JP2007129991A - Method for identifying, separating and/or recovering mammalian a-type (undifferentiated type) spermatogonium - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for identifying, separating and recovering an A type (undifferentiated type) spermatogonium in an exceedingly high fixation degree in a testis of adult mammal. <P>SOLUTION: The method for identifying an A type (undifferentiated type) spermatogonium comprises using one or more kinds of antibodies having specific affinity for a marker protein selected from the group consisting of c-Kit, E-cadherin, Ep-CAM, Notch-3, VLA-6 and Ret. Preferably the antibody contains at least one or both of anti-c-Kit antibody and anti-E-cadherin antibody. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for identifying, separating and / or recovering mammalian type A (undifferentiated) spermatogonia cells, and uses of the cells.

  In mammals, old cells are always replaced with new cells except for some tissues such as nerve tissue. In addition, when a cell or organ is damaged due to injury or disease, rapid repair is performed. In such a case, stem cells provided in the tissue play a major role. Therefore, stem cells are attracting attention as a central material for regenerative medicine. Stem cells remain in the tissue in a state of being differentiated to a certain stage, and have both the property of reproducing themselves and the property of differentiating into functional cells. Stem cells are known to have large plasticity that differentiates into many types of cells and those that differentiate into cells of specific tissues that have already been determined, such as skin, blood cells, and muscles. Therefore, stem cells have the above-mentioned common properties and different characteristics depending on tissues. Here, A type (undifferentiated) spermatogonia cells including male germ cells and sperm stem cells are particularly treated. Since spermatozoa are always produced in large quantities, the original spermatogonial stem cells and the type A (undifferentiated) spermatogonia that contain them are important, and their characterization is essential for elucidating the cause of infertility and treating them. It is. It is known that spermatogonia cells undergo a plurality of differentiation stages (A type → intermediate type → B type, etc.). And it is thought that the most original spermatogonial stem cell is contained in the A type (undifferentiated) spermatogonia. But most of the details are still unclear. In order to analyze and utilize the properties of these cells, it is first necessary to accurately identify and sort A type (undifferentiated) spermatogonia.

  In mammals, type A (undifferentiated) spermatogonia cells are homogenous in the nucleus among the spermatogonia existing on the innermost outer periphery of the seminiferous tubule, do not contain heterochromatin, and form clusters. It is known and its property analysis has been partially reported (Non-Patent Documents 1 and 2). However, there are no successful examples of accurate identification and sorting. In addition, no examples of in vitro culture / differentiation induction have been reported.

The process of differentiation and maturation of sperm stem cells contained in spermatogonia is a complicated process involving several stages, and among them, the identification of the original type A (undifferentiated) spermatogonia has not yet been established. For example, there is a report that Ep-CAM ⁺ is expressed in spermatogonia cells (Non-patent Document 3), whereas c-Kit antigen, which is a marker for stem cells and young cells, is type A (undifferentiated). There is also a report that it is not expressed in (type) spermatogonia (Non-patent Document 4). We also reported that Notch3 is activated in spermatogonia (Non-patent Document 5). On the other hand, the expression of E-cadherin antigen was confirmed histochemically in the seminiferous tubule of the 8th day mouse (Non-patent Document 6), but the subsequent expression has not been confirmed histochemically. Even analysis by RT-PCR is undetectable and negative after 21 days of age (Non-patent Document 7). In addition, Ret positive cells, which are markers for stem cells, may be included in type A (undifferentiated) spermatogonia (Non-patent Document 8).

Oakberg, EF., Anat. Rec. (1971), 169, 515-532. de Rooji, DG., Russell, LD., J. Androl. (2000) 21, 776-798. Anderson, R., 2 others, J. Reprod. Fertil. (1999) 116, 379-384. Yoshinaga, K., 6 others, Development (1991), 113, 689-699. Mori, S. 3 others, Devlop. Growth & Differ. (2003), 45, 7-13. Wu, J., 2 others, Biol. Reprod. (1993), 49, 1353-1361. Munro, S., & Blashuk, OW. Biol. Reprod. (1993), 55, 822-827. Meng, X., 13 others, Science (2000), 287,1489-1493.

  As described above, none of the reported prior arts can still sufficiently identify and isolate type A (undifferentiated) spermatogonia. In addition, the marker proteins of stem cells of other tissues are different depending on each tissue, and they have not been sufficiently clarified yet. Therefore, discovery of a marker protein that can efficiently isolate stem cells, particularly type A spermatogonia cells, is desired.

  Under such circumstances, the present invention provides a method for solving conventional technical problems, identifying A-type (undifferentiated) spermatogonia cells in mammalian testis, and efficiently separating and recovering them. With the goal.

The cells of E-cadherin ⁺ and c-Kit ⁻ on the seminiferous tubule membrane include type A (undifferentiated) spermatogonia , and the inventors have made extensive and careful studies for the above purpose. Cells on the adult seminiferous tubule basement membrane are mostly VLA-6 ⁺ , Notch3 ⁺ , and Ep-CAM ⁺ , of which E-cadherin ⁺ (and c-Kit ⁻ ) has been missed so far. It was found that a small amount of cells were contained, and more than 80% of them formed a single or a cluster of 2, 4, 8, 16 cells. This cluster fraction was highly likely to be type A (undifferentiated) spermatogonia, and it was further confirmed that Ret positive cells were contained in this cluster fraction. Based on these results, the possibility of identification and separation of type A (undifferentiated) spermatogonia is dramatically improved by combining one or more of the above-mentioned markers including E-cadherin from cells on the seminiferous tubule basement membrane. The present inventors have found that it can be improved and have completed the present invention.

  That is, the present invention is a method for identifying, separating and / or recovering type A (undifferentiated) spermatogonia in mammalian testis tissue, which comprises c-Kit, E-cadherin, Ep-CAM, One or more antibodies having specific affinity for a marker protein selected from the group consisting of Notch-3, VLA-6 and Ret are used. In a preferred embodiment, the antibody includes at least one or both of an anti-c-Kit antibody and an anti-E-cadherin antibody.

  In a different aspect, the present invention provides a type A (undifferentiated) spermatogonia isolated and / or recovered from testis tissue by the above method and a pharmaceutical composition for diagnosis and / or treatment of infertility comprising the same. It is characterized by that.

  According to the present invention, type A (undifferentiated) spermatogonia in the testis of mammals can be identified, separated and recovered more efficiently than in the conventional technique. From the above, the present invention is expected to contribute greatly in the field of elucidation of the cause of infertility and its treatment.

  In the present specification, “mammal” includes bovine, horse, dog, guinea pig, mouse, rat and the like in addition to primates including human.

  The c-Kit protein is a receptor for stem cell factor, has tyrosine kinase activity, and is also a proto-oncogene product. Ret protein is a receptor for GDNF (glial cell line-derived neurotrophic factor) and has a cadherin motif and is present in the cell membrane. E-cadherin is one of the members of the large cadherin family. It is a calcium-dependent adhesion molecule that plays the most important role in cell-cell adhesion. Ep-CAM is one of allophilic adhesion proteins and is known to play an important role in the germline developmental stage. Notch-3 is one of four Notch families in mammals. It is a cell membrane-bound receptor molecule that transmits signals such as Delta and Jagged, which are also membrane-bound ligands, to the nucleus, and is involved in stem cell maintenance and differentiation. VLA-6 is a dimer composed of α6-integrin and β1-integrin and is present in the cell membrane, and transmits a signal from the extracellular matrix into the cell as a specific receptor for laminin. In the present specification, the “marker protein” is a protein expressed on the surface of such a cell, and shows a characteristic expression pattern in testis tissue. “Having specific affinity” for these marker proteins refers to the ability to specifically recognize and bind to these proteins through an antigen-antibody reaction. As the “antibody”, any of a polyclonal antibody, a monoclonal antibody, or a functional fragment thereof may be used. These antibodies or fragments thereof may be labeled with a fluorescent substance, an enzyme, a radioisotope, or the like. Furthermore, these can be used commercially, and can be obtained from, for example, Chemicon International, UK-Serotec, Santa Cruz Biotech, Developmental Studies Hybridoma Bank (University Iowa), R & Dsystems, and Takara Bio Inc. Is possible.

The method for identifying, separating and / or recovering type A (undifferentiated) spermatogonia of the present invention is at least one, preferably 2 of the respective antibodies having specific affinity for the six marker proteins. It is carried out by examining the expression of the marker protein in combination of two or more. Among the expression modes of such marker proteins, c-Kit non-expression (c-Kit ⁻ ), E-cadherin expression (E-cadherin ⁺ ), Ep-CAM expression (Ep-CAM ⁺ ), Notch-3 expression ( Notch-3 ⁺ ), VLA-6 expression (VLA-6 ⁺ ) and Ret expression (Ret ⁺ ) cells satisfying at least one, preferably two or more, are A-type (undifferentiated) spermatogonia. Can be identified, separated and / or recovered.

  The method for separating / collecting cells showing the expression mode of each marker protein using an antibody having specific affinity for the marker protein is a method carried out in the art and a combination thereof. Used. For example, a method using an antibody-binding bead described in Examples described later, a panning method, or a cell sorter can be used for scaling up. For example, the target cells are automatically separated and collected by labeling the antibody with an appropriate dye or fluorescent substance and using a fluorescence activated cell sorter (FACS). A fluorescently labeled secondary antibody may be used for labeling the antibody.

  In the present invention, what kind of factors and environment are proliferating of spermatogonia cells through analysis of proliferation and maturation process of the A type (undifferentiated) spermatogonia cells identified and separated by such a method in vivo and in vivo. It is possible to analyze whether it is necessary for differentiation. In addition, according to the present invention, type A (undifferentiated) spermatogonia can be obtained selectively in a viable state with high efficiency. When treatment is required, a method for collecting A type (undifferentiated) spermatogonia for a period of time and storing it outside the body can be developed and used clinically. That is, A-type (undifferentiated) spermatogonia cells separated and recovered by the method of the present invention, or cells induced to differentiate therefrom, can be used for diagnosis and treatment of infertility. These cells are suspended in an aqueous medium such as a medium or in the form of an injectable preparation mixed in a pharmaceutically acceptable carrier or diluent such as sterile water or buffered saline. It is possible. In addition to the above carriers, additives such as substantially non-toxic excipients, auxiliaries, and stabilizers can be blended. The pharmaceutical composition containing the above components can be sterilized by a method such as filtration, if necessary, and filled in a sterile ampoule container. It may also be a dosage form suitable for other uses. The dosage of the pharmaceutical composition of the present invention should be appropriately increased or decreased depending on conditions such as the patient's age, weight, symptoms, and administration route. It is possible to set appropriately.

Furthermore, the present invention provides a method for screening a substance that induces mammalian differentiation using type A (undifferentiated) spermatogonia isolated and recovered by the method of the present invention. The method includes at least the following steps.
(A) a step of bringing a type A (undifferentiated) spermatogonia cell into contact with a candidate substance;
(B) one or more marker proteins selected from the group consisting of c-Kit, E-cadherin, Ep-CAM, Notch-3, VLA-6 and Ret for the cells contacted with the candidate substance Measuring expression.
The “contacting” is not particularly limited as long as it is a condition that allows the A-type (undifferentiated) spermatogonia cells to interact with the candidate substance. For example, the candidate compound is added to the cell culture medium. And cultured in a CO ₂ incubator at 33 to 37 ° C. for several minutes to several tens of days. Whether or not type A (undifferentiated) spermatogonia have differentiated can be confirmed by measuring the expression of the marker protein. Alternatively, other appropriate marker proteins may be used. Candidate substances used in the screening method of the present invention are not particularly limited. For example, various known compounds (including peptides) registered in a chemical file, combinatorial chemistry techniques, or ordinary synthesis techniques are used. The obtained compound group or a random peptide group created by applying the phage display method can be used. Further, microorganism culture supernatants, natural substances derived from plants or marine organisms, or animal tissue extracts can also be used as screening candidate substances. Furthermore, a compound obtained by chemically or biologically modifying a substance obtained by the screening method of the present invention can also be used.

  Next, specific and detailed examples will be given to describe the present invention in detail, but the present invention is not limited only to the following examples.

VLA-6 ⁺ and E-cadherin ⁺ and c-Kit ⁻ type A (undifferentiated) spermatogonia cells of the 8-week-old ICR mouse are closely attached to the inner periphery of the seminiferous tubule, and are in close contact with the basement membrane. It was discovered that there are a small number of cells exhibiting the properties of ⁺ , c-Kit ⁻ and VLA-6 ⁺ (FIGS. 1, 2, and 3). A positive reaction was shown for anti-α6-integrin antibody and anti-β1-integrin antibody (FIG. 3a, b), but negative for term c-Kit antibody (FIG. 3c). It was strongly suggested that this cell does not contain heterochromatin in the nucleus (Fig. 2), and the morphological characteristics of the cell may have been described as a type A (undifferentiated) spermatogonia. .

  The immunohistochemical method used at this time is a normal method. Specifically, samples were anesthetized with diethyl ether, fixed by perfusion with 10% neutral buffered formalin solution containing 10% methanol, immersed in 10-18% sucrose solution, embedded in liquid nitrogen, and sliced with a cryostat. Was used.

Analysis in the maturation process As a result of observing E-cadherin ⁺ cells over time following the maturation stage of mice, it was consistent with the expected kinetics of type A (undifferentiated) spermatogonia cells. That is, all of the stage of gonocyte located in the middle of the seminiferous tubule immediately after birth (1 day old), 4 days old that started moving around, and 7 days old that adhered to the basement membrane of the seminiferous tubule showed E-cadherin positivity. (FIG. 4-a, b, c). At 2 weeks of age when meiosis begins, some cells on the basement membrane become negative for E-cadherin, and positive cells gradually decrease at 4 and 8 weeks of age. Only one positive cell was detected (FIGS. 4-d, e, f, g). During this period, all spermatogonia cell populations located on the basement membrane were Ep-CAM positive, and the proportion of E-cadherin ⁺ and Ret ⁺ cells decreased.

Analysis using mutants
Steel factor (stem cell factor) receptor is a c-Kit mutants, W / ^{W v} and mutant loci steel factor, undifferentiated by dysfunction of c-Kit in the Sl / Sl ^d mice spermatogonia It is known that the maturation process stops at the cellular stage. In both of these strains of 8-week-old (adult) mice, germ cells in all seminiferous tubules were positive for E-cadherin (FIGS. 5-a and b).

Isolation of E-cadherin-positive cells and transplantation E-cadherin-positive cells in the seminiferous tubules of 8 week-old (adult) mice (which are consistent with type A (undifferentiated) spermatogonia as described in Example 5 below) )), E-cadherin positive cells were isolated and confirmed to contain spermatogonial stem cells. Although c-Kit mutant mouse (W / W ^v ) that can normally synthesize steel factor as a host but is defective in its receptor, c-Kit normal mouse expressing GFP as E-cadherin positive cells Was prepared using anti-E-cadherin antibody-coupled magnetic beads. W / W ^v mice were transplanted at a rate of about 1-4 × 10 ⁴ cells / testis. The number of GFP positive colonies in the testis was counted 8 weeks after transplantation. As a control, the same number of E-cadherin negative cells were transplanted into the opposite testis. As a result, colonies were observed in all testes transplanted with GFP positive type A (undifferentiated) spermatogonia cells, and were not detected at all in the control transplanted testis (FIGS. 6-a and b and Table 1). The colony formation rate obtained here is the highest reported so far (see Shinohara, T. et al., Proc. Natl. Acad. Sci. USA, (1999), 96, 5504-5509). It was more than 10 times.

E-cadherin-positive cells form clusters on the seminiferous tubule basement membrane An immunohistochemical experiment was conducted by the seminiferous whole-mount method for the purpose of examining the presence of E-cadherin-positive cells in 8-week-old (adult) mouse seminiferous tubules . To clarify the relationship with other surface antigens, double staining with anti-c-Kit antibody was performed. It was shown that the E-cadherin positive cells were c-Kit negative and connected to adjacent positive cells in a cluster via cell protrusions (FIG. 7). Further, when double staining with an anti-Ep-CAM antibody was performed, E-cadherin positive cells were Ep-CAM positive, and most of the E-cadherin positive cells were single or 2, 4, 8, 16 cells. A cluster was formed (FIG. 8). In addition, when double staining with an anti-c-Ret antibody was performed, about 20% of the E-cadherin positive cells were c-Ret positive. This existence state suggests that E-cadherin positive cells of adult seminiferous tubules are almost identical to type A (undifferentiated) spermatogonia cells, and further include Ret positive spermatogonia stem cells. .

  As described above, among the Ep-CAM positive cells clarified in the present invention, E-cadherin positive (and c-Kit negative) cells almost coincide with A type (undifferentiated) spermatogonia. It is extremely useful for the identification, separation and recovery of cells, and will greatly contribute to future research on type A (undifferentiated) spermatogonia and other stem cells.

Staining of testis of 8-week-old ICR mice prepared in Example 1 with anti-E-cadherin antibody. E-cadherin ⁺ cells (arrows) are in close contact with only the basement membrane inside the seminiferous tubule and are slightly present. The white line is 10 μm. Staining of testis of 8-week-old ICR mice prepared in Example 1 with anti-E-cadherin antibody (a) and DAPI (b) and double staining of both (c). The nuclei of E-cadherin positive cells (center of b) are oval and free of heterochromatin. Arrows indicate E-cadherin negative cells, and arrowheads indicate spermatocytes. The white line is 10 μm. With the anti-E-cadherin antibody of the seminiferous tubule of the 8-week-old ICR mouse prepared in Example 1 and the VLA-6 subunit, α6-integrin (a), β1-integrin (b) or c-Kit (c) antibody Double staining. The left figure shows E-cadherin (green), the middle figure shows each antibody, and the right figure shows double-stained images. The white line is 10 μm. Staining of testis during development of normal mice prepared in Example 2 with anti-E-cadherin antibody. 1 day old (a), 4 days old (b), 7 days old (c), 2 weeks old (d), 4 weeks old (e), 8 weeks old (f), 480 days old (g). The white line is 10 μm. Staining with testicular anti E-cadherin antibody of the mutant mice produced in Example ^{3, Sl / Sl d (a} ) and ^{W /} W v (b). The white line is 10 μm. The transplantation experiment of the E-cadherin positive cell in the seminiferous tub conducted in Example 4. E-cadherin positive cells (a) or negative cells (b) isolated and collected from the testis of GFP mice were transplanted into the testis of 8-week-old W / W ^v mice, and the testis after 8 weeks was photographed under a fluorescence actual microscope. did. Many GFP positive colonies were observed in the testis. Colonies were not formed by transplantation of E-cadherin negative cells. The white line is 10 μm. Whole-mount staining for E-cadherin and c-Kit in 8-week-old ICR mouse seminiferous tubules shown in Example 5. E-cadherin (a, green), c-Kit (b, red), double staining (c), E-cadherin positive cells form 1, 2 and 8 clusters and are c-Kit negative. The white line is 10 μm. Whole mount staining for E-cadherin and Ep-CAM in 8-week-old ICR mouse seminiferous tubules shown in Example 5. E-cadherin (a, green), Ep-CAM (b, red), double staining (c), E-cadherin positive cells form a cluster and are all Ep-CAM positive. The white line is 10 μm.

Claims (8)

  Mammals using one or more antibodies having specific affinity for a marker protein selected from the group consisting of c-Kit, E-cadherin, Ep-CAM, Notch-3, VLA-6 and Ret To identify type A (undifferentiated) spermatogonia in testicular tissue.   The method according to claim 1, wherein the antibody comprises at least one or both of an anti-c-Kit antibody and an anti-E-cadherin antibody.   Mammals using one or more antibodies having specific affinity for a marker protein selected from the group consisting of c-Kit, E-cadherin, Ep-CAM, Notch-3, VLA-6 and Ret Of isolating and / or recovering type A (undifferentiated) spermatogonia in testicular tissue.   4. The method of claim 3, wherein the mammal is a human.   A type (undifferentiated) spermatogonia cells isolated and / or recovered from testis tissue by the method according to claim 3 or 4. The cell marker protein exhibits at least one expression pattern selected from the group consisting of c-Kit ⁻ , E-cadherin ⁺ , Ep-CAM ⁺ , Notch-3 ⁺ , VLA-6 ⁺ and Ret ^+. A type (undifferentiated) spermatogonia described in 1.   A pharmaceutical composition for diagnosis and / or treatment of infertility, comprising the type A (undifferentiated) spermatogonia cell according to claim 5 or 6, or a cell differentiated from the cell. A type A (undifferentiated) spermatogonia cell and a candidate substance are contacted, and the cell is selected from the group consisting of c-Kit, E-cadherin, Ep-CAM, Notch-3, VLA-6 and Ret A method for screening a substance that induces differentiation of a mammalian spermatogonia cell, comprising measuring the expression of one or more marker proteins.