stem cell separation <br/> mouse embryonic 13 using (clone 3A2) and magnetic beads. 5 days of liver cells (4.2 x 10 ⁷ cells) as described above, ITM2A positive cells and ITM2A negative using AutoMACS As a result, the cells were separated into Itm2A-positive cells (1.3 × 10 ⁶ cells; 11% after separation) and Itm2A-negative cells (1.21 × 10 ⁷ cells; 89% after separation). As a result of preparing cytospin specimens of each cell fraction after separation and double-staining with albumin and dlk, which are hepatocyte markers, 93% of Itm2A-positive cells are albumin / dlk-positive hepatocytes, 89% of Itm2A-negative cells were albumin / dlk-negative blood cells.

Discussion Itm2A, a type 2 membrane protein, was originally identified as a molecule expressed in immature cartilage and bone progenitor cells, but its expression in the liver was not known at all (Deleersnijder, W. et al (1996). ) Journal of Biological Chemistry 271 (32): 19475-82). Here, by comparing gene expression in embryonic hepatocytes between embryonic day 12.5 and embryonic day 17.5, it was revealed that the gene was highly expressed in embryonic liver cells from embryonic day 10.5 to embryonic day 12.5. Hepatocytes at this time are called hepatoblasts and have the ability to differentiate into both hepatocytes and bile duct epithelial cells. Furthermore, it is considered that the hepatoblasts include hepatic stem cells that also have a self-renewal ability. Also, in adult liver, hepatic stem cells called oval cells appear in a certain pathological state (severe liver disease) when the division and proliferation of mature hepatocytes is suppressed. . There are many unclear points about the origin of this oval cell, but there have been several reports suggesting that it is derived from bone marrow. In other words, it has been suggested that it is caused by transdifferentiation of bone marrow cells to hepatocytes. Expression of Itm2A was not induced at all during the normal liver regeneration process, and was induced in the liver regeneration model in which oval cells appeared, together with the appearance time. This result indicates that Itm2A is a novel cell surface antigen expressed not only on fetal liver stem cells during development but also on liver stem cells in adult liver, which is said to be derived from bone marrow. Based on the above results, Itm2A is a good marker for hepatic stem cells during development, hepatic stem cells in adults, and hepatic stem cells generated by transdifferentiation from bone marrow and ES cells. Can be identified and separated and recovered with high purity. The hepatic stem cells isolated in this manner can be used for hepatocyte transplantation, which is expected as a next-generation regenerative medicine replacing organ transplantation, hybrid artificial liver, drug metabolism experiments in vitro, and the like.

Claims (12)

  A method for detecting or separating hepatic stem cells, comprising detecting or separating hepatic stem cells in a sample separated in vitro using the Itm2A protein or mRNA encoding the same as an index.   The method according to claim 1, wherein a monoclonal antibody that reacts with the extracellular region of the Itm2A protein as an antigen-antibody reacts with the cells, and the monoclonal antibody bound on the cell surface is used as an index.   2. The method according to claim 1, wherein the presence or production amount of mRNA of Itm2A protein in the cell is used as an index.   The method according to any one of claims 1 to 3, wherein hepatic stem cells contained in a cell population in fetal liver are detected or separated.   The method according to any one of claims 1 to 3, wherein hepatic stem cells contained in a cell population in a regenerating adult liver are detected or separated.   6. The method of claim 5, wherein the regenerating adult liver is an adult liver to which liver toxin has been administered and a portion of the liver has been removed.   The method according to any one of claims 1 to 3, wherein hepatic stem cells contained in a cell population in a tissue other than the liver or in embryonic stem cells are detected or separated.   The method according to any one of claims 1 to 7, wherein the Itm2A is human Itm2A.   The method according to any one of claims 1 to 8, which is a method for separating hepatic stem cells from a cell population.   A monoclonal antibody for detecting or separating hepatic stem cells, which reacts with an antigen-antibody with the extracellular region of Itm2A.   A nucleic acid for measuring Itm2A mRNA or cDNA, which hybridizes with Itm2A mRNA or cDNA. The nucleic acid according to claim 11, which is a probe or a primer.