JP2007055965A - Monoclonal antibody selective to human nerve stem cell and screening method using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new antibody which is highly selective to the surface antigen of human nerve stem cells, and to provide a method for identifying and separating nerve stem cells and/or nerve precursor cells, Nestin high expression cells in live cell states by an immunological method using the new antibody. <P>SOLUTION: This monoclonal antibody produced from a hybridoma of a myeloma with the antibody-producing cells of a mammal immunized with a homogenate of cultured human neurospheres. The monoclonal antibody was screened with a human brain tissue, and was consequently found to be a new antibody (HFB 184, HFB 25 antibody) highly selective to nerve stem cells. The antibody expresses in undifferentiated nerve stem cells, and selectively reacts with a surface antigen disappearing in the subsequent differentiation process. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a novel antibody that selectively recognizes cell surface antigens of neural stem cells and / or neural progenitor cells (hereinafter collectively referred to as “NSPC” when they are not distinguished from each other), a hybridoma that produces the same, and A method for screening and selectively sorting human NSPCs from human neurospheres obtained by in vitro culture using this monoclonal antibody and heterogeneous cell populations containing human NSPCs collected from living bodies, The present invention relates to a method for evaluating the content of NSPC and a screening reagent used for carrying out the method.

  Neural stem cells are undifferentiated cells characterized by self-renewal and pluripotency, and it has been found that they exist not only in the fetal brain but also in the adult brain. In addition, clinical applications in the field of regenerative medicine are expected as useful cells for transplantation for generating new neurons and new glial cells in the central nervous system.

For clinical application, it is important to separate from easily available cell sources and establish a selective culture method.
Neural cells, which are tissues containing a large amount of neural stem cells, contain not only undifferentiated cells such as neural stem cells and neural progenitor cells, but also many differentiated neural cells. In addition, cells other than the nervous system have been reported to contain neural stem cells, and umbilical cord blood cells and bone marrow cells are considered as cell sources that are relatively easily available. However, most of these sources are various cells other than the nervous system. Therefore, in order to use such a heterogeneous cell population as a cell source and to isolate neural stem cells therefrom, it is necessary to establish a separation method and a NSPC screening method.

  Currently, the neurosphere method is most widely used as a selective method for culturing neural stem cells, but the cell population amplified by the neurosphere method is also a heterogeneous cell population including cells that have undergone differentiation. Therefore, isolation and screening methods for neural stem cells and neural progenitor cells are important.

  As a method for identifying neural stem cells, Non-Patent Document 1 proposes a method using a Nestin marker that is an intermediate filament protein called Nestin identified by Lendahl and McKay et al. However, since Nestin exists in the cell, it is difficult to apply it to the operation of identifying and isolating neural stem cells as living cells.

  In addition to Nestin, Musashi1, which is an RNA binding protein having a molecular weight of about 38 kDa, identified by Nakamura and Okano et al. Is known as an expression marker with high selectivity for neural stem cells.

  Musashi1 is also a protein that exists in the cell, like Nestin. Therefore, in the case of a living cell, it is a normal method to grasp the expression status of these proteins from the outside while the cell is alive and visualize it. Then it is impossible.

  As a method for visualizing the expression level of Nestin and Musashi1 in living cells and efficiently separating neural stem cells based on the expression level, GFP (green fluorescent protein) was incorporated downstream of the musashi1 promoter or nestin enhancer. It has been reported that a reporter gene is artificially introduced into neural stem cells artificially in advance by various methods, and GFP-expressing cells are separated using FACS according to the expression intensity (fluorescence intensity) (Non-patent Document 2, Patent) Literature 1, Patent Literature 2).

  However, since this method involves the introduction of a GFP-expressing gene that does not exist in the human body, the neural stem cells obtained by this separation operation are actually transplanted to the human body even if they are efficiently separated and expanded. Such clinical applications have problems in terms of safety.

  Therefore, a separation technique using a cell surface marker that can detect neural stem cells by a method not involving gene transfer is desired.

In recent years, non-patent document 3 reports a method for separating human-derived neural stem cells using a CD133 antibody as a method for performing separation by FACS using a selective cell surface marker. CD133 ⁺ / CD34 ⁻ / CD45 ⁻ cells have been reported to form neurospheres at a high rate. In addition, Patent Document 3 discloses a method of using a monoclonal antibody AC133 or monoclonal antibody 5E12 to recognize a cell surface marker as a method for producing a highly enriched population of central nervous system stem cells (CNS-SC). Is disclosed. CD133 antibody (AC133 antibody) is expected to be an antibody that recognizes prominin homologues.

Furthermore, in Patent Document 4, as a cell surface marker of neural stem cells, anti-HFB115 monoclonal antibody and four kinds of artificial antibodies (No. 16 antibody (FERM P-18778), No. 27 antibody (FERM P-18879), No. 115 are used. A method of separating using an antibody (FERM P-18780) and an antibody No. 211 (FERM P-18881) has been proposed.
These antibodies are hybridoma production antibodies prepared from mouse antibody-producing cells and myeloma cells immunized with fetal human brain tissue homogenate as an immunogen.

Furthermore, in Patent Document 5, the identification of neural stem cells from mammalian peripheral nervous system, as isolation method, using anti-p75 antibody as a positive marker, has been proposed to use anti-P ₀ as a negative marker.

  Furthermore, in Patent Document 6 and Non-Patent Document 4, expression of GD2 ganglioside, MHC class I, β2 microglobulin, CD8, CD9, CD15, CD34, CD38, CD56, CD81, CD95, and CD152 are used as positive markers of neural stem cells. , MHC class II, HLA-DR, Glycophorin-A, CD3, CD5, CD7, CD10, CD11b, CD13, CD14, CD16, CD19, CD20, CD22, CD23, CD25, CD31, CD33, CD41, CD45, CD54, CD80 , CD83, CD86, CD117, CD133, and CD154 have been proposed as techniques for isolating neural stem cells using negative markers for neural stem cells.

JP 2001-292768 A JP2002-34580 JP2004-2350 Special table 2002-536023 Special Table 2002-537802 WO02 / 086082 Lehndahl et al., Cell 60: 585-595 (1990). H. M.M. Keyung et al., “High-yield selection and extraction of two promoters-defined phenotypes of neural cells from the human brain.” Biotechnol. , 19, 843-850 (2001) Uchida N et al., "Direct isolation of human central system system cells", Proc Natl Acad Sci USA 2000; 97; 14720-25). Klassen H, Schwartz MR, Bailey AH, Young MJ, et al. 26; 312 (3): 180-2 (2001)

Any of the above methods using cell surface antigens and cell surface markers can be used to identify and isolate enriched populations of neural stem cells or neural progenitor cells in a living cell state without introducing foreign genes that do not exist in the living body. However, the selectivity for human NSPC is still insufficient, and it is insufficient as a method for separating and collecting high-purity neural stem cell and neural progenitor cell populations from heterogeneous cell populations.
The present invention has been made in view of the circumstances as described above, and an object thereof is a highly selective antibody capable of identifying and separating human NSPC in a living cell state, and heterogeneous cells using the same. The object is to provide a method capable of detecting and separating human NSPC from a population, and a screening reagent used in the method.

  The present inventors produced a novel antibody having high selectivity for cell surface antigen of human NSPC using a mammal immunized with a suspension of high-purity cultured human NSPC, and completed the present invention. .

  That is, the monoclonal antibody of the present invention is a monoclonal antibody produced from a hybridoma of a mammalian antibody-producing cell immunized with a human NSPC homogenate and myeloma, and binds to the surface antigen of human NSPC, but differentiated nerves. It is a monoclonal antibody that does not substantially react with the surface antigen of the cell line. The antigen is preferably a non-reduced protein antigen.

  A monoclonal antibody according to another aspect of the present invention is a monoclonal antibody produced from a hybridoma of a mammalian antibody-producing cell immunized with a human NSPC homogenate and myeloma, wherein the cerebral ventricular wall in the early fetal period It is a monoclonal antibody that reacts with cells in the lower layer and ventricular wall. Furthermore, it is preferable that it does not substantially react with the cortical plate of the cerebrum in the early fetal period.

  The monoclonal antibody of the present invention is preferably one in which cultured cells are used as the human NSPC used for immunization, and specifically, human neurosphere homogenate is preferred. Further, when screening the monoclonal antibody-producing cells of the present invention from the obtained hybridoma, at least one selected from the group consisting of human fetal brain tissue, cultured human neural stem cells, and a cell population in which human neurospheres are made into single cells is used. It is preferable that they have been screened.

  Furthermore, the monoclonal antibody according to another aspect of the present invention was found to have a positive reaction with 80% or more of the single cell population in which human neurospheres are dispersed and NETRA2 in the analysis by flow cytometry, and U-87MG, T98G, TKG- 1, U251MG, and 293T cells are monoclonal antibodies that show less than 5% positive reaction.

  The present invention is also directed to a hybridoma that produces the monoclonal antibody of the present invention.

  The hybridoma of the present invention is a hybridoma having the accession number FERM P-20606 or the accession number FERM P-20607, and monoclonal antibodies produced from these hybridomas are also the subject of the present invention.

  The present invention also relates to a monoclonal antibody that recognizes an antigenic determinant recognized by a monoclonal antibody (HFB25 antibody) produced from a hybridoma having the deposit number FERM P-20606, and a monoclonal antibody produced from the hybridoma having the deposit number FERM P-20607. Monoclonal antibodies that recognize antigenic determinants recognized by (HFB184 antibody) are also targeted.

  The monoclonal antibody of the present invention can be used for identification, detection, separation, and sorting of human neural stem cells, human neural progenitor cells, and nestin high-expressing cells.

  That is, the human NSPC identification method of the present invention is a method for identifying cells having the characteristics of human NSPC and the localization of the cells from a heterogeneous cell population. It is a method comprising the steps of performing immunohistochemical staining with an antibody and identifying the stained portion as human NSPC.

  Further, a method for identifying human NSPC according to another aspect of the present invention is a method comprising a step of detecting a cell exhibiting an immunological reaction for an antigenic determinant recognized by an HFB25 antibody or an HFB184 antibody from a heterogeneous cell population. There may be.

  The screening method for cells expressing nestin of the present invention comprises a step of reacting any of the above monoclonal antibodies with a cell population containing neural stem cells, and detecting a cell positive for the reaction with the monoclonal antibody. The nestin high-expressing cell may be human NSPC.

  The nestin high expression screening method of the present invention preferably further comprises a step of detecting a cell that is negative for the reaction with the anti-JAM-1 antibody with respect to the monoclonal antibody-positive cell. It becomes possible.

  The cell population may be a population containing human umbilical cord blood-derived cells, a population containing human peripheral blood-derived cells, or a cell population obtained by dispersing neurospheres into single cells. Good.

  The method for evaluating a cell population of the present invention comprises detecting a cell positive in reaction with any of the monoclonal antibodies of the present invention in a cell population containing human NSPC, and Including a step of calculating a ratio.

  About the said monoclonal antibody positive cell detected at the said process, the cell with a negative reaction with an anti-JAM-1 antibody is detected, The cell with the said monoclonal antibody reaction positive and the anti-JAM-1 antibody reaction negative in this cell population The step of calculating the ratio may be included.

  A screening reagent of the present invention is a reagent for screening a human NSPC-enriched population from a human NSPC-containing cell population, characterized by containing any of the monoclonal antibodies of the present invention and a solvent. To do.

  In the present invention, “react with antigen” means an antigen-antibody reaction in immunology, and means that an antibody and an antigen bind. Here, the antigen-antibody bond includes a chemically weak bond mode such as hydrogen bond, ionic bond (Coulomb force), van der Waals force, and hydrophobic bond, and may be a sum of these, so-called It is a bond that depends on the relationship of the three-dimensional structure such as the relationship between the key and the keyhole.

  In addition, “react with tissue” is a target monoclonal antibody, and when the corresponding tissue is stained by direct method, indirect method or ABC (avidin-biotin complex) method and developed, color ( When a fluorescent dye is used, it means that fluorescence having a specific wavelength) is observed.

  The “neural stem cell” referred to in the present invention means a pluripotent stem cell having a self-replicating ability and capable of forming a neurosphere when cultured in a growth medium by a neurosphere method. Refers to an undifferentiated cell having a proliferation ability slightly differentiated from a neural stem cell.

  In addition, “differentiated neural cells” express various cell differentiation markers (GFAP, tubulin βIII, etc.) created by inducing differentiation of neural stem cells, and do not express neural stem cell marker molecules (nestin, musashi1, etc.). A cell is mentioned, and specifically, a neuron cell, a glial cell, etc. are mentioned.

  In addition, the “positive reaction in the analysis by flow cytometry” referred to in the present invention is determined as a positive reaction by measuring the cell population reacted with the labeled primary antibody with a flow cytometer. When the fluorescence intensity of the specific wavelength emitted by the fluorescent dye used for labeling is observed, the fluorescence intensity higher than the background intensity measured without using the primary antibody is judged to be positive. .

“Substantially non-reactive” means not only when there is no reaction at all, but also when the staining intensity of the immunostaining indicates the degree of background staining intensity evaluated without using the primary antibody, or when the macroscopic evaluation is sufficient. In the case of weak staining that is not possible, the case where positive in the analysis reaction by flow cytometry is not recognized is also included.

Since the novel antibody of the present invention has high selectivity for cell surface antigens expressed in human NSPC, it can be used from a solid tissue mass or a cell suspension mixed with various cells, which is a heterogeneous cell population, Neural stem cells and / or neural progenitor cells can be detected, identified, screened, separated and sorted without disrupting the cells or introducing an exogenous reporter gene or the like.
Furthermore, the content rate of neural stem cells, neural progenitor cells, etc. can be evaluated for the above cell population.

[Method for producing monoclonal antibody and hybridoma]
First, the method for producing the antibody of the present invention and the hybridoma producing the same will be described.
The antibody of the present invention is produced by preparing a normal monoclonal antibody production technique, that is, by preparing a hybridoma that produces an antibody of interest by fusing an antibody-producing cell taken from an immunized mammal and myeloma. However, it is characterized by using cultured human neural stem cells and neural progenitor cells as immunogens.

  That is, the immunogen used in the present invention is a homogenate of cultured human neural stem cells and neural progenitor cells. Specifically, it is a homogenate obtained by rapidly freezing and homogenizing a neurosphere obtained by subculturing human NSPC derived from the human fetal forebrain.

  In the case of brain tissue homogenate, there are many differently differentiated nervous system cells in addition to neural stem cells, and many interstitial components other than cell components are also present. For this reason, as disclosed in the above-mentioned Patent Document 4, when brain tissue homogenate is used as an immunogen, the efficiency of immunization with a component selective only to undifferentiated human NSPC is deteriorated, The selectivity of the obtained antibody for neural stem cells was insufficient as a tool for identifying, detecting, separating and sorting neural stem cells from a cell population containing various cells other than neural stem cells. In this respect, the neurosphere homogenate used in the present invention has almost no extra parts other than cellular components, and most of the contained cells are human NSPCs. The probability of obtaining an antibody to be produced was greatly increased, and an antibody having high selectivity for human NSPC could be obtained.

  In immunization, it is preferable to use a suspension in which an immunogen is mixed with an adjuvant or the like.

  As a mammal to be immunized, for example, a mouse, a rat, a hamster or the like can be used. For the production of an antibody against a human antigen, it is preferable to use a mouse, particularly a 6-8 week old Balb / C mouse.

  The immunization method is performed, for example, by immunizing a mouse by injecting the immunogen subcutaneously, intraperitoneally, intravenously, intramuscularly, intradermally or the like. The inoculation interval, the inoculation amount, and the like vary depending on the inoculation method. For example, when inoculating the mouse subcutaneously, the antigen was inoculated every 3 to 4 days for about 2 weeks. Boost if necessary. Thus, antibody-producing cells such as lymphocytes are removed from the immunized mammal and subjected to cell fusion operation.

  As the myeloma cell used for cell fusion, a HGPRT-deficient strain required for the salvage pathway is preferably used. As a preparatory operation before cell fusion, myeloma is added to 10% CS (calf serum), 10% FCS (fetal calf serum) in basic media such as Eagle's minimum basic medium (MEM), Dulbecco's modified MEM, and PRMI 1640, which are usually used. A medium that has been cultured in advance is used.

Cell fusion is performed by mixing myeloma and antibody-producing cells in a ratio of 1: 5 to 1:20. At this time, a fusing agent such as polyethylene glycol or Sendai virus is used.
After cell fusion, only hybridomas fused with antibody-producing cells can be grown by culturing in a HAT selective medium.

  As a method for screening a hybridoma that produces the target antibody from the obtained hybridoma, a method such as radioimmunoassay or enzyme immunoassay can be used.

  For example, a homogenate used as an immunogen, for example, is coupled to a carrier such as cellulose beads according to a conventional method, and a culture supernatant of the hybridoma is added thereto and reacted for a predetermined time. After washing, an enzyme-labeled anti-mouse Ig antibody (secondary antibody) is added and reacted to remove unreacted antibody, followed by washing. The enzyme substrate is then added. After the enzyme reaction, antibody-producing hybridomas can be screened by measuring the absorbance or fluorescence of the reaction-generated color product and examining the presence or absence of antibody production (primary screening).

  The monoclonal antibody produced from the antibody-producing hybridoma screened in the primary screening was further reacted with a human brain tissue section and cultured human NSPC to screen for antibody-producing hybridomas that showed a positive reaction (secondary screening). For detection of a positive reaction, a cell bound with an antibody labeled with an antibody that can bind to an antibody produced from an antibody-producing cell, such as an anti-mouse Ig antibody as a secondary antibody (labeled secondary antibody) is flowed. Detection is performed by detecting the label of the labeled secondary antibody using a cytometer or the like. When positive in the secondary screening, the monoclonal antibody produced from the antibody-producing hybridoma is an antibody that can selectively bind to the cell surface antigen of human NSPC.

  By the primary screening and the secondary screening as described above, an antibody that can selectively bind to a cell surface antigen of human NSPC and a hybridoma that produces the antibody can be selected.

The hybridoma thus selected is cloned by a limiting dilution method or the like to obtain the hybridoma of the present invention. This hybridoma is deposited under the accession number FERM P-20606 and the accession number FERM P-20607 at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology.
The target antibody can be obtained by growing the obtained hybridoma in a liquid medium or in the peritoneal cavity of a mammalian cell.

[Monoclonal antibody]
The monoclonal antibody of the present invention is an antibody produced from a hybridoma designated by the accession number FERM P-20606, designated as HFB25 antibody, and an antibody produced from a hybridoma designated by the accession number FERM P-20607, designated HFB184 antibody. . The monoclonal antibody of the present invention also includes a monoclonal antibody that recognizes an antigenic determinant recognized by the HFB25 antibody or an antigenic determinant recognized by the HFB184 antibody, and each has the following characteristics.
Hereinafter, the HFB25 antibody and the HFB184 antibody will be described as representatives of the monoclonal antibody of the present invention.

  The monoclonal antibodies of the present invention typified by the HFB25 antibody and the HFB184 antibody react with cells in the cerebral ventricle wall layer and the subventricular wall layer in the early early human fetal period without brain development abnormalities or genetic diseases. Although it may react with cells in the intermediate layer and molecular layer, it does not substantially react with cells in the cortical plate. The fetal stage is the period from the cell stage, which is 2 weeks until the fertilized egg becomes the germ layer, from the third week following the cell stage to the birth stage excluding the embryonic stage from the end of 2 months. Refers to the period from 8 to 18 weeks of age.

  The layer structure in the fetal cerebral tissue can be recognized by hematoxylin / eosin staining, Nissl staining, Golgi staining, or the like. The cerebral ventricular wall layer is a cell layer formed by cells in contact with the ventricle on one side, and usually nuclei are arranged in several layers by hematoxylin and eosin staining to define the layer structure. The ventricular wall lower layer is a part in contact with the ventricular wall layer, and is usually defined as a region composed of several rows of converging cell layers in contact with the ventricular wall layer by hematoxylin and eosin staining for defining the layer structure. The cortical plate is a part where cells that differentiate into neurons (neuroblasts) line up in high density in the future, and is usually an area where several rows of cells are densely accumulated by staining with hematoxylin and eosin to define the layer structure. The part that appears. The intermediate layer is the outside of the ventricular wall lower layer, the region where the cell density from the lower ventricular wall layer to the cortical plate is relatively sparse, and the molecular layer is the region outside the cortical plate and inside the cerebral buffy coat. In the early fetal period, particularly in the ventricular wall layer and the lower ventricular wall layer of about 8 to 10 weeks of age, cell proliferation is prominent and neural stem cells are localized in large quantities. On the other hand, the cortical plate has many differentiated neuroblasts and almost no neural stem cells. Therefore, HFB25 antibody and HFB184 antibody are surface antigens that are expressed when undifferentiated neural stem cells and neural progenitor cells, and are selective for surface antigens that disappear as differentiation progresses.

  Here, the reactivity with the tissue is determined by staining the relevant tissue with the target monoclonal antibody by the direct method, the indirect method or the ABC (avidin-biotin complex) method, and using a fluorescence microscope, a laser microscope or an immune microscope. Observation with an electron microscope or the like is made based on the presence or absence of color development (fluorescence with a specific wavelength when a fluorescent dye is used).

  As used herein, “selective for an antigen” is a characteristic of an antibody that can serve as an index for selecting neural stem cells from a large number of cell populations, and is a concept wider than specificity. That is, even if the antigen is expressed in other than the neural stem cell, it means the degree that the neural stem cell can be selectively identified to such an extent that it can be used for the neural stem cell separation operation.

  The monoclonal antibody of the present invention is not limited to the HFB25 antibody and the HFB184 antibody, and is at least an antigenic determinant recognized by the HFB25 antibody (indicating an antigen-antibody reaction) or an antigenic determinant recognized by the HFB184 antibody (indicating an antigen-antibody reaction). Antibodies that react with either antigenic determinant are included. In other words, as long as it is a monoclonal antibody having an antigen-binding site common to the HFB25 antibody or HFB184 antibody, the other part may have a structure different from that of the HFB25 antibody or HFB184 antibody.

  Here, “react” means an antigen-antibody reaction as referred to in immunology, and means that an antibody binds to an antigen. The antigen-antibody bond includes chemically weak bonds such as hydrogen bond, ionic bond (Coulomb force), van der Waals force, and hydrophobic bond, and may be the sum of these, so-called key and keyhole. It is a bond that depends on the relationship of each other's three-dimensional structure, such as

  Antigen determinants recognized by HFB25 antibody (indicating antigen-antibody reaction) and antigenic determinants recognized by HFB184 antibody (indicating antigen-antibody reaction) are expressed on the cell surface of neural stem cells. The expression level changes (decreases), and the percentage of positive cells decreases when viewed as a whole cell population. Specifically, the percentage of positive cells decreases by 70% or more, preferably 80% or more after 7 weeks from the start of differentiation induction.

  The antigenicity of the antigen recognized by the monoclonal antibody of the present invention is not lost by glycosylation by glycosidase or glycosaminoglycan, degreasing by chloroform treatment, etc., but Western blot using a normal reducing agent Then it will be lost. Therefore, it is considered to be a non-reducing protein antigen.

  Further, the monoclonal antibody of the present invention showed positive reaction with 80% or more of the single cell population in which human neurospheres were dispersed and NETRA2 by the analysis by flow cytometry, and U-87MG, T98G, TKG-1, U251MG. And less than 5% positive with 293T cells. NETRA2 is a teratoma-derived human embryonic cancer cell that is similar to a neural progenitor cell having differentiation potential. U-87MG, T98G, YKG-1, and U251MG are all neuronal cells derived from human glioblastoma and are cells having characteristics similar to astrocytes (differentiated cells).

  In the analysis by the flow cytometry, the antibody is directly or indirectly or fluorescently labeled by the ABC method, the target cell population is measured with a flow cytometer, and the percentage of cells showing a positive reaction is calculated. . A positive reaction by flow cytometry means that the fluorescence intensity of the specific wavelength emitted by the fluorescent dye used for labeling is higher than the background intensity measured without using the primary antibody. When included.

[Neural stem cell screening method]
The method of the present invention uses an immunological method using the monoclonal antibody of the present invention including HFB25 or HFB184 antibody to identify and detect human NSPC or Nestin high-expressing cells from a heterogeneous cell population. It is a method of separation and fractionation.

  That is, a heterogeneous cell population is immunostained using the monoclonal antibody of the present invention, and the stained portion is detected, whereby the stained portion is identified with high probability that the portion is human NSPC, or the cell It is a method of knowing the localization of human NSPC in a population.

  Further, it is a method of screening the above-mentioned monoclonal antibody (HFB25 or HFB184 antibody) positive cells of the present invention from a cell population containing neural stem cells, and separating and collecting them as necessary.

  Furthermore, since human NSPC is considered to be equivalent to Nestin high-expressing cells, Nesti high-expressing cells are identified, detected, separated, and sorted from a heterogeneous cell population by the above identification, screening, separation, and sorting methods. This method can also be applied.

 The cell population to which the method of the present invention can be applied is not particularly limited, and is not limited to a single cell population, but may be a tissue mass section by biopsy, but is preferably a single cell population. Examples of cell populations containing human NSPC include cell populations such as neurospheres; solid tissues collected from adult brain and spine; and humoral cell populations such as umbilical cord blood and human adult peripheral blood. Even in tissues other than the nervous system, since there are cell populations containing human NSPC, these are also subject to application of the method of the present invention in order to know the presence, localization, and content of human NSPC. Further, in the case of a humoral cell population, not only a single tissue-derived cell population but also a mixed system of heterogeneous tissue-derived cell populations may be used.

  It is noted that human adult peripheral blood also contains pluripotent stem cells, such as “Ilham Saleh Abuljadayel”, “Induction of stem cell cell-like plasticity in mononuclear cells depleted from human cells. 2003, Vol 19, no. 5,355-375).

  Immunostaining is a reaction between a labeled antibody and a target cell population, and the labeled antibody reacts with and binds to an antigen contained in the cell population, thereby staining the corresponding cells contained in the cell population. That means.

  The antibody may be labeled by directly binding the label to the antibody (directly) or indirectly by binding the monoclonal antibody of the present invention as the primary antibody to the labeled secondary antibody. Good (indirect method). Moreover, you may utilize ABC method using the fluorescence labeled avidin which has the property couple | bonded specifically with biotin instead of a secondary antibody.

  As the label, those conventionally used for labeling can be used. Specific examples include fluorescent dyes, radioactive materials, gold colloids, enzymes, chemiluminescence, and the like. The detection method of these labels is appropriately selected from conventionally known methods according to the type of label. For example, in the case of enzyme labeling, a substrate is added instead of the secondary antibody, and the color development is observed. In the case of radioactive substances, the radioactivity level is measured with a scintillation counter or the like. In the case of a fluorescent dye, excitation light is applied and the intensity of the emitted fluorescence is measured.

  When a section of a fixed tissue mass is used as the cell population, endogenous peroxidase is inactivated and non-specific adsorption is blocked, and then reacted with an antibody. The section may be a paraffin section, a microslide section, or a frozen section. For staining, an enzyme antibody method or a fluorescent antibody method is preferably used, and the presence or absence of staining may be determined by observing with an optical microscope, a fluorescence microscope, a laser microscope, or the like.

When the cell population is a single cell population, analysis by flow cytometry is preferably used.
Analysis by flow cytometry means that a monoclonal antibody is labeled with a fluorescent dye by a direct method, an indirect method, or an ABC method, reacted with a cell population, measured with a flow cytometer, and measured on the surface of individual cells. This is a measurement method in which fluorescence obtained from an antibody bound to an antigen is optically captured and further converted into an electrical signal to detect and record a label. Among the fluorescence intensities at the specific wavelengths of the fluorescent dye used for the labeling, those that are included in the fluorescence region having a higher intensity than the background intensity measured without using the primary antibody are judged as positive.

The cell population applied to the flow cytometry analysis is preferably a population containing 1 × 10 ⁵ cells or more, and if the number of cells that can be judged as a positive group is 80% or more, preferably 90% or more, it is selectively used. It is thought that it reacted.

  Cells that are determined to be positive by analysis by flow cytometry include flow cytometry (FACS) having a sort function, MACS (Magnetic Cell Sorting) system using a combination of surface antigen-specific antibodies and magnetic beads, HFB25 or HFB184. Separation and separation can be performed by a method of separating cells bound to secondary antibody-bound cells by passing through an antibody column to which an antibody capable of specifically binding to the antibody is bound.

  In FACS, positive cells and negative cells can be separated and collected according to the fluorescence intensity used as a label. Separation by the MACS system is a method in which cells are labeled with magnetic beads, passed through a separation column placed on a strong permanent magnet, and separated by holding only the magnetically labeled cells on the column. In the method using an antibody column, a secondary antibody that can specifically bind to the effector site of the monoclonal antibody of the present invention (particularly HFB25 or HFB184 antibody) or an antibody that can specifically bind to the monoclonal antibody of the present invention itself (for example, anti-antibody). Separating cells (antibody positive cells) that are not bound to cells that are bound with anti-HFB25 antibody or anti-HFB184 antibody (antibody positive cells) by passing through a column that is bound to HFB25 antibody or anti-HFB184 antibody) Can do.

  Monoclonal antibody positive cells of the present invention, including HFB25 or HFB184 antibody positive cells, are nestin high expressing cells. Since Nestin is an intracellular protein recognized as a marker for neural stem cells, Nestin high-expressing cells, that is, by separating and sorting out the monoclonal antibody positive cells of the present invention such as HFB25 or HFB184 antibody positive cells, Human NSPC can be separated and fractionated.

  In the screening method of the present invention, neural stem cells are selectively detected by detecting cells judged to be negative by further reacting with anti-JAM-1 antibody for cells judged to be HFB25 or HFB184 antibody positive. be able to.

  Here, JAM-1, which is an antigen of the anti-JAM-1 antibody used in the method of the present invention, is a cell surface molecule that is expressed in the state of neural cells differentiated from human NSPC, and is almost expressed in human NSPC. Although it is not expressed, it is expressed in the middle of differentiation, and the expression decreases as further differentiation progresses. In other words, it is a cell surface protein molecule that appears transiently during the differentiation process of neural cells. Such an anti-JAM-1 antibody that specifically binds to JAM-1 is negative for neural stem cells and mature differentiated cells (astrocytes), but transiently positive in the course of neural stem cell differentiation. Show. Therefore, anti-JAM-1 antibody negative cells, i.e., more undifferentiated cells, from a cell population determined to be HFB25 or HFB184 antibody positive, i.e., a mixed population of human neural progenitor cells exhibiting a differentiation tendency and more undifferentiated human NSPC. It is possible to select human NSPC.

The anti-JAM-1 antibody used in the method of the present invention recognizes a cell surface antigen of JAM-1, which is a monoclonal antibody that recognizes the cell surface protein JAM-1 (Junctional-Adhesion-Molecular 1, Swiss Prot ID: Q9Y624). It is a concept including an antibody fragment (for example, F (ab) ₂ , Fab, etc.) and an immunoglobulin having an antigen binding site. As a commercially available product, a mouse-produced anti-JAM-1 IgG monoclonal antibody manufactured by Hycut biotechnology or the like can be used.

  Thus, by using the HFB25 or HFB184 antibody of the present invention in combination with an anti-JAM-1 antibody, it is possible to separate and collect Nestin high-expressing cells, that is, human NSPC-enriched population from the cell population, By reacting with the JAM-1 antibody and separating and collecting cells determined to be negative, it becomes possible to separate and collect a neural stem cell enriched population. The same applies to all the monoclonal antibodies of the present invention capable of showing an antigen-antibody reaction with the antigen-binding site recognized by the HFB25 or HFB184 antibody instead of the HFB25 or HFB184 antibody.

  The human NSPC screening reagent of the present invention contains the monoclonal antibody of the present invention including HFB25 or HFB184 antibody as an essential component, and as a solvent, a medium used for neural stem cell culture (DMEM / F12) or PBS is used. Can be used. On the other hand, it is preferable that the screening reagent does not contain serum or surfactant. This is because serum has an effect of inducing differentiation of neural stem cells, and a surfactant may interfere with recognition of cell surface molecules with an antibody.

  It is preferable that the neural stem cell screening reagent of the present invention further contains an anti-JAM-1 antibody. By screening for cells that are positive for the HFB25 or HFB184 antibody and negative for the anti-JAM-1 antibody, it is possible to isolate and collect a cell population with high purity of neural stem cells.

  The cell population evaluation method of the present invention is a method for evaluating the content of human NSPC in a cell population containing human NSPC. The evaluation method of the present invention is a method in which a cell population is reacted with the screening reagent of the present invention, that is, the monoclonal antibody of the present invention including the HFB25 or HFB184 antibody, and evaluated based on the content of positive cells. This method is based on the fact that HFB25 or HFB184 antibody-positive cells are correlated with cells that highly express Nestin, and can be applied to the target cell population in the screening method of the present invention.

  Furthermore, by using the reactivity with anti-JAM-1 antibody in combination, that is, detecting HFB25 or HFB184 antibody-positive and anti-JAM-1 antibody-negative cells, the content of human NSPC and neural stem cells in the cell population Can be evaluated.

  The above evaluation method uses HFB25 antibody (or HFB184 antibody and, if necessary, further anti-JAM-1 antibody) by fluorescence labeling and profiles such as histograms and cytograms (dot plots) obtained using flow cytometry and the like. From the above, it may be carried out by determining the content ratio of HFB25 antibody (or HFB184 antibody) positive cells (or anti-JAM-1 antibody negative cells if anti-JAM-1 antibody is included), or a cell population to be a sample Alternatively, it may be performed by attaching immunohistochemistry or tissue to a slide glass, or by ELISA, RIA, or the like.

[Production of hybridoma]
(1) About immunogens Under the approval of the National Hospital Organization Osaka Medical Center Ethics Committee and the National Institute of Advanced Industrial Science and Technology, the NSPCs derived from the human forebrain at 9 weeks of gestation were used in the following neural stem cell growth medium. The human neurospheres obtained by subculture using the method of Kanemura et al. (Kanemura et.al. Journal of Neuroscience Research 69, 869-879, 2002) were rapidly frozen and homogenized. The resulting homogenate was used as an immunogen.

The composition of the medium used for the culture of the human neurosphere method is as follows.
DMEM / F12 (1: 1 mixture, Sigma)
Human recombination (hereinafter abbreviated as “hr-”) EGF (Invitrogen) 20 ng / ml
hr-FGF2 (Pepro Tech) 20 ng / ml
hr-LIF (Chemicon International) 10 ng / ml
Heparin (Sigma) 5mg / ml
B27 (Invitrogen)
HEPES15mM
Antibiotic-antimycotic (Invitrogen)

(2) Immunization of animals Production of monoclonal antibodies was performed using inguinal lymph node cells according to a modification by Orlik, O & Alterer, C (J. Immunol. Methods. 115: 55-59, 1998).

  That is, the immunogen (200 μg) prepared in (1) was mixed with an equal amount of RIBI adjuvant (Funakoshi Co., Ltd.) at a volume of 0.2 ml once on both foot soles of 6-week-old female Balb / c mice. Were inoculated three times every 3-4 days. After another 5 days, the same amount was immunized as a boost. Mice 3 days after the last boost were used for the cell fusion operation.

(3) Cell fusion Remove both inguinal lymph nodes of mice (5) immunized by (2) and place them in RPMI1640 medium (GIBCO Life Technology) and sandwich them between two pieces of ground glass. Then, lymphocytes were taken out (about 1 × 10 ⁸ ). On the other hand, the established myeloma (FO-1 cell) was cultured at 37 ° C. in RPMI 1640 medium containing 15% fetal calf serum (FCS) and 8 azaguanine (20 μg / ml), and further, for 2 to 3 days before cell fusion. The cells were prepared by culturing in the same composition medium from which 8 azaguanine was removed (about 4 × 10 ⁷ cells).
The mouse lymphocytes taken out and the established myeloma cells were mixed in 50 ml of growth medium (RPMI1640 medium + 15% FCS), centrifuged at 1000 rpm (number of revolutions / min) for 5 minutes at room temperature, the supernatant was discarded and 20 ml of growth was performed. Cells were suspended in the medium. To 20 ml of this cell suspension, 100 μl of 50% polyethylene glycol aqueous solution was added, and the mixture was kept in a CO ₂ incubator at 37 ° C. for 90 minutes. Thereafter, the mixture was centrifuged at 1000 rpm (number of revolutions / minute) for 5 minutes at room temperature, the supernatant was discarded, and the mixture was further centrifuged at a number of revolutions of about several seconds to absorb as much of the supernatant as possible. The centrifugal precipitate was loosened while warming the container containing the cell sample lightly against the water surface of a 37 ° C. constant temperature water bath. 1 ml of a 50% aqueous polyethylene glycol solution pre-warmed at 37 ° C. was slowly added over 1 minute and shaken, and another 1 ml was added slowly over 1 minute and shaken. Further, 8 ml was added over 2-3 minutes and mixed, and then centrifuged at 1000 rpm (revolutions / minute) for 5 minutes.

50 ml of HAT medium (2 ml of 50 × HAT supplement per 100 ml of growth medium) was added to the resulting precipitate (cells). This suspension was seeded in a 96-well plate at 0.1 ml / well and cultured (5 plates). At this time, thymocytes were added at a rate of about 1 × 10 ⁵ cells / well as Feeder cells (other types of cells that coexist for helping cell growth). After culturing for 3 days, 50 μl / well of HAT medium was added, and then, while adding and replacing the HAT medium, the cells were cultured until the hybridoma growth colonies were about ¼ to １ ／ of the wells. When the colonies became an appropriate size, the culture supernatant was collected and subjected to the following screening.

(4) Selection and Cloning of Hybridoma For the hybridoma-derived antibody obtained in (3) above, a well containing a hybridoma that immunoreacts with a human fetal brain tissue specimen was selected by the following enzyme antibody method.

  Paraffin-embedded sections of human fetal autopsy brains without serious complications such as brain malformations were used for immunohistochemical screening. A 5 μm section was prepared from the paraffin-embedded tissue piece, attached to a silane coating slide, and dried overnight in a 37 ° C. incubator. Prior to staining, deparaffinization was performed with histoclear and ethanol. The sections were washed with PBS containing 0.2% Tween-20, and immersed in 2% hydrogen peroxide-containing methanol for 30 minutes at room temperature to inactivate endogenous peroxidase. After blocking non-specific adsorption with 10% goat serum / 0.01% Triton-X for 1 hour, 100 μl of the hybridoma culture supernatant obtained in (3) was added dropwise without dilution and reacted at 4 ° C. overnight. I let you. The next day, a biotinylated secondary antibody (anti-mouse IgG antibody + anti-mouse IgM antibody) was added dropwise and allowed to react at room temperature for 1 hour. Then, using ABC kit (Nichirei Multi stain), color was developed with DAB. Nuclear staining was performed as necessary, and after dehydration and penetration, it was sealed with an oykit.

For human fetal brain tissue sections, well-positive wells were limiting diluted to approximately 0.5 hybridomas / well, and then the culture medium was replaced with HT medium + Bricklone (growth factor, Dainippon Pharmaceutical). Thus, a cell population derived from the target hybridoma was obtained.
The hybridomas thus obtained were deposited at the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology, and have accession numbers FERM P-20606 and FERM P-20607.

(5) Production of mouse ascites Using a small needle (23G TERRUMO), a small syringe (1 ml TERRUMO) and pristine (tetramethylpentadecane; Tokyo Kasei) at a volume of 0.5 ml / mouse (4 weeks old female BALB) / C mouse, SCL) was pre-treated. Two weeks after the pretreatment, hybridoma cells (0.5-1 × 10 ⁷ cells / ml) suspended in PBS [PBS (−) pH 7.4 filter sterilized] with a small syringe (5 ml TERUMO) using a small needle. / Mouse) was administered intraperitoneally. Several days later, after confirming that the abdomen of the mouse was swollen, ascites was collected using a syringe (16G TERRUMO) with a syringe. The obtained ascites was dispensed in 2 ml portions in a small tube and stored at minus 80 ° C.
The obtained monoclonal antibodies were named HFB25 antibody and HFB184 antibody, respectively. These are all IgM type antibodies.

[Property test of HFB25 antibody and HFB184 antibody]
(1) Immunohistochemical staining of fetal brain A 5 μm section was prepared from a paraffin permanent tissue specimen of the cerebrum of a human 8-week-old fetus, attached to a silane-coated slide, and dried overnight in a 37 ° C. incubator. Prior to staining, deparaffinization was performed with histoclear and ethanol. The sections were washed with PBS containing 0.2% Tween-20, and immersed in 2% hydrogen peroxide-containing methanol for 30 minutes at room temperature to inactivate endogenous peroxidase. After blocking non-specific adsorption with 10% goat serum / 0.01% Triton-X for 1 hour, the mouse ascites (HFB25 and HFB184 antibodies) obtained in (5) was diluted 500-fold, and 4 ° C. Reacted overnight. The next day, a biotinylated secondary antibody (anti-mouse IgM antibody) was added dropwise and allowed to react at room temperature for 1 hour. Then, using ABC kit (Nichirei Multi stain), color was developed with DAB. Nuclear staining was performed as necessary, and after dehydration and penetration, it was sealed with an oykit.

  FIG. 1 shows a photomicrograph showing the reaction result of the HFB25 antibody-containing solution with a cerebral tissue section of a human 8-week-old fetus. FIG. 1 shows a photomicrograph showing the reaction result of the HFB184 antibody-containing solution with a cerebral tissue section of a human 8-week-old fetus. Each is shown in FIG.

  From FIG. 1, for the HFB25 antibody, in the human fetal 8-week-old cerebrum, the ventricular wall layer (VZ) and the subventricular wall layer (SVZ) were strongly stained, and the intermediate layer (Intermediate zone; IMZ ) And molecular layer (ML) were also positive, but there was no obvious staining in the cortical plate (CP) (FIG. 1).

  From FIG. 2, the HFB184 antibody exhibited a fibrous stained image over almost all layers in the human fetus 8-week-old cerebrum (FIG. 2). Among them, a relatively strong positive finding was seen in ML.

(2) Reactivity to human neurospheres The subcultured human neurospheres were fixed with 4% paraformaldehyde at 4 ° C. for 20 minutes. Then, it was immersed in 30% sucrose / PBS at 4 ° C. for 30 minutes, and frozen and embedded in OCT-compound. Thereafter, a 12 μm section was prepared using a cryostat, and immunohistological analysis was performed. The frozen sections were reacted overnight at 4 ° C. using HFB25 antibody and HFB184 antibody diluted 100 times as primary antibodies. On the next day, as a secondary antibody, a goat-producing anti-mouse IgM-polyclonal antibody (1: 500, Molecular Probes) labeled with Alexa Floor 488 was reacted, and TO-PRO-3 iodide (1 μM: Molecular Probes) for nuclear staining and Reacted. The staining results were observed with a confocal laser scanning microscope (LSM510, Carl Zeiss).

The results of nuclear staining and HFB25 antibody staining are shown in FIG. Moreover, the nuclear staining result and the staining result of HFB184 antibody are shown in FIG.
In both FIG. 3 and FIG. 4, the antibody staining results and the nuclear staining results almost coincided. Therefore, it can be seen that most cells constituting the human neurosphere, that is, human NSPC, are positive cells of both antibodies.

(3) Analysis using FACS Human NSPC was dispersed using trypsin, and a cell suspension of a single cell was prepared using a cell strainer. These cell suspensions were each reacted with HFB25 antibody and HFB184 antibody (mouse ascites) diluted 100 times on ice for 30 minutes, and then washed with a culture solution. Then, Alexa488-labeled anti-mouse-IgM antibody (1: 500, goat-producing polyclonal antibody, Molecular Probes) was reacted on ice for 30 minutes, washed with culture medium, and further PI (propidium iodide) staining was performed. It was. The analysis was performed using the 4-color formula of FACS Calibur Flow Cytometer (trade name: Catalog No. 343202) of Becton Dickinson (San Jose, Calif.), And labeled with each monoclonal antibody (positive cell) and non-labeled cells. The ratio of labeled cells (negative cells) was calculated.

  PI is a DNA stain that intercalates into DNA with a double helix structure, whereby the total number of cells can be determined. HFB25 antibody-positive cells can be known from the fluorescence intensity of the green fluorescent dye Alexa488, which is a label for the secondary antibody bound to the HFB25 antibody.

  As control cells, 10 types of cultured human cell lines, ie, U-87MG, T98G, YKG-1, U251MG (derived from human glioblastoma) Daoy, D283Med (derived from human medulloblastoma); nervous system having differentiation ability NTERA2 which is a progenitor cell (human embryo carcinoma derived from teratoma); Jurkat which is a blood cell line, K562 (derived from human leukemia); , Calculate the ratio of HFB25 positive cells and HFB25 negative cells using FACS Calibur I put it out.

  The results are shown in FIG. In FIG. 5, the vertical axis represents the cell count, the horizontal axis represents the fluorescence intensity of green fluorescence, and those showing 10 or more signals correspond to HFB25 antibody positive cells. Table 1 summarizes the ratios of HFB25 antibody positive cells and HFB25 antibody negative cells in each cell population.

  Regarding NTERA2, which is a human NSPC and human EC cell, the percentage of HFB25 antibody-positive cells is very high, and there is a theory that other neural cells and non-neural nervous cell lines are originally similar to human NSPC There were relatively many HFB25 positive cells in Daoy (54%) and blood cell line Jurkat (42.5%), but the rate of HFB25 positivity was low in other neural cells and non-neural cells.

When the analysis by FACS was similarly performed using HFB184 antibody, the ratio of HFB184 positive cells was 99.1% for human NSPC, 0.2% for U251MG, and 46.6% for Jurkat, and analysis using HFB25 antibody The result was almost the same as the result.
From the above, the HFB25 antibody and HFB184 antibody of the present invention are antibodies that selectively recognize surface antigens expressed in undifferentiated neural cells and do not react with surface antigens of differentiated neural cells and non-neural cells. It turns out that it is an antibody.

[Neural stem cell differentiation level and antibody reactivity]
(1) Neural stem cell differentiation induction Differentiation induction method A:
Differentiation is induced in a medium to which growth factors have been removed from the medium used in the neurosphere method and 10% FBS is added. After induction of differentiation, the cells are dispersed in single cells using a 0.05% trypsin solution every 1-2 weeks. The operation of subculture was repeated several times. By this method, a cell population of almost 100% of glial cells can be obtained.
Differentiation induction method B:
Growth factors were removed from the medium used in the neurosphere method, and the cells were cultured in a medium supplemented with 1% FBS for 2 weeks to induce differentiation. In this method, a mixed culture of nerve cells and glial cells (hereinafter referred to as “N / G cells”) population (approximately 20% of tubulin βIII-positive neurons and approximately 80% of GAFP-positive glial cells) is obtained.

(2) Analysis using FACS Two lots (lot Nos. 1 and 2) of human NSPC cell populations were each induced to differentiate by the method A. For the cell populations after 2 weeks and 7 weeks after induction of differentiation, the ratio of HFB25 antibody positive cells was examined by FACS using the same method as described above.
FIG. 6 (a), (b), and (c) show human NSPCs that were not induced to differentiate, and the results after 2 weeks and 7 weeks after differentiation induction, respectively. In FIG. 6, the horizontal axis represents the fluorescence intensity, and the vertical axis represents the cell number count. An intensity of 10 or more corresponds to a HFB25 antibody positive cell. Table 2 summarizes the ratio of HFB25 antibody-positive cells in each cell population.

  As can be seen from Table 2, the proportion of positive HFB25 antibody gradually decreases with the progress of human NSPC differentiation. That is, it can be seen that the HFB25 antibody is an antibody selective for a cell surface antigen that exists on the surface of undifferentiated human NSPC and then disappears during the differentiation process.

(3) Relationship between anti-tubulin βIII antibody and HFB25 antibody-positive cells Anti-tube that is a neuronal marker for each cell population 2 weeks after differentiation induction and 7 weeks after differentiation induction method B Double staining of phosphorus βIII antibody and HFB25 antibody was performed. The cells were fixed with 4% paraformaldehyde, blocked with 10% normal goat serum for 1 hour at room temperature, blocked, and then HFB25 antibody (mouse ascites) diluted 100-fold as the primary antibody and anti-tubulin βIII (TuJ1) antibody (1 : 500; mouse IgG monoclonal, BabCO) and 10% normal goat serum / 0.01% Triton-X / PBS was reacted overnight at 4 ° C. After the reaction, secondary antibody (goat-producing anti-rabbit IgM antibody labeled with Alexa Fluor 488 and goat-producing anti-rabbit IgG antibody labeled with Alexa Fluor 568, both from Molecular Probes) and TO-PRO- for nuclear staining 3 Iodide (1 μM, Molecular Probes) was reacted at room temperature for 1 hour. Observation after staining was performed using a confocal laser microscope (LSM510; Carl Zeiss).

  FIG. 7 (a) (stained with HFB25 antibody) and (b) (stained with anti-tubulin βIII antibody) show fluorescence micrographs after 2 weeks of differentiation induction. Further, micrographs after 7 weeks of induction of differentiation are shown in FIGS. 8 (a) (HFB25 antibody staining) and (b) (anti-tubulin βIII antibody staining). The scale bar in FIGS. 7 and 8 is 100 μm.

  7 and 8, in the examination using the nervous system marker (anti-tubulin βIII antibody), HFB25 (+) / tubulin βIII (+) cells are confirmed at the time of early differentiation induction (2 weeks). It can be seen that in the late stage of differentiation induction (7 weeks), HFB25 (+) cells almost disappeared, and cells co-expressed with tubulin βIII were hardly seen.

(4) Relationship between anti-GFAP antibody and HFB25 positive cell Double staining of anti-GAFP antibody and HFB25 antibody, which are markers for glial cells, was performed on each cell population after 2 weeks and 7 weeks after induction of differentiation by method A. It was. In the same manner as described above, after fixing and blocking the cells, the method includes HFB25 antibody (mouse ascites) diluted 1: 500 as a primary antibody and rabbit-produced anti-GFAP polyclonal antibody (Sigma) (1:80). % Normal goat serum / 0.01% Triton-X / PBS was reacted at 4 ° C. overnight. After the reaction, secondary antibodies (goat-producing anti-mouse IgM antibody labeled with Alexa Fluor 488 and goat-producing anti-mouse IgG antibody labeled with Alexa Fluor 568, both from Molecular Probes) and TO-PRO-3 iodide (1 μM, Molecular (Probes) was allowed to react at room temperature for 1 hour. Observation after staining was performed using a confocal laser microscope (LSM510; Carl Zeiss).

  Fluorescence micrographs after 2 weeks of induction of differentiation are shown in FIGS. 9 (a) (HFB25 antibody staining) and (b) (anti-GAFP antibody staining). Further, micrographs after 7 weeks of induction of differentiation are shown in FIGS. 10 (a) (HFB25 antibody staining) and (b) (anti-GAFP antibody staining). The scale bar in FIGS. 9 and 10 is 100 μm.

  In FIG. 9, HFB25 (+) / GAFP (+) cells were confirmed, but in FIG. 10, HFB25 (+) cells almost disappeared, and cells co-expressed with GAFP were hardly seen. From these results, it can be seen that the HFB25 antibody does not react with glial cells.

[Relationship between various enzyme treatments and antigenicity]
(1) Effect of degreasing by chloroform treatment In order to examine whether or not it has the characteristics of lipidic antigens, the effect of degreasing by chloroform treatment was examined.

  A cell suspension was prepared by dispersing human neurospheres subcultured in 0.05% trypsin solution and dispersing them in single cells. The cell suspension was fixed with 4% paraformaldehyde for 4 minutes at 4 ° C., and then sections were prepared for immunohistochemical analysis. The sections were degreased with a solution of chloroform and methanol mixed at a ratio of 2: 1 for 10 minutes at room temperature, washed with PBS (−), and blocked by reacting with 10% goat normal serum for 1 hour at room temperature. 10% normal goat serum / 0.01% Triton-X / PBS containing HFB25 antibody (mouse ascites) diluted 100-fold as a primary antibody was reacted at 4 ° C. overnight. The next day, as a secondary antibody, it was reacted with Alexa Floor488-labeled goat-producing anti-mouse IgM-polyclonal antibody (1: 500, Molecular Probes) and TO-PRO-3 (1 μM: Molecular Probes) for nuclear staining. It was. The staining results were observed with a confocal laser scanning microscope (LSM510, Carl Zeiss).

As a control, A2B5 antibody that recognizes lipidic antigen was reacted with chloroform-treated cells in the same manner as above, and the same treatment as above except that each antibody before and after chloroform treatment (FHB25 antibody and A2B5 antibody) was not used. This was used and observed under a microscope.
A photomicrograph of the reaction result with the untreated HFB25 antibody is shown in FIG. 11, and a photomicrograph of the reaction result with the chloroform-treated HFB25 antibody is shown in FIG.
As can be seen from the comparison between FIG. 11 and FIG. 12, even when the HFB25 antibody was treated with chloroform, there was almost no effect on cell staining and no effect on the antigen binding site. on the other hand. Since the defatted antibody binding site was destroyed in the A2B5 antibody, the cell staining portion was almost lost.

(2) Effects of glycosidase and glycosaminoglycan degrading enzyme treatment In order to examine the antigenicity of sugar chains, the effects of treatment with various sugar chain cleaving enzymes were examined.
A cell suspension was prepared by dispersing human neurospheres subcultured with a 0.05% trypsin solution treatment solution and dispersing them into single cells. The cell suspension was fixed with 4% paraformaldehyde for 4 minutes at 4 ° C., and then sections were prepared for immunohistochemical analysis. To the sections, N-glycosidase, O-glycosidase, chondroitinase ABC, keratanase, heparitinase, or heparinase was added to a concentration as shown in Table 3, and reacted for the time shown in Table 3. These enzymes cleave the parts shown in Table 3. After enzyme treatment, blocking was performed by reacting with normal serum of 10% goat at room temperature for 1 hour. 10% normal goat serum / 0.01% Triton-X / PBS containing HFB25 antibody (mouse ascites) diluted 100 times as a primary antibody was reacted at 4 ° C. overnight. On the next day, goat-producing anti-mouse IgM-polyclonal antibody (1: 500, Molecular Probes) labeled with Alexa Floor 488 and TO-PRO-3 (1 μM: Molecular Probes) for nuclear staining at room temperature 1 Reacted for hours. The staining results were observed with a confocal laser scanning microscope (LSM510, Carl Zeiss).

FIGS. 13 to 18 show micrographs of staining results of N-glycosidase treatment, O-glycosidase treatment, chondroitinase ABC treatment, keratanase treatment, heparitinase treatment, and heparinase treatment, respectively.
All were stained to the same extent as in the case of untreated (see FIG. 11), and it was found that the antigen-binding site of the HFB25 antibody was not affected by these enzyme treatments.

[Separation and collection of neural stem cells and progenitor cells from human umbilical cord blood]
(1) Separation of nucleated cell population from human umbilical cord blood Lymphoprep (AXIS-SHIELD) in an amount equal to human umbilical cord blood is placed in a centrifuge tube, and human umbilical cord blood is layered thereon, and 800 g at 20 ° C. for 20 minutes. Centrifugation and nucleated sphere layers were collected. The isolated cells were cryopreserved (banbanker) in the same manner as human NSPC, and used for the following analysis.

(2) HFB25 antibody reactivity Cell suspensions (UCB1-UCB3) were prepared by diluting 3 lots of human umbilicus-derived cells prepared in (1) to 1 × 10 ⁷ cells / 100 μl, respectively.
To this cell suspension, 2 μl of mouse ascites producing HFB25 antibody was added and mixed, and reacted on ice for 30 minutes. Next, it was reacted with a goat-producing anti-mouse IgM-polyclonal antibody (1: 500, Molecular Probes) labeled with Alexa 488 as a secondary antibody, further PI-stained, and FACS Calibur of Becton Dickinson (San Jose, Calif.). A 4-color system of Flow Cytometer (trade name: catalog number 343202) was measured using a flow cytometer.

The results of UCB1 to UCB3 are shown in FIG. In FIG. 19, the horizontal axis represents the fluorescence intensity, and the vertical axis represents the cell number count. An intensity of 10 or more is HFB25 antibody positive cells.
FIG. 19 shows that although there are variations among lots, HFB25 antibody-positive cells are contained in an amount of about 2 to 6.7%.
Further, using a cell sorter (FACS Vantage-SE, manufactured by Becton-Dixon), human umbilical cord blood-derived HFB25 positive cells (labeled cells) and HFB25 negative cells (unlabeled cells) were separated.

(3) Examination of Nestin expression in human umbilical cord blood-derived HFB25-positive cells After fixing each of human umbilical cord blood-derived HFB25-positive cells and negative cells sorted in (2) with 4% paraformaldehyde, cytospin3 (Thermo Shandon) Was attached to a slide glass. After blocking by reacting with normal serum of 10% goat for 1 hour at room temperature, a rabbit-produced anti-human Nestin polyclonal antibody (1: 500) was reacted overnight at 4 ° C. as a primary antibody, followed by Alexa Floor 568 as a secondary antibody. (Red) labeled goat-producing anti-rabbit IgG (H + L) conjugate high cross-adsorbed (final concentration 4 μg / ml, Molecular Probes) was reacted at room temperature for 1 hour to prepare an immune cell-stained specimen. The secondary antibody recognizes mouse H and L chains by goat production and does not recognize human, bovine, goat, rabbit and rat antibodies.

  For the purpose of quantitatively examining the expression level of Nestin protein in each cell population of human umbilical cord blood-derived HFB25 antibody positive cells and negative cells, an immunostained specimen using an anti-Nestin antibody was analyzed by a laser scanning cytometer ( LSC2, Olympus Corporation). The results are shown in FIG. In FIG. 20, the horizontal axis represents the anti-Nestin antibody fluorescent labeling intensity, and the vertical axis represents the number of cells. The HFB25 antibody-positive cell population showed a stronger fluorescence intensity distribution than the HFB25 antibody-negative cell population, and it was found that cells that were positive for the HFB25 antibody in human umbilical cord blood-derived cells also had a higher expression level of Nestin.

  Furthermore, based on the results of FIG. 20, the results of the laser scanning cytometer for the human umbilical cord blood-derived HFB25 antibody positive cells and negative cells, and the fluorescence label intensity of 0 to 50000 based on the Nestin expression level are Nestin negative (−). , 50,000 to 100,000 were classified as Nestin positive (+), and 100,000 to 150,000 were classified as Nestin positive (++). The results are shown in Table 4.

  As can be seen from Table 4, in human umbilical cord blood cells, 88.3% of HFB25-positive cells are nestin-expressing cells, 18.2% of them strongly express nestin, and the ratio is HFB25-negative cells (8. 7%).

[Separation and collection of neural stem cells and progenitor cells from human adult peripheral blood]
(1) Separation of nucleated cell population from human adult peripheral blood Lymphoprep (AXIS-SHIELD) equivalent to human adult peripheral blood is placed in a centrifuge tube, and human adult peripheral blood is layered thereon and 800 g at 20 ° C. And centrifuged for 20 minutes, and a nucleated sphere layer was collected. The isolated cells were cryopreserved (banbanker) in the same manner as human NSPC, and used for the following analysis.

(2) HFB25 antibody reactivity In the same manner as in the case of umbilical cord blood, a 4-color expression of FACS Calibur Flow Cytometer (trade name: catalog number 343202) manufactured by Becton Dickinson (San Jose, Calif.) Was used using a flow cytometer. The ratio of HFB25 antibody positive cells was measured.
The results are shown in FIG. In FIG. 21, the horizontal axis represents the fluorescence intensity, and the vertical axis represents the cell number count. An intensity of 10 or more is HFB25 antibody positive cells.
FIG. 21 shows that human adult peripheral blood contains about 25% of HFB25 antibody positive.
Further, human adult peripheral blood-derived HFB25 positive cells (labeled cells) and HFB25 negative cells (unlabeled cells) were separated using a cell sorter (FACS Vantage-SE, manufactured by Becton-Dixon).

(3) Examination of Nestin expression in HFB25 positive cell As a result of examining Nestin expression level like the case of cord blood, it became as shown in FIG.

  As can be seen from Table 5, 74.5% of HFB25-positive cells in human adult peripheral blood are Nestin-expressing cells, 14.6% of which are strongly expressing Nestin, and the ratio is HFB25-negative cells (7 0.7%).

[Relationship between cell differentiation level and JAM-1 expression]
(1) Screening of protein expressed in neural stem cells using antibody chip The antibody chip analysis was performed using BD Clontech ™ Ab Microarray 500 (BD Biosciences Clontech) to which 512 types of known antibodies were immobilized. The glass slide was scanned using a glass slide scanner (manufactured by Applied Precision).
Proteins are extracted from human NSPC and the glial cell population obtained by the differentiation induction method of A with the reagent attached to BD Clontech ™ Ab Microarray 500, and each sample is labeled with Cy3 or Cy5 fluorescent dye (manufactured by Amersham Bioscience). Then, antibody chip analysis was performed to examine the expression level of JAM-1 in each cell population.
The JAM-1 expression level (human NSPC / glia cells) of the human NSPC cell population relative to the JAM-1 expression level in the glial cell population was 0.6737772.

(2) Examination by Western Blot To further examine the expression pattern of JAM-1, the expression pattern of JAM-1 in a total of 13 types of human cell lines was similarly examined by Western blot.
As neuronal cells, U-251MG, U-87MG, U-373MG, T98G, YKG-1 (derived from human gliblastoma), D283 Med, Daoy (derived from human medulloblastoma); NTERA2 (teratomato as a neural progenitor cell having differentiation ability) Derived from human embryo carcinoma); Jurkat (derived from human accurate T cell leukemia), 293T (human fetal transformed form of H13, He-Gel epithelial cell) Cultures of human cell lines using standard methods. Cells were prepared. As controls, the N / G cell population (N / G) obtained by the differentiation induction method of B and the glial cell population (AS) differentiated by the differentiation induction method of A were used.

  Proteins were extracted from each cell and electrophoresed using SDS polyacrylamide gel (5-20% gradient gel, ATTO) at 10 μg / lane (SDS-PAGE). After SDS-PAGE, it was transferred to a nitrocellulose membrane (Advantec) by a wet method. After blocking with 5% bovine serum albumin (BSA) for 1 hour, anti-JAM-1 antibody (1: 500 diluted with 1% BSA, Becton Dickinson) was reacted as a primary antibody at room temperature for 1 hour. After washing with PBS containing 0.1% Tween 20 (hereinafter referred to as “PBS-T”) for 15 minutes and 3 times, anti-mouse IgG antibody (diluted 1: 10000 with 1% BSA) as a secondary antibody for 1 hour at room temperature Reacted. After washing, chemiluminescence was performed according to the method of ECL Plus kit (Amersham Bioscience). This film was placed in a cassette with X-ray film, exposed and developed.

The results are shown in FIG. JAM-1 appears at the position of a 38 kDa band (arrow in the figure).
The expression of JAM-1 was hardly observed in U-373MG with respect to nervous system cells, and the expression was lower in glial cells (AS) in D283Med and T98G. The expression of JAM-1 in the remaining neural cells was higher than glial cells (AS). The expression of JAM-1 for non-neural cells was slightly lower for glare cells (AS) for Jurkat, but glial cells (AS) for the remaining non-neural cells (293T, HepG2, HUH-6, HUH-7). ) Was higher.

(3) Relationship between differentiation induction level of human NSPC and anti-JAM-1 antibody positive cells In order to evaluate the presence rate of JAM-1 positive cells in differentiation induction of human NSPC, human NSPC and glial cells differentiation-induced therefrom were Using the 4-color method of FACS Calibur Flow Cytometer (trade name: Catalog No. 343202) of Becton Dickinson (San Jose, Calif.), The presence rate of JAM-1 positive cells was evaluated.
The human NSPC cell population of the same lot was divided into three groups, and two of them were induced to differentiate by the method A. Cell suspensions were prepared by treatment with 0.05% trypsin solution for the cell populations after 4 weeks and 7 weeks after differentiation induction.

The cell suspension was reacted with anti-JAM-1 antibody (Hycut biotechnology) diluted 10 times on ice for 30 minutes, and then washed with the culture solution. Then, Alexa488-labeled anti-mouse-IgG antibody (1: 500, goat-producing polyclonal antibody, Molecular Probes) was reacted on ice for 30 minutes, then washed with culture medium, and further PI (propidium iodide) staining was performed. It was. The analysis was performed using a 4-color expression of FACS Calibur Flow Cytometer (trade name: Catalog No. 343202) of Becton Dickinson (San Jose, Calif.), And each labeled cell (positive cell) and unlabeled with a monoclonal antibody. The percentage of cells (negative cells) was calculated.
24 (a), (b), and (c) show the human NSPC that was not induced to differentiate, and the results after 4 weeks and 7 weeks after differentiation induction, respectively. In FIG. 24, the horizontal axis represents the fluorescence intensity, and the vertical axis represents the cell number count. The horizontal axis of 10 or more corresponds to anti-JAM-1 antibody positive cells. Table 6 summarizes the ratio of anti-JAM-1 antibody-positive cells in each cell population.

In the state of human NSPC, JAM-1 positive cells were only 6.0%, but in glial cells (4 weeks after differentiation induction), about 40.8% of the cells were JAM-1 positive. However, the positive rate decreased again (10.5%) by further differentiation induction (7-week culture).
From these results, the anti-JAM-1 antibody selectively binds to a surface antigen that appears temporarily from the neural stem cell in the process of complete differentiation. Therefore, in a mixed population of neural progenitor cells and neural stem cells, if the anti-JAM-1 antibody is negative, it can be determined that it is a neural stem cell.

  Since the novel monoclonal antibody of the present invention binds to a surface antigen selective for neural stem cells, it is possible to screen neural stem cells or neural stem cell-enriched populations as living cells from heterogeneous cell populations containing neural stem cells. . Therefore, cell populations containing neural stem cells collected from living organisms for regenerative medicine, specifically from neural stem cell-containing populations such as fetal brain tissue, umbilical cord blood, and human peripheral blood, neural stem cell enriched populations, and thus neural stem cell It can be used as a method for detection and further separation. This makes it possible to use a relatively wide range of sources as neural stem cell supply sources for neural stem cells that have had problems in terms of cell source supply.

  The screening reagent of the present invention can be used as a reagent for screening neural stem cells for human body transplantation.

It is a microscope picture (70 times) which shows the HFB25 antibody reaction result in the cerebral tissue section | slice of a human 8 week-old fetus. It is a microscope picture (32 times) which shows the HFB184 antibody reaction result in the cerebral tissue section | slice of a human 8-week-old fetus. (A) is a photomicrograph (200 times) showing the result of nuclear staining in the neurosphere, and (b) is a photomicrograph (200 times) showing the result of staining the HFB25 antibody in the neurosphere. (A) is a photomicrograph (200 times) showing the result of nuclear staining in the neurosphere, and (b) is a photomicrograph (200 times) showing the result of staining the HFB184 antibody in the neurosphere. It is a profile which shows the measurement result of the flow cytometer which shows the HFB25 antibody reactivity in the single cell population of a neurosphere. (A) Human NSPC cell population, (b) Profile obtained by flow cytometer analysis showing results showing reactivity with HFB25 antibody 2 weeks after differentiation induction and (c) 7 weeks after differentiation induction. (A) is a photomicrograph showing the HFB25 antibody staining result of the cell population after 2 weeks of differentiation induction, and (b) is a photomicrograph showing the anti-tubulin βIII antibody staining result of the cell population after 2 weeks of differentiation induction. (A) is a photomicrograph showing the HFB25 antibody staining result of the cell population 7 weeks after differentiation induction, and (b) is a photomicrograph showing the anti-tubulin βIII antibody staining result of the cell population 7 weeks after differentiation induction. (A) is a photomicrograph showing the HFB25 antibody staining result of the cell population after 2 weeks of differentiation induction, and (b) is a photomicrograph showing the anti-GAFP antibody staining result of the cell population after 2 weeks of differentiation induction. (A) is a photomicrograph showing the HFB25 antibody staining result of the cell population after 7 weeks of differentiation induction, and (b) is a photomicrograph showing the anti-GAFP antibody staining result of the cell population after 7 weeks of differentiation induction. It is a microscope picture which shows the reaction result of an untreated human NSPC cell population and HFB25 antibody. It is a microscope picture (20 time) which shows the reaction result of the human NSPC cell population which carried out chloroform treatment, and HFB25 antibody. It is a microscope picture (20 time) which shows the reaction result of the human NSPC cell population which processed N-glycosidase, and HFB25 antibody. It is a microscope picture (20 time) which shows the reaction result of the human NSPC cell population which processed O-glycosidase, and HFB25 antibody. It is a microscope picture (20 times) which shows the reaction result of the human NSPC cell population which processed chondroitinase ABC, and HFB25 antibody. It is a microscope picture (20 times) which shows the reaction result of the human NSPC cell population treated with keratanase and HFB25 antibody. It is a microscope picture (20 times) which shows the reaction result of the human NSPC cell population treated with heparitinase and HFB25 antibody. It is a microscope picture (20 time) which shows the reaction result of the human NSPC cell population which processed heparinase, and HFB25 antibody. It is the profile obtained by the flow cytometer analysis which shows the reactivity with the HFB25 antibody of the cell population derived from human cord blood. It is a graph which shows the measurement result in LSM of the expression level of Nestin protein in the human umbilical cord blood-derived HFB25 antibody positive cell population and negative cell population. It is the profile obtained by the flow cytometer analysis which shows the reactivity with the HFB25 antibody of the cell population derived from a human adult peripheral blood. It is a graph which shows the measurement result in LSM of the expression level of the Nestin protein in the HFB25 antibody positive cell population derived from a human adult peripheral blood, and a negative cell population. It is a photograph which shows the result of the Western blot about the expression of JAM-1 in each cell. (A) Profile of flow cytometer analysis results for anti-JAM-1 antibody staining of human NSPC, (b) Flow cytometer analysis results for anti-JAM-1 antibody staining in cells 4 weeks after differentiation induction. Profile (c) is a profile of the analysis result of a flow cytometer for anti-JAM-1 antibody staining in cells 7 weeks after differentiation induction.

Claims (24)

A monoclonal antibody produced from a hybridoma of an antibody-producing cell of a mammal immunized with a homogenate of human neural stem cells and / or human neural progenitor cells, and myeloma,
A monoclonal antibody that reacts with surface antigens of neural stem cells and / or neural progenitor cells but does not substantially react with surface antigens of differentiated neural cells. The monoclonal antibody according to claim 1, wherein the antigen is a protein antigen in a non-reducing state. A monoclonal antibody produced from a hybridoma of an antibody-producing cell of a mammal immunized with a homogenate of human neural stem cells and / or human neural progenitor cells, and myeloma,
Monoclonal antibody that reacts with cerebral ventricular wall layer and lower ventricular wall cells in early fetal period. The monoclonal antibody according to claim 3, which does not substantially react with a cortical plate of the cerebrum in the early fetal period. The monoclonal antibody according to any one of claims 1 to 4, wherein the homogenate is a homogenate of cultured human neural stem cells and / or cultured human neural progenitor cells. The monoclonal antibody is screened using at least one selected from the group consisting of human fetal brain tissue, cultured human neural stem cells, and a cell population in which human neurospheres are single cells, for monoclonal antibodies produced from the obtained hybridomas. The monoclonal antibody according to any one of claims 1 to 3. In analysis by flow cytometry, 80% or more of the single cell population in which human neurospheres are dispersed and NETRA2 show a positive reaction, and all of U-87MG, T98G, TKG-1, U251MG, and 293T cells are 5 Monoclonal antibodies that show less than a positive response. The hybridoma which produces the monoclonal antibody in any one of Claims 1-7. The hybridoma of accession number FERM P-20606. The hybridoma of accession number FERM P-20607. A monoclonal antibody produced from a hybridoma having the deposit number FERM P-20606 or FERM P-20607. A monoclonal antibody that recognizes an antigenic determinant recognized by a monoclonal antibody (HFB25 antibody) produced from a hybridoma having the accession number FERM P-20606. A monoclonal antibody that recognizes an antigenic determinant recognized by a monoclonal antibody (HFB184 antibody) produced from a hybridoma having the accession number FERM P-20607. A method for identifying a cell having characteristics of a human neural stem cell and / or neural progenitor cell and the localization of the cell from a heterogeneous cell population, comprising:
A neural stem cell comprising a step of performing immunohistochemical staining using the monoclonal antibody according to any one of claims 1 to 7 and 11 to 13, and identifying the stained portion as a neural stem cell and / or neural progenitor cell; A method for identifying neural progenitor cells. A method for identifying a cell having characteristics of a human neural stem cell and / or neural progenitor cell and the localization of the cell from a heterogeneous cell population, comprising:
A method for identifying neural stem cells and / or neural progenitor cells, comprising a step of detecting a cell that shows an immunological reaction with an antigenic determinant recognized by an HFB25 antibody or an HFB184 antibody. A process comprising: reacting a cell population containing human neural stem cells with the monoclonal antibody according to any one of claims 1 to 7 and 11 to detecting cells positive for the reaction with the monoclonal antibody. A screening method for high-expressing cells. The screening method according to claim 16, wherein the nestin high-expressing cells are neural stem cells and / or neural progenitor cells. Furthermore, the screening method of the neural stem cell including the process of detecting the cell with a negative reaction with an anti- JAM-1 antibody about the monoclonal antibody positive cell detected by Claim 16. The screening method according to claim 16, wherein the cell population is a population containing human umbilical cord blood-derived cells. The screening method according to claim 16, wherein the cell population is a population containing human peripheral blood-derived cells. The screening method according to claim 16, wherein the cell population is a cell population obtained by dispersing human neurospheres into single cells. In a cell population containing human neural stem cells and neural progenitor cells, cells that are positive for reaction with the monoclonal antibody according to any one of claims 1 to 7 and 11 to 13 are detected,
A method for evaluating a cell population, comprising calculating a ratio of the monoclonal antibody positive cells in the cell population. In a cell population containing human neural stem cells and neural progenitor cells, cells that are positive for reaction with the monoclonal antibody according to any one of claims 1 to 7 and 11 to 13 are detected,
About the detected monoclonal antibody positive cells, cells that are negative in reaction with anti-JAM-1 antibody are detected,
A method for evaluating a cell population, comprising a step of calculating a proportion of cells positive for the monoclonal antibody reaction and negative for an anti-JAM-1 antibody response in the cell population. A reagent for screening an enriched population of neural stem cells and / or neural progenitor cells from a human neural stem cell and neural progenitor cell-containing cell population,
A reagent for screening neural stem cells and neural progenitor cells, comprising the monoclonal antibody according to any one of claims 1 to 7 and 11 to 13, and a solvent.