WO2017022472A1

WO2017022472A1 - Marker for undifferentiated mesenchymal stem cells and use thereof

Info

Publication number: WO2017022472A1
Application number: PCT/JP2016/071190
Authority: WO
Inventors: 篤榎本; 高橋　雅英; 啓子前田; 昭壽原; 水谷　泰之
Original assignee: 国立大学法人名古屋大学
Priority date: 2015-08-03
Filing date: 2016-07-19
Publication date: 2017-02-09
Also published as: JPWO2017022472A1; JP6841429B2

Abstract

The present invention addresses the problem of providing a marker molecule that is highly specific for mesenchymal stem cells in an undifferentiated state and enables identification of mesenchymal stem cells at a high sensitivity or preparation of highly pure mesenchymal stem cells. Provided are a marker for undifferentiated mesenchymal stem cells, said marker comprising immunoglobulin superfamily containing leucine-rich repeat, and a use of the same.

Description

Undifferentiated mesenchymal stem cell marker and use thereof

The present invention relates to a mesenchymal stem cell marker. Specifically, the present invention relates to a molecule (biomarker) having high specificity for mesenchymal stem cells in an undifferentiated state and use thereof. This application claims priority based on Japanese Patent Application No. 2015-153712 filed on Aug. 3, 2015, the entire contents of which are incorporated by reference.

Mesenchymal stem cells (mesenchymal stem cell or mesenchymal stromal cell; hereinafter referred to as “MSC”) are cells that are present in many organs including bone marrow, adipose tissue, dental pulp, umbilical cord, and endometrium It is known that when cultured on culture dishes under specific conditions, it differentiates into bone, cartilage and fat. In addition, mesenchymal stem cells are also known to have the effect of promoting the proliferation and differentiation of other cell groups (trophic effect) and immunomodulation, and are now GVHD (graft-versus-host disease), spinal cord injury, after kidney transplantation Application to the treatment of various diseases such as renal failure, myocardial infarction and peripheral occlusive arterial disease is expected.

It is known that mesenchymal stem cells exist around blood vessels such as sinusoids and small arteries in the bone marrow, and around capillaries in other organs (Non-patent Document 1). The bone marrow functions as a niche cell for maintaining hematopoietic stem cells or as a bone stem cell for bone regeneration (Non-patent Document 2). In organs other than the bone marrow, they exist as pericytes (vascular pericytes) or fibroblasts around the blood vessels, and are considered to be involved in vascular maturation (Non-patent Document 3). There is also a theory that the cause of interstitial fibrosis in cancer and various fibrotic diseases is perisite proliferation (Non-patent Document 4).

Internationally, cells that satisfy the following conditions (1) to (3) are defined as mesenchymal stem cells.
(1) Cells that adhere on the culture dish (2) CD105 positive, CD73 positive, CD90 positive, CD45 negative, CD34 negative, CD14 or CD11b negative, CD79a or CD19 negative, HLA-DR negative (3) Having the ability to differentiate into osteoblasts, chondroblasts and adipocytes in vitro (in vitro)

In addition to the molecules listed in (2) above, Stro-1, PDGF receptor, Sca-1, CD271, CD146, etc. have been reported as markers for mesenchymal stem cells. A method for isolating a stem cell has been developed (see, for example, Patent Document 1). However, all markers including CD105, CD73, and CD90 are expressed in epithelial cells, cancer cells, nerve cells, etc., and are not specific for mesenchymal stem cells.

International Publication No. 2009/031678 Pamphlet US Patent Application Publication No. 2005/0260639 A1

Many molecules have been reported as markers for mesenchymal stem cells, but all are expressed in cells other than mesenchymal stem cells and have low specificity. Therefore, the present invention provides a marker molecule that is highly specific for mesenchymal stem cells in an undifferentiated state and enables high-sensitivity mesenchymal stem cell identification and preparation of high-purity mesenchymal stem cells, and uses thereof It is an issue to provide.

While conducting research under the above-mentioned problems, the present inventors focused on the leucine-rich repeat-containing immunoglobulin superfamily (ISLR), a cell membrane-bound / secreted molecule, and conducted various experiments. Went. ISLR was cloned as a molecule expressed in the retina of the eye in 1997, but its function has not yet been clarified (Non-Patent Documents 5 and 6). In a paper on gene expression analysis published in 2009, it has been reported that it is expressed in the mesenchymal tissue of the arch, limb bud, body segment, head and trunk in mouse fetuses (non-patent literature) 7). It has also been reported that ISLR2 (also known as Linx), which is a homologous gene of ISLR, is expressed mainly in nervous system tissues (Non-patent Document 8). There is also a report that ISLR expression is higher than normal in lung cancer, breast cancer, pancreatic cancer and the like (see, for example, Patent Document 2, Non-Patent Documents 9 and 10). However, in either report, ISLR is only listed as one of many genes that exhibit similar behavior, and there is no mention of ISLR function. On the other hand, in a report of a study that comprehensively examined gene expression in mesenchymal stem cells, ISLR is listed in a list of genes that recognize increased expression (Non-Patent Documents 11 and 12). However, the significance of ISLR expression in mesenchymal stem cells and its function are unknown.

As shown in the examples described later, as a result of the study by the present inventors, it was revealed that ISLR is a marker molecule specific for mesenchymal stem cells. ISLR is highly specific for undifferentiated mesenchymal stem cells, and is distinct from the previously reported markers of mesenchymal stem cells. Moreover, ISLR has been found to be a marker for mesenchymal stem cells in mice as well as humans, and its utility value is high.

By the way, there is a theory that mesenchymal stem cells have the property of accumulating at the site of tumor or cancer occurrence or metastasis. However, there are various theories about the significance of accumulation and the role of accumulated cells (particularly involved in the progression of cancer), and their elucidation is eagerly desired. By obtaining a tool called ISLR that specifically detects mesenchymal stem cells, the present inventors can visually grasp the accumulation of mesenchymal stem cells and detect the abundance of mesenchymal stem cells. I can do it now. As a result of further research based on this result, it was found that the expression of ISLR is related to the progression of cancer and the prognosis of cancer patients. In addition, an experiment using a model animal of myocardial infarction shows a phenomenon in which mesenchymal stem cells accumulate at the infarct site of myocardial infarction, and ISLR knockout mice show that the prognosis after myocardial infarction is significantly worse, It has been found that ISLR is also useful for the progression and prognosis evaluation or estimation of fibrotic diseases such as myocardial infarction.
The following invention is mainly based on the above results.
[1] An undifferentiated mesenchymal stem cell marker consisting of a leucine-rich repeat-containing immunoglobulin superfamily (ISLR).
[2] The undifferentiated mesenchymal stem cell marker according to [1], wherein ISLR comprises any one of the amino acid sequences of SEQ ID NOS: 1 to 3.
[3] A method for preparing undifferentiated mesenchymal stem cells, comprising a step of selecting and collecting ISLR-expressing cells from a cell population containing mesenchymal stem cells.
[4] The method according to [3], wherein the cell population containing mesenchymal stem cells is derived from bone marrow, dental pulp, adipose tissue, endometrium, umbilical cord, skeletal muscle, or peripheral blood.
[5] A method for detecting undifferentiated mesenchymal stem cells, characterized by detecting the expression state of ISLR as an index.
[6] A method for evaluating the undifferentiation of a mesenchymal stem cell, comprising the step of examining the expression state of ISLR in a test mesenchymal stem cell.
[7] The method according to any one of [3] to [6], wherein the mesenchymal stem cell is a human cell.
[8] A reagent for detecting undifferentiated mesenchymal stem cells, comprising an anti-ISLR antibody.
[9] An undifferentiated mesenchymal stem cell detection kit comprising the reagent according to [8].
[10] A method for detecting undifferentiated mesenchymal stem cells that accumulate in a diseased site of cancer or fibrotic disease, wherein the expression state of ISLR is detected as an index.
[11] The method according to [10], wherein the cancer is pancreatic cancer, colon cancer, or breast cancer, and the fibrosis is myocardial infarction.
[12] A biomarker for prognosis estimation of cancer or fibrotic disease, comprising ISLR.
[13] The biomarker for prognosis estimation according to [12], wherein the cancer is pancreatic cancer, colon cancer, or breast cancer, and the fibrosis is myocardial infarction.
[14] A method for estimating the prognosis of a patient with cancer or fibrosis using the expression level of ISLR as an index.
[15] The prognosis estimation method according to [14], wherein the cancer is pancreatic cancer, colon cancer, or breast cancer, and the fibrosis is myocardial infarction.
[16] A reagent for estimating the prognosis of a patient with cancer or fibrosis, comprising an anti-ISLR antibody.
[17] The prognostic reagent according to [16], wherein the cancer is pancreatic cancer, colon cancer, or breast cancer, and the fibrotic disease is myocardial infarction.
[18] A kit for estimating the prognosis of a patient with cancer or fibrosis, comprising the reagent according to [16] or [17].

ISLR expression in human bone marrow-derived mesenchymal stem cells and dermal fibroblasts. The expression of ISLR was examined by Western blot. Note 1: Human skin fibroblasts transfected with ISLR-specific shRNA ISLR expression in human adipose-derived stem cells. The expression of ISLR was examined by Western blot. Note 1: Glyceraldehyde-3- Phosphate Dehydrogenase (endogenous control) ISLR expression in mouse bone marrow-derived mesenchymal stem cells. The expression of ISLR was examined by Western blot. Note 1: Endogenous control Decreased ISLR expression associated with the differentiation of mouse mesenchymal stem cells. The expression of ISLR and various marker molecules on differentiation induction day 0, day 1, day 7, and day 14 was examined by Western blotting. Note 1: Glyceraldehyde-3-Phosphate Dehydrogenase (endogenous control), Note 2: fatty-acid binding protein 4 (adipocyte marker), Note 3: Chondroblast marker, Note 4: Osteoblast marker Decreased ISLR expression associated with human mesenchymal stem cell differentiation. The expression of ISLR and various marker molecules on differentiation induction day 0, day 1, day 7, and day 14 was examined by Western blotting. Note 1: Glyceraldehyde-3-Phosphate Dehydrogenase (control) Decreased expression of ISLR gene mRNA with mesenchymal stem cell differentiation. The expression level of ISLR mRNA on differentiation induction day 7 was examined by quantitative RT-PCR. Verification of the effect of high ISLR expression on differentiation of mesenchymal stem cells. Differentiation induction of C3H10T1 / 2 in which ISLR was forcibly expressed was induced, and the expression of ISLR, Sox9 (cartilage differentiation marker), Osteopontin, and Runx2 (both bone differentiation markers) were examined by Western blot on day 0 and day 7. It was. Note 1: Chondrocyte marker, Note 2: Osteoblast marker, Note 3: Osteoblast marker ISLR expression in various cultured cells. The expression of ISLR in human fibroblasts, vascular smooth muscle cells, vascular endothelial cells and various epithelial cells (purchased from Lonza Japan Co., Ltd. or ZenBio) was examined by Western blotting. ISLR expression in mesenchymal stem cells growing in the stroma of pancreatic cancer. Mesenchymal stem cells accumulate in the stroma of moderately differentiated pancreatic cancer and poorly differentiated pancreatic cancer. The presence of mesenchymal stem cells was examined by in situ hybridization using ISLR expression as an index. ISLR expression in colon cancer tissue. Mesenchymal stem cells accumulate in the stroma of moderately differentiated colorectal cancer and poorly differentiated colorectal cancer. The presence of mesenchymal stem cells was examined by in situ hybridization using ISLR expression as an index. ISLR expression in breast cancer tissue. Mesenchymal stem cells accumulate in the stroma of invasive ductal carcinoma. The presence of mesenchymal stem cells was examined by in situ hybridization using ISLR expression as an index. ISLR expression at the infarct site in a mouse myocardial infarction model. Mesenchymal stem cells accumulate at the myocardial infarction site of the mouse myocardial infarction model. The presence of mesenchymal stem cells was examined by in situ hybridization using ISLR expression as an index. Difference in survival rate after myocardial infarction between wild-type and ISLR knockout mice. ISLR knockout mice were prepared and the survival rate after myocardial infarction was compared with wild type mice.

1. Undifferentiated Mesenchymal Stem Cell Marker The first aspect of the present invention relates to a marker molecule that has been specifically expressed in undifferentiated mesenchymal stem cells, that is, an “undifferentiated mesenchymal stem cell marker”. An “undifferentiated mesenchymal stem cell marker” refers to a molecule that serves as an indicator of mesenchymal stem cells that retain undifferentiated properties. Detection, measurement, labeling, and preparation (sorting) of undifferentiated mesenchymal stem cells using the undifferentiated mesenchymal stem cell marker of the present invention (hereinafter sometimes referred to as “the marker of the present invention” for short)・ Concentration), evaluation, etc. become possible. In particular, the marker of the present invention has high utility value in preparing undifferentiated mesenchymal stem cells from a cell population containing mesenchymal stem cells. “Undifferentiated mesenchymal stem cells” have the ability to differentiate into osteoblasts, chondroblasts, and adipocytes. If undifferentiated mesenchymal stem cells are cultured under specific induction conditions, they differentiate along the cell lineage corresponding to the induction conditions.

The marker of the present invention consists of leucine-rich repeat-containing immunoglobulin superfamily (ISLR: immunoglobulin-superfamily-containing leucine-rich-repeat). ISLR is a cell membrane-bound or secreted molecule, and is highly expressed in lung cancer, breast cancer, pancreatic cancer and the like (see, for example, Patent Document 2, Non-Patent Documents 9 and 10).

The amino acid sequences of ISLR registered in public databases and the gene sequences encoding them are shown in the attached sequence listing. The correspondence between the sequence numbers and the sequences is as follows.
SEQ ID NO: 1: human ISLR amino acid sequence (NCBI Reference Sequence: NP_005536.1, immunoglobulin superfamily containing leucine-rich repeat protein precursor [Homo sapiens].)
SEQ ID NO: 2 amino acid sequence of mouse ISLR (NCBI Reference Sequence: NP_036173.1, immunoglobulin superfamily containing leucine-rich repeat protein precursor [Mus musculus].)
SEQ ID NO: 3: Amino acid sequence of rat ISLR (NCBI Reference Sequence: NP_001119772.1 immunoglobulin superfamily containing leucine-rich repeat protein precursor [Rattus norvegicus])
SEQ ID NO: 4: cDNA sequence of human ISLR (GeneID: 3671, NCBI Reference Sequence: NM_005545.3, homosapiens immunoglobulin superfamily containing leucine-rich repeat (ISLR), transcript variant 1, mRNA.)
SEQ ID NO: 5: cDNA sequence of mouse ISLR (GeneID: 26968, NCBI Reference Sequence: NM_012043.4, Mus musculus immunoglobulin superfamily containing leucine-rich repeat (Islr), transcript variant 1, mRNA.)
SEQ ID NO: 6: Rat ISLR cDNA sequence (GeneID: 686539, NCBI Reference Sequence: XM_006243176.2, NM_001126300.1, Rattus norvegicus immunoglobulin superfamily containing leucine-rich repeat (Islr), mRNA)

Human ISLR has a known variant (variant 2) sequence (Homo sapiens immunoglobulin superfamily containing leucine-rich repeat (ISLR), transcript variant 2, mRNA, amino acid sequence: NP_958934.1, cDNA sequence: NM_201526.1) . The amino acid sequence of the variant is identical to the above amino acid sequence (SEQ ID NO: 1).

Also for mice, the variant (variant 2) sequence (Mus musculus immunoglobulin superfamily containing leucine-rich repeat (Islr), transcript variant 2, mRNA, amino acid sequence: NP_001182360.1, cDNA sequence: NM_001195431.1) is known. The amino acid sequence of the variant is identical to the above amino acid sequence (SEQ ID NO: 2).

In the case of application to humans, the marker of the present invention is used not as an indicator of undifferentiated mesenchymal stem cells present in the human body but as an indicator of undifferentiated mesenchymal stem cells in a state separated from the human organism. Is done.

2. Preparation of Undifferentiated Mesenchymal Stem Cells The second aspect of the present invention relates to the use of the marker of the present invention and provides a method for preparing undifferentiated mesenchymal stem cells (hereinafter referred to as “preparation method of the present invention”). . When the cell population before carrying out the preparation method of the present invention (that is, the cell population subjected to the preparation method of the present invention) and the cell population obtained by the preparation method of the present invention are compared, the latter is less than the former. Since the content of differentiated mesenchymal stem cells is high, “preparation of undifferentiated mesenchymal stem cells”, “concentration of undifferentiated mesenchymal stem cells”, or “purity (ratio) of undifferentiated mesenchymal stem cells” It can be paraphrased as “improvement”.

Undifferentiated mesenchymal stem cells are useful per se, and are expected to be used as transplantation materials for regenerative medicine, for example. Various cells (osteoblasts, chondroblasts, etc.) obtained by inducing differentiation thereof can also be used for reconstruction of a specific tissue. Furthermore, chemokines, cytokines, growth factors and the like produced by mesenchymal stem cells are expected to be applied to the treatment of various diseases. On the other hand, it is known that undifferentiated mesenchymal stem cells are important for maintenance of hematopoietic stem cells, and undifferentiated mesenchymal stem cells can also be used as support cells when maintaining hematopoietic stem cells in vitro. Therefore, undifferentiated mesenchymal stem cells are also useful in the field of bone marrow transplantation. According to the preparation method of the present invention, since highly specific markers are used, undifferentiated mesenchymal stem cells that are extremely useful in this way can be prepared with high purity. Also, efficient preparation is possible.

The preparation method of the present invention uses the marker of the present invention. Specifically, in the preparation method of the present invention, the following step is performed, that is, “the step of selecting and collecting ISLR-expressing cells from a cell population containing mesenchymal stem cells”. Hereinafter, details of the step will be described.

A cell population containing mesenchymal stem cells is prepared in advance. The cell population can be obtained by a conventional method from bone marrow, dental pulp, adipose tissue, endometrium, umbilical cord, umbilical cord blood, skeletal muscle, peripheral blood or the like of a human or non-human animal (eg, mouse, rat). Usually, physical processing (cutting, pipetting, filtering, etc.) and enzymes on samples (eg, bone marrow fluid, aspirated fat, skeletal muscle tissue, blood) collected from these cell sources (cell sources, origins) Treatment (for example, using trypsin, dispase, collagenase, elastase, papain, etc.) is performed alone or in combination to separate cells. Preferably, the cell mass is not present. When preparing a cell population from peripheral blood or the like, it is preferable to remove blood cell components by hemolysis or the like.

The sample collected from the cell source or the cells separated therefrom may be seeded and cultured in a culture vessel, and the cells showing adhesion may be used as “cell population including mesenchymal stem cells”. In other words, in one embodiment of the present invention, a cell population obtained by a method similar to the conventional method for preparing mesenchymal stem cells, in which adherent cells are selected, is used as the “cell population containing mesenchymal stem cells”. .

In the preparation method of the present invention, ISLR-expressing cells are selected and collected from a cell population containing mesenchymal stem cells. That is, an undifferentiated mesenchymal stem cell, which is a target cell, is obtained using ISLR expression as an index. Selection and collection using ISLR as an indicator can be performed, for example, by flow cytometry and cell sorting using an anti-ISLR antibody. According to flow cytometry and cell sorting, it is possible to sort ISLR positive cells specifically and efficiently. Devices for performing flow cytometry and cell sorting (flow cytometer and cell sorter) are commercially available from, for example, Beckman Coulter Co., Ltd., Nippon Becton Dickinson Co., Ltd., and the like. Basic operating methods, sorting conditions, etc. may be in accordance with the instruction manual attached to the device.

In addition to flow cytometry, ISLR positive cells can be selected and collected by affinity chromatography using anti-ISLR antibodies or magnetic cell separation using magnetic beads.

Anti-ISLR antibodies used for flow cytometry, cell sorting, magnetic cell separation, etc. are polyclonal antibodies and oligoclonal antibodies (mixtures of several to several tens of antibodies) as long as they can be used for selection and recovery of ISLR positive cells. And any of monoclonal antibodies. As a polyclonal antibody or an oligoclonal antibody, an anti-serum-derived IgG fraction obtained by animal immunization, or an affinity-purified antibody using an antigen can be used. Anti-ISLR antibodies can be prepared using immunological techniques, phage display methods, ribosome display methods, and the like. Preparation of a polyclonal antibody by an immunological technique can be performed by the following procedure. An antigen (ISLR or a part thereof) is prepared and used to immunize animals such as rabbits. An antigen can be obtained by purifying a biological sample. A recombinant antigen can also be used. Recombinant ISLR is prepared by, for example, introducing a gene encoding ISLR (which may be a part of the gene) into a suitable host using a vector and expressing it in the resulting recombinant cell. can do.

In order to enhance the immunity-inducing action, an antigen to which a carrier protein is bound may be used. As the carrier protein, KLH (KeyholeHLimpet） Hemocyanin), BSA (Bovine Serum Albumin), OVA (Ovalbumin) and the like are used. The carbodiimide method, the glutaraldehyde method, the diazo condensation method, the MBS (maleimidobenzoyloxysuccinimide) method, etc. can be used for the coupling | bonding of carrier protein. On the other hand, an antigen in which ISLR (or a part thereof) is expressed as a fusion protein with GST, β-galactosidase, maltose-binding protein, histidine (His) tag or the like can also be used. Such a fusion protein can be easily purified by a general method.

Immunization is repeated as necessary, and blood is collected when the antibody titer has sufficiently increased, and serum is obtained by centrifugation or the like. The obtained antiserum is affinity purified to obtain a polyclonal antibody.

On the other hand, monoclonal antibodies can be prepared by the following procedure. First, an immunization operation is performed in the same procedure as described above. Immunization is repeated as necessary, and antibody-producing cells are removed from the immunized animal when the antibody titer sufficiently increases. Next, the obtained antibody-producing cells and myeloma cells are fused to obtain a hybridoma. Subsequently, after this hybridoma is monoclonalized, a clone producing an antibody having high specificity for the antigen is selected. The target antibody can be obtained by purifying the culture medium of the selected clone. On the other hand, the desired antibody can be obtained by growing the hybridoma to a desired number or more, then transplanting it into the abdominal cavity of an animal (for example, a mouse), growing it in ascites, and purifying the ascites. For purification of the culture medium or ascites, affinity chromatography using protein G, protein A or the like is preferably used. Alternatively, affinity chromatography in which an antigen is immobilized may be used. Furthermore, methods such as ion exchange chromatography, gel filtration chromatography, ammonium sulfate fractionation, and centrifugation can also be used. These methods can be used alone or in any combination.

Since the preparation method of the present invention uses a marker highly specific to undifferentiated mesenchymal stem cells, it becomes possible to specifically and efficiently prepare undifferentiated mesenchymal stem cells alone. However, other marker molecules that can be used to select undifferentiated mesenchymal stem cells (for example, CD105, CD73, CD90, CD45, CD34, CD14, CD11b, CD79a, CD19, known as markers for mesenchymal stem cells, HLA-DR etc.) is not excluded or restricted. That is, the steps of constructing the method of the present invention (using ISLR) for the purpose of further improving purity or homogeneity, enriching a specific cell population (eg, characterized by expression of a specific marker molecule), etc. In addition to (Step), a step of selection and collection with other marker molecules may be performed. This step is performed before or after the sorting and collection step using ISLR.

3. Detection of Undifferentiated Mesenchymal Stem Cells The third aspect of the present invention relates to detection of undifferentiated mesenchymal stem cells, a method for detecting undifferentiated mesenchymal stem cells (hereinafter referred to as “the detection method of the present invention”), Reagents and kits used in the method are provided. The detection method of the present invention has the greatest feature in that undifferentiated mesenchymal stem cells are detected using the expression state of ISLR as an index. According to the detection method of the present invention, undifferentiated mesenchymal stem cells in a sample can be detected specifically and with high sensitivity. Therefore, the detection method of the present invention is useful as an experimental means (research tool) that enables identification, visualization, extraction and the like of undifferentiated mesenchymal stem cells.

The detection method of the present invention is applied to a sample containing undifferentiated mesenchymal stem cells. The sample is not particularly limited as long as it may contain undifferentiated mesenchymal stem cells. For example, a tissue piece or a cell population collected or separated from a living body, or a cell population extracted from them can be used as a sample.

The detection method of the present invention is typically carried out in vitro, but in the case of using a sample from a non-human animal or detecting ISLR expression in a human sample transplanted to a non-human animal, Can also be implemented.

In the detection using “ISLR expression state” as an index, ISLR mRNA or ISLR protein is a detection target. That is, in the detection method of the present invention, the expression of ISLR mRNA or ISLR protein is detected. In the present invention, the “expression state” means the degree of expression (level), and is used as a term encompassing the presence / absence of expression and the expression level. Therefore, in the detection method of the present invention, qualitative or quantitative detection is performed on the expression of ISLRIS mRNA or ISLR protein.

Detection of ISLR mRNA expression can be performed by various methods using primers or probes specific to ISLR mRNA, such as RT-PCR, quantitative PCR, in situ hybridization, Northern blotting, and the like. On the other hand, for the detection of ISLR protein expression, a substance exhibiting specific binding property to ISLR protein is used. As the substance, an anti-ISLR antibody is preferably employed. Anti-ISLR antibody enables highly specific detection. If a labeled antibody is used, the amount of bound antibody can be directly detected using the amount of label as an index. Therefore, a simpler detection method can be constructed. On the other hand, in addition to the need to prepare an anti-ISLR antibody to which a labeling substance is bound, there is a problem that the detection sensitivity is generally lowered. Therefore, it is preferable to use an indirect detection method such as a method using a secondary antibody to which a labeling substance is bound or a method using a polymer to which a secondary antibody and a labeling substance are bound. The secondary antibody here is an antibody having a specific binding property to the anti-ISLR antibody. For example, when an anti-ISLR antibody is prepared as a rabbit antibody, an anti-rabbit IgG antibody can be used. Labeled secondary antibodies that can be used against various types of antibodies such as rabbits, goats, and mice are commercially available (for example, Funakoshi Co., Ltd., Cosmo Bio Co., Ltd., etc.), depending on the configuration of the detection method of the present invention. Appropriate ones can be appropriately selected and used.

As is clear from the above explanation, the anti-ISLR antibody is useful for detecting undifferentiated mesenchymal stem cells. Therefore, the present invention also provides a reagent for detecting undifferentiated mesenchymal stem cells containing an anti-ISLR antibody. In one aspect, the anti-ISLR antibody is labeled. Labeling substances used for labeling include, for example, fluorescent dyes such as fluorescein, rhodamine, Texas red, oregon green, enzymes such as horseradish peroxidase, microperoxidase, alkaline phosphatase, β-D-galactosidase, luminol, acridine dye, and other chemicals. Alternatively, bioluminescent compounds, radioisotopes such as ³² P, ¹³¹ I and ¹²⁵ I, and biotin can be mentioned.

The present invention further provides a kit for detecting undifferentiated mesenchymal stem cells comprising the reagent of the present invention as a constituent element. By using the kit, the detection method of the present invention can be carried out more easily. Other reagents (buffers, reaction reagents, enzymes, enzyme substrates, etc.) and / or devices or instruments (containers, reaction devices, fluorescent readers, etc.) used in carrying out the detection method of the present invention are included in the kit. May be. Usually, an instruction manual is attached to the kit of the present invention.

4). Evaluation of Undifferentiation of Mesenchymal Stem Cells As described above, ISLR is useful as a marker for undifferentiated mesenchymal stem cells, and the expression state of ISLR is an indicator of undifferentiation. Therefore, a further aspect of the present invention provides a method for evaluating the undifferentiation of mesenchymal stem cells, characterized by determining the expression state of ISLR as an index. In the evaluation method of the present invention, the expression state of ISLR is examined for a sample (test mesenchymal stem cell), and whether or not the undifferentiated state is maintained is determined based on the result. In order to examine the expression state of ISLR, ISLR mRNA or ISLR protein is detected. In the evaluation method of the present invention, for example, when the expression of ISLR mRNA or ISLR protein is observed, it is determined that the undifferentiation is maintained. The level of undifferentiation may be determined from the expression level of ISLR mRNA or ISLR protein. In addition, the description of the said (item 3 column) is used about the detection of ISLR mRNA or ISLR protein.

By using the evaluation method of the present invention, it becomes possible to identify or select mesenchymal stem cells that maintain undifferentiated properties (that is, undifferentiated mesenchymal stem cells). That is, the evaluation method of the present invention is useful as a means for identifying and selecting mesenchymal stem cells. On the other hand, the undifferentiated nature of mesenchymal stem cells also represents the effectiveness of applying mesenchymal stem cells to various uses, in other words, “quality”. Therefore, the evaluation method of the present invention is also useful as a means for evaluating or ensuring the quality of mesenchymal stem cells.

5). Detection of mesenchymal stem cells that accumulate (infiltrate) in the affected area of cancer or fibrotic disease and estimate the prognosis of the patient (evaluation)
Based on the knowledge that the expression of ISLR is related to the progression of cancer and the prognosis of cancer patients, and the knowledge that ISLR is useful for the evaluation or estimation of the progression and prognosis of fibrotic diseases such as myocardial infarction, the present invention As a further aspect, there are provided a method for detecting undifferentiated mesenchymal stem cells accumulated at the affected site of cancer or fibrotic disease, a prognosis estimation method for patients with cancer or fibrotic disease, and a reagent / kit used therefor.

In the detection method of this aspect, the ISLR expression state of the sample is examined, and it is determined whether or not mesenchymal stem cells are accumulated in the affected site based on the result. The detection result can be used for determination of disease progression (for example, the degree of differentiation of cancer tissue) and estimation of a patient's prognosis (details of prognosis estimation will be described later). When cancer is targeted, cancerous tissue is the affected site. On the other hand, a diseased site in the case of fibrosis is a site where inflammation or fibrosis occurs.

Typically, a pathological tissue extract or a patient-derived serum, exosome, etc., which uses a pathological tissue or pathological tissue specimen separated from a living body (patient) by biopsy (biopsy) or at the time of surgery as a sample. It may be used as a sample.

Cancer is not particularly limited. Examples of cancer include pancreatic cancer, colon cancer, breast cancer, lung cancer, kidney cancer, prostate cancer, and melanoma. Fibrotic diseases are not limited in the same manner, and typical examples include myocardial infarction, pulmonary fibrosis (interstitial pneumonia), cirrhosis, and chronic nephropathy.

For the detection means (detection of ISLR mRNA or ISLR protein), the explanation of the above (Item 3) is used, but a specific example of a detection method applicable when a pathological tissue or a pathological tissue specimen is used as a sample. One example is an in situ hybridization method.

The present invention also provides a prognostic estimation method for cancer or fibrotic disease patients as an application of the above detection method. In the prognostic estimation method of the present invention, ISLR is used as a biomarker. That is, in the present invention, the prognosis of a patient is estimated using ISLR as a marker for prognosis of cancer or fibrotic disease prognosis. According to the prognosis estimation method of the present invention, information useful for prognosis estimation of cancer patients or fibrotic disease patients can be obtained. The information is used, for example, for determining a treatment policy (selecting an effective treatment method, etc.). By using the determination result, improvement of treatment results, improvement of prognosis, improvement of patient's quality of life (QOL) and the like are brought about.

In this specification, the “biomarker for prognosis estimation” refers to a biomolecule that serves as an index for estimating the prognosis of a patient. In the prognosis estimation method of the present invention, a step of detecting the ISLR expression level in a patient-derived sample (detection step) and a step of estimating the prognosis based on the detection result (prognosis estimation step) are performed. The detection step is in accordance with the above detection method. In the prognosis estimation step, the prognosis is estimated using the detected ISLR (ie, ISLR expression level) as an index. Basically, a criterion is adopted that the prognosis is good when the ISLR expression level is large. Hereinafter, specific examples of evaluation based on the ISLR expression level are shown. First, a plurality of evaluation categories in which ISLR expression level and prognosis are associated are set in advance. Then, based on the ISLR expression level obtained in the detection step, the corresponding evaluation category is determined. As specific examples for setting the evaluation category, an example focusing on the presence or absence of ISLR expression (Example 1), an example focusing on the level of ISLR expression (Example 2), and an example focusing on changes in the expression level (Example 3) ) Is shown below. In the case of Example 3, detection is usually performed at least twice (at different times). In addition, it describes in order of classification name: ISLR expression level linked | related with the said classification | category: Evaluation result linked | related with the said classification | category.
<Example 1>
Category 1: ISLR positive: good prognosis Category 2: ISLR negative: poor prognosis <Example 2>
Category 1: No ISLR expression: poor prognosis Category 2: Low ISLR expression: relatively poor prognosis Category 3: Moderate ISLR expression: relatively good prognosis Category 4: ISLR Strong expression: good prognosis <Example 3>
Category 1: Increase in ISLR expression level: good prognosis Category 2: Decrease in ISLR expression level: poor prognosis

The number of evaluation categories, ISLR expression levels and evaluation results associated with each evaluation category can be arbitrarily set through preliminary experiments and the like, without being limited to the above example. Note that the determination / evaluation in the present invention can be automatically / mechanically performed regardless of the judgment of a person having specialized knowledge such as a doctor or a laboratory technician.

The present invention further provides a prognostic estimation reagent and a prognostic estimation kit. The prognosis estimation reagent enables detection of ISLR, and one of the specific examples is an anti-ISLR antibody. Regarding the anti-ISLR antibody, the above description (column in item 3) is incorporated. The prognostic estimation kit uses prognostic estimation as an essential component, and conforms to the kit used for the above-mentioned “detection of undifferentiated mesenchymal stem cells”.

1. ISLR expression in human bone marrow-derived mesenchymal stem cells and dermal fibroblasts (1) Method and results (Fig. 1)
Western using anti-ISLR antibody developed by the laboratory, preparing cell extracts from human bone marrow-derived mesenchymal stem cells (hereinafter human bone marrow MSC) and skin fibroblasts (both obtained from Lonza Japan KK) The expression of ISLR was examined by blotting. Anti-ISLR antibody was prepared by the following method. First, the following peptides for antigen 1 to 3 were synthesized.
Peptide for antigen 1: amino acids 229-251 of human ISLR (CSAPSVQLSYQPSQDGAELRPGF: SEQ ID NO: 7)
Peptide for antigen 2: amino acids 344-368 of human ISLR (LATPGEGGEDTLGRRFRFKKAVEGKG: SEQ ID NO: 8
Peptide for antigen 3: amino acids 341-359 of mouse ISLR (NVALATPGEGGEDAVGHKF: SEQ ID NO: 9)
Next, the antigen peptide was combined with the carrier protein KLH, and then rabbits (antigen peptides 1, 3) or guinea pigs (antigen peptide 2) were immunized. Serum was collected after immunization, and the specific antibody was purified using an affinity column to which a peptide for antigen was covalently bound. In this way, three types of anti-ISLR antibodies were obtained. For convenience of explanation, an antibody obtained by immunization with peptide 1 for antigen is anti-ISLR antibody 1, an antibody obtained by immunization with peptide 2 for antigen is anti-ISLR antibody 2, and an antibody obtained by immunization with peptide 3 for antigen is anti-ISLR antibody. Called 3 respectively.

As a result of Western blotting using anti-ISLR antibody 2, a band confirmed at a size of about 50 kDa was observed, and the expression of ISLR in human bone marrow MSCs and fibroblasts was proved. ISLR was also detected in the culture supernatant (medium) of human bone marrow MSCs and fibroblasts, indicating that ISLR is a secretory factor. In the fibroblasts in which ISLR expression was suppressed (knocked down) by RNA interference, no ISLR band was observed, indicating the specificity of the antibody used in this experiment. PDGF receptors α and β-actin are used as endogenous controls.

(2) Discussion The expression of ISLR in human bone marrow MSCs and dermal fibroblasts was confirmed. It has also been reported that fibroblasts isolated from various organs have the ability to differentiate into bone, cartilage, and fat in the same way as MSC, and at least some of the cells called fibroblasts have the characteristics of MSC. It has been reported that The results of this Western blot suggest that ISLR may be expressed not only in human bone marrow MSCs but also in MSCs contained in dermal fibroblasts.

2. ISLR expression in human adipose-derived stem cells (1) Methods and results (Fig. 2)
Adipose tissue is known to contain cells with characteristics and functions equivalent to bone marrow-derived mesenchymal stem cells, and is called adipose tissue-derived stem cells (ADSC) or simply MSCs. . A cell extract was prepared from human ADSC (Lonza Japan KK), and the expression of ISLR was examined by Western blotting using anti-ISLR antibody 2. As a result, the expression of ISLR in human ADSC was confirmed. GAPDH is used as an endogenous control.

3. ISLR expression in mouse bone marrow-derived mesenchymal stem cells (1) Method and results (Fig. 3)
Cell extracts were prepared from mouse bone marrow-derived mesenchymal stem cells (Cyagen), and the expression of ISLR was examined by Western blotting using anti-ISLR antibody 3. As a result, it was confirmed that ISLR was expressed in mouse bone marrow-derived mesenchymal stem cells. It was also confirmed that ISLR expression increased when the cells were cultured at high density. Β-tubulin is used as an endogenous control.

4). Decrease in ISLR expression associated with differentiation of mesenchymal stem cells (validation using mouse mesenchymal stem cell line C3H10T1 / 2)
(1) Method and results (Fig. 4)
Using C3H10T1 / 2, a cell line of mouse mesenchymal stem cells, we examined changes in ISLR expression during differentiation into fat, cartilage, and bone. After the addition of each differentiation-inducing medium (Lonza Japan Co., Ltd.), the expression of ISLR and various marker molecules on the 0th, 1st, 7th, and 14th days was detected by Western blotting (anti-ISLR antibody 3 was used for ISLR detection) ). In all of fat, cartilage, and bone differentiation, a significant decrease in ISLR expression was observed one day after differentiation induction. FABP4 is used as an adipocyte marker, Collagen IIa is used as a chondroblast marker, and Osteopontin is used as an osteoblast marker.

(2) Discussion In any of fat, cartilage, and bone differentiation, a significant decrease in ISLR expression was observed immediately after induction of differentiation, suggesting that ISLR is a marker molecule for undifferentiated mesenchymal stem cells. .

5). Decrease in ISLR expression associated with differentiation of mesenchymal stem cells (validation using human bone marrow MSC)
(1) Method and results (Fig. 5)
Human bone marrow MSCs were used to examine changes in ISLR expression during differentiation into fat, cartilage, and bone. After the addition of each differentiation-inducing medium (Lonza Japan Co., Ltd.), the expression of ISLR on the 0th, 1st, 7th, and 14th was verified by Western blotting (using anti-ISLR antibody 2). In any of fat, cartilage, and bone differentiation, a marked decrease in ISLR expression was observed on the first day after differentiation induction.

(2) Consideration As in the experiment of FIG. 4, in all of fat, cartilage, and bone differentiation, a marked decrease in ISLR expression was observed immediately after induction of differentiation. ISLR is an undifferentiated human bone marrow MSC marker molecule. It was suggested that there is.

6). Decreased expression of ISLR gene messenger RNA (mRNA) accompanying mesenchymal stem cell differentiation (validation using mesenchymal stem cell line C3H10T1 / 2)
(1) Method and results (Fig. 6)
Using the mesenchymal stem cell line C3H10T1 / 2, changes in the expression level of ISLR mRNA during differentiation into fat, cartilage, and bone were examined. After adding each differentiation-inducing medium (Lonza Japan Co., Ltd.), the expression level of ISLR mRNA 7 days later was verified by a quantitative RT-PCR method. In any of fat, cartilage, and bone differentiation, a significant decrease in the expression level of ISLR mRNA was observed 7 days after differentiation induction. The vertical axis of the graph shows ISLR and the ratio of GAPDH mRNA used as an endogenous control.

(2) Consideration In all of fat, cartilage, and bone differentiation, a significant decrease in the expression level of ISLR mRNA was observed after differentiation induction, suggesting that ISLR is a marker molecule for undifferentiated mesenchymal stem cells. It was done.

7). Verification of the effect of high ISLR expression on differentiation of mesenchymal stem cells (1) Method and results (Fig. 7)
The effect of exogenous forced expression of ISLR on mesenchymal stem cell line C3H10T1 / 2 on fat, cartilage, and bone differentiation was examined. ISLR was forcibly expressed in C3H10T1 / 2 using a retrovirus expression system, and then each differentiation-inducing medium (Lonza Japan Co., Ltd.) was added. The expression of bone differentiation marker) and Runx2 (bone differentiation marker) was verified by Western blotting (anti-ISLR antibody 3 was used to detect ISLR). As a result, it became clear that forced expression of ISLR suppressed the expression of Sox9, Osteopointin and Runx2.

(2) Consideration In both cartilage and bone differentiation, suppression of the expression of various differentiation markers of Sox9, Osteopontin and Runx2 was observed by forced expression of ISLR, and ISLR maintains the mesenchymal stem cells in an undifferentiated state. The possibility of being a necessary molecule was shown.

8). Expression of ISLR in various cultured cells (1) Method and results (Fig. 8)
Cell extracts of human fibroblasts, vascular smooth muscle cells, vascular endothelial cells and various epithelial cells (purchased from Lonza Japan Co., Ltd. or ZenBio) were prepared, and Western blotting using antibodies against ISLR and various marker molecules ( Anti-ISLR antibody 2 was used for ISLR detection). As a result, it was revealed that ISLR is not expressed in cells other than fibroblasts.

(2) Discussion ISLR is expressed in mesenchymal stem cells, fibroblasts, and adipose-derived stem cells, but is not expressed in other cells, and ISLR is a marker molecule highly specific for MSC. It was shown that.

9. ISLR expression in mesenchymal stem cells infiltrating the pancreatic cancer stroma (1) Method and results (Fig. 9)
The expression of ISLR in moderately differentiated pancreatic cancer and poorly differentiated pancreatic cancer was examined by in situ hybridization. As indicated by the arrowheads, the expression of ISLR was high in mesenchymal stem cells that accumulated or infiltrated into the stroma of moderately differentiated pancreatic cancer, and it was detected that mesenchymal stem cells were accumulated or infiltrated. On the other hand, ISLR expression was not observed in poorly differentiated pancreatic cancer. Moreover, as shown in the right figure, the expression of ISLR correlated with a good patient prognosis. The specimen was collected as follows.

From the histopathological specimen obtained by surgery of a patient with pancreatic cancer, the region with the most tumor components was selected under microscopic examination, and the expression of ISLR was examined by an in situ hybridization method. Among the examined tissues, more than 20% of the cells with fibroblast-like morphology that infiltrate the cancer stroma in the medium magnification field of view (20x objective lens) have high ISLR expression and less ISLR positive. Groups were grouped as low expression and the post-operative cumulative progression-free survival was plotted for each group. Since it was detection of messenger RNA by the in situ hybridization method, it was determined as positive when a signal was observed even in part of the cytoplasm.

(2) Discussion It was shown that ISLR is expressed in mesenchymal stem cells infiltrating cancer tissues, and the degree of mesenchymal stem cells detected by ISLR reflects the good prognosis of cancer-bearing patients.

10. Expression of ISLR in mesenchymal stem cells infiltrating the colorectal cancer stroma (1) Method and results (FIG. 10)
The expression of ISLR in moderately differentiated colorectal cancer and poorly differentiated colorectal cancer was examined by in situ hybridization. As indicated by the arrowheads, the expression of ISLR was high in mesenchymal stem cells that accumulated or infiltrated into the stroma of moderately differentiated colorectal cancer, and it was detected that mesenchymal stem cells were accumulated or infiltrated. On the other hand, ISLR expression was not observed in poorly differentiated colorectal cancer.

(2) Discussion ISLR is expressed in mesenchymal stem cells infiltrating colon cancer tissues in addition to pancreatic cancer, and is a useful marker for detecting mesenchymal stem cells infiltrating various cancer tissues. The possibility was suggested.

10. Expression of ISLR in mesenchymal stem cells infiltrating breast cancer stroma (1) Method and results (FIG. 11)
The expression of ISLR in invasive ductal carcinoma was examined by in situ hybridization. As indicated by the arrowhead, ISLR expression was observed in mesenchymal stem cells that accumulated or infiltrated into the stroma of invasive ductal carcinoma.

(2) Discussion ISLR is expressed in mesenchymal stem cells that infiltrate breast cancer tissues in addition to pancreatic cancer and colon cancer, and is a useful marker for detecting mesenchymal stem cells infiltrating various cancer tissues. It was suggested that

11. Expression of ISLR at the infarct site of mouse myocardial infarction model (1) Method and results (Fig. 12)
The expression of ISLR at the myocardial infarction site of the mouse myocardial infarction model was examined by in situ hybridization method (using RNAscope method). An 8-week-old male myocardial infarction model was used. As indicated by the arrowhead, ISLR is found around the epicardium 7 days after onset. That is, it was detected that the mesenchymal stem cells were infiltrated even at the myocardial infarction site.

(2) Discussion ISLR is expressed in mesenchymal stem cells that accumulate at the site of myocardial infarction, suggesting that ISLR is a useful marker even in fibrotic diseases such as myocardial infarction.

12 Difference in survival rate after myocardial infarction between wild-type mice and ISLR knockout mice (1) Methods and results (FIG. 13)
ISLR knockout mice were prepared and the survival rate after myocardial infarction was compared with wild type mice. ISLR knockout mice were found to have a significantly worse prognosis after myocardial infarction than wild-type mice.

(2) Discussion It was suggested that ISLR is also related to the prognosis of fibrotic diseases such as myocardial infarction.

The present invention provides a marker for undifferentiated mesenchymal stem cells. The marker of the present invention shows high specificity for undifferentiated mesenchymal stem cells. According to the preparation method using the marker, undifferentiated mesenchymal stem cells that are particularly important and useful in the field of regenerative medicine can be prepared with high purity. The marker is also useful for detection of undifferentiated mesenchymal stem cells, evaluation of undifferentiation, and the like.

The present invention is not limited to the description of the embodiments and examples of the above invention. Various modifications may be included in the present invention as long as those skilled in the art can easily conceive without departing from the description of the scope of claims. The contents of papers, published patent gazettes, patent gazettes, and the like specified in this specification are incorporated by reference in their entirety.

Claims

An undifferentiated mesenchymal stem cell marker consisting of leucine-rich repeat-containing immunoglobulin superfamily (ISLR).
The undifferentiated mesenchymal stem cell marker according to claim 1, wherein the ISLR comprises any one of the amino acid sequences of SEQ ID NOS: 1 to 3.
A method for preparing undifferentiated mesenchymal stem cells, comprising a step of selecting and collecting ISLR-expressing cells from a cell population containing mesenchymal stem cells.
The method according to claim 3, wherein the cell population containing mesenchymal stem cells is derived from bone marrow, dental pulp, adipose tissue, endometrium, umbilical cord, skeletal muscle, or peripheral blood.
A method for detecting undifferentiated mesenchymal stem cells, characterized by detecting the expression state of ISLR as an index.
A method for evaluating the undifferentiation of mesenchymal stem cells, comprising the step of examining the expression state of ISLR in a test mesenchymal stem cell.
The method according to any one of claims 3 to 6, wherein the mesenchymal stem cells are human cells.
A reagent for detecting undifferentiated mesenchymal stem cells containing an anti-ISLR antibody.
A kit for detecting undifferentiated mesenchymal stem cells, comprising the reagent according to claim 8.
A method for detecting undifferentiated mesenchymal stem cells that accumulate in affected sites of cancer or fibrotic diseases, characterized by detecting the expression state of ISLR as an index.
The method according to claim 10, wherein the cancer is pancreatic cancer, colon cancer, or breast cancer, and the fibrosis is myocardial infarction.
Biomarker for prognosis estimation of cancer or fibrotic disease consisting of ISLR.
The biomarker for prognosis estimation according to claim 12, wherein the cancer is pancreatic cancer, colon cancer, or breast cancer, and the fibrotic disease is myocardial infarction.
A method for estimating the prognosis of patients with cancer or fibrosis using the expression level of ISLR as an index.
The prognostic estimation method according to claim 14, wherein the cancer is pancreatic cancer, colon cancer, or breast cancer, and the fibrosis is myocardial infarction.
A reagent for estimating the prognosis of patients with cancer or fibrotic diseases, including anti-ISLR antibodies.
The reagent for prognosis estimation according to claim 16, wherein the cancer is pancreatic cancer, colon cancer, or breast cancer, and the fibrotic disease is myocardial infarction.
A kit for estimating the prognosis of a patient with cancer or fibrosis, comprising the reagent according to claim 16 or 17.