WO2012147992A1 - ANTI-iPS/ES CELL-SPECIFIC ANTIBODY AND USE THEREOF - Google Patents

Abstract

This invention provides a monoclonal antibody capable of recognizing iPS and ES cells, wherein said antibody recognizes a keratan sulfate on podocalyxin present on the surface of iPS and ES cells as an epitope, and wherein said antibody does not recognize EC cells. Also provided is a method of detecting an iPS or ES cell, comprising bringing a cell sample into contact with the monoclonal antibody mentioned above, and detecting a cell bound with the antibody in the sample.

Description

DESCRIPTION

ANTI-iPS/ES CELL-SPECIFIC ANTIBODY AND USE THEREOF

Technical Field of the Invention

The present invention relates to a monoclonal antibody specifically binding to induced pluripotent stem cells (iPS cells) and embryonic stem cells (ES cells) and use thereof. More specifically, the present invention relates to a monoclonal antibody recognizing a keratan sulfate on podocalyxin on the surface of iPS and ES cells different from those recognized by known anti-iPS/ES antibodies, as well as to use thereof as a marker antibody of human iPS/ES cells.

Background of the Invention

Carbohydrate recognizing antibodies are very useful

experimental tools for monitoring the changes of cell surface glycan structures as well as for the identification of specific glycans on a specific cell type with high sensitivity and sharp specificity. This is true also in the case of iPS cells. Namely, among the conventional human iPS/ES cell marker antibodies,

SSEA-3 and SSEA-4 specifically recognize globosides, and TRA-1- 60, TRA-1-81, GCTM2 and GCTM343 recognize keratan sulfate.

However, one problem here is that most of these antibodies were generated against human embryonal carcinoma' (EC) cells. In other word, these antibodies are not specific to human iPS/ES cells, rather they recognize those glycans, which are common to human iPS/ES and human EC cells (non-patent reference 1) .

Recently, Choo reported an anti-human ES cell monoclonal antibody (designated as mAb84) generated using a human ES cell as an immunogen (patent reference 1) . However, it is unknown whether or not mAb84 also recognizes human iPS cells. In fact, the Experimental Examples described below have revealed that this anti-human ES cell antibody hardly reacts with human iPS and EC cells. In addition, mAb84 is a cytotoxic antibody and may be useful in eliminating residual human ES cells from differentiated cells populations for clinical purpose. Hence, there remains a need for an anti-iPS/ES cell monoclonal antibody capable of discriminating iPS and EC cells. Prior Art References

[Patent reference 1] WO 2007/102787

[Non-patent reference 1] Wright, A.J. and Andrews, P.W., Stem Cell Res., 3(1): 3-11 (2009) Summary of the Invention

It is an object of the present invention to provide a monoclonal antibody specific to iPS/ES cells, namely binding to iPS/ES cells but not to other pluripotent stem cells such as EC cells.

To accomplish the object above, the present inventors used a differential screening method to generate antibodies specific to human iPS cells. Thus, the present inventors first selected the human iPS cell-positive hybridomas, from which human EC cell-positive hybridomas were excluded. By this procedure we have obtained hybridomas, which is essentially specific to human iPS/ES cells. The epitopes of these clones should be novel, since there is no report on human iPS cell-recognizing

antibodies discriminating human iPS and human EC cells. Among these novel mAbs, the present inventors have chosen one

hybridoma, R-10G, as the first target, and biochemical

properties of the monoclonal antibody (mAb) produced by the hybridoma and physiological significance of the antigen molecule recognized by the mAb has been investigated. As a result, the present inventors have found that mAb R-10G recognizes a keratan sulfate on podocalyxin, which keratan sulfate is present on the surface of human iPS and ES cells but absent on the surface of EC cells, as an epitope.

The present inventors conducted further investigations based on these findings, and have developed the present

invention. Accordingly, the present invention provides:

[1] a monoclonal antibody capable of recognizing iPS and ES cells, wherein said antibody recognizes a keratan sulfate on podocalyxin present on the surface of iPS and ES cells as an epitope, and wherein said antibody does not recognize EC cells;

[2] the monoclonal antibody according to [1] above, wherein the iPS and ES cells are derived from human;

[3] the monoclonal antibody according to [1] or [2] above, wherein said antibody is not cytotoxic to its target cells;

[4] the monoclonal antibody according to any of [1] to [3] above, which is a monoclonal antibody produced by hybridoma R- 10G ( FERM BP-11301) or a monoclonal antibody recognizing the same region as that recognized by the antibody produced by R-10G as an epitope;

[5] a method of detecting an iPS or ES cell, comprising bringing a cell sample into contact with the monoclonal antibody

according to any of [1] to [4] above, and detecting a cell bound with the antibody in the sample; and

[6] a method of making iPS or ES cell population homogeneous, comprising sorting iPS or ES cells recognized by the monoclonal antibody according to any of [1] to [4] above, and excluding iPS or ES cells recognized by an antibody selected from the group consisting of SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, GCT 2 and GCTM3 3 from the sorted cells.

It is clear that the anti-iPS/ES cell antibody of the present invention is a novel marker mAb recognizing iPS/ES cells and should be useful not only as a new molecular probe to disclose the roles of glycans on the surface of iPS/ES cells in the maintenance of self-renewal and pluripotency, and during the process of differentiation, but also as a potent tool for the evaluation and standardization of iPS cells from different tissue origin and different history in regenerative medicine.

Brief Description of the Drawings Fig. 1 shows screening of hybridomas by Western blotting. Tic cell lysates in the complete RIPA buffer (15 μg protein) were resolved by SDS-PAGE on a 4-15% gradient gel under non- reducing conditions and then protein were transferred to a

PVDF membrane, followed by immunoblot detection with mAbs secreted into the culture supernatant of the respective

hybridomas as described under MATERIALS and METHODS in Example.

(A) Protein staining of Tic cell lysates by Gel code blue.

Cell lysate, Tic cell lysates in the complete RIPA buffer.

Lane M, molecular weight markers.

(B) Western blotting of Tic cell lysates by mAb R10-G. Numbers on the top of the lanes indicate the clone numbers of

hybridomas. Lane M, molecular weight markers.

Fig. 2 shows localization of R-IOG, TRA-1-60 and TRA-1-81 epitopes in cultured Tic cells observed by laser confocal microscopy.

(A) Tic cells cultured in Millipore EZ slides were double stained first with R-10G (I) and Alexa Fluor 488-conjugated secondary antibody, followed by TRA-1-60 (II) and Alexa Fluor 555-conjugated secondary antibody. Cells expressing antigens for R-IOG and those for TRA-1-60 were visualized by laser confocal microscopy. (Ill) Merged image of (I) and (II) . (IV) Nuclear counterstain by TO-PR03.

(B) Cultured Tic cells were double stained first with R-IOG (I) and Alexa Fluor 488-conjugated secondary antibody,

followed by TRA-1-81 (II) and Alexa Fluor 555-conjugated

secondary antibody. Cells expressing antigens for R-IOG and those for TRA-1-81 were visualized by laser confocal

microscopy. (Ill) Merged image of (I) and (II) . (IV) Nuclear counterstain by TO-PR03.

Scale bar, 100 um.

Fig. 3 shows isolation and identification of R-IOG

antigen protein.

(A) R-IOG antigen proteins were isolated from Tic cell lysates with an R-IOG-Sepharose 4B Column and resolved by SDS-PAGE on

A a 4-15% gradient SDS-polyacrylamide gel as described under MATERIALS AND METHODS. The positions of molecular weight markers are shown in the left margin. The protein bands, which corresponded to those of the immunoblot bands, were divided into three segments as indicated by A, B and C as shown in Sypro Ruby positive bands and used for the identification of antigen proteins by mass spectroscopy as described under

MATERIALS AND METHODS.

(B) The results of a search against the human protein database of the National Center for Biotechnology Information based on the acquired fragmentation spectra of peptides. The identified peptides in segment A are shown in white against black

background, those in segment B are either shaded or underlined with a wavy line and those in segment C are underlined black. Potential N-glycosylation sites (position 33, 43, 104, 144 and 360) are indicated by blocked n.

(C) Schematic presentation of membrane topology of human podocalyxin. Potential N-glycosylation sites are indicated by "X" .

Fig. 4 shows identificatin of mAb R-10G epitope as keratan sulfate.

(A) Tic cell lysates (12 g protein, corresponding to 1 x 10⁵ cells) and isolated R-10G antigens (derived from 1 x 10⁵ cells) were subjected to SDS-PAGE and Western blotting on a 4-15% gradient SDS-polyacrylamide gel as described under MATERIALS AND METHODS. Tic cell lysates reacted not only with R-10G but also with TRA-1-60 and TRA-1-81 and showed broad but single bands at somewhat different positions in a high molecular region (>250kDa) , respectively. The isolated R-10G antigens also reacted with TRA-1-60 slightly and TRA-1-81 considerably at their respective positions.

(B) The isolated R-10G antigens (derived from 1 x 10⁵ cells) were digested with increasing amounts (0 to 10 munits) of keratanase II and subjected to SDS-PAGE and Western blotting with mAbs, R-10G and TRA-1-81, respectively, as described under MATERIALS AND METHODS.

Fig. 5 shows schematic presentation of keratan- sulfate/podocalyxin recognizing mAbs specific to pluripotent cells .

R-10G, TRA-1-60 and TRA-1-81 recognizes different structures of keratan-sulfate expressed on podocalyxin polypeptide and mAb84 is reported to recognize podocalyxin polypeptide.

Fig. 6 shows identification of R-10G as a keratan-sulfate recognizing antibody.

The mAb R-IOG (1 μg/ml) was added to biotinylated

glycosaminoglycans (GAGs, lmg/ml) of various kinds, which had been fixed on a streptavidin coated plate, and the amounts of R-IOG bound to the GAGs were assayed by incubation with HRP- labeled second antibody (anti-mouse IgG antibody) and TMB

(3, 3 ' , 5, 5 ' -tetramethyl benzidine).

GAGs used: HA (hyaluronic acid from pig skin) , Ch (chondroitin) , CSA(W) (chondroitin sulfate from whale cartilage), CSA(S)

(chondroitin sulfate from the Spinal Column of Acipenser

medirostris) , CSB (chondroitin sulfate B from pig skin) , CSC (chondroitin sulfate C from shark cartilage) , CSD (chondroitin sulfate D from shark cartilage) , CSE (chondroitin sulfate E from squid cartilage) , HS (heparan sulfate from bovine kidney) , KS (keratan sulfate from bovine cornea) . All these GAGs were obtained from Seikagaku Biobusiness (Tokyo, Japan) .

Detailed Description of the Invention

The present invention provides a monoclonal antibody capable of specifically recognizing iPS and ES cells

[hereinafter also referred to as the " (anti-iPS/ES cell)

antibody of the present invention"] . This antibody is further characterized in that (a) it does not recognize EC cells and (b) it recognizes a keratan sulfate on podocalyxin present on the surface of iPS and ES cells. Since known anti-iPS/ES antibodies recognizing a keratan sulfate on podocalyxin such as TRA-1-60 and TRA-1-81 also recognize EC cells, the anti-iPS/ES cell antibody of the present invention recognizes a keratan sulfate structure on podocalyxin different from those recognized by the known antibodies (see Fig. 5) .

Although the isotype of the antibody of the present invention is not subject to limitation, it is preferably IgG, IgM or IgA, particularly preferably IgG.

The antibody of the present invention is not subject to limitation, as long as it has at least a complementality determining region (CDR) for specifically recognizing and binding to the target antigen; in addition to the whole antibody molecule, the antibody may, for example, be a

fragment such as Fab, Fab', or F(ab')₂, Fv, V_H, a genetically engineered conjugate molecule such as scFv, scFv-Fc, dsFv, minibody, or diabody, or a derivative thereof modified with a molecule having protein stabilizing action, such as

polyethylene glycol (PEG), or the like, and the like.

The antibody of the present invention can be produced by a method of antibody production known per se. Hereinafter, a method of preparing an immunogen (iPS/ES cell) for an antibody of the present invention, and a method of producing the antibody are described.

(1) Preparation of antigen

As an antigen used to prepare an antibody of the present invention, any of iPS and ES cells or a fraction thereof containing cell surface keratan sulfate-modified podocalyxin molecules (e.g., membrane fraction) and the like, can be used.

An iPS cell can be produced by reprogramming a somatic cell obtained from a mammal according to any known methods [see, for example, Cell 2007;131:861-72, Science 2007;318:1917-20 (human); Cell 2006;126:663-76 (mouse); Cell Stem Cell

2008;3 (6) :587-90 (Rhesus monkey); Cell Stem Cell 2008; (1) : 11-5, Cell Stem Cell 2008; 4 (1) : 16-9 (rat); J Mol Cell Biol

2009; 1 (1) : 6-54 (pig); Mol Reprod Dev 2010; 77(1): 2 (dog); Stem Cell Res 2010; 4 (3) : 180-8, Genes Cells 2010; 15 (9) : 959-69

(marmoset); J Biol Chem 2010;285 (41) : 31362-9 (rabbit)]. Also, iPS cells can be obtained from various public and private depositories and are commercially available. For example, human iPS cell lines 201B7 and 235G1 can be obtained from CELL BANK of RIKEN BIORESOURCE CENTER and Tic (JCRB1331) can be obtained from National Institute of Biomedical Innovation.

An ES cell can be produced by any known methods . For example, available methods of preparing ES cells include, but are not limited to, methods in which a mammalian inner cell mass in the blastocyst stage is cultured [see, for example, Manipulating the Mouse Embryo: A Laboratory Manual, Second

Edition, Cold Spring Harbor Laboratory Press (1994)] and

methods in which an early embryo prepared by somatic cell nuclear transfer is cultured (Nature 1997; 385 : 810, Science

1998;280: 1256, Protein Nucleic Acid and Enzyme 1999; 4 : 892, Nat Biotechnol 1999; 17: 456, Nature 1998 ; 394 : 369, Nat Genet

1999;22:127, Proc Natl Acad Sci USA 1999; 96: 14984, Nat Genet 2000 ; 24 : 109) . Also, ES cells can be obtained from various public and private depositories and are commercially available. For example, human ES cell lines HI and H9 can be obtained from WiCell Institute of University of Wisconsin and KhES-1, - 2 and -3 can be obtained from Institute for Frontier Medical Sciences, Kyoto University.

Intact iPS or ES cells may be used for immunization, or freeze-thawed, irradiated or glutaraldehyde-treated iPS or ES cells also may be used.

Alternatively, a cell membrane fraction of the iPS or ES cells can be used as an immunogen for producing an antibody of ^'the present invention. The cell membrane fraction can be prepared by homogenizing iPS or ES cells, removing the cell debris by low speed centrifugation, thereafter precipitating a cell membrane-containing fraction by high speed centrifugation of the supernatant (and, where necessary, purifying the cell membrane fraction by density gradient centrifugation and the like) . The cell membrane fraction can be fused with liposomes to give proteolipsomes in which a keratan sulfate-modified podocalyxin is embedded, or the glycoprotein can be purified from the membrane fraction, for example, using an anti- podocalyxin antibody such as mAb84, and mixed with an

appropriate surfactant to prepare a complex whose hydrophobic (trans-membrane) region is covered with a surfactant micelle. In another embodiment, a recombinant keratan sulfate-modified podocalyxin can be purified from iPS or ES cells into which an expression cassette of podocalyxin with His-, GST- or Myc-tag at N- or C-terminus is introduced, using an affinity column onto which Ni, glutathione or anti-Myc antibody is immobilized. (2) Preparation of monoclonal antibody

(a) Preparation of monoclonal antibody-producing cells

The immunogen prepared as. mentioned above is administered as is, or along with a carrier or a diluent, to a warm-blooded animal at a site enabling antibody production by the methods such as intraperitoneal injection, intravenous injection, subcutaneous injection, intradermal injection and the like. In order to increase antibody productivity upon the

administration, Freund' s complete adjuvant or Freund' s

incomplete adjuvant may be administered. Dosing is normally performed about 2 to 10 times in total every 1 to 6 weeks. As examples of the warm-blooded animal to be used, mouse, rat rabbit, goat, monkey, dog, guinea pig, sheep, donkey and

chicken, preferably mouse, rat and rabbit can be mentioned.

Alternatively, the immunogen can be subjected to in vitro immunization method. As the animal cells used in the in vitro immunization method, lymphocytes, preferably B-lymphocytes and the like, isolated from peripheral blood, spleen, lymph node and the like of a human and the above-described warm-blooded animals (preferably mouse or rat) can be mentioned. For

example, in the case of mouse or rat cells, the spleen is extirpated from an about 4- to 12-week-old animal, and

splenocytes are separated and rinsed with a appropriate medium [e.g., Dulbecco's modified Eagle medium (DMEM) , RPMI1640

medium,. Ham's F12 medium and the like], after which the splenocytes are suspended in an antigen-containing medium supplemented with fetal calf serum (FCS; about 5 to 20%) and cultured using a C0₂ incubator and the like for about 4 to 10 days. It is preferable to prepare a culture supernatant of thymocytes of an animal of the same strain (preferably at about 1 to 2 weeks of age) according to a conventional method, and to add the supernatant to the medium.

Because it is difficult to obtain a thymocyte culture supernatant in in vitro immunization of human cells, it is preferable to perform immunization by adding, to the medium, several kinds of cytokines such as IL-2, IL-4, IL-5, and IL-6 and the like, and if necessary, an adjuvant substance (e.g., muramyldipeptide and the like) along with the antigen.

In preparing a monoclonal antibody, it is possible to establish an antibody-producing hybridoma by selecting an individual or cell population showing an elevated antibody titer from among antigen-immunized warm-blooded animals (e.g., mice, rats) or animal cells (e.g., human, mouse, rat),

respectively; collecting spleens or lymph nodes at 2 to 5 days after the final immunization or collecting the cells after 4 to 10 days of cultivation after in vitro immunization to isolate antibody-producing cells; and fusing the isolated cells with myeloma cells. A measurement of serum antibody titer can be performed by, for example, reacting a labeled antigen and an antiserum, and thereafter determining the activity of the label bound to the antibody.

Although the myeloma cells are not subject to limitation, as long as they are capable of producing a hybridoma that secretes a large amount of antibody, those that do not produce or secrete the antibody per se are preferable, with greater preference given to those of high cell fusion efficiency. To facilitate hybridoma selection, it is preferable to use a cell line that is susceptible to HAT (hypoxanthine, aminopterin, thymidine) . As examples of the mouse myeloma cells, NS-1, P3U1, SP2/0, AP-1 and the like can be mentioned; as examples of the rat myeloma cells, R210.RCY3, Y3-Ag 1.2.3 and the like can be mentioned; as examples of the human myeloma cells, SKO-007, GM 1500-6TG-2, LICR-LON-HMy2, UC729-6 and the like can be

mentioned.

Fusion operation can be performed according to a known method, for example, the method of Koehler and Milstein

[Nature, 256, 495 (1975)]. As a fusion promoter, polyethylene glycol (PEG) , Sendai virus and the like can be mentioned, and PEG and the like are preferably used. Although the molecular weight of PEG is not subject to limitation, PEG1000 to PEG6000, which are of low toxicity and relatively low viscosity, are preferable. As examples of the PEG concentration, about 10 to 80%, preferably about 30 to 50%, can be mentioned. As the solution for diluting PEG, various buffers such as serum-free medium (e.g., RPMI1640) , complete medium comprising about 5 to 20% serum, phosphate buffered saline (PBS) , and Tris buffer can be used. DMSO (e.g., about 10 to 20%) can also be added as desired. As examples of the pH of the fusion solution, about 4 to 10, preferably about 6 to 8 can be mentioned.

The ratio by number of antibody-producing cells

(splenocytes) and myeloma cells is preferably about 1:1 to

20:1, and the cell fusion can be efficiently performed by incubation normally at 20 to 40°C, preferably at 30 to 37°C, normally for 1 to 10 minutes.

An antibody-producing cell line can also be obtained by infecting antibody-producing cells with a virus capable of transforming lymphocytes to immortalize the cells. As such viruses, for example, Epstein-Barr (EB) virus and the like can be mentioned. Although the majority of persons have immunity because they have ever been infected with this virus in an asymptomatic infection of infectious mononucleosis, virion is also produced when the ordinary EB virus is used; therefore, appropriate purification must be performed. As an EB system free from the possibility of viral contamination, it is also preferable to use a recombinant EB virus that retains the capability of immortalizing B lymphocytes but lacks . the capability of replicating virion (for example, deficiency of the switch gene for transition from latent infection state to lytic infection state and the like) .

Because marmoset-derived B95-8 cells secrete EB virus, B lymphocytes can be easily transformed by using a culture supernatant thereof. An antibody-producing B cell line can be obtained by, for example, culturing these cells using a medium supplemented with serum and penicillin/streptomycin (P/S) (e.g., RPMI1640) or a serum-free medium supplemented with a cell growth factor, thereafter separating the culture

supernatant by filtration or centrifugation and the like, suspending therein antibody-producing B lymphocytes at a suitable concentration (e.g., about 10⁷ cells/mL) , and

incubating the suspension normally at 20 to 40°C, preferably at 30 to 37°C, normally for about 0.5 to 2 hours. When human antibody-producing cells are provided as mixed lymphocytes, it is preferable to previously remove T lymphocytes by allowing them to form an E rosette with, for example, sheep

erythrocytes and the like, to increase transformation

frequency of EB virus, because the majority of persons have T lymphocytes which exhibit cytotoxicity to cells infected with EB virus. It is also possible to select lymphocytes specific for the target antigen by mixing sheep erythrocytes,

previously bound with a soluble antigen, with antibody- producing B lymphocytes, and separating the rosette using a density gradient of percoll and the like. Furthermore, because antigen-specific B lymphocytes are capped by adding the antigen in large excess so that they no longer present IgG to the surface, mixing with sheep erythrocytes bound with anti- IgG antibody results in the formation of rosette only by antigen-nonspecific B lymphocytes. Therefore, by collecting a layer of cells that don't form rosette from this mixture using a density gradient of percoll and the like, it is possible to select antigen-specific B lymphocytes. Human antibody-secreting cells having acquired the capability of proliferating indefinitely by the transformation can be back fused with mouse or human myeloma cells in order to stably sustain the antibody-secreting ability. As the myeloma cells, the same as those described above can be used.

Hybridoma screening and breeding are normally performed using a medium for animal cells (e.g., RPMI1640) containing 5 to 20% FCS or a serum-free medium supplemented with cell growth factors, with the addition of HAT (hypoxanthine, aminopterin, thymidine) . As examples of the concentrations of hypoxanthine, aminopterin and thymidine, about 0.1 mM, about 0.4 μΜ and about 0.016 mM and the like, respectively, can be mentioned. For selecting a human-mouse hybridoma, ouabain resistance can be used. Because human cell lines are more susceptible to ouabain than mouse cell lines, it is possible to eliminate unfused human cells by adding ouabain at about 10^{" 7} to 10^"3 M to the medium.

In selecting a hybridoma, it is preferable to use feeder cells or culture supernatants of certain cells. As the feeder cells, an allogenic cell species having a lifetime limited so that it dies after helping the emergence of hybridoma, cells capable of producing large amounts of a growth factor useful for the emergence of hybridoma with their proliferation potency reduced by irradiation and the like, and the like are used. For example, as the mouse feeder cells, splenocytes, macrophage, blood, thymocytes and the like can be mentioned; as the human feeder cells, peripheral blood mononuclear cells and the like can be mentioned. As examples of the cell culture supernatant, primary culture supernatants of the above- described various cells and culture supernatants of various established cell lines can be mentioned.

Moreover, a hybridoma can also be selected by reacting a fluorescein-labeled antigen with fusion cells, and thereafter separating the cells that bind to the antigen using a

fluorescence-activated cell sorter (FACS) . In this case, efforts for cloning can be lessened significantly because a hybridoma that produces an antibody against the target antigen can be directly selected.

For cloning a hybridoma that produces a monoclonal

antibody against the target antigen, various methods can be used.

It is preferable to remove aminopterin as soon as

possible because it inhibits many cell functions. In the case of mice and rats, aminopterin can be removed 2 weeks after fusion and beyond because most myeloma cells die within 10 to 14 days. However, a human hybridoma is normally maintained in a medium supplemented with aminopterin for about 4 to 6 weeks after fusion. It is desirable that hypoxanthine and thymidine be removed more than one week after the removal of aminopterin. That is, in the case of mouse cells, for example, a complete medium (e.g., RPMI1640 supplemented with 10% FCS) supplemented with hypoxanthine and thymidine (HT) is added or exchanged 7 to 10 days after fusion. About 8 to 14 days after fusion, visible clones emerge. Provided that the diameter of clone has reached about 1 mm, the amount of antibody in the culture supernatant can be measured.

A measurement of the amount of antibody can be performed by, for example, a method comprising adding the hybridoma culture supernatant to a solid phase (e.g., microplate) to which the target antigen is adsorbed directly or with a

carrier, subsequently adding an anti-immunoglobulin (IgG) antibody (an antibody against IgG derived from an animal of the same species as the animal from which the original

antibody-producing cells are derived is used) or protein A, which had been labeled with a radioactive substance (e.g., ¹²⁵I, ¹³¹I, ³H, ¹C) , enzyme (e.g., β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase) ,

fluorescent substance (e.g., fluorescamine, fluorescein

isothiocyanate) , luminescent substance (e.g., luminol, luminol derivative, luciferin, lucigenin) and the like, and detecting the antibody against the target antigen bound to the solid phase, a method comprising adding the hybridoma culture supernatant to a solid phase to which an anti-IgG antibody or protein A is adsorbed, adding the target antigen labeled with the same labeling reagent as described above, and detecting the antibody against the target antigen bound to the solid phase and the like.

Although limiting dilution is normally used as the cloning method, cloning using soft agar and cloning using FACS (described above) are also possible. Cloning by limiting dilution can be performed by, for example, the following procedures, which, however, are not to be construed as limiting .

The amount of antibody is measured as described above, and positive wells are selected. Selected suitable feeder cells are previously added to a 96-well plate. Cells are collected from the antibody-positive wells and suspended in complete medium (e.g., RMPI1640 supplemented with 10% FCS and P/S) to obtain a density of 30 cells/mL; 0.1 mL (3 cells/well) of this suspension is added to the well plate with feeder cells added thereto; a portion of the remaining cell

suspension is diluted to 10 cells/mL and sown to other wells (1 cell/well) in the same way; the still remaining cell

suspension is diluted to 3 cells/mL and sown to other wells (0.3 cells/well). The cells are cultured for about 2 to 3 weeks until a visible clone appears, when the amount of antibody is measured to select positive wells, and the

selected cells are recloned in the same way. In the case of human cells, cloning is relatively difficult, so that a plate in which cells are seeded at 10 cells/well is also prepared. Although a monoclonal antibody-producing hybridoma can be obtained normally by two times of subcloning, it is desirable to repeat recloning regularly for several more months to confirm the stability thereof.

(b) Differential screening The hybridomas producing a monoclonal antibody against iPS or ES cells obtained as described above are then subjected to the second screening, in which ES or iPS cells, pluripotent stem cells such as EC cell, EG cell, mGS cell and the like, and somatic cells are used as probes as well as iPS or ES cells used as the immunogen. As a result of the second

screening, a hybridoma producing a monoclonal antibody that reacted with iPS and ES cells but not with pluripotent stem cells other than iPS and ES cells such as EC cells, and

somatic cells can be selected as a hybridoma producing an anti-iPS/ES cell antibody of the present invention.

Hybridomas thus obtained can be cultured in vitro or in vivo. As a method of in vitro culture, a method comprising gradually scaling up a monoclonal antibody-producing hybridoma obtained as described above, from a well plate, while keeping the cell density at, for example, about 10⁵ to 10⁶ cells/mL, and gradually lowering the FCS concentration, can be mentioned.

As a method of in vivo culture, for example, a method comprising an intraperitoneal injection of a mineral oil to a mouse (a mouse that is histocompatible with the parent strain of the hybridoma) to induce plasmacytoma (MOPC) 5 to 10 days later, to which intraperitoneally injecting about 10⁶ to 10⁷ cells of hybridoma, and collecting ascites fluid under

anesthesia 2 to 5 weeks later, can be mentioned.

(c) Purification of monoclonal antibody

Separation and purification of the monoclonal antibody are performed according to a method known per se, for example, a method of immunoglobulin separation and purification [e.g., salting-out, alcohol precipitation, isoelectric point

precipitation, electrophoresis, adsorption-desorption with an ion exchanger (e.g., DEAE, QEAE) , ultracentrifugation, gel filtration, specific purification comprising selectively

collecting the antibody by means of an antigen-coupled solid phase or an active adsorbent such as protein A or protein G, and dissociating the linkage to obtain the antibody, and the like] .

As described above, a monoclonal antibody can be produced by culturing a hybridoma in or outside the living body of a warm-blooded animal, and harvesting an antibody from the body fluid or culture thereof.

As an example of the anti-iPS/ES cell antibody of the present invention obtained as described above, mouse anti- human iPS/ES cell antibody mAb R-10G, described in Examples below, is exemplified. A hybridoma that produces this antibody (R-10G) has been deposited at the International Patent

Organism Depositary (IPOD) , National Institute of Advanced

Industrial Science and Technology (Chuo 6, 1-1-1, Higashi, Tsukuba, Ibaraki, 305-8566 Japan) since October 6, 2010

(accession number: FERM BP-11301) . The depositary name has been changed to "IPOD, National Institute of Technology and Evaluation (NITE) " ([email protected])

(d) Production of recombinant antibody

In another embodiment, cDNAs that encode the heavy chain and light chain of an anti-iPS/ES cell antibody thus obtained can be isolated from cDNA library derived from a hybridoma producing the antibody and cloned into appropriate expression vector (s) functional in a host cell of interest by

conventional methods. Then, a host cell is introduced with the heavy chain and light chain expression vector (s) thus obtained. Useful host cells include animal cells, for example, mouse myeloma cells as described above, as well as Chinese hamster ovary (CHO) cells, monkey-derived COS-7 cells, Vero cells, rat-derived GHS cells and the like. Although this introduction can be achieved by any method that is applicable to animal cells, it is preferable to use electroporation or a method based on a cationic lipid and the like. After the host cell is cultured in a suitable medium for a given period, the

conditioned medium is recovered, and the antibody protein is purified by a conventional method, whereby the antibody of the present invention can be isolated. Alternatively, by producing a transgenic animal by a conventional method using a germline cell of an animal as a host cell for which transgenic

technology has been established, and for which know-how for mass propagation for a domestic animal (poultry) has been compiled, such as bovine, goat or chicken, it is also possible to obtain a large amount of the antibody of the present

invention from the milk or egg of-.- the animal thus obtained.

Furthermore, it is also possible to obtain a large amount of the antibody of the present invention from seeds, leaves and the like obtained from a transgenic plant prepared by

microinjection or electroporation for protoplast, the particle gun method, the Ti vector method and the like for intact cells, using as the host cell a cell of a plant for which transgenic technology has been established, and which is cultured in large amounts as a major crop, such as corn, rice, wheat, soybean or tobacco.

(e) Cytotoxicity of the antibody of the invention

An anti-iPS/ES cell antibody of the present invention may be or may not be cytotoxic to target iPS and cells. For

example, the mAb R-10G mentioned above is not cytotoxic to human iPS and ES cells. It can be examined whether or not an anti-iPS/ES cell antibody is cytotoxic to target cells by the methods known per se (for example, see WO 2007/102787). One of ordinary skill in the art can choose either cytotoxic or non- cytotoxic antibody according to his/her object of use.

(3) Use of the antibody of the present invention

, Because an antibody of the present invention is capable of specifically recognizing iPS and ES cells, it can be used for detection and quantitation of iPS cells or ES cells in a test cell sample, particularly for detection and quantitation by immunocytochemistry . For these purpose, the antibody

molecule itself may be used, and any fragment thereof,, such as the F(ab')₂, Fab' or Fab fraction of the antibody molecule, may also be used. The method for measurement using an antibody against iPS/ES cells is not to be limited particularly, any

13 , method of measurement can be used.

As the labeling agent to be used for the measurement method using a labeling substance, for example, a radioisotope, an enzyme, a fluorescent substance, a luminescent substance and the like can be used. As the radioisotope, for example,

[¹²⁵I], [¹³¹I], [³H], [¹⁴C] and the like can be used. The above- described enzyme is preferably stable and has a high specific activity and, for example, β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase and the like can be used. As the fluorescent substance, for example, fluorescamine, fluorescein isothiocyanate (FITC) ,

phycoerythrin (PE) and the like can be used. As the

luminescent substance, for example, luminol, luminol

derivative, luciferin, lucigenin and the like can be used.

The antibody of the present invention may be directly or indirectly labeled with a labeling agent. In a preferable embodiment, the anti-iPS/ES cell antibody is an unlabeled antibody and can be detected by the labeled second antibody such as anti-serum or anti-Ig antibody against the animal from which the anti-iPS/ES cell antibody was produced.

Alternatively, the biotinylated second antibody can be used and a conjugate of iPS or ES cell-the antibody of the present invention-the second antibody can be visualized using a

labeled streptavidin.

For example, a test cell sample can be fixed and

permealized with glutaraldehyde, paraformaldehyde or the like, washed with a buffer such as PBS, blocked with BSA or the like and incubated with an anti-iPS/ES cell antibody of the present invention. After washing with a buffer such as PBS to remove unreacted antibody, the cells reacted with the anti-iPS/ES cell antibody can be visualized with the labeled second

antibody and analyzed using a confocal laser scanning

microscope, a flexible automated cell imaging system INCell Analyzer (Amarsham/GE) and the like.

In another embodiment, the antibody of the present invention can be used to isolate or remove iPS or ES cells from a sample containing the same. For this purpose, for example, the antibody of the present invention may be

immobilized on a solid phase comprising any suitable matrix such as agarose, acrylamide, Sepharose, Sephadex and the like. The solid phase may also be any suitable culture vessel such as a microtiter plate. iPS or ES cells in a sample is

immobilized on the solid phase when the sample is brought into contact with the solid phase. The cells can be released from the solid phase using an appropriate elution buffer.

In a preferable embodiment, the antibody of the present invention is immobilized on magnetic beads such that iPS or ES cells can be separated from the rest of the sample upon provision of a magnetic field (i.e., magnetic activated cell sorting; MACS) . In another preferable embodiment, the antibody of the present invention is directly or indirectly labeled with any suitable fluorescent molecule as exemplified above and iPS or ES cells are isolated using a fluorescence

activated cell sorter (FACS) .

As shown in the Example below, double-staining of the cells of a single iPS clone using an antibody of the present invention and a known anti-iPS/ES cell antibody that also recognizes EC cells revealed that an iPS cell line cloned by the conventional procedures is a heterogeneous cell population in distribution of cell surface components. To be specific, many of cells dominantly express podocalyxin containing a keratan sulfate recognized by either an antibody of the present invention or a known anti-iPS/ES cell antibody on their surface, while some cells express podocalyxin containing both of the keratan sulfates. Although the significance of such heterogeneity in glycosylation patterns of cell surface protein is unclear, the antibody of the present invention can sort heterogeneous iPS or ES cells into homogenous subsets in combination with a known anti-iPS/ES cell antibody such as SSEA-3, SSEA-4, TRA-1-60, TRA-1-81 or the like. The present invention is hereinafter described in further detail by means of the following examples, to which, however, the invention is never limited. Examples

[MATERIALS and METHODS]

Antibodies :

Antibodies: anti-human TRA-1-60 (Clone # TRA-1-60, mouse IgM) mAb, anti-human TRA-1-81 (Clone # TRA-1-81, mouse IgM) , anti-human/mouse SSEA-4 (clone# MC813, mouse IgG3) mAbs were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) , and anti-human/mouse SSEA-1 (clone#MC480, mouse IgM) and anti- human/mouse SSEA-3 (clone#MC631, rat IgM) and anti-human podocalyxin (clone# 222328, mouse IgG_2A) mAbs were obtained from R & D Systems, Inc. (Minneapolis, MN) . Anti-human podocalyxin- like protein I (Cytotoxic) (clone mAb 84, mouse IgM) was obtained from Millipore, Billerica, Hercules, CA) , Anti-human Nanog mAb and anti-human Oct4 mAb were obtained from ReproCELL (Kanagawa, Japan ) and Abeam (Cambridge, UK) , respectively.

Cells and Cell Culture:

Human iPS cell lines, Tic (JCRB1331) was obtained from the Japanese Collection of Research Bioresources (JCRB) , National Institute of Biomedical Innovation (Osaka, Japan) , 201B2 and 201B7 were provided from the Center for iPS Cell Research and Application (CiRA) , Kyoto University (Kyoto, Japan) . Human ES cell line, KhES-3 was provided from the Institute for Frontier Medical Sciences, Kyoto University (Kyoto, Japan) . These cells were cultured at 37°C/5% C0₂ on mitomycin C-inactivated feeders (mouse embryonic fibroblasts (MEF) , 5 x 10³ cells/cm²) in rectangular canted neck cell culture flask with vent cap (25cm², Corning, Corning, NY) . Human embryonal carcinoma cell line, 2102Ep, was a generous gift from Prof. Peter Andrews (University of Sheffield) and MRC-5 (JCRB9008) , fibroblast-like cell line derived from human lung tissue of a 14-week-old male fetus was obtained from JCRB. Preparation of human iPS cells for immunization and for

screening:

Human iPS cell line, Tic, which was generated from MRC-5 (Toyoda et al., 2011), human embryonic fibroblasts, by

transduction of four defined factors: Oct3/4, Sox2, Klf4, and c-Myc (Takahashi et al., 2007), was used as immunogenic

antigens and also as the screening probe. Tic cells maintained on mitomycin C-inactivated mouse embryonic fibroblasts (MEF, B6) in a serum free cell culture media, S medium, which

consisted of KNOCKOUT DMEM/F-12 (400 ml, Invitrogen-Life

technologies, Carlsbad, CA) , MEM non-essential amino acids solution (4.0 ml, Invitrogen-Life technologies, Carlsbad, CA) , 200 mM L-glutamine (5.0 ml), KNOCKOUT Serum Replacement (100 ml, Invitrogen-Life technologies, Carlsbad, CA) , and 55 mM 2- mercaptoethanol (0.925 ml), were transferred to hESF9 medium, which comprises ESF basal medium (Cell Science and Technology Institute, Sendai, Japan, Furue et al., 2005) without HEPES supplemented with nine defined factors: Asc 2-P, 6-factors (human recombinant insulin, human transferrin, 2- mercaptoethanol, 2-ethanolamine, sodium selenite, oleic acid conjugated with fatty acid-free bovine serum albumin (FAF- BSA) ) , bovine heparan sulfate sodium salt, and human

recombinant FGF-2 (Katayama Chemical Industries, Osaka, Japan) , as described previously (Furue et al., 2008). After incubation at 37°C for 4-5 days, the cells in one group of flasks (3 x 10⁵ ~ 1 x 10⁶ cells/25cm² flask) were harvested by treating with 0.1% EDTA-4Na (lml/flask) (in phosphate-buffered saline (PBS)), collected by centrifugation at 1,000 rpm for 2 min, washed with PBS and stored at -80°C until just before use as

immunogens. The cells in other group of flasks were used for the preparation of cell screening plates. To these flasks ROCK inhibitor (10 μΜ, Y27632, Wako Pure Chemical, Osaka, Japan) was added to permit survival of dissociated cells (Watanabe et al., 2007). After incubation at 37°C for 1 h, the cells were harvested with accutase (1 ml, Millipore, Billerica, MA) collected by centrifugation, washed with S-medium, suspended in hESF9 medium, and seeded in fibronectin coated 96-well plates (5 x 10³ cells/well, BD, Franklin Lakes, NJ) . Cells were fixed with 1% acetic acid/ethanol (100 μΐ/well) for 15-30 min. After washing with PBS, the plates were stored at -80°C until just before use.

Immunization:

Two different protocols were used for the immunization of mice with human iPS cells. In protocol A, the freeze-thawed Tic cells (1.5 x 10⁷ cells in 0.5 ml PBS) were emulsified with an equal volume of Freund' s Complete Adjuvant (CFA, Thermo Fisher Scientific, Rockford, IL) and injected into three 8- week old female C57BL/6 mice (200 ml/mice) intraperitoneally on day 0, followed by the booster injection on day 25, and the mice were sacrificed on day 28. In protocol B, FCA emulsion of Tic cells was injected subcutaneously into three mice (200 ml/mice) and the mice were sacrificed after 2 weeks.

Cell fusion and cloning:

Lymphocytes from the spleen of the protocol A mice and lymph nodes from the protocol B mice were mixed and fused with P3U1 mouse myeloma cells using polyethylene glycol. Fused cells were seeded in ten 96-well tissue culture plates, and hybridoma were selected by adding the hybridoma medium (S- Clone cloning medium CM-B containing hypoxanthine, aminopterin and thymidine (HAT) , Sanko Junyaku, Tokyo, Japan) . On the day 7 after plating, the first screening was performed using Tic cell fixed plates. Culture supernatant from each hybridoma was added to Tic cell-fixed screening plates, which had been pretreated with blocking solution containing 0.1% ¾(¾ (Blocker Casein, Pierce-Thermo Fisher Scientific, Rockford, IL) , overnight. The hybridoma culture supernatant was incubated in the cell plates at room temperature for 2 hour. After washing the plates with PBS, 1:2000 diluted HRP-conj ugated anti mouse IgG (Takra-bio, Shiga, Japan) was added in each well and incubated for 1 hour. After final washing, DAB (Metal Enhanced DAB Substrate Kit, Pierce-Thermo Fisher Scientific, Rockford, IL) was added to the plates and colored for 10-15min, followed by observing the stained plates under the light microscope (Olympus IX 7, Olympus, Tokyo, Japan) .

. he human iPS positive antibody producing hybridomas were then subjected to the second cell screening, in which human EC cells (2102Ep) , original human fibroblasts (MRC-5) and MEF cells were used as probes as well as human iPS cells (Tic) . Isotype of mAb was analyzed by using mouse monoclonal antibody isotyping test kit (AbD Serotec (Kidlington, UK) ) .

Immunocytochemistry:

InCell analyzer 2000

Cells seeded in 24 well plates were fixed in 4%

paraformaldehyde (PFA) at room temperature for 15 min, blocked with 3% FBS/PBS for 1 hour, permeabilized with 0.1% Triton

X/FBS · PBS for inernatl antigen, and then incubated with various mAbs (R-10G, TRA-1-60, TRA-1-81, SSEA-4, SSEA-3, SSEA-1, mAb84, Nanog, 0ct4 and anti-PODXL, ) at 4°C overnight. After washing with 0.1% PBS three times each for 5 min, localization of antibodies was visualized by incubation with Alexa Fluor 647- conjugated chicken anti-mouse IgG antibody (Invitrogen-Life technologies, Carlsbad, CA) as the second antibody at room temperature for 1 hour, followed by staining with Hoechst 33342 (1:5000 in PBS, Dojindo Laboratories, Kumamoto, Japan), then cells were analyzed using InCell analyzer 2000 (GE Healthcare, Buckinghamshire, UK) and Developer Toolbox verl.8.

Laser confocul scanning microscope

Tic cells were seeded to Millipore EZ slides (Millipore, Billerica, MA) , which had been coated with gelatin, and plated with MEF (B6) . After a couple of day's culture, cells were fixed in 4% PFA at room temperature for 10 minutes, blocked with 3% FBS/PBS for 1 hour and then incubated with mAb R-10G (as a first primary antibody) at 4°C overnight. After washing with 0.1% PBS three times, cells were incubated with Alexa Fluor 488- conjugated goat anti-mouse IgGl antibody as the secondary antibody in 1% FBS/PBS at room temperature for 30-60 min. For double staining with the second primary antibody (TRA-1-60 and TRA-1-81) , cells were washed and blocked in the same way as described above and incubated with the second primary antibody at 4°C overnight. Then, the cells were incubated with Alexa Fluor 555- conjugated goat anti-mouse IgM antibody as the secondary antibody as described above. After washing with 0.1% FBS/PBS three times, the cells were fixed with 0.1% Triton X- 100/4% PFA at room temperature for 10 min, followed by staining with TO-PR03 (1:500 in PBS, Invitrogen-Life technologies,

Carlsbad, CA) and monitored using confocal laser scanning microscope FV1000 (Olympus, Tokyo, Japan) .

Purification of mAb R-10G from mouse ascites fluid:

R-10G hybridoma cell line was injected intraperitoneally into pristane-treated SCID mice (CB-17/Icr-scid Jcl) . A couple of weeks later, the ascites fluid (2.5 mL) were collected from the mice and subjected to a Protein A-Sepharose column (1 x 6.0 cm) (GE Healthcare, Buckinghamshire, UK) . mAb R-10G bound to the column in 1.5 M Glycine-NaOH buffer, pH 8.9/3M NaCl and eluted with 0.1 M citric acid-phosphate buffer, pH 4.0. The eluate containing mAb R-10G was immediately neutralized to pH 7~8 by adding 3M Tris-HCl buffer, pH 9.0.

Preparation of mAb R-10G affinity column:

R-10G mAb (3mg protein) was coupled to BrCN-activated Sepharose 4B (1.0 ml, GE Healthcare, Buckinghamshire, UK) in 0.1 M NaHC0₃ buffer, pH 8.3/0.5 M NaCl, according to the

manufacturer's instructions.

Isolation of mAb R-10G antigens from human iPS cells:

Human iPS cells lysates were prepared by dissolving the Tic cells (1.5 mg protein/1.2 x 10⁷ cells as determined by Micro BCA protein assay kit (Pierce-Thermo Fisher Scientific, Rockford, IL) ) in the complete RIPA buffer (0.5 ml) under sonication, which consists of RIPA lysis buffer (6 mM Tris-HCl pH 8.0, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 0.004% sodium azide) , protease inhibitor cocktail, PMSF and sodium orthovanadate (Santa Cruz Biotechnology, Santa Cruz, CA) . The lysates were centrifuged to remove insoluble residues and the supernatant was subjected to an R-IOG-Sepharose 4B

Column. After washing the column with, the complete RIPA lysis buffer, the protein bound to the column was eluted with the eluting buffer, consisting of RIPA buffer (1:10 diluted), protease inhibitor cocktail, PMSF and sodium orthovanadate, 0.1 M diethylamine (pH 11.2). The eluates containing R-10G antigens were immediately neutralized by adding 1 M Tris-HCl buffer, pH 6.8.

SDS-PAGE and Western Blotting:

SDS-PAGE and Westen blotting were performed according to the methods of Laemmli (1970) and Towbin et al. (1992),

respectively. Briefly, samples were resolved by electrophoresis on a 4-15% gradient SDS-polyacrylamide gel (Mini-PROTEAN TGX-gel, BioRad Laboratories, Hercules, CA) under non-reducing conditions and followed by either Westen blotting or protein staining. For Western blotting, resolved proteins were transferred onto

Immobilion Transfer membrane (Millipore, Billerica, MA) ,

followed by immunoblot detection with specific Abs. For

visualization, a chemiluminescent substrate kit (Pierce-Thermo Scientific, Rockford, IL) was used with HRP-conjugated rabbit anti-mouse immunoglobulins (DAKO Cytomation, Denmark A/S) , followed by analysis with Luminolmage Analyzer, Las 4000 mini (GE Healthcare, Buckinghamshire, UK) . Protein was stained by Coomassie brilliant Blue G-250, (GelCode Blue, Invitrogen-Life technologies, Carlsbad, CA) .

Identification of the R-10G antigen protein:

Following SDS-PAGE, gels were stained by SYPRO Ruby

Protein Gel Stain ( Invitrogen-Life Technologies, Carlsbad, CA) , and protein bands corresponding to the Western blotting bands were excised from the gel and subjected to in-gel trypsin digestion. The peptides released from the gel were subjected to liquid chromatography (LC) /MS/MS analysis with a linear ion trap mass spectrometer (Finnigan LTQ, Thermo Electron Corp., San Jose, CA) interfaced on-line with a capillary HPLC

(Paradigm, Michrom BioResources , Auburn, CA) . The eluents consisted of H₂0 containing 2% CH3CN and 0.1% formic acid (Pump A) and 90% CH₃CN and 0.1% formic acid (Pump B) , and the.

peptides were eluted with a linear gradient of 5-95% of Pump B. Data-dependent MS/MS acquisitions were performed for the most intense ions as precursors. Proteins were identified by

searching the MS Protein Sequence Database (MSDB) and NCBI database (human) using the Mascot search engine (Matrixscience, London, UK) and TurboSEQUEST search engine (Thermo Electron, Waltham, MA) , respectively.

Digestion of Tic cell lysates and R-10G antigens with

keratanase II:

The reaction mixture consisting of the cell lysates and the R-10G antigens in complete RIPA buffer, aqueous solution of 0.5-10 mU keratanase II {Bacillus sp. Ks 36, Seikagaku

Biobusiness, Tokyo, Japan) and 0.04 M sodium acetate buffer, pH 6.0 in the ratio of 2:1:1 by volume was incubated for 18 hour at 37°C, followed by boiling for 2 min. The digest was subjected to SDS-PAGE and Western blotting.

[RESULTS]

Example 1. Generation of mAb to human iPS cells

In order to raise a panel of mAbs to cell surface markers on human iPS cells, freeze-thawed Tic cells in PBS were mixed with FCA and used to immunize C57BL/6 mice intraperitoneally or subcutaneously. Primary screening of a total of 960

hybridomas using Tic cell fixed plates and MRC-5 cell fixed plates (control) indicated that 29 clones produced mAbs that had reactivity to surface antigens on Tic cells. Secondary screening was performed for these 29 clones to determine the cross reactivity of the mAbs with human EC cells such as

2102Ep and mouse feeders (MEF) . As shown in Table 1, there was essentially no mAb reactivity with the mouse feeders that human iPS were cultured on prior to immunization. In contrast, many of the mAb panel had reactivity with 2102 Ep, an EC cell line. Interestingly, however, mAb Nos. 10, 11 and 17 had no or weak reactivity with 2102Ep, indicating clearly that there are differences in antigen expression between human iPS and human EC cells.

Table 1

The binding of the mAbs to human iPS cells was confirmed by Western blotting, in which Tic cell lysates were resolved by SDS-PAGE and the culture supernatant of hybridomas were tested as primary antibodies. Some of the representative profiles of Western blotting were shown in Fig. 1. The

positions and the intensity of the positive bands were diverse depending upon the respective hybridomas, but their apparent molecular sizes of antigens may be assorted into three groups: one is between 35 kDa to 50kDa (clones #25, #27), second between 75kDa to 100 kDa (#25), and third over 250kDa (#10, #26) . Such mAbs that showed faint or essentially no band on Western blotting despites their strong binding on cell plates assay may react to cell surface components other than proteins (#11, #12, #17) . Among these mAbs, we first focused on clones #10 (designated as mAb R10-G) , which was shown to be IgGi.

Experimental Example 1. Cell binding properties of the mAb R-10G

The reactivity of mAb R-10G on human iPS cells, Tic, was compared with those of conventional human iPS/ES cell marker antibodies, TRA-1-60, TRA-1-81, SSEA-4, SSEA-3, SSEA-1, Nanog and Oct-4 and also with those of mAb84, a mouse monoclonal antibody raised against human ES cell line HES-3 (Choo et al., 2008) and anti-podocalyxin antibody raised against recombinant human podocalyxin . The results are summarized in Fig. 2 and

Table 2.

Table 2

R-10G Tra-1-60 Tra-1-81 SSEA-4 SSEA-3 SSEA-1 mAb84 Nanog Oct4 aPODXL

Tic +++ ++++ ++++ ++++ ++++ + + +++ ++++ ++++

KhES-3 +++ ++++ ++++ ++++ +++ + +++ +++ ++++ ++++

2102Ep + ++++ ++++ +++ +++ + +/- +++ +++ ++++

NCR-G3 + ++++ ++++ ++++ +++ ++ + ++++ ++++ ++++

Most of the Tic cells were reactive to R-10G. Similar or even stronger reactivity was observed for TRA-1-60, TRA-1-81, SSEA-4, Nanog and Oct-4. Most intriguingly, R-10G as well as mAb84 did not show significant binding to human EC cells, 2101Ep, while the other human iPS/ES cell marker antibodies, TRA-1-60, TRA-1-81, SSEA-3, SSEA-4, Nanog and Oct-4 bound to 2101Ep

strongly. In contrast, SSEA-1, which is known to be negative to human iPS cells, did not show any significant binding to Tic (human iPS) , KhES-3 (human ES) and 2102Ep (human EC) cells.

Interestingly, mAb84 bound strongly to human ES cells, KhES-3 cells, but not significantly to Tic cells, suggesting that there are differences in antigen expression between human iPS and human ES cells and the molecular properties of epitopes

recognized by R-IOG are different from those recognized by mAb84, although R-IOG is reactive to Tic cells and KhES-3 cells with similar intensity, 68% and 83%, respectively. These binding properties were confirmed by using different clones of human iPS cells (201B7 and 201B2; data not shown).

The most intriguing observations in the cell staining experiments were shown in Fig. 2. Here, it appears that R-IOG stained almost all Tic cells ubiquitously by green color (A-I) and TRA-1-60 stained also almost all Tic cells ubiquitously by red color (A-II) . However, if these two images were merged into a single color image (A-III) , it became clear that some Tic cells were stained by green color, while other cells were

stained by red color, and there were cells or portions of cells definitely stained by yellow color, being indicative of the co- localization of two epitopes in the close vicinity. These

results demonstrated clearly that human iPS cells prepared and cultured by conventional procedures are in fact very

heterogeneous in their distribution of cell surface components from one cell to another. The same was true between R-IOG and TRA-1-81 (see Fig. 4B) , indicating the heterogeneous but

characteristic distribution of R-IOG and TRA-1-81 epitopes on the surface of human iPS cells.

How and why they are so heterogeneous in this otherwise pure and homogenous population of cells is a vital and critical question with regard to the biology of these pluripotent cells.

Experimental Example 2. Identification of mAb R-IOG antigen molecule as podocalyxin

On the basis of the results of Western-blotting of Tic cell lysates (Fig.l B) , which showed a very broad but a single band reactive to R-IOG, we tried to isolate a presumptive antigen molecule by using an affinity column of R-IOG. Freeze- thawed iPS cells (1.5 mg protein) were solubilized in 0.5 ml of the complete RIPA buffer (containing 0.1% SDS, 0.5% DOC and 1% Nonidet P-40, see MATERIALS AND METHODS) and the lysates were subjected to an mAb R-IOG-Sepharose 4B Column. Proteins bound to the column and eluted with pH 11.2 buffer were analyzed with Western blotting. As shown in Fig. 3A, a single but a broad R-10G positive band were observed at the same position as that with the whole cell lysate (Fig. IB, also Fig. 4A) , suggesting that R-10G antigens present in the cell lysates were recovered almost quantitatively in this isolated fraction. This is confirmed by the presence of no R-10G positive band in the pass through fraction of the

chromatography. Since this isolated antigen fraction still contained several lower molecular size contaminants as shown by SyproRuby staining in Fig. 3A, the isolated antigen

fraction were subjected to SDS-PAGE and the resolved protein bands that correspond to those of the Western blotting bands were excised into three fractions (A,B,C in SyproRuby staining in Fig. 3A) and they were subjected to in-gel trypsin

digestion and the peptides released were analyzed by LC-MS/MS. The results obtained are shown in Fig. 3B. Three fractions of the major Western blotting bands generated several peptide sequences, all of which corresponding to the partial sequences of human podocalyxin.

Podocalyxin is a heavily glycosylated type-1

transmembrane protein belonging to the CD34 family of

sialomucins (see Fig. 3C) . Podocalyxin was originally

described as the major sialoprotein on podocytes of the kidney glomerulus (Kajaschki et al., 1984) but was later found to be expressed on vascular endothelial cells and early

hematopoietic progenitors (Kershaw et al., 1995 & Doyonnas et al., 2005), and is recently implicated as an indicator of tumor aggressiveness in breast, liver and prostate cancers (Casey et al., 2006, Chen et al . , 2004 & Somasiri et al . , 2004). Human podocalyxin gene encodes for a protein of 528 amino acids. However, because of the extensive glycosylation of the extracellular domain, the approximate molecular weight of podocalyxin is 160-165 kDa (Kershaw et al . , 1997). In undifferentiated human ES cells, podocalyxin was identified transcriptionally to be highly expressed (Brandenberger et al . , 2004 & Cai et al. , 2006) .

Experimental Example 3. Identification of mAb R-10G epitopes as keratan sulphate

Because of the unusual broad shape of the R-10G reactive band, the band was presumed to represent glycoproteins. First, we digested the cell lysate and the purified R-10G antigen with PNGase F prior to SDS-PAGE, which release IV-linked

glycans from the core protein. These digestions, however, resulted in essentially no change in their R-10G binding

activity on Western blotting and also the migrating positions of the main protein bands, indicating that N-linked glycans are not the major epitopes on the antigen (data not shown) .

Then we tested the cross-reactivity between R-10G and TRA-1-60 or TRA-1-81, since they have clearly distinct but similar expression profiles in the pluripotent cells (human iPS and ES cells with the former and human iPS, ES and EC cells with the latter) and they seem to share common protein backbone i.e., podocalyxin (Choo et al . , 2008 & Schopperle et al, 2007).

As shown in Fig. 4A, upon Western blotting, Tic cell lysates reacted with TRA-1-60 or TRA-1-81 and showed a broad but a single band at a high molecular region (>250kDa) ,

although the positions of the respective bands were slightly different from each other and apparent molecular sizes of the respective antigens for R-10G, TRA-1-60 and TRA-1-81 decreased in this order. Then we examined the reactivity of the isolated R-10G antigen to TRA-1-60 and TRA-1-81 with the results shown in Fig. 4A. Interestingly, TRA-1-60 and TRA-1-81 reacted with the isolated R-10G antigen and showed a single band at their respective positions expected from those with the Tic cell lysates, although the intensity of the bands relative to the respective bands with the Tic cell lysates were different from each other and the band for TRA-1-81 was stronger than that for TRA-1-60.

These results suggest the possibility that these three mAbs share the same family of carbohydrate molecule, keratan sulfate, as the epitope. This was tested by digestion with keratanase II, which degrade keratan sulfate specifically. As shown in Fig. 4B, upon digestion of the isolated R-10G antigen with an increasing amount of keratanase II, the R-10G epitope was very sensitive to the enzyme and disappeared rapidly by the digestion with a small amounts of the enzyme, whereas TRA- 1-81 epitope was relatively resistant to the digestion and degraded only by the digestion with a large amount of the enzyme. These results indicated that the epitope of mAb R-10G consists of keratan sulfate, which is structurally similar to those of TRA-1-60 and TRA-1-81, but the detailed structures of the keratan sulfate in the epitope structure are clearly

distinct from each other for the respective mAbs. The

relationship of the mAb members recognizing keratan sulfae on human pluripotent cells is illustrated schematically in Fig. 5.

Experimental Example 4. Identification of R-10G as a keratan- sulfate recognizing antibody

Binding specificity of R-10G was further investigated using various glycosaminoglycan specimens in an ELISA system with the results shown in Fig. 6. Keratan sulfate isolated from bovine cornea reacted strongly with R-10G, while other glycosaminoglycans, hyaluronic acid from pig skin, chondroitin, chondroitin sulfate from whale cartilage, chondroitin sulfate from the spinal column of acipenser medirostris, chondroitin sulfate B from pig skin, chondroitin sulfate C from shark cartilage, chondroitin sulfate D from shark cartilage,

chondroitin sulfate E from squid cartilage and heparan sulfate from bovine kidney, did not show any binding activity to R-10G. These results demonstrate very clearly that binding

specificity of R-10G is very strict and this antibody can effectively and precisely discriminate between

glycosaminoglycans having extraordinary heterogeneous

sequences with strong negative charges.

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While the present invention has been described with

emphasis on preferred embodiments, it is obvious to those

skilled in the art that the preferred embodiments can be

modified. The present invention intends that the present

invention can be embodied by methods other than those described in detail in the present specification. Accordingly, the present invention encompasses all modifications encompassed in the gist and scope of the appended "CLAIMS."

The contents disclosed in any publication cited herein, including patents and patent applications, are hereby

incorporated in their entireties by reference, to the extent that they have been disclosed herein.

This application is based on US provisional patent application No. 61/478,704, the contents of which are

incorporated by reference in full herein.

Claims

1. A monoclonal antibody capable of recognizing iPS and ES cells, wherein said antibody recognizes a keratan sulfate on podocalyxin present on the surface of iPS and ES cells as an epitope, and wherein said antibody does not recognize EC. cells.

2. The monoclonal antibody according to clam 1, wherein the iPS and ES cells are derived from human.

3. The monoclonal antibody according to claim 1 or 2, wherein said antibody is not cytotoxic to its target cells.

4. The monoclonal antibody according to any one of claims 1 to

3. which is a monoclonal antibody produced by hybridoma R-10G (FERM BP-11301) or a monoclonal antibody recognizing the same region as that recognized by the antibody produced by R-10G as an epitope.

5. A method of detecting an iPS or ES cell, comprising bringing a cell sample into contact with the monoclonal antibody

according to any one of claims 1 to 4, and detecting a cell bound with the antibody in the sample.

6. A method of making iPS or ES cell population homogeneous, comprising sorting iPS or ES cells recognized by the monoclonal antibody according to any one of claims 1 to 4, and excluding iPS or ES cells recognized by an antibody selected from the group consisting of SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, GCTM2 and GCTM343 from the sorted cells.