JP2006115767A - Method for growing spermatogonial stem cells in the absence of feeder cells - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for proliferating spermatogonial stem cells in which the influence of uncertain factors is eliminated. The present invention relates to spermatogonial cells, comprising culturing spermatogonial stem cells in a feeder-free or serum-free condition. A method for proliferating stem cells is provided. By using the proliferation method of the present invention, it is possible to efficiently proliferate spermatogonial stem cells even in the absence of feeder cells and serum, and to eliminate the influence of undetermined factors. It is possible to proliferate spermatogonial stem cells in a more stable and simple manner while maintaining the ability as spermatogonial stem cells. Moreover, in the clinical application of spermatogonial stem cells, the risk of infectious diseases derived from different animals can be avoided. Therefore, the propagation method of the present invention includes the production of genetically modified animals (gene-transfected animals, gene-deficient animals, etc.), the development of treatment methods and treatment agents for male infertility, research and drugs for gene therapy at the germ cell level. It is useful for the development of
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Description

  The present invention relates to a method of proliferating spermatogonial stem cells, comprising culturing spermatogonial stem cells in a feeder-free or serum-free condition, spermatogonial stem cells proliferated in vitro by the method, spermatozoa derived from the spermatogonial stem cells, The present invention relates to a method for producing embryos, non-human progeny, an agent for proliferating spermatogonial stem cells by culturing them under feeder-free conditions, and the like.

  Spermatogonial stem cells are a unique population with the ability to self-renew. Unlike other spermatogonia that are destined to differentiate, spermatogonial stem cells can continually self-renew and produce differentiated progenitor cells (de Rooij DG, Russell LD. All you wanted to know about spermatogonia but were afraid to ask.J Androl 2000; 21: 776-798, Meistrich ML, van Beek MEAB.Spermatogonial stem cells.In: Desjardins, C, Ewing, LL (eds.), Cell and Molecular Biology of the Testis, New York: Oxford University Press; 1993: 266-295). Spermatogonial stem cells interact closely with Sertoli cells and are thought to be distributed non-randomly in tissues (Chiarini-Garcia H, Raymer AM, Russell LD. Non-random distribution of spermatogonia in rats: evidence of niches in the seminiferous tubules. Reproduction 2003; 126: 669-680). In general, stem cells are a specialized microenvironment that provides factors that control the growth or differentiation of a stem cell population, namely Schofield R. The relationship between the spleen colony-forming cell and the heamopoietic stem cells. Blood Cells 1978 ; 4: 7-25). This niche was originally hypothetical, but recent research at Drosophila has elucidated the molecular mechanism of stem cell-Niche interaction in the testis (Kiger AA, White-Cooper H, Fuller MT. Somatic support Nature 2000; 407: 750-754, Tran J, Brenner TJ, DiNardo S. Somatic control over the germline stem cell lineage during Drosophila spermatogenesis.Nature 2000; 407: 754-757 cells restrict germline stem cell self-renewal and promote differentiation. ). For example, stem cells adhere to Nische via E-cadherin (Yamashita YM., Leanne Jones D., Fuller MT. Orientation of asymmetric stem cell division by the APC tumor suppressor and centrosome. Science 2003; 301: 1547-1550 ), And the undifferentiated state of stem cells is maintained by Jak-Stat signaling (Kiger AA, Jones DL, Schulz C, Rogers MB, Fuller MT. Stem cell self-renewal specified by JAK-STAT activation in response to a support cell cue. Science 2001; 294: 2542-2545, Tulina N, Matunis E. Control of stem cell self-renewal in Drosophila spermatogenesis by JAK-STAT signaling. Science 2001; 294: 2546-2549). Similar studies in other self-renewing tissues revealed a close interaction between stem cells and stromal cells that make up the niche (Spradling A, Drummond-Barbosa D, Kai T. Stem cells find their niche. Nature 2001; 414: 98-104). Although these studies have established an important role for the microenvironment in stem cell biology, most of the factors are involved in spermatogonial stem cell self-renewal in mammals and how it affects stem cell behavior unknown.

  A valuable approach to study this problem would be to reproduce the self-renewal division of stem cells in vitro. This will reveal the minimal external requirements of the self-renewal process and provide important information about stem cell-Niche interactions in vivo. In recent years, the present inventors have described a method for culturing mouse spermatogonial stem cells (Non-patent Document 1, PCT / JP2004 / 004612). In this method, spermatogonial stem cells from the neonatal testis were able to proliferate over 5 months in the presence of glial cell-derived neurotrophic factor (GDNF), leukemia inhibitory factor (LIF), and the like. After transplantation into the infertile recipient testis, the cultured cells retained stem cell activity, caused spermatogenesis, and produced offspring. A system for embryonic stem cells (ES cells) and embryonic germ cells (EG cells) (Evans MJ, Kaufmann MH. Establishment in culture of pluripotential cells from mouse embryos. Nature 1981; 292: 154-156, Martin GR. Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells.Proc Natl Acad Sci USA 1981; 78: 7634-7638, Matsui Y, Zsebo K, Hogan BLM.Derivation of pluripotential embryonic stem cells from murine primordial germ Cell 1992; 70: 841-847, Resnick JL, Bixler LS, Cheng L, Donovan PJ. Long-term proliferation of mouse primordial germ cells in culture. Nature 1992; 359: 550-551) The method is a third method for growing germline cells. Based on these results, the inventors named this cell a germline stem cell (GS cell) to distinguish it from the other two cell types. This GS cell culture system supports the self-renewal of spermatogonial stem cells and opens up a promising route to analyze stem cell-Niche interactions in vitro.

  Here, in order to further promote the proliferation of spermatogonial stem cells in the culture system, it was preferable to culture the spermatogonial stem cells in the presence of serum and feeder cells. However, the use of serum and feeder cells in culture is limited by our knowledge of the factors that control spermatogonial stem cells. Serum is complex and contains undefined substances, which sometimes affect cell differentiation (Barnes D, Sato G. Serum-free culture: a unifying approach. Cell; 1980: 22: 649-655). For example, neural stem cells can differentiate into progenitor cells when cultured in a culture medium containing serum (Reynolds BA, Weiss S. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 1992; 255: 1707-1710). In addition to providing physical support for stem cell attachment, feeder cells also affect stem cells by producing a variety of undetermined factors through interaction with stem cells. Thus, the presence of serum or feeder cells complicates the culture conditions and makes it uncontrollable.

As a method for growing mammalian stem cells without using feeder cells, Patent Document 1 discloses a method for growing primate-derived primordial stem cells in a culture that does not contain feeder cells. The method includes culturing primate-derived primordial stem cells on the extracellular matrix of fibroblasts using a fibroblast conditioned medium or the like, but is not disclosed for spermatogonial stem cells.
In Non-Patent Document 2, freshly prepared spermatogonial stem cells adhere preferentially to laminin, and the stem cell concentration is concentrated about 3 to 8 times after selection on a laminin-coated plate, Although β ₁ -and α ₆ -integrins have been disclosed to be surface markers for mouse spermatogonial stem cells, there is no mention of proliferation of spermatogonial stem cells.
WO99 / 20741 pamphlet Kanatsu-Shinohara, M. et al., Biology of Reproduction, (USA), Vol. 69, No. 2, p. 612-616, 2003 Shinohara, T et al., Proceedings of the National Academy of Science of the United States of America (Proc Natl Acad Sci USA), (USA), Vol. 96, p. 5504-5509, 1999

  In view of the above circumstances, an object of the present invention is to establish an efficient and stable culture of spermatogonial stem cells in serum-free or feeder-free cells.

  As a result of earnest research to achieve the above-mentioned purpose, spermatogonial stem cells such as GS cells cannot efficiently proliferate in the absence of both serum and feeder cells. In the absence, it has been found that it can continue to grow efficiently and stably, and the present invention has been completed.

That is, the present invention relates to the following.
(1) A method for proliferating spermatogonial stem cells, comprising culturing spermatogonial stem cells in the absence of feeder cells in a state of being adhered to an insoluble carrier.
(2) The method according to (1) above, wherein the spermatogonial stem cell adheres to an insoluble carrier via an extracellular matrix.
(3) The method according to (2) above, wherein the extracellular matrix is laminin.
(4) The method according to (1) above, wherein the spermatogonial stem cells are cultured in a medium containing a GDNF receptor agonist compound.
(5) The method according to (4) above, wherein the medium further contains LIF.
(6) The method according to (4) above, wherein the medium further contains at least one of EGF and bFGF.
(7) The method according to (1) above, wherein the spermatogonial stem cells are cultured in a medium containing serum.
(8) The method according to (1) above, wherein the spermatogonial stem cells are cultured in a medium not containing a conditioned medium for feeder cells.
(9) The method according to (1) above, wherein the proliferated spermatogonial stem cells have an embryonic phenotype.
(10) The method according to (9) above, wherein the embryonic phenotype is positive expression of SSEA-1.
(11) A method for expanding spermatogonial stem cells, comprising culturing spermatogonial stem cells in a serum-free medium.
(12) The method according to (11) above, wherein the medium contains a GDNF receptor agonist compound.
(13) The method according to (12) above, wherein the medium further contains LIF.
(14) The method according to (12) above, wherein the medium further contains at least one of EGF and bFGF.
(15) The method according to (11) above, wherein the medium contains B27.
(16) The method according to (11) above, comprising culturing spermatogonial stem cells in the presence of feeder cells.
(17) The spermatogonial stem cell to be cultured is in a differentiation stage capable of growing in a medium containing substantially only a GDNF receptor agonist compound as a spermatogonial stem cell growth cofactor, (1) or (11 ) The method described.
(18) The method according to (17) above, wherein the spermatogonial stem cells to be cultured are in a differentiation stage in contact with the basement membrane of the seminiferous tubule.
(19) A spermatogonial stem cell to be cultured is a spermatogonial stem cell in a differentiation stage requiring a GDNF receptor agonist compound and LIF as a growth factor for in vitro growth in a medium containing the GDNF receptor agonist compound and LIF. The method according to (17) above, wherein the method is obtained by culturing in the above.
(20) The method according to (17) above, wherein the spermatogonial stem cells are cultured in a medium containing a GDNF receptor agonist compound.
(21) The method according to (20) above, wherein the medium does not contain LIF.
(22) The growth method according to (20) above, wherein the medium contains substantially only a GDNF receptor agonist compound as a growth cofactor for spermatogonial stem cells.
(23) A spermatogonial stem cell grown in vitro using the method according to (1) or (11) above.
(24) A fertility treatment agent comprising the spermatogonial stem cell according to (23).
(25) A method for producing a non-human animal that forms sperm derived from transplanted spermatogonial stem cells, comprising the following steps:
a) proliferating spermatogonial stem cells by culturing spermatogonial stem cells in the absence of feeder cells in a state of being attached to an insoluble carrier;
b) A step of transplanting the spermatogonial stem cells grown in a) into the seminiferous tubule of an infertile non-human animal to obtain a non-human animal that has caused spermatogenesis derived from the spermatogonial stem cell.
(26) A method for producing sperm, including the following steps:
a) proliferating spermatogonial stem cells by culturing spermatogonial stem cells in the absence of feeder cells in a state of being attached to an insoluble carrier;
b) transplanting the spermatogonial stem cells grown in a) into the seminiferous tubule of an infertile non-human animal to obtain a non-human animal that has caused spermatogenesis derived from the spermatogonial stem cell;
c) A step of obtaining sperm from the non-human animal.
(27) A method for producing an embryo derived from spermatogonial stem cells, comprising the following steps:
a) proliferating spermatogonial stem cells by culturing spermatogonial stem cells in the absence of feeder cells in a state of being attached to an insoluble carrier;
b) transplanting the spermatogonial stem cells grown in a) into the seminiferous tubule of an infertile non-human animal to obtain a non-human animal that has caused spermatogenesis derived from the spermatogonial stem cell;
c) obtaining sperm from said non-human animal;
d) A step of fertilizing the sperm into an egg to obtain an embryo.
(28) A method for producing non-human progeny derived from spermatogonial stem cells, comprising the following steps:
a) proliferating spermatogonial stem cells by culturing spermatogonial stem cells in the absence of feeder cells in a state of being attached to an insoluble carrier;
b) transplanting the spermatogonial stem cells grown in a) into the seminiferous tubule of an infertile non-human animal to obtain a non-human animal that has caused spermatogenesis derived from the spermatogonial stem cell;
c) obtaining sperm from said non-human animal;
d) fertilizing the sperm into an egg to obtain an embryo;
e) transferring the embryo into the uterus or fallopian tube of the host female animal to obtain non-human offspring.
(29) A method for producing non-human progeny derived from spermatogonial stem cells, comprising the following steps:
a) proliferating spermatogonial stem cells by culturing spermatogonial stem cells in the absence of feeder cells in a state of being attached to an insoluble carrier;
b) transplanting the spermatogonial stem cells grown in a) into the seminiferous tubule of an infertile non-human animal to obtain a non-human animal that has caused spermatogenesis derived from the spermatogonial stem cell;
c) A step of naturally mating the non-human animal with a female to obtain non-human offspring.
(30) A method for producing a non-human animal that forms sperm derived from transplanted spermatogonial stem cells, comprising the following steps:
a) proliferating spermatogonial stem cells by culturing spermatogonial stem cells in a serum-free medium;
b) A step of transplanting the spermatogonial stem cells grown in a) into the seminiferous tubule of an infertile non-human animal to obtain a non-human animal that has caused spermatogenesis derived from the spermatogonial stem cell.
(31) A method for producing sperm, including the following steps:
a) proliferating spermatogonial stem cells by culturing spermatogonial stem cells in a serum-free medium;
b) transplanting the spermatogonial stem cells grown in a) into the seminiferous tubule of an infertile non-human animal to obtain a non-human animal that has caused spermatogenesis derived from the spermatogonial stem cell;
c) A step of obtaining sperm from the non-human animal.
(32) A method for producing an embryo derived from spermatogonial stem cells, comprising the following steps:
a) proliferating spermatogonial stem cells by culturing spermatogonial stem cells in a serum-free medium;
b) transplanting the spermatogonial stem cells grown in a) into the seminiferous tubule of an infertile non-human animal to obtain a non-human animal that has caused spermatogenesis derived from the spermatogonial stem cell;
c) obtaining sperm from said non-human animal;
d) A step of fertilizing the sperm into an egg to obtain an embryo.
(33) A method for producing non-human progeny derived from spermatogonial stem cells, comprising the following steps:
a) proliferating spermatogonial stem cells by culturing spermatogonial stem cells in a serum-free medium;
b) transplanting the spermatogonial stem cells grown in a) into the seminiferous tubule of an infertile non-human animal to obtain a non-human animal that has caused spermatogenesis derived from the spermatogonial stem cell;
c) obtaining sperm from said non-human animal;
d) fertilizing the sperm into an egg to obtain an embryo;
e) transferring the embryo into the uterus or fallopian tube of the host female animal to obtain non-human offspring.
(34) A method for producing non-human progeny derived from spermatogonial stem cells, comprising the following steps:
a) proliferating spermatogonial stem cells by culturing spermatogonial stem cells in a serum-free medium;
b) transplanting the spermatogonial stem cells grown in a) into the seminiferous tubule of an infertile non-human animal to obtain a non-human animal that has caused spermatogenesis derived from the spermatogonial stem cell;
c) A step of naturally mating the non-human animal with a female to obtain non-human offspring.
(35) An agent for culturing spermatogonial stem cells in the absence of feeder cells in an adherent state to an insoluble carrier via an extracellular matrix, the agent comprising an extracellular matrix and an insoluble carrier.
(36) The agent according to (35) above, wherein the extracellular matrix is laminin.
(37) The agent according to (35) above, wherein the extracellular matrix is bound to the surface of an insoluble carrier.
(38) A method for producing spermatogonial stem cells, comprising culturing spermatogonial stem cells in a state in which they are adhered to an insoluble carrier in the absence of feeder cells.
(39) A method for producing spermatogonial stem cells, comprising culturing spermatogonial stem cells in a serum-free medium.
(40) Use of the spermatogonial stem cell according to (23) above for producing an infertility therapeutic agent.
(41) A method for treating infertility using the spermatogonial stem cell according to (23) above.

By using the proliferation method of the present invention, it is possible to efficiently proliferate spermatogonial stem cells even in the absence of feeder cells and serum, and to eliminate the influence of undetermined factors. It is possible to proliferate spermatogonial stem cells stably and easily while maintaining the ability as spermatogonial stem cells. Moreover, in the clinical application of spermatogonial stem cells, the risk of infectious diseases derived from different animals can be avoided. Therefore, the propagation method of the present invention includes the production of genetically modified animals (gene-transfected animals, gene-deficient animals, etc.), the development of treatment methods and treatment agents for male infertility, research and drugs for gene therapy at the germ cell level. It is useful for the development of
Furthermore, since the proliferation method of the present invention does not include the influence of feeder cells and serum-derived uncertain factors, it enables a more definitive test for evaluating factors that control the proliferation and differentiation of spermatogonial stem cells, It provides a new opportunity to understand the regulatory mechanisms governing spermatogonial stem cells.

  Hereinafter, the present invention will be described in detail. First, the meaning of terms used in this specification will be described, and then each aspect of the present invention will be described.

  Testicular cells include all cells that make up the testis, such as spermatogonial stem cells, male gonocytes, spermatogonia, sperm cells, spermatogonia, spermatocytes, sperm cells, sperm, Leydig Examples include cells, Sertoli cells, intercellular cells, germ cells and the like.

A spermatogonial stem cell is capable of self-renewal and differentiation into a sperm or a progenitor cell thereof (for example, a spermatogonia cell, a sperm cell, a spermatogonia cell, a spermatocyte, a spermatogonia cell) Germline cells with
Examples of spermatogonial stem cells include primordial germ cells, male germ cells (gonocytes), spermatogonia that are stem cells (cells that have the ability as spermatogonial stem cells), germ line stem cells (GS cell) and the like. In the present invention, the spermatogonial stem cell is preferably a male gonocyte, a spermatogonial cell that is a stem cell, or a germ line stem cell (GS cell). The spermatogonial stem cell used in the present invention is not particularly limited as long as it is a vertebrate cell.

  The GS cell refers to a spermatogonial stem cell proliferated in vitro in a GDNF receptor agonist compound-dependent manner, for example, a spermatogonial stem cell proliferated by the method described in Non-Patent Document 1.

  Whether or not the cell is a spermatogonial stem cell can be determined, for example, according to the method described in (Proc Natl Acad Sci USA, Vol. 91, p.11298-11302, 1994) and the like in the seminiferous tubule of an infertile male animal. Can be determined by transferring the cells and testing whether the cells have the ability to form spermatogenic colonies in the seminiferous tubules. As a result of the test, cells having the ability to form spermatogenic colonies are identified as spermatogonial stem cells.

Vertebrate animals include, for example, mammals, birds, fish, amphibians and reptiles. Examples of mammals include, but are not limited to, laboratory animals such as rodents such as mice, rats, hamsters, guinea pigs, and rabbits, domestic animals such as pigs, cows, goats, horses, sheep, minks, dogs, cats, etc. Primates such as pets, humans, monkeys, rhesus monkeys, marmosets, orangutans and chimpanzees. Examples of birds include chickens, quails, ducks, geese, turkeys, ostrichs, emu, guinea fowls, and pigeons.
In the present invention, the vertebrate is preferably a mammal.

A spermatogonial stem cell growth cofactor refers to a substance (for example, cytokine) that assists the proliferation of spermatogonial stem cells while maintaining the ability as a spermatogonial stem cell, and more specifically, the ability as a spermatogonial stem cell. A substance that induces the proliferation of spermatogonial stem cells in the state of maintaining spermatogonial stem cells, a substance that enhances the proliferation of spermatogonial stem cells while maintaining the ability as spermatogonial stem cells, and a substance that contributes to maintaining the ability as spermatogonial stem cells Etc. are included.
Examples of the growth cofactor include GDNF receptor agonist, epidermal growth factor (EGF), growth factor such as basic fibroblast growth factor (bFGF), differentiation inhibitory cytokine such as leukemia inhibitory factor (LIF), and the like. Can be mentioned.
The growth cofactor that can be contained in the medium in the growth method of the present invention is not particularly limited as long as it is derived from an animal, preferably from the above-mentioned mammal.

  The GDNF receptor agonist compound means a compound that can bind to a GDNF receptor or an auxiliary receptor of the receptor and activate a cell or a living body through the receptor. For example, it refers to a substance that, when used in the proliferation method of the present invention, can assist the proliferation of spermatogonial stem cells via the GDNF receptor and achieve the proliferation of the cells.

  Examples of the GDNF receptor agonist compound include glial cell-derived neurotrophic factor (GDNF), Nordrin (Neurturin), Persephin (Persephin), Artemin (Artemin) and the like. The GDNF receptor agonist compound is preferably GDNF.

  Examples of GDNF include GDNF such as human and rat (WO93 / 06116 pamphlet), mouse (see, for example, Gene 203, 2, 149-157, 1997) and the like.

  The GDNF receptor means a compound that is a GDNF binding substance and can transmit a GDNF signal into cells or living bodies. Examples of the receptor include cRet receptor tyrosine kinase, which is a signal-mediated receptor for GDNF.

  The GDNF co-receptor means a compound that does not directly transmit a signal of a GDNF receptor agonist compound into a cell or a living body but activates a receptor that transmits a signal of a GDNF receptor agonist compound. Such compounds are in particular receptors whose members are referred to as GDNF family receptor α: s (GFRα). They are also related to the signaling receptor complex of GDNF, percefin, artemin and northrin. The family of receptors includes 4 members (GFRα1-4) (Jing, S., et al., Cell, 85, 9-10 (1996); Jing, SQ, et al., J. Biol. Chem., 272 , 33111-33117 (1997); Trean or, JJ, et al., Nature, 382, 80-83 (1996); Subanto, P., et al., Human Molecular Genetics, 6, 1267-1273 (1997)) Is known. They can signal independently, but all are essential for ligand binding and cRet activation.

  The GDNF receptor agonist compound includes an antibody that specifically recognizes the GDNF receptor or an auxiliary receptor of the receptor, a binding fragment thereof, and the like.

  Examples of LIFs include humans (Japanese National Publication No. Hei 1-502985), mice (Japanese National Publication No. Hei 1-502985), sheep (Japanese National Publication No. Hei 4-502554), pigs (Japanese National Publication Hei 4-502554). And LIF such as cattle (JP-A-854681).

  Examples of EGF include EGF such as mouse (see Nature, 257, 325-327, 1975), human (see Proc Natl Acad Sci USA, 88, 415, 1991), and the like.

  Examples of bFGF include human bFGF (see, for example, Endocrine Rev., 8, 95, 1987), bovine bFGF (see, for example, Proc. Natl. Acad. Sci. USA, 81, 6963, 1984), and mouse bFGF (see, for example, Dev. Biol., 138, 454-463, 1990), rat bFGF (see, for example, Biochem. Biophys. Res. Commun., 157, 256-263, 1988) and the like.

  In addition, the above-mentioned spermatogonial stem cell growth cofactor, when used in the proliferation method of the present invention, assists in the proliferation of spermatogonial stem cells and is purified in the range where the proliferation of the cells can be achieved, Includes synthetic or recombinant proteins, mutant proteins (including insertion, substitution and deletion mutants), fragments, and chemically modified derivatives thereof. Further, within the same range, a protein substantially homologous to the wild-type amino acid sequence of each of the above-mentioned growth cofactors is included.

  The number of amino acids to be inserted, substituted or deleted in the mutant protein is usually 1 to 20, preferably 1 to 10, more preferably 1 to 5, and most preferably 1 or 2.

“Substantially homologous” means that the degree of homology to the wild-type amino acid sequence is preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more. Means that. The percent homology is to aid sequence alignment as described in (Atlas of Protein Sequence and Structure v.5, p.124, National Biochemical Research Foundation, Washington, DC (1972)). When four gaps can be introduced in the length of 100 amino acids, the percentage of amino acid residues that are the same amino acid residues in the compared sequences and are present in the smaller of the two sequences aligned (% ). In addition, any protein that can be isolated based on cross-reactivity with an antibody against each of the above cytokines having a wild type amino acid sequence, or a stringent gene or gene segment that encodes the wild type amino acid sequence of each of the above cytokines Proteins encoded by genes isolated by hybridization under mild conditions are included as being substantially homologous.
Examples of the stringent conditions include Sambrook, J., "Expression of cloned genes in E. coli (Molecular Cloning: A laboratory manual (1989)) Cold Spring harbor. Laboratory Press, New York, USA, 9. 47-9.62 and 11.45-11.61 ", etc. (for example, hybridization buffer (6.0 × SSC, 0.5% SDS, 100 μg / ml denatured fragmented salmon sperm DNA) after hybridization at 68 ° C. and then washed under the following conditions: washing solution (2 × SSC, 0.5% SDS) at room temperature for 5 minutes, washing solution (2 × SSC, 0. 1 minute SDS) at room temperature for 15 minutes, washing solution (0.1 × SSC, 0.5% SDS) at 37 ° C. for 1 hour, washing solution (0.1 × SSC, 0.5% SDS) at 68 ° C. for 1 hour) Illustrated.

Serum refers to a portion of blood excluding coagulation components. The serum is not particularly limited as long as it is a vertebrate-derived serum, but is preferably the above-mentioned mammal-derived serum (for example, fetal calf serum (FCS), human serum, etc.). The serum is preferably subjected to a treatment such as deactivation.
In the present invention, serum supplements (for example, Knockout Serum Replacement (KSR) (manufactured by Invitrogen)) are also included in the serum.

  A feeder cell refers to a non-spermatogonial stem cell that provides an environment for assisting the proliferation of spermatogonial stem cells while maintaining the ability as a spermatogonial stem cell when cultured with spermatogonial stem cells. Although it does not specifically limit as a feeder cell, For example, a fibroblast (mouse embryo fibroblast (MEF), mouse | mouth fibroblast cell line STO etc.) etc. are mentioned. The feeder cells are preferably inactivated by a method known per se, for example, irradiation with radiation (eg gamma rays) or treatment with an anticancer agent (eg mitomycin C).

1. Method of proliferating spermatogonial stem cells under feeder-free conditions (Method I)
The proliferation method (Method I) of the present invention comprises culturing spermatogonial stem cells in the absence of feeder cells in a state of being adhered to an insoluble carrier.

  “Absence of feeder cells” refers to an environment in which the substantial absence of feeder cells, that is, the number of feeder cells in the culture assists the proliferation of spermatogonial stem cells while maintaining the ability as a spermatogonial stem cell. A state that is insufficient to provide.

  The insoluble carrier is not particularly limited as long as it can achieve proliferation of spermatogonial stem cells when used in the proliferation method (Method I) of the present invention, and is usually non-cytotoxic, sterilizable, A member having affinity can be used. In general, plastic or glass members are preferred. However, the insoluble carrier may be metal or ceramic and is not limited to a certain material.

  Plastic members are thermosetting and thermoplastic polymers having excellent moldability, such as polystyrene, methacrylic resin, polymethylpentene, ethylene vinyl alcohol copolymer, polypropylene, cellulose, polyethylene, polysulfone, polyacrylonitrile. However, it is not limited to these.

  The glass-based member means a material in which silicate, borate, phosphate and the like are vitrified without being crystallized. Silicate glass is preferred because of its strong vitrification tendency. In addition, crystallized glass, which is a kind of composite material produced by heat-treating silicate glass, is more preferable because of its high formability and high impact resistance.

  The above insoluble carrier is usually provided to culture after being formed into a culture device (apparatus or material for use in culture) such as a petri dish, a plate, a flask, a bottle, a bead, or a hollow fiber.

  The strength of adhesion of spermatogonial stem cells to an insoluble carrier is not particularly limited in the range in which proliferation of spermatogonial stem cells can be achieved in the proliferation method (Method I) of the present invention, but usually physical and / or chemical treatment. The strength is such that it does not dissociate unless it is applied. Examples of the physical processing include processing by pipetting and tapping. Examples of the chemical treatment include treatment with a chelating agent such as EDTA and EGTA, treatment with a proteolytic enzyme such as trypsin and collagenase, and the like.

The mode of adhesion of the spermatogonial stem cell to the insoluble carrier is not particularly limited in the range in which the proliferation of the spermatogonial stem cell can be achieved in the proliferation method (Method I) of the present invention. Although it can adhere to an insoluble carrier, preferably the cells adhere indirectly to the insoluble carrier via a substrate.
In other words, the spermatogonial stem cells adhere indirectly to the insoluble carrier, preferably by attaching to a substrate bound to the surface of the insoluble carrier.

  The substrate is not particularly limited as long as it can mediate indirect adhesion of spermatogonial stem cells to an insoluble carrier and achieve proliferation of spermatogonial stem cells in the proliferation method (Method I) of the present invention. Examples of the substrate include an extracellular matrix and a polycation (polylysine, etc.), and an extracellular matrix is preferable.

  The extracellular matrix refers to a biopolymer that constitutes the space outside the cell. Examples of the extracellular matrix include fibrous proteins such as collagen and elastin, glucosaminoglycans such as hyaluronic acid and chondroitin sulfate, and cell adhesion proteins such as proteoglycan, fibronectin, vitronectin, and laminin, and are insoluble in spermatogonial stem cells. Although it is not particularly limited as long as it can mediate adhesion to a carrier and achieve proliferation of spermatogonial stem cells, it is preferably a cell adhesion protein, more preferably laminin.

  When used in the proliferation method of the present invention (Method I), the extracellular matrix mediates the adhesion of spermatogonial stem cells to an insoluble carrier, and is purified to the extent that spermatogonial stem cell proliferation can be achieved. Synthetic, recombinant proteins, mutant proteins (including insertion, substitution and deletion mutants), fragments, and chemically modified derivatives thereof. In addition, within the same range, a protein substantially homologous to the wild-type amino acid sequence of the extracellular matrix is also included.

  The number of amino acids to be inserted, substituted or deleted in the mutant protein is usually 1 to 20, preferably 1 to 10, more preferably 1 to 5, and most preferably 1 or 2.

“Substantially homologous” means that the degree of homology to the wild-type amino acid sequence is preferably 70% or more, more preferably 80% or more, still more preferably 90% or more, and most preferably 95% or more. Means that. The percent homology is to aid sequence alignment as described in (Atlas of Protein Sequence and Structure v.5, p.124, National Biochemical Research Foundation, Washington, DC (1972)). When four gaps can be introduced in the length of 100 amino acids, the percentage of amino acid residues that are the same amino acid residues in the compared sequences and are present in the smaller of the two sequences aligned (% ). Any protein that can be isolated based on cross-reactivity with an antibody against the extracellular matrix having a wild-type amino acid sequence, or a gene or gene segment encoding the wild-type amino acid sequence of each extracellular matrix; Proteins encoded by genes isolated by hybridization under stringent conditions are included as being substantially homologous.
Examples of the stringent conditions include Sambrook, J., "Expression of cloned genes in E. coli (Molecular Cloning: A laboratory manual (1989)) Cold Spring harbor. Laboratory Press, New York, USA, 9. 47-9.62 and 11.45-11.61 ", etc. (for example, hybridization buffer (6.0 × SSC, 0.5% SDS, 100 μg / ml denatured fragmented salmon sperm DNA) after hybridization at 68 ° C. and then washed under the following conditions: washing solution (2 × SSC, 0.5% SDS) at room temperature for 5 minutes, washing solution (2 × SSC, 0. 1 minute SDS) at room temperature for 15 minutes, washing solution (0.1 × SSC, 0.5% SDS) at 37 ° C. for 1 hour, washing solution (0.1 × SSC, 0.5% SDS) at 68 ° C. for 1 hour) Illustrated.

  Laminin is a protein that is usually localized outside the basal layer in vivo, and three subunit chains of α, β, and γ are associated in a cross-like manner via a coiled-coil domain. Heterotrimeric protein.

  There are several types of the three subunit chains constituting laminin. That is, there are five types of α chain (α1, α2, α3, α4 and α5), three types of β chain (β1, β2 and β3), and three types of γ chain (γ1, γ2 and γ3). Exists. For example, as an α chain, access numbers P25391 (human laminin α1), P19137 (mouse laminin α1), P24043 (human laminin α2), Q60675 (mouse laminin α2), Q16787 (human laminin α3), NCBI public protein database, Proteins whose amino acid sequences are registered as Q61789 (mouse laminin α3), AAB17053 (rat laminin α3), Q16363 (human laminin α4), CAA70970 (mouse laminin α4), CAC22310 (human laminin α5), Q61001 (mouse laminin α5), etc. For the β chain, access numbers P07794 (human laminin β1), AAA39407 (mouse laminin β1), P55268 (human laminin) have been added to NCBI's official protein database. 2), proteins with amino acid sequences registered as Q61292 (mouse laminin β2), P15800 (rat laminin β2), BAA22263 (human laminin β3), Q61087 (mouse laminin β3), etc. Amino acid sequences are registered as access numbers AAA59488 (human laminin γ1), P02468 (mouse laminin γ1), Q13753 (human laminin γ2), Q61092 (mouse laminin γ2), AAD36991 (human laminin γ3), and AAF08893 (mouse laminin γ3). The protein etc. currently being mentioned can be mentioned, respectively. As long as the type of laminin subunit chain can mediate adhesion of spermatogonial stem cells to an insoluble carrier and achieve proliferation of spermatogonial stem cells when used in the proliferation method of the present invention (Method I), There is no particular limitation.

In addition, twelve types of isoforms having different types of subunit chain combinations are known for laminin. That is, laminin includes laminin 1 (α1β1γ1), laminin 2 (α2β1γ1), laminin 3 (α1β2γ1), laminin 4 (α2β2γ1), laminin 5 (α3β3γ2), laminin 6 (α3β1γ1), laminin 7 (α3β2γ1) Isoforms comprising (α4β1γ1), laminin 9 (α4β2γ1), laminin 10 (α5β1γ1), laminin 11 (α5β2γ1) and laminin 12 (α2β1γ3) are known. The laminin isoform is not particularly limited as long as it can mediate adhesion of spermatogonial stem cells to an insoluble carrier and achieve proliferation of spermatogonial stem cells when used in the proliferation method (Method I) of the present invention. .
In addition, new laminin isoforms that are not currently found but that may be found in the future also mediate adhesion of spermatogonial stem cells to insoluble carriers when used in the growth method of the present invention (Method I). As long as proliferation of spermatogonial stem cells can be achieved, it is within the scope of the present invention.

  Laminin, when used in the proliferation method of the present invention (Method I), was isolated and purified to the extent that it can mediate adhesion of spermatogonial stem cells to an insoluble carrier and achieve proliferation of spermatogonial stem cells. Or a mixture with another compound (for example, the other extracellular matrix described above).

  Examples of the mixture of laminin and other compounds include an extracellular matrix of laminin producing cells (for example, fibroblasts). The extracellular matrix of laminin-producing cells can be obtained by, for example, culturing laminin-producing cells in a cell culture container, removing the cells with an EDTA-containing buffer solution, and washing the residue in the cell culture container with a buffer solution. Can be prepared. Alternatively, using commercially available products such as Matrigel (Becton Dickinson) (preparation of extracellular matrix components secreted by mouse chondrosarcoma EHS, including laminin and basement membrane components such as type IV collagen as main components) Also good.

  The mode of attachment of the spermatogonial stem cells to the substrate is not particularly limited in the range in which the proliferation of the spermatogonial stem cells can be achieved in the proliferation method (Method I) of the present invention. It adheres to the substrate through a substrate-specific receptor on the cell surface of the progenitor stem cell. Examples of the receptor include cell adhesion molecules such as integrins when the substrate is an extracellular matrix.

If the extracellular matrix is laminin, spermatogonial stem cells can attach to laminin via the laminin receptor.
The laminin receptor is not particularly limited. For example, integrin such as α ₁ β ₁ , α ₂ β ₁ , α ₃ β ₁ , α ₆ β ₁ , α ₇ β ₁ , α _V β ₃ , α ₆ β _{4 and the} like can be used. Can be mentioned. The laminin receptor is preferably an integrin of α ₃ β ₁ , α ₆ β ₁ , α ₆ β ₄ , and more preferably an α ₆ β ₁ integrin. Spermatogonial stem cells highly express α ₆ β ₁ integrin (see, for example, Non-Patent Document 2).

  The surface of an insoluble carrier refers to a portion that enables adhesion of cultivated spermatogonial stem cells to the insoluble carrier when culturing spermatogonial stem cells in a state adhered to the insoluble carrier. The part that comes into contact with the culture medium.

  When spermatogonial stem cells are cultured in a state where they are attached to a substrate bound to the surface of an insoluble carrier and indirectly adhered to the insoluble carrier, the insoluble carrier includes an insoluble carrier having the substrate bound to the surface, or It can be provided as the above-described culture equipment comprising such an insoluble carrier.

  For example, when the culture equipment is a petri dish or a plate, it may be a plastic or glass member having a substrate bonded to the bottom. In the case where the culture equipment is beads, a plastic or glass culture container or the like that is usually used is filled with beads bound to a substrate, filled with a medium, and spermatogonial stem cells can be cultured. Alternatively, spermatogonial stem cells can be cultured by incorporating a hollow fiber to which a substrate is bound into a hollow fiber module. As the hollow fiber material, cellulose (regenerated cellulose, acetate) or synthetic polymer (polysulfone, polyacrylonitrile, polymethyl methacrylate, ethylene vinyl alcohol copolymer, polyester polymer alloy) can be used. The form of the culture equipment is not particularly limited as long as it can be used so that the cultured spermatogonial stem cells adhere to the bound substrate and can be proliferated. Not.

  As a method for binding the substrate to the insoluble carrier, generally, a method using a non-covalent bond (hydrogen bond, ionic bond, hydrophobic bond, etc.), a covalent bond or the like can be used.

  Examples of the method for binding the substrate to the insoluble carrier by non-covalent bonding include a method in which the insoluble carrier is allowed to stand in an appropriate buffer solution (for example, a phosphate buffer solution) containing the substrate. The conditions of the method (type of buffer solution, concentration of substrate in buffer solution, standing time, etc.) are as follows when the insoluble carrier bound to the substrate is used in the growth method of the present invention (Method I). It can be set as appropriate as long as the proliferation of the progenitor stem cells can be achieved. For example, when the substrate is an extracellular matrix such as laminin and the insoluble carrier is plastic, the insoluble carrier is added to 0.05 to 24 in neutral phosphate buffered saline containing the substrate at a concentration of 0.05 to 200 μg / mL. By allowing to stand for about an hour, the substrate binds to the insoluble carrier.

  Examples of the covalent bonding method include a method of treating the surface of an insoluble carrier with a silane coupling agent having a functional group. As examples of functional groups, amino groups, aldehyde groups, epoxy groups, carboxyl groups, hydroxyl groups, and thiol groups can be introduced. Examples of silane coupling agents include γ-aminopropyltriethoxysilane, N-β- (aminoethyl) γ-aminopropyltrimethoxysilane, N-β- (aminoethyl) γ-aminopropylmethyldimethoxysilane, and the like. It can be used (for example, refer to JP2003-189843A).

  As a specific method for binding the functional group introduced to the insoluble carrier and the substrate, for example, a method using a cross-linking agent may be mentioned. Examples of crosslinking agents include diethylene glycol diglycidyl ether, 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide, N, N′-carbodidiimidazole, glutaraldehyde, succinic anhydride, phthalic anhydride, hexamethylene And diisothianate.

  Furthermore, as an example of a substrate binding method, a method using a photochemical reaction detailed in Japanese Patent No. 3056512 can be used. According to this method, first, a compound having a functional group such as an azide group or a substance containing such a compound is coated on the surface of an insoluble carrier. On top of this coating is present the substance to be bound, in the present invention the substrate. Next, the surface is irradiated with ultraviolet rays, and the nitrogen atom of the azide group and the carbon atom present in the substrate can be covalently bonded by a photochemical reaction.

  In this case, examples of the compound having an azide group include, but are not limited to, polyacrylic acid, polymethacrylate, polyallylamine and the like into which an azide group is introduced. In addition, polyamino acids such as poly-L-lysine into which an azide group is introduced, proteins such as gelatin, and biopolymers such as polysaccharides such as alginic acid and hyaluronic acid can also be used. Coating of such a compound can be easily performed by dropping a solution in which a compound having a functional group such as an azide group is dissolved on the surface of the carrier and drying. Moreover, it is preferable to irradiate ultraviolet rays at a wavelength of 320 nm or more using a light source such as a mercury lamp.

  As another example of the substrate binding method, after forming a thin film of a low molecular compound having an azide group and a functional group capable of covalently bonding to the substrate on the surface of an insoluble carrier, the low molecular compound is immobilized by ultraviolet irradiation. In addition, there is a method of covalently bonding a substrate. As the functional group at this time, succinimide, maleimide, iodoacetic acid and the like can be used. However, these are examples of substrate binding methods and are not limited thereto.

  As the basal medium of the medium used in the growth method (Method I) of the present invention, a per se known medium can be used, and is not particularly limited. For example, DMEM, EMEM, RPMI-1640, α-MEM, F-12, F-10, M-199, HAM, ATCC-CRCM30, DM-160, DM-201, BME, SFM-101, Fischer, McCoy's 5A, Leibovitz's L-15, RITC80-7, HF-C1, MCDB107, NCTC135, Waymout's MB752 / 1, StemPro-34 SFM, etc. are mentioned. Further, a medium modified for ES cell culture or the like may be used, or a mixture of the above basal medium may be used.

The medium used in the growth method (Method I) of the present invention may contain additives known per se used in normal cell culture. The additive is not particularly limited, but for example, growth factors (such as insulin), iron sources (such as transferrin), polyamines (such as putrescine), minerals (such as sodium selenate), saccharides (such as glucose) , Organic acids (eg pyruvic acid, lactic acid etc.), serum proteins (eg albumin etc.), amino acids (eg L-glutamine etc.), reducing agents (eg 2-mercaptoethanol etc.), vitamins (eg ascorbic acid, d-biotin etc.) Etc.), steroids (eg β-estradiol, progesterone etc.), antibiotics (eg streptomycin, penicillin, gentamicin etc.), buffers (eg HEPES etc.), nutritional additives (eg StemPro-Nutrient Supplement etc.) and the like.
The additive is preferably contained in the medium within a concentration range known per se.

  The medium used in the growth method (Method I) of the present invention may contain a spermatogonial stem cell growth cofactor.

That is, the medium used in the growth method (Method I) of the present invention preferably contains a GDNF receptor agonist compound. The GDNF receptor agonist compound is extremely useful for assisting the proliferation of spermatogonial stem cells, particularly for inducing or enhancing proliferation, maintaining the ability as a spermatogonial stem cell, and the like.
The concentration of the GDNF receptor agonist compound contained in the medium is not particularly limited as long as it is a concentration capable of assisting the proliferation of spermatogonial stem cells and achieving the proliferation of the cells in the proliferation method of the present invention (Method I), Usually 0.05 ng / ml to 100 mg / ml, for example 0.5 ng / ml to 100 μg / ml, preferably 0.5 ng / ml to 10 μg / ml, more preferably 0.5 ng / ml to 1 μg / ml, more preferably 0.5 to 200 ng / ml, more preferably 0.5 to 50 ng / ml, most preferably 2 to 20 ng / ml.

The medium used in the growth method (Method I) of the present invention may further contain LIF. When LIF is contained in the medium, the concentration thereof is not particularly limited as long as it is a concentration that assists the proliferation of spermatogonial stem cells and can achieve the proliferation of the cells, but usually 10 to 10 ⁶ units / ml, for example 10 -10 ⁵ units / ml, preferably 10 ² to 10 ⁴ units / ml, more preferably 3 × 10 ^{2 to} 5 × 10 ³ units / ml.

  The medium used in the growth method (Method I) of the present invention can further contain at least one of EGF and bFGF. EGF and bFGF are useful for assisting proliferation of spermatogonial stem cells, particularly for inducing or enhancing proliferation, and by adding the factor to the medium, spermatogonial stem cells can be more stably and efficiently proliferated.

  When bEGF is contained in the medium, the concentration thereof is not particularly limited as long as it is a concentration that assists the proliferation of spermatogonial stem cells and can achieve the proliferation of the cells, but a normal concentration of 0.05 ng / ml to 100 mg / ml For example, 0.5 ng / ml to 100 μg / ml, preferably 0.5 ng / ml to 10 μg / ml, more preferably 0.5 ng / ml to 1 μg / ml, still more preferably 0.5 to 200 ng / ml, and more Preferably it is 0.5 to 50 ng / ml, most preferably 2 to 30 ng / ml.

  When bFGF is contained in the medium, the concentration thereof is not particularly limited as long as it is a concentration capable of assisting the proliferation of spermatogonial stem cells and achieving the proliferation of the cells, but a normal concentration of 0.05 ng / ml to 100 mg / ml For example, 0.5 ng / ml to 100 μg / ml, preferably 0.5 ng / ml to 10 μg / ml, more preferably 0.5 ng / ml to 1 μg / ml, still more preferably 0.5 to 200 ng / ml, and more Preferably it is 0.5 to 50 ng / ml, most preferably 2 to 20 ng / ml.

The medium used in the growth method (Method I) of the present invention preferably contains serum. In the absence of feeder cells, serum is useful for assisting proliferation of spermatogonial stem cells, particularly for inducing or enhancing proliferation, and by adding serum to the medium, spermatogonial stem cells can be more stably and efficiently grown. I can do it.
The concentration of serum is not particularly limited, but is usually in the range of 0.1 to 30 (v / v)%.

  Here, when serum is contained in the medium at a high concentration of, for example, 2 to 30 (v / v)%, preferably 10 to 30 (v / v)%, either or both of EGF and bFGF are contained in the medium. Even without the inclusion of, a very stable proliferation of spermatogonial stem cells can be achieved.

  In the growth method (Method I) of the present invention, although not particularly limited, it is not necessary to add a conditioned medium of feeder cells to the medium. This makes it possible to proliferate spermatogonial stem cells stably and easily while avoiding the risk of heterologous animal-derived infections more reliably.

  As used herein, “feeder cell conditioned medium” refers to a medium containing soluble factors derived from feeder cells, and can be prepared by collecting the supernatant of the medium after culturing the feeder cells.

  As long as the spermatogonial stem cells used in the proliferation method (Method I) of the present invention are cells derived from vertebrates, they may be cells directly isolated from living organisms, or after being isolated from living organisms, It may be a cell cultured outside (for example, a GS cell).

In the growth method (Method I) of the present invention, when spermatogonial stem cells isolated directly from a living body are used for culture, first, testicular cells containing spermatogonial stem cells are collected from male testis by a method known per se. .
In this case, the male animal to be used may be before birth or after birth, but an animal after birth is preferable because it is easily available. The age of the animal after birth is not particularly limited and may be any of a newborn, an infant, an adult, and an old body.

  For example, the testis is removed, and the extracted testis is digested with a degrading enzyme such as collagenase, trypsin, or DNase to disperse testis cells (see, for example, Non-Patent Document 1). Testicular cells are washed with a medium or the like and used for the growth method (Method I) of the present invention.

  Alternatively, spermatogonial stem cells may be concentrated from the testis cells to obtain concentrated spermatogonial stem cells, which may be used in the proliferation method (method I) of the present invention. Examples of the concentration method include a method of concentrating the cells using a cell sorter, an antibody magnetic microbead, or the like using an antibody that recognizes a cell surface antigen specifically expressed in spermatogonial stem cells. For example, spermatogonial stem cells can be concentrated using cell surface antigens such as α6-integrin, c-kit, and CD9 as an index (see, for example, Proc Natl Acad Sci USA, 97, 8346-8351, 2001). Alternatively, spermatogonial stem cells can be concentrated using a dye such as Hoechst (see, for example, Development, 131, 479-487, 2004, etc.).

Next, the testis cells described above are suspended in the medium, seeded in a cell culture vessel, and cultured (first culture).
When spermatogonial stem cells are cultured in a state where they are adhered to an insoluble carrier via a substrate (for example, laminin), a cell-coated vessel coated with the substrate is used. The same applies to the cell culture vessel used for the following culture. As a result, the spermatogonial stem cells contained in the seeded testis cells adhere to the substrate and adhere to the insoluble carrier (cell culture container) through the substrate.

Although it is possible to proliferate spermatogonial stem cells only by continuing the first culture, it is preferable that about 6 to 18 hours after the start of the first culture (for example, after overnight culture), The cultured cells in the first culture, preferably the cultured cells (including at least spermatogonial stem cells) are dispersed by EDTA treatment, trypsin treatment, etc., and subcultured to another cell culture vessel (second Culture).
Although the passaged cells differ depending on the culture conditions, colonies are usually formed on the bottom surface of the cell culture vessel within one week after the passage. Colony formation can be confirmed using a microscope or the like. The colony has a form characterized by an intercellular bridge and a morula-like structure.

  Preferably, usually 5 to 14 days after the start of the second culture, the cells are dispersed by trypsin treatment or the like, resuspended in the medium, and further subcultured into a new cell culture container (third culture). By repeating the passage in the same manner, flat somatic cells usually disappear after 4 weeks from the start of culture. The interval between passages and the dilution rate of the cells are appropriately determined depending on the culture conditions. Examples include an interval of 2 to 5 days and a dilution of 1 to 1/4 (preferably a dilution of 1 to 1/2 at the beginning of the culture). The In addition, examples of the passage interval of established GS cells (ie, GS cells having stable colony formation) and the cell dilution rate include, for example, intervals of 2 to 5 days, and dilution of 1/4 to 1/10. .

  The spermatogonial stem cell colony is stabilized usually after about 3 to 6 weeks after the start of the culture.

Here, when serum is contained in the medium, the serum is preferably contained in the medium at the start of spermatogonial stem cell culture (for example, during the first culture) at a concentration of 0.1 to 5 (v / v)%, More preferably, it is contained at a relatively low concentration of 0.3 to 3 (v / v)%. In the medium after passage of the spermatogonial stem cells (for example, after the second culture), the concentration is preferably 2 It may be contained at a concentration of about ˜30 (v / v)%, more preferably about 5 to 20 (v / v)%.
By doing in this way, proliferation of cells other than spermatogonial stem cells, such as a fibroblast contained in culture | cultivation with a spermatogonial stem cell at the time of a culture | cultivation start, can be suppressed.

  It should be noted that the serum concentration contained in the medium may be relatively low even after the passage, but in this case, it is preferable that either or both of EGF and bFGF are contained in the medium.

In the proliferation method (Method I) of the present invention, when spermatogonial stem cells cultured in vitro are used, for example, testis cells containing spermatogonial stem cells are first collected from the testes of male animals as described above. Then, after culturing this in the presence of feeder cells by the method described in Non-Patent Document 1, etc., spermatogonial stem cells are recovered and used for the proliferation method (Method I) of the present invention. The recovered spermatogonial stem cells can be GS cells.
The culture conditions of spermatogonial stem cells in the presence of feeder cells can be the same as the conditions of the growth method (Method I or II) of the present invention except that the above-mentioned substrate is not used and feeder cells are used. .

  By using the proliferation method of the present invention (Method I), spermatogonial stem cells can be used as spermatogonial stem cells in the absence of feeder cells, usually for 2 months or more, preferably 4 months or more, more preferably 6 months or more. It can grow stably while maintaining its ability.

Furthermore, using the method of growth present invention (Method I), spermatogonial stem cells, in the absence of feeder cells, 2-10 ⁶ times the usual 50 days, preferably about 4 to 10 ⁶ fold in 50 days Until the spermatogonial stem cell is maintained, it can be proliferated.

In addition, the cell culture conditions in the growth method (Method I) of the present invention can be those normally used in cell culture technology. For example, the culture temperature is usually in the range of about 30 to 40 ° C, preferably about 37 ° C. The CO ₂ concentration is usually in the range of about 1 to 10%, preferably about 5%. The humidity is usually in the range of about 70 to 100%, preferably about 95 to 100%.

  The spermatogonial stem cells grown by the proliferation method (Method I) of the present invention maintain the ability as a spermatogonial stem cell with high efficiency, but have a trait different from that of spermatogonial stem cells grown in the presence of feeder cells. Can have.

  For example, spermatogonial stem cells grown by the growth method (Method I) of the present invention have an embryonic phenotype. An embryonic phenotype is a trait specific to embryonic cells (eg, primordial germ cells) and does not usually have somatic cells after birth.

  The spermatogonial stem cells proliferated in the presence of feeder cells are usually composed of β1-integrin, α6-integrin, EE2, EpCAM and c-kit, for example, in the case of mouse cells, according to analysis by a flow cytometer. At least any cell surface antigen selected from the group is positive, preferably all are positive. Moreover, c-kit is preferably weakly positive. In addition, the spermatogonial stem cell is negative for at least one cell surface antigen selected from the group consisting of SSEA-1 and Forssman antigen, preferably negative for any antigen (see, for example, Non-Patent Document 1). .

  In contrast, spermatogonial stem cells grown by the proliferation method of the present invention (Method I) are positive for SSEA-1. Moreover, the expression level of c-kit is low, and the expression level can be about 1/2 to 1/100 of spermatogonial stem cells grown in the presence of feeder cells as peak fluorescence intensity by a flow cytometer. The expression of other cell surface antigens is similar to spermatogonial stem cells grown in the presence of feeder cells.

  Here, the expression “positive” of the cell surface marker means that the cell surface marker is expressed on the cell surface, and specific binding by a specific antibody against the cell surface marker can be confirmed. “Weak positive” means that the expression level of the cell surface marker is relatively weak compared to other cells, the population whose expression level of the cell surface marker is relatively weak, or the cell surface marker is expressed. It means that the ratio of the cell population is relatively small.

  In spermatogonial stem cells of animal species other than mice, the expression pattern of cell surface markers is the same as in mice. However, if there is a marker that the animal species does not inherently possess, the species difference is taken into account, for example, the marker is excluded from the analysis.

  The positive expression of SSEA-1 indicates that the proliferation method of the present invention (Method I) changes the expression of cell surface markers on spermatogonial stem cells, induces embryonic phenotype, and spermatogonial stem cells have more primitive characteristics. Suggest that it may be possible to gain.

In addition, spermatogonial stem cells grown in the presence of feeder cells usually form massive colonies, but spermatogonial stem cells grown by the proliferation method of the present invention (Method I) have a fibroblast-like appearance. Various patterns of colonies including lumps can be formed. Some of the colonies form chains or form a network, which is similar to the spermatogonial stem cell proliferation pattern observed in vivo after spermatogonia transplantation (eg, Nagano M, Avarbock MR, Brinster RL. Pattern and kinetics of mouse donor spermatogonial stem cell colonization in recipient testes. See Biol Reprod 1999; 60: 1429-1436).
Therefore, the proliferation method (Method I) of the present invention enables colony growth in a state closer to in vivo of spermatogonial stem cells, and also more accurately reproduces the proliferation pattern of spermatogonial stem cells in vivo. This is useful in stem cell research and the like.

  The spermatogonial stem cells proliferated by the proliferation method (Method I) of the present invention can be cryopreserved semipermanently, and can be used after thawing and sleeping as needed. The spermatogonial stem cells maintain their ability as spermatogonial stem cells even after cryopreservation and thawing. In cryopreservation, cells are suspended in a cell cryopreservation composition known per se such as a cell banker (manufactured by DIA-IATRON) containing dimethyl sulfoxide and fetal bovine serum albumin, and is preferably -80 to -200 ° C. Preserve cells at -196 ° C (in liquid nitrogen).

  When the spermatogonial stem cells grown by the growth method (Method I) of the present invention are cryopreserved and then put to sleep, they are thawed and suspended in a solvent according to a conventional method to obtain a cell suspension. The method of thawing is also not particularly limited, and for example, it can be performed using DMEM (DMEM / FCS) containing 10% fetal calf serum in a constant temperature bath at 37 ° C. Specifically, a freezing tube is floated on a thermostatic bath, and DMEM / FCS is dropped and thawed on the frozen cells. The cells are washed by centrifugation and then resuspended in the medium.

  Even if the spermatogonial stem cells once asleep are cultured and then frozen again, the ability of the cells as spermatogonial stem cells is maintained.

  By using the proliferation method (Method I) of the present invention, it is possible to efficiently and stably proliferate spermatogonial stem cells while maintaining the ability as spermatogonial stem cells even in the absence of feeder cells.

  The present invention also relates to an agent for culturing spermatogonial stem cells in an adherent state to an insoluble carrier via an extracellular matrix, the agent comprising an extracellular matrix and an insoluble carrier.

  In one embodiment, the agent may be provided as the above-described culture device comprising or consisting of an insoluble carrier having an extracellular matrix bound to the surface. The types of extracellular matrix and insoluble carrier, the types of culture equipment, the binding mode of the extracellular matrix to the surface of the insoluble carrier and the like are as described above.

  Further, the extracellular matrix and the insoluble carrier may be separated and packaged and provided in the form of a kit. In this case, the extracellular matrix can be in a state of a buffer solution containing the extracellular matrix or a powder. The extracellular matrix further includes physiologically acceptable carriers, excipients, preservatives, stabilizers, binders, solubilizers, nonionic surfactants, buffers, preservatives, antioxidants, etc. It may be provided as a composition comprising.

  The content of the extracellular matrix in the agent is not particularly limited as long as spermatogonial stem cells can be proliferated when used in the proliferation method (Method I) of the present invention. It can be set easily.

  In addition, a kit for culturing spermatogonial stem cells adhered to an insoluble carrier via an extracellular matrix by combining the above-mentioned agent with the above-mentioned basal medium, additives, growth cofactors, serum, etc. You can also These components can be mixed in advance as required.

  By using the above agent (or kit) in the proliferation method (Method I) of the present invention, it is possible to easily proliferate spermatogonial stem cells in the absence of feeder cells.

2. Method of proliferating spermatogonial stem cells under serum-free conditions (Method II)
The growth method (Method II) of the present invention includes culturing spermatogonial stem cells in a serum-free medium.

  The basal medium of the medium used in the growth method (Method II) of the present invention, the additive that can be added to the medium, and the growth cofactor for spermatogonial stem cells are the same as in Method I.

  The medium used in the growth method (Method II) of the present invention is preferably supplemented with B27 in order to enhance the proliferation of spermatogonial stem cells.

B27 is a culture additive used for serum-free culture of nerve cells and the like, and its composition is, for example, (J. Neurosci. Res., Vol. 35, p. 567-576, 1993) or (Brain Res , vol. 494, p. 65-74, 1989) and the like. More specifically, B27 is d-biotin, albumin (BSA, etc.), catalase, L-carnitine hydrochloride, corticosterone, ethanolamine hydrochloride, D-galactose (anhydrous), glutathione (reduced), insulin (bovine etc., Zn), linoleic acid, linolenic acid, progesterone, putrescine dihydrochloric acid, sodium selenate (1000 ×, etc.), superoxide dismutase, T-3 / albumin complex, DL α-tocopherol (vitamin E), DL α-tocopherol acetic acid (Vitamin E acetic acid), transferrin (human etc., Iron-poor) and the composition containing vitamin A acetic acid. Here, one or more components selected from the group consisting of DL α-tocopherol (vitamin E), DL α-tocopherol acetic acid (vitamin E acetic acid), superoxide dismutase, catalase, glutathione and vitamin A acetic acid from the above-mentioned composition The composition excluding the elements is also included in B27.
The content of each component in B27 is not particularly limited as long as the proliferation of spermatogonial stem cells can be enhanced in the proliferation method (Method II) of the present invention. For example, (Brewer, GJ, Torricelli, JR, Evage, EK, Price, PJ, Optimized Survival of Hipocampal Neurons in B27 Supplemented Neurobasal.A new Serum-free Medium Combination., J. Neurosci. Res., Vol. 35, p. 567-576, 1993) and (Brain Res. , vol. 494, p. 65-74, 1989) and the like.
As B27, commercial products such as B-27 Supplement (trade name), B-27 Supplement Minus AO (trade name), and B-27 Supplement Minus Vitamin A (trade name) (above, manufactured by Invitrogen) may be used. Good.
The amount of B27 added to the medium is not particularly limited as long as the proliferation of spermatogonial stem cells can be enhanced in the proliferation method (Method II) of the present invention. For example, B-27 Supplement (manufactured by Invitrogen) can be used. 1-100 μl / ml, preferably about 5-40 μl / ml is added to the medium.

In the proliferation method (Method II) of the present invention, spermatogonial stem cells are preferably cultured in the presence of feeder cells. By culturing spermatogonial stem cells in the presence of feeder cells, spermatogonial stem cells can adhere to the feeder cells and more efficiently form proliferating colonies.
When using feeder cells, for example, about 10 ⁵ to 10 ⁶ cells are usually used per well of a 6-well plate.

The spermatogonial stem cells used in the proliferation method (method II) of the present invention may be cells directly isolated from a living body, as long as they are cells derived from vertebrates, or after being isolated from a living body, It may be a cell cultured outside (for example, a GS cell).
Preferably, the spermatogonial stem cells subjected to the proliferation method (Method II) of the present invention are cells that have been isolated from a living body and then cultured in a medium containing serum in vitro.

In the growth method (Method II) of the present invention, when spermatogonial stem cells cultured in vitro are used, for example, testis cells containing spermatogonial stem cells are first collected from the testes of male animals as described above. Then, after culturing it in a medium containing serum by the method described in Non-Patent Document 1 or the like, or the above method I or the like, spermatogonial stem cells are recovered and used for the proliferation method (method II) of the present invention. . The recovered spermatogonial stem cells can be GS cells.
The culture conditions of spermatogonial stem cells in the presence of serum can be the same as the conditions of the culture method (Method I or Method II) of the present invention except that a serum-containing medium is used.

  By using the proliferation method of the present invention (Method II), spermatogonial stem cells can be used as spermatogonial stem cells in the absence of serum, usually for 2 months or more, preferably 4 months or more, more preferably 6 months or more. It is possible to grow stably while maintaining

Furthermore, using the method of growth present invention (Method II), spermatogonial stem cells, in the absence of serum, 2 to 10 ⁹ times the usual 50 days, preferably 10 to 10 ⁹ times at 50 days, and more Preferably, it can be expanded to about 10 ² to 10 ⁹ times in 50 days while maintaining the ability as a spermatogonial stem cell.
Surprisingly, by culturing spermatogonial stem cells in a serum-free medium by the growth method of the present invention (Method II), compared to culture in a serum-containing medium, the efficiency is very high (eg 1.5 It is possible to achieve growth that is twice or more, preferably twice or more efficient.

  As the cell culture conditions in the growth method (Method II) of the present invention, the culture conditions usually used in the cell culture technique can be used as in Method I above.

  When the spermatogonial stem cells grown by the proliferation method of the present invention (method II) are, for example, mouse cells, according to analysis by a flow cytometer, they are usually β1-integrin, α6-integrin, EE2, EpCAM and c. -At least one cell surface antigen selected from the group consisting of kits is positive, preferably all are positive. Moreover, c-kit is preferably weakly positive. In addition, the spermatogonial stem cell is negative for at least one cell surface antigen selected from the group consisting of SSEA-1 and Forssman antigen, preferably negative for any antigen.

  In the spermatogonial stem cells of animal species other than mice, the expression pattern of cell surface markers is the same as that of mice. However, if there is a marker that the animal species does not inherently possess, the species difference is taken into account, for example, the marker is excluded from the analysis.

  The spermatogonial stem cells grown by the proliferation method (Method II) of the present invention can be cryopreserved semipermanently as in Method I above, and are used after thawing and sleeping as needed. be able to.

  By using the proliferation method (Method II) of the present invention, it is possible to stably proliferate spermatogonial stem cells with extremely high efficiency while maintaining the ability as spermatogonial stem cells even under serum-free conditions.

3. Differentiation stages of spermatogonial stem cells subjected to culture It is disclosed that the following two differentiation stages may exist in the spermatogonial stem cells subjected to the proliferation method (Method I or II) of the present invention.
(A) A differentiation stage requiring a GDNF receptor agonist compound and LIF as growth factors for in vitro growth (hereinafter referred to as differentiation stage (A)).
(B) A differentiation stage (hereinafter referred to as differentiation stage (B)) capable of growing in a medium containing substantially only a GDNF receptor agonist compound as a growth cofactor for spermatogonial stem cells.

  In other words, in vitro GDNF receptor agonist-dependent compound-dependent proliferation of spermatogonial stem cells in differentiation stage (A) requires LIF, but in vitro GDNF receptor agonism of spermatogonial stem cells in differentiation stage (B) Compound dependent growth does not require LIF.

Which differentiation stage the spermatogonial stem cell is in is determined by, for example, the spermatogonial stem cell by GDNF receptor agonist compound and LIF by the above-described proliferation method (method I or II) or the proliferation method described in Non-Patent Document 1. Is cultured in a medium containing medium (referred to as medium (A)) or a medium containing substantially only a GDNF receptor agonist compound as a growth cofactor for spermatogonial stem cells (referred to as medium (B)). A person skilled in the art can easily determine whether or not the cells proliferate.
The spermatogonial stem cells in the differentiation stage (A) can proliferate in the medium (A), but cannot proliferate in the (B).
On the other hand, the spermatogonial stem cells in the differentiation stage (B) can be grown in the medium (A) and the medium (B).

  In the above test, in order to amplify the sensitivity, at least one or both of EGF and bFGF may be added to the media (A) and (B).

“A medium containing substantially only a GDNF receptor agonist compound as a spermatogonial stem cell growth cofactor” means that only a GDNF receptor agonist compound is purified except for the components derived from the above-mentioned basal medium, additives, and serum. As a prosthetic cell growth cofactor, a medium containing a concentration capable of assisting the proliferation of spermatogonial stem cells.
That is, the medium does not contain growth cofactors other than GDNF receptor agonist compounds such as LIF, EGF, bFGF, etc., at a concentration capable of supporting the proliferation of spermatogonial stem cells.
Examples of the “medium containing substantially only a GDNF receptor agonist compound as a spermatogonial stem cell growth cofactor” include, for example, the above-mentioned basal medium, additives, serum and a medium comprising a GDNF receptor agonist compound.

  In addition, spermatogonial stem cells are collected from the testes of male animals at various developmental (growth) stages or ages, and the cells are obtained by the proliferation method described above (method I or II) or the proliferation method described in Non-Patent Document 1. Are cultured in the medium (A) and the medium (B) and tested for the presence or absence of proliferation of spermatogonial stem cells, so that the development (growth) stage or age of a male animal containing the spermatogonial stem cells in each differentiation stage described above Can be easily determined by those skilled in the art.

In one embodiment, a testis tissue section of a male animal is observed under a microscope (see, for example, Int. J. Androl., Vol. 4, p. 475-493 (1981), Cell Tiss. Res., Vol. 154, p. 443-470 (1974) etc.), and the histological characteristics of the spermatogonial stem cells contained therein can be used as an index to determine which differentiation stage the spermatogonial stem cells are in .
Histologically, spermatogonial stem cells are present in the lumen of the seminiferous tubule before and after the birth of male animals, and the spermatogonial stem cells at this time are called male gonocytes. Thereafter, male gonocytes move to the basement membrane of the seminiferous tubule, come into contact with the basement membrane, differentiate into spermatogonia, and initiate spermatogenesis.
Here, the spermatogonial stem cells can be considered to be in the differentiation stage (A) by using as an indicator that the spermatogonial stem cells are in the differentiation stage existing in the lumen of the seminiferous tubule.
Moreover, it can be said that the spermatogonial stem cell is in the differentiation stage (B), using as an index the differentiation stage in which the spermatogonial stem cell is in contact with the basement membrane of the seminiferous tubule.

That is, it can be assumed that the spermatogonial stem cell is in the differentiation stage (A) with the index that the spermatogonial stem cell is a male gonocyte.
In addition, it can be assumed that the spermatogonial stem cell is in the differentiation stage (B), using the spermatogonial stem cell as an index.

More specifically, the spermatogonial stem cells in the differentiation stage (A) are, for example, 4 to 6 days (for example, 4 days) after birth in mice and 4 to 6 days (for example, 4 days) after birth in rats. Mention may be made of spermatogonial stem cells in the differentiation stage.
Examples of the spermatogonial stem cells in the differentiation stage (B) include spermatogonial stem cells in the differentiation stage after the above-mentioned period.

In the proliferation method (Method I or II) of the present invention, spermatogonial stem cells in any of the differentiation stages (A) and (B) described above can be used.
The spermatogonial stem cells in the differentiation stage (A) or (B) that can be subjected to the proliferation method of the present invention are males containing the spermatogonial stem cells by the method described in Method I based on the above-described indicators and the like, respectively. By collecting testicular cells from the testis of an animal by the above method, a cell population containing the spermatogonial stem cells can be obtained.

  Here, “the testis containing spermatogonial stem cells in the differentiation stage (A) (or (B))” means at least 10%, preferably 50% or more, more preferably 80% of the spermatogonial stem cells contained. % Or more, more preferably 90% or more, and most preferably 100% is a testis which is a spermatogonial stem cell in the differentiation stage (A) (or (B)).

  The spermatogonial stem cells in the differentiation stage (B) can be obtained by culturing the spermatogonial stem cells in the differentiation stage (A) in a medium containing a GDNF receptor agonist compound and LIF.

The culture conditions for culturing the spermatogonial stem cells in the differentiation stage (A) and obtaining the spermatogonial stem cells in the differentiation stage (B) are the above-described proliferation method (method I) of the present invention except for the following points. Or II), or the culture conditions in the method described in Non-Patent Document 1.
(1) The medium contains a GDNF receptor agonist compound and LIF.
(2) A culture period of about 1 to 30 days is sufficient.

  Whether or not the spermatogonial stem cells obtained by the method are in the differentiation stage (B) is determined according to the above-described culture method, by using only the GDNF receptor agonist compound as a growth factor of spermatogonial stem cells. A person skilled in the art can easily determine whether or not the spermatogonial stem cells have been grown by culturing in a medium containing

  In the proliferation method (Method I or II) of the present invention, when spermatogonial stem cells are proliferated in a GDNF receptor agonist-dependent manner, when the spermatogonial stem cells in the differentiation stage (A) are used, the medium contains LIF. Is preferred.

On the other hand, in the proliferation method (Method I or II) of the present invention, when spermatogonial stem cells are proliferated in a GDNF receptor agonist-dependent manner, when the spermatogonial stem cells in the differentiation stage (B) are used, the medium is The LIF may not be included.
A medium not containing LIF refers to a medium that does not substantially contain LIF, that is, a medium that does not contain LIF at a concentration capable of assisting the growth of spermatogonial stem cells.

  In the proliferation method (Method I or II) of the present invention, when spermatogonial stem cells are proliferated in a GDNF receptor agonist compound-dependent manner, when the spermatogonial stem cells in the differentiation stage (B) are used, the medium is substantially A medium containing only a GDNF receptor agonist compound as a growth cofactor for spermatogonial stem cells may be used.

  A new spermatogonia obtained as a result of growing the spermatogonial stem cells in the differentiation stage (B) by culturing them in a medium containing a GDNF receptor agonist compound according to the proliferation method (Method I or II) of the present invention. Progenitor stem cells also maintain the differentiation stage (B).

  In the proliferation method (Method I or II) of the present invention, particularly when spermatogonial stem cells in the differentiation stage (B) are used, the culture stability does not depend on feeder cells or serum, and does not depend on LIF. Very stable, efficient, operable and economical culture can be achieved.

4). Method for producing sperm, embryo, non-human progeny, etc. derived from the proliferated spermatogonial stem cell The spermatogonial stem cell proliferated by the proliferating method of the present invention (method I or II) has a high efficiency as a spermatogonial stem cell. Therefore, a non-human animal that forms sperm derived from the transplanted cell can be produced by transplanting the cell into the seminiferous tubule of an infertile non-human animal. The transplanted spermatogonial stem cells grow in the seminiferous tubule and form a spermatogenic colony.

  Examples of infertile non-human animals include non-human male animals sterilized by administration of anticancer agents such as busulfan, and non-human male animals (eg, W mice) that are genetically deficient in spermatogenic ability. (See Patent Document 1, Proc Natl Acad Sci USA, vol. 91, No. 24, p. 11298-11302, 1994).

  From the viewpoint of engrafting the transplanted spermatogonial stem cells with high efficiency, the animal species of the infertile non-human animal is preferably the same as the animal species of the spermatogonial stem cell to be transplanted. Further, it is more preferable that the infertile non-human animal strain is the same as the transplanted spermatogonial stem cell strain.

If the animal species and / or lineage is different between infertile non-human animals and transplanted spermatogonial stem cells, the infertile non-human animals are immune in order to reduce the possibility of transplanted cells being rejected. A deficient animal is preferred.
Examples of the immunodeficient animal include administration of an antibody against a cell surface antigen (eg, CD4) of a non-human animal (eg, a nude mouse) that is genetically deficient in immune function or an immunocompetent cell such as a T cell. Non-human animals in which immune tolerance has been induced by such as.

  In addition, the spermatozoa proliferated by the proliferation method (method I or II) of the present invention by collecting spermatozoa from non-human animals that form spermatozoa derived from transplanted spermatogonial stem cells obtained by the above-described method. Progenitor stem cell-derived sperm can be produced.

Furthermore, the spermatogonial stem cell-derived embryo propagated by the proliferation method (Method I or II) of the present invention can be produced by fertilizing the sperm obtained by the above-described method into an egg and obtaining an embryo.
An egg is a female gamete that can be fertilized by a sperm. Examples of eggs include egg cells and oocytes.
Fertilization of sperm and eggs can be performed by well-known methods such as microinsemination and IVF.

  In addition, the spermatogonial stem cells propagated by the proliferation method (Method I or II) of the present invention by transferring the embryo obtained by the above-described method into the uterus or fallopian tube of a host female animal to obtain non-human offspring. Non-human progeny of origin can be produced.

The host female animal is not particularly limited, but is preferably a pseudopregnant animal. A pseudopregnant animal can be obtained by mating a female animal having a normal cycle with a male animal castrated by vagina ligation or the like.
The host female animal into which the embryo has been transferred becomes pregnant and gives birth to non-human progeny derived from spermatogonial stem cells that have been propagated by the propagation method of the present invention (Method I or II).

The proliferation method (method) of the present invention can also be obtained by naturally mating a non-human animal that forms a sperm derived from a transplanted spermatogonial stem cell obtained by the above-described method with a female to obtain a non-human offspring. Non-human progeny derived from spermatogonial stem cells grown by I or II) can be produced.
The female used for the natural mating is usually a wild type female animal.

  By using the proliferation method (Method I or II) of the present invention, it is possible to stably proliferate spermatogonial stem cells over a long period of time while maintaining the ability as spermatogonial stem cells. The gene of a spermatogonial stem cell proliferated by the proliferation method of the present invention is modified, for example, a spermatogonial stem cell into which a specific foreign gene has been introduced, a spermatogonial stem cell lacking a specific gene, etc. Stem cells can be produced.

  As a method for introducing a gene into a spermatogonial stem cell, for example, a vector constructed so that a specific gene can be functionally expressed is introduced into a spermatogonial stem cell. As the vector, a plasmid vector, a viral vector, or the like can be used. Examples of viral vectors include retrovirus, adenovirus, centivirus, herpes virus, adeno-associated virus, parvovirus, Semliki Forest virus, vaccinia virus and the like.

  Examples of the method for introducing a vector into spermatogonial stem cells include general gene introduction methods such as the calcium phosphate method, the DEAE dextran method, the electroporation method, and the lipofection method. When a virus is used as a vector, the virus genome may be introduced into the cell by the general gene transfer method described above, or the virus genome may be introduced into the cell by infecting the cell with a virus particle. Can be introduced.

  Further, by using the growth method (Method I or II) of the present invention, stable genetically modified spermatogonial stem cells into which a foreign gene has been stably introduced can be selected. For example, a marker gene may be introduced into a cell simultaneously with a vector, and the cell may be cultured by a method according to the nature of the marker gene. For example, when the marker gene is a gene that confers drug resistance to a selected drug exhibiting lethal activity on the host cell, the cell into which the vector has been introduced may be cultured using a medium containing the drug. Examples of combinations of a drug resistance-conferring gene and a selective drug include, for example, a combination of a neomycin resistance-conferring gene and neomycin, a combination of a hygromycin resistance-conferring gene and hygromycin, and a blasticidin S resistance-conferring gene and blasticidin S. Combinations can be given.

  It is also possible to obtain spermatogonial stem cells lacking a specific gene using the same method. Examples of a method for obtaining spermatogonial stem cells lacking a specific gene include homologous recombination (gene targeting method) using a targeting vector. That is, a chromosomal DNA of a specific gene is isolated and a neomycin resistance gene, a drug resistance gene typified by a hygromycin resistance gene, or lacZ (β-galactosidase gene) or cat (chloramphenicol acetyltransferase gene) ) Is inserted into the intron part between exons to insert a DNA sequence that terminates gene transcription (eg, polyA addition signal, etc.) Obtained by introducing a DNA strand (targeting vector) having a DNA sequence constructed so as to result in gene disruption by making it impossible to synthesize messenger RNA into the chromosome of spermatogonial stem cells by homologous recombination. The cell PCR using Southern hybridization analysis using the DNA sequence of or near the DNA of the gene as a probe, or the DNA sequence of the targeting vector other than the DNA sequence of the specific gene used for the targeting vector and the targeting vector. It can be obtained by analyzing by the method and selecting spermatogonial stem cells lacking a specific gene. Alternatively, a Cre-loxP system or the like that deletes a specific gene in a tissue-specific or developmental stage-specific manner may be used (Marth, JD (1996) Clin. Invest. 97: 1999-2002; Wagner, KU et al. (1997) Nucleic Acids Res. 25: 4323-43 30).

  Using the genetically modified spermatogonial stem cells obtained in this manner, the same method as described above, non-human animals that form sperm derived from genetically modified spermatogonial stem cells, sperm derived from genetically modified spermatogonial stem cells ( Genetically modified sperm), embryos derived from genetically modified spermatogonial stem cells (gene modified embryos), non-human progeny derived from genetically modified spermatogonial stem cells (genetically modified non-human progeny or genetically modified non-human animals), etc. I can do it.

  The proliferation method (Method I or II) of the present invention and the spermatogonial stem cells grown by the method are useful for the treatment of male infertility.

For example, spermatogonial stem cells are collected by biopsy from the testes of infertile patients and cultured in a test tube by the proliferation method (Method I or II) of the present invention. Then, the spermatogonial stem cells proliferated in the seminiferous tubules of the infertile patient are injected (microinjection), and the spermatogenesis derived from the cultured cells is caused to occur in the seminiferous tubules of the patient's testis. it can. This technique is particularly effective when infertility is caused by, for example, chemotherapy or radiotherapy.
Thus, since the said spermatogonial stem cell can be utilized for the infertility treatment of a male animal, the fertility treatment agent especially for males containing the said spermatogonial stem cell is also within the scope of the present invention.

  In addition, the proliferation method (Method I or II) of the present invention and the spermatogonial stem cells grown by the method are useful for gene therapy at the germ cell level.

  For example, spermatogonial stem cells are collected by biopsy from a patient having a mutation in a specific gene and cultured in a test tube by the growth method of the present invention (Method I or II). Then, a spermatogonial stem cell in which the mutated gene is replaced with a gene that functions normally by the above-described gene modification method or the like is prepared, and the mutation is prevented from being transmitted to offspring. Such a treatment method also requires the maintenance and proliferation of spermatogonial stem cells. In this case, the proliferation method (Method I or II) of the present invention can be used effectively.

  EXAMPLES Hereinafter, although an Example is shown and this invention is demonstrated more concretely, this invention is not limited at all by the Example shown below.

Example 1
[1] Materials and Methods (1) Animals GS cells are transgenic mouse strain C57BL6 / Tg14 (act-EGFP-OsbY01) (referred to as Green) crossed to DBA / 2 background (provided by Dr. Okabe, Osaka University) From existing protocols (Kanatsu-Shinohara M, Ogonuki N, Inoue K, Miki H, Ogura A, Toyokuni S, Shinohara T. Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol Reprod 2003; 69 : 612-616). Although the level of EGFP expression gradually decreases after meiosis, the spermatogonia and spermatocytes of the mouse express the EGFP gene (Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune Y. 'Green mice' as a source of ubiquitous green cells. FEBS Lett 1997; 407: 313-319). Thus, donor cells are easily distinguished after transplantation. All GS cells used in this example were derived from 0-2 day old newborn mice.
For feeder-free culture, fully established GS cells were cultured on laminin-coated plates. Laminin-coated plates were prepared by treating culture dishes with PBS containing laminin (BD Biosciences) at a concentration of 20 μg / ml overnight at 37 ° C., then removing the solution and washing twice with PBS.
The basal culture medium was StemPro supplement (Invitrogen), 25 μg / ml insulin, 100 μg / ml transferrin, 60 μM putrescine, 30 nM sodium selenate, 6 mg / ml D-(+)-glucose, 30 μg / ml pyruvic acid, 1 μl / ml DL Lactic acid (Sigma), 5 mg / ml bovine albumin (ICN Biomedicals), 2 mM L-glutamine, 5 × 10 ⁻⁵ M 2-mercaptoethanol, MEM vitamin solution (Invitrogen), MEM non-essential amino acid solution (Invitrogen), 10 ⁻ StemPro-34 SFM (Invitrogen) supplemented with ⁴ M ascorbic acid, 10 μg / ml d-biotin, 30 ng / ml β-estradiol, and 60 ng / ml progesterone (Sigma). The growth cofactors used were 20 ng / ml mouse EGF (BD Biosciences), 10 ng / ml human bFGF (BD Biosciences), 10 ³ U / ml ESGRO (mouse leukemia inhibitory factor: LIF, Invitrogen), and 10 ng / ml recombination. Rat GDNF (R & D Systems). Growth cofactors were added as needed. MEF conditioned medium was prepared as previously described (Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, Carpenter MK. Feeder-free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 2001; 19: 971-974). Serum supplemented media was prepared by adding FCS (JRH Biosciences) to serum free media. In serum-free culture experiments on MEF, the medium was supplemented with the required growth cofactors for B27 (Invitrogen) 20 μl / ml in the basal culture medium.
The cells were maintained at 37 ° C. in air containing 5% carbon dioxide.

(2) Antibody and staining The primary antibodies used were rat anti-EpCAM (G8.8), mouse anti-SSEA-1 (MC-480) (Developmental Research Hybridoma Bank, University of Iowa), rat anti-human α6-integrin (CD49f). ) (GoH3), biotinylated hamster anti-rat β1-integrin (CD29) (Ha2 / 5), biotinylated rat anti-CD9 antigen (KMC8), allophycocyanin (APC) -conjugated rat anti-mouse c-kit (CD117) (2B8 ) (BD Biosciences). APC-conjugated goat anti-rat IgG (Cedarlane Laboratories), APC-conjugated streptavidin (BD Biosciences), and Alexa Fluor 633-conjugated goat anti-mouse IgM (Molecular Probes) were used as secondary antibodies. The cell staining technique is as described above (Shinohara T, Avarbock MR, Brinster RL. Β1- and α6-integrin are surface markers on mouse spermatogonial stem cells. Proc Natl Acad Sci USA 1999; 96: 5504-5509). Cells were analyzed using a FACS-Calibur system and 10,000 events were collected (BD Biosciences).

(3) Analysis of marker gene expression Total RNA was isolated using Trizol (Invitrogen). First-strand cDNA for RT-PCR was synthesized by Superscript ^™ II (RNase H ^- reverse transcriptase, Invitrogen) and PCR was performed using rTaq (Takara). RT-PCR for Oct-4, Rex-1, neurogenin3 (ngn3), c-ret, Mvh, Fragilis and Stella was performed as described (Keller G, Kennedy M, Papayannopoulou T, Wiles MV. Hematopoietic commitment during embryonic stem cell differentiation in culture.Mole Cell Biol 1993; 13: 473-486, Feng LX, Chen Y, Dettin L, Reijo Pera RA, Herr JC, Goldberg E, Dym M. Generation and in vitro differentiation of a spermatogonial cell line. Science 2002; 297: 392-395, Goolsby J, Marty MC, Heletz D, Chiappelli J, Tashko G, Yarnell D, Fishman PS, Dhib-Jalbut S, Bever Jr CT, Trisler D. Hematopoietic progenitors express neural genes.Proc Natl Acad Sci USA 2003; 100: 14926-14931, Yoshida S, Takakura A, Ohbo K, Abe K, Wakabayashi J, Yamamoto M, Suda T, Nabeshima Y. Neurogenin3 delineates the earliest stages of spermatogenesis in the mouse testis. Dev Biol 2004; 269: 447-458, Toyooka Y, Tsunekawa N, Akasu R, Noce T. Embryonic stem cells can form germ cells in vitro. Proc Natl Acad Sci USA 2003; 100: 11457-11462).

(4) Transplantation and analysis Approximately 5 μl of donor cell suspension was injected through the export tube into the seminiferous tubule of the recipient testis (referred to as WBB6F1-W / W ^v, purchased from Japan SLC) (Silvers WK. Dominant spotting, Patch, and Rump-White.In: Silvers WK (ed.), The Coat Colors of Mice, New York: Springer; 1979: 206-223, Ogawa T, Arechaga JM, Avarbock MR, Brinster RL.Transplantation of testis germinal cells into mouse seminiferous tubules. Int J Dev Biol 1997; 41: 111-122). In each recipient's testis, the infusion accounted for 75-80% of the tubules. 50 μg of anti-CD4 antibody (GK1.5) was administered intraperitoneally to recipient mice at 0, 2, 4 days after transplantation to induce tolerance to heterogeneous donor cells (Kanatsu-Shinohara M Ogonuki, N, Inoue K, Ogura A, Toyokuni S, Honjo T, Shinohara T. Allogeneic offspring produced by male germ line stem cell transplantation into infertile mouse testis. Biol Reprod 2003; 68: 167-173).
To count colonies, recipient mouse testes were collected 2 months after donor cell transplantation and analyzed by observing fluorescence under UV light (Okabe M, Ikawa M, Kominami K, Nakanishi T, Nishimune). Y. 'Green mice' as a source of ubiquitous green cells. FEBS Lett 1997; 407: 313-319). Donor germ cells were specifically identified because host testis cells do not have intrinsic fluorescence. A germ cell cluster is defined as a colony if it occupies the entire circumference of the tubule and has a length of at least 0.1 mm (Nagano M, Avarbock MR, Brinster RL. Pattern and kinetics of mouse donor spermatogonial stem cell colonization in recipient testes. Biol Reprod 1999; 60: 1429-1436). All animal experiment protocols were approved by the Kyoto University Animal Protection and Use System Committee.

(5) Micro-insemination The seminiferous tubule of the recipient testis was carefully dissected and the germ cells were mechanically recovered. Microinsemination was performed as previously described (Kimura Y, Yanagimachi R. Intracytoplasmic sperm injection in the mouse. Biol Reprod 1995; 52: 709-720). Embryos that reached the 4-cell stage after 24 hours of culture were transferred to the fallopian tubes of day-1 pseudopregnant ICR female mice. Growing fetuses removed on the 19.5th day were raised by lactating ICR foster mice.

[2] Results (1) Serum-free culture of GS cells In order to establish a serum-free culture system of spermatogonial stem cells, the inventors of the present application first determine whether serum is required to establish GS cells. (Fig. 1A, upper left). Testicular cells have been established by the establishment protocol [Kanatsu-Shinohara M, Ogonuki N, Inoue K, Miki H, Ogura A, Toyokuni S, Shinohara T. Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol Reprod 2003; 69: 612-616] prepared from neonatal Green mice and cultured in gelatin-coated plates. The medium was supplemented with EGF, bFGF, LIF, and GDNF. In the absence of serum, few cells attached to the plate, most of which were suspended in the medium. The cells occasionally formed aggregates, but no obvious germ cell proliferation was observed. However, in the presence of serum, some of the cells attached to the gelatin-coated plate by the next day and could be recovered by vigorously pipetting the floating cells. These cells were relatively rich in gonocytes and were transferred to new culture plates for further culture. When these cells were cultured in low concentrations (0.3-2%) of serum, fibroblastic somatic cells started to grow on the culture plate by 2-3 days after the start of culture. The majority of germ cells attached to somatic cells and began to form germ cell colonies by 5-7 days. In contrast, when high concentrations (5-15%) of serum were added to the medium, germ cells proliferated further on somatic cells and formed colonies. However, these colonies eventually disappeared due to the massive proliferation of fibroblasts. These results indicate that, in order to establish GS cells, it is desirable to add serum to the medium, but the presence of a high concentration of serum suppresses the growth of germ cell colonies.

  The inventors then determined whether the established GS cells require serum for in vitro growth. EGFP-expressing GS cells were established from neonatal Green mouse testis in the presence of 1% serum. When the cultured cells became confluent, they were passaged (diluted 1-1 / 2) to a new plate. Within 3-4 weeks after the start of culture, few fibroblastic somatic cells were present with repeated passages, and GS cells were transferred to mitomycin C-treated MEF for further expansion. After 63 days from the start of culture, the present inventors transferred EGFP-expressing GS cells to serum-free culture. In this culture, the inventors removed serum from the medium and supplemented the medium with B27 to increase stem cell proliferation. When transferred to MEF, GS cells attached to MEF and these cells maintained the characteristic morphology of GS cells (FIG. 1A, middle top). Similar to GS cells in serum-containing cultures, these cells were passaged every 4-6 days under serum-free conditions. However, cell growth was even more dramatic in serum-free conditions; GS cells grew approximately 5-fold after 5 days in medium containing 1% serum, but up to 12-fold in the same culture period in serum-free medium Proliferated. Flow cytometric analysis showed no significant change in cell surface marker expression due to serum removal: GS cells were expressed in EpCAM [Anderson R, Schaible K, Heasman J, Wylie CC. Expression of the homophilic adhesion molecule, J Reprod Fertil. 1999; 116: 379-384], CD9 [Kanatsu-Shinohara M, Toyokuni S, Shinohara T. CD9 is a surface marker on mouse and rat male germline stem cells. Biol Reprod 2004; 70: 70-75], α6-integrin and β-1 integrin [Shinohara T, Avarbock MR, Brinster RL. Β1- and α6-integrin are surface markers on mouse spermatogonial stem cells. Proc Natl Acad Sci USA 1999; 96: 5504-5509]; these cells are c-kit [Yoshinaga K, Nishikawa S, Ogawa M, Hayashi SI, Kunisada T, Fujimoto T, Nishikawa SI. Role of c-kit in mouse spermatogenesis: identification of spermatogonia as a specific site of c-kit expressi on and function. Development 1991; 113: 689-699], and SSEA-1 [Solter D, Knowles BB. Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA-1). Proc Natl Acad Sci USA 1978; 75: 5565-5569] (FIG. 2A). RT-PCR analysis shows that cultured cells may be other primordial germ cell (PGC) markers or spermatogonia markers (including Oct-4) [Pesce M, Scholer HR. Oct-4: gatekeeper in the beginnings of mammalian development. Stem Cells 2001; 19: 271-278] were expressed (FIG. 2B).

  In order to test whether cultured cells have the ability to cluster seminiferous tubules, the inventors of the present invention have introduced a spermatogonia transplant technique [Brinster RL, Zimmermann JW. Spermatogenesis following male germ-cell transplantation. Proc Natl Acad Sci USA 1994; 91: 11298-11302] was used. This technique allows competent spermatogonial stem cells to repopulate the empty seminiferous tubules of infertile animals and differentiate into mature sperm. EGFP-expressing GS cultured in serum-free culture for 6 to 25 days was immunosuppressed W mice [Kanatsu-Shinohara M, Ogonuki, N, Inoue K, Ogura A, Toyokuni S, Honjo T, Shinohara T. Allogeneic offspring It was transplanted into the seminiferous tubule of produced by male germ line stem cell transplantation into infertile mouse testis. Biol Reprod 2003; 68: 167-173] (FIG. 3A). W is the result of mutation of the c-kit gene [Geissler EN, Ryan MA, Housman DE. The dominant white spotting (W) locus of the mouse encodes the c-kit proto-oncogene. Cell 1988; 55: 185-192] It is deficient in differentiating germ cells (FIG. 3B); therefore, all spermatogenesis in the recipient testis is derived from cultured donor cells. After various numbers of passages, the cells were re-implanted and the increase in the number of stem cells during this period was measured. Two months after transplantation, recipient mice were sacrificed and colonies in the testes were counted under UV light irradiation. The results are shown in Table 1.

  Table 1 shows the proliferation of spermatogonial stem cells in serum-free culture. The values in the table are mean ± standard error. Results from at least 4 recipient testes for each transplant. a is the number of days from the start of serum-free culture to transplantation. b is the increase in total cell number or stem cell number between the two time points. In addition, the value of the increase in the number of cells and the number of stem cells is the value when the value after 6 days from the start of serum-free culture is 1.

As shown in Table 1, proliferation of about 6.5 × 10 ⁷ times the number of stem cells was observed during 46 days in culture (from 6 to 52 days after the start of culture). During this time, the total cell number increased by 8.5 × 10 ⁷ times (FIG. 1B). Assuming that the transplant colony survival rate is 10% [Nagano M, Avarbock MR, Brinster RL. Pattern and kinetics of mouse donor spermatogonial stem cell colonization in recipient testes. Biol Reprod 1999; 60: 1429-1436], in culture The stem cell concentration ranged from 6.6 × 10 ^{−3 to} 1.3 × 10 ⁻² . Histological analysis of the recipient's testis confirmed the presence of spermatogenesis that appeared normal (FIG. 3C). Mature spermatogenic cells were observed in the seminiferous tubules. The cells continued to grow in serum-free conditions for at least 3 months. These results indicate that GS cells can grow in serum-free conditions and that these cells have the ability to cause normal spermatogenesis when transferred into the seminiferous tubule environment.

(2) Non-feeder culture of GS cells Next, the present inventors investigated the role of feeder cells in GS cell proliferation. To test whether laminin can replace MEF in supporting GS cell proliferation, EGFP-expressing GS cells cultured on MEF for 60 days were transferred onto laminin.
In this experiment, the inventors tested various combinations of cytokines, and in some experiments also tested the effect of conditioned media from MEF feeder cells. Because it is undifferentiated under feeder-free medium conditions, it is essential for human ES cells [Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, Carpenter MK. Feeder -Free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 2001; 19: 971-974]. Table 2 shows the results.

  Table 2 shows the proliferation of spermatogonial stem cells in feeder-free culture. The values in the table are mean ± standard error. Show results from at least 4 recipient testes. E represents EGF, F represents bFGF, G represents GDNF, L represents LIF, and CM represents conditioned medium. a is the number of days from the start of feeder-free culture to transplantation. b is the increase in total cell number or stem cell number between the two time points. In addition, the value of increase in the number of cells and the number of stem cells in Test 1 is the value when the value on the 31st from the start of feeder-free culture is 1.

  As shown in Table 2, GS cells were able to grow under all five conditions tested. These cells continued to grow as long as the medium contained GDNF and either EGF or bFGF was sufficient to promote GS cell growth in medium containing 1% FCS. However, the cells could not grow in the absence of GDNF, even though the medium was supplemented with both EGF and bFGF. Only after replenishment of GDNF cells resumed growth. The inventors have not found a specific effect of the culture supernatant of LIF or MEF on GS cell proliferation. In feeder-free culture, it was preferable to add serum to the medium. This is because GS cells did not adhere to laminin when cultured in serum-free medium.

  When transferred to laminin, GS cells gradually changed their morphology. GS cells generally formed clumps on MEF feeder cells (FIG. 1A, upper left), but these cells formed various patterns on the laminin, including clumps (FIG. 1A, upper right), and fibroblast-like (Fig. 1A, lower left). Occasionally cells formed chains or formed a network (FIG. 1A, middle bottom). This was similar to the spermatogonial stem cell proliferation pattern observed in vivo after spermatogonia transplantation [Nagano M, Avarbock MR, Brinster RL. Pattern and kinetics of mouse donor spermatogonial stem cell colonization in recipient testes. Reprod 1999; 60: 1429-1436]. Cell morphology in feeder-free culture appeared to depend on cell density. In general, cells tend to form clumps at a low cell density, but at high cell densities, they become fibroblasts. When seeded at a relatively high density, GS cells grew and dispersed at the bottom of the well and covered the entire surface of the well (FIG. 1A, lower right).

  To confirm the phenotype, cultured cells were analyzed for expression of cell surface markers using flow cytometry (FIG. 2A). Cells on laminin had a phenotype similar to GS cells on MEF; however, not only was c-kit expression decreased, but SSEA-1 expression was initiated. GS cells on MEF do not express SSEA-1 [Kanatsu-Shinohara M, Ogonuki N, Inoue K, Miki H, Ogura A, Toyokuni S, Shinohara T. Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol Reprod 2003; 69: 612-616], whereas a significant proportion of cells on laminin expressed this molecule. Regardless of the type of cytokine, there was no significant difference in cell surface marker expression (data not shown). RT-PCR analysis shows that cultured cells not only express PGC markers (Fragilis, Stella) but also spermatogonia markers (ngn3) [Yoshida S, Takakura A, Ohbo K, Abe K, Wakabayashi J, Yamamoto M, Suda. T, Nabeshima Y. Neurogenin3 delineates the earliest stages of spermatogenesis in the mouse testis. Dev Biol 2004; 269: 447-458, Saito M, Barton SC, Surani MA.A molecular program for the specification of germ cell fate in mice. 2002; 418: 293-300] was also expressed (FIG. 2B). Since SSEA-1 is expressed on PGC, but not normally on spermatogonia, this indicates that feeder-free culture changes cell surface marker expression and a partial embryonic phenotype. Suggested to induce.

EGFP-expressing GS cells cultured for various periods (range 9-39 days) to test whether cultured cells still retain the ability to cluster seminiferous tubules and produce spermatogenesis Was recovered using trypsin and microinjected into the seminiferous tubules of immunosuppressed W mice (FIG. 3D). Cells were harvested again 35-186 days after passage 4 to 31 and the increase in the number of stem cells during this period was measured. At least two different cultures with the same cytokine combination were transplanted. Analysis of the number of colonies in the recipient's testis showed that the number of stem cells increased in all five experiments, regardless of the cytokine combination. Assuming that the colony formation efficiency of stem cells is 10% [Nagano M, Avarbock MR, Brinster RL. Pattern and kinetics of mouse donor spermatogonial stem cell colonization in recipient testes. Biol Reprod 1999; 60: 1429-1436] The range was 4.3 × 10 ^{−4 to} 1.3 × 10 ⁻² . The total cell number increased approximately 1.2 × 10 ⁹ fold during the 6 month culture period (FIG. 1B). Tissue sections confirmed the presence of spermatogenesis in the recipient's testis and all stages of spermatogenic cells were found (FIG. 3E).

  In order to further confirm that these germ cells are functionally normal, the inventors of the present invention have used microinsemination (a technique commonly used to derive offspring from infertile animals and humans [Kimura Y, Yanagimachi R Intracytoplasmic sperm injection in the mouse. Biol Reprod 1995; 52: 709-720, Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992; 340: 17-18 ]) To try to make offspring from the cultured cells. EGFP-expressing GS cells were cultured for 4 months under feeder-free conditions and transplanted into 3 immunosuppressed W mice. Two recipients were sacrificed 4 months after transplantation. Their testis were mechanically isolated and live spermatogenic cells were recovered by repeated pipetting of the clustered tube segments; EGFP expression was confirmed under UV light. The cell suspension was frozen and stored in liquid nitrogen. After 21 days of storage, mature sperm or elongated spermatide were microinjected into oocytes from C57BL / 6 × DBA / 2 F1 mice. A total of 83 eggs were constructed and 56 eggs grown to the 2-cell stage were transferred to 5 pseudopregnant female recipients the day after microinsemination. These recipient females gave a total of 11 offspring and 9 of these grew to adulthood (4 males and 5 females). These offspring were fertile and were able to transfer the EGFP gene to the next generation (FIG. 3F). These results therefore indicate that GS cells in feeder-free culture retain the ability to colonize seminiferous tubules and can differentiate normally and produce fertile offspring.

[3] Discussion In Example 1, the present inventors have shown that spermatogonial stem cells can proliferate in vitro in the complete absence of serum or somatic feeder cells. Serum and feeder cells contain many undefined components that mask the effects of added substances and affect stem cell differentiation (Barnes D, Sato G. Serum-free culture: a unifying approach. Cell ; 1980: 22: 649-655). The inventors used serum in previous studies (Kanatsu-Shinohara M, Ogonuki N, Inoue K, Miki H, Ogura A, Toyokuni S, Shinohara T. Long-term proliferation in culture and germline transmission of mouse male. germline stem cells. Biol Reprod 2003; 69: 612-616). This is because it is preferable to add serum to the medium for efficient induction of early development of GS cells from newborn male gonocytes. Many attempts have been made to culture spermatogonial stem cells under serum-free conditions (Kubota H, Avarbock MR, Brinster RL. Culture conditions and single growth factors affect fate determination of mouse spermatogonial stem cells. Biol Reprod 2004; 71: 722-731, Dirami G, Ravindranath N, Pursel V, Dym M. Effects of stem cell factor and granulocyte macrophage-colony stimulating factor on survival of porcine type A spermatogonia cultured in KSOM. Biol Reprod 1999; 61: 225-230 , Creamers LB, den Ouden K, van Pelt AMM, de Rooij DG.Maintenance of adult mouse type A spermatogonia in vitro: influence of serum and growth factors and comparison with prepubertal spermatogonial cell culture.Reproduction 2002; 124: 791-799), It was impossible to induce long-term proliferation of spermatogonial stem cells. In Example 1, the inventors have found that the initiation and maintenance of GS cell culture appears to be a different process and that no serum is required to maintain a fully established GS cell. If GS cell culture begins with neonatal male gonocytes and the established GS cells have spermatogonia characteristics (Kanatsu-Shinohara M, Ogonuki N, Inoue K, Miki H, Ogura A , Toyokuni S, Shinohara T. Long-term proliferation in culture and germline transmission of mouse male germline stem cells. Biol Reprod 2003; 69: 612-616), male germ cells (gonocytes) It seems that some characteristic is acquired, and some component in the serum is thought to cause this conversion. The development of spermatogonia from male gonocytes probably involves a number of differentiation stages, which are reflected in changes in phenotypic and stem cell surface markers (Ryu BY, Orwig KE, Kubota H, Avarbock MR, Brinster RL. Phenotypic and functional characteristics of male germline stem cells in rats.Dev Biol 2004; 274: 158-170, Detin L, Ravindranas N, Hofmann MC, Dym M. Morphological characterization of the spermatogonial subtypes in neonatal mouse testis Biol Reprod 2003; 69: 1565-1571, McLean DJ, Friel PJ, Johnston DS, Griswold MD. Characterization of spermatogonial stem cell maturation and differentiation in neonatal mice. Biol Reprod 2003; 69: 2085-2091). Recent studies have also shown that prepubertal germ cells are twice as viable in vitro (Creemers LB, den Ouden K, van Pelt AMM, de Rooij DG. Maintenance of adult mouse type A spermatogonia in in vitro: influence of serum and growth factors and comparison with prepubertal spermatogonial cell culture. Reproduction 2002; 124: 791-799). Although further research is needed to determine factors that affect the conversion of male gonocytes to spermatogonia or GS cells, serum-free culture of established GS cells is the growth of spermatogonial stem cells. And may be useful to characterize the mechanisms that control differentiation. Importantly, the results of Example 1 showed that GS cell growth was stimulated even under serum-free conditions. Consistent with other stem cell culture reports, serum-free conditions are improved culture conditions for spermatogonial stem cells that not only increase our understanding of the GS cell self-renewal process, but also increase the efficiency of GS cells. Will be possible.

  Stem cells usually require stromal cells to maintain an undifferentiated state in vivo (Spradling A, Drummond-Barbosa D, Kai T. Stem cells find their niche. Nature 2001; 414: 98-104). Stromal cells also enhance stem cell survival in vitro (Spradling A, Drummond-Barbosa D, Kai T. Stem cells find their niche. Nature 2001; 414: 98-104); classical studies with hematopoietic stem cells Have shown that stem cell activity is rapidly lost without bone marrow feeder (Dexter ™, Spooncer E. Growth and differentiation in the hematopoietic system. Annu Rev Cell Biol 1987; 3: 423-441). Similarly, PGC requires a fibroblast feeder for survival or conversion to EG cells (Matsui Y, Zsebo K, Hogan BLM. Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 1992; 70: 841-847, Resnick JL, Bixler LS, Cheng L, Donovan PJ. Long-term proliferation of mouse primordial germ cells in culture. Nature 1992; 359: 550-551). In spermatogonial stem cell culture, mouse spermatogonial stem cells can survive in vitro on STO feeders for approximately 4 months, but were initially reported to disappear within one week when cultured directly on culture dishes (Nagano M, Avarbock MR, Leonida EB, Brinster CJ, Brinster RL. Culture of mouse spermatogonial stem cells. Tissue Cell 1998; 30: 389-397), which requires a feeder layer to maintain spermatogonial stem cells in vitro showed that. However, subsequent studies revealed that even on STO feeders, the number of spermatogonial stem cells in the culture was reduced and only 10-20% of the stem cells could be recovered after 1 week in vitro. (Nagano M, Ryu BY, Brinster CJ, Avarbock MR, Brinster RL. Maintenance of mouse male germ line stem cells in vitro. Biol Reprod 2003; 68: 2207-2214). Similar results were recently reported for rats (Orwig KE, Avarbock MR, Brinster RL. Retrovirus-mediated modification of male germline stem cells in rats. Biol Reprod 2002; 67: 874-879, Kent Hamra F, Schultz N, Chapman KM, Grellhesl DM, Cronkhite JT, Hammer RE, Garbers DL. Defining the spermatogonial stem cell. Dev Biol 2004; 269: 393-410). Feeder cell type affects stem cell survival; rat spermatogonial stem cells have been reported to survive longer on Sertoli cells than on STO cells, but have been reported (Kent Hamra F, Schultz N, Chapman KM, Grellhesl DM, Cronkhite JT, Hammer RE, Garbers DL. Defining the spermatogonial stem cell. Dev Biol 2004; 269: 393-410), in other studies, Sertoli cells have a negative effect on spermatogonial stem cells in mice (Nagano M, Ryu BY, Brinster CJ, Avarbock MR, Brinster RL. Maintenance of mouse male germ line stem cells in vitro. Biol Reprod 2003; 68: 2207-2214). Although these culture conditions cannot induce long-term proliferation of spermatogonial stem cells, the development of GS cell culture conditions provides an opportunity to assess the need for feeder cells in the self-renewal division of spermatogonial stem cells in vitro. provide. The results of Example 1 demonstrate that laminin can be used in place of the MEF feeder layer, suggesting that the feeder layer is not essential for induction of self-renewal division of spermatogonial stem cells.

  Interestingly, we notice that there are many differences between feeder-free culture conditions and MEF-based culture. For example, GS cells on laminin tend to form various types of colonies, ranging from chain to clump, whereas GS cells on MEF usually form clump. . The formation of chain colonies reminds us of spermatogenic colonies after spermatogonia transplantation into the seminiferous tubule environment (Nagano M, Avarbock MR, Brinster RL. Pattern and kinetics of mouse donor spermatogonial stem cell colonization in recipient testes. Biol Reprod 1999; 60: 1429-1436). Conversely, formation of clump-type colonies has also been observed in vivo when GDNF is overexpressed in Sertoli cells (Yomogida K, Yagura Y, Tadokoro Y, Nishimune Y. Dramatic expansion of germinal stem cells by ectopically). expressed human glial cell line-derived neurotrophic factor in mouse Sertoli cells. Biol Reprod 2003; 69: 1303-1307). GDNF is an essential factor for spermatogonial stem cell self-renewal division (Meng X, Lindahl M, Hyvonen ME, Parvinen M, de Rooij DG, Hess MW, Raatikainen-Ahokas A, Sainio K, Rauvala H, Lakso M , Pichel JG, Westphal H, Saarma M, Sariola H. Regulation of cell fate decision of undifferentiated spermatogonia by GDNF. Science 2000; 287: 1489-1493); overexpression of GDNF causes loss of germ cell differentiation and in vivo Induces mass formation and proliferation of spermatogonial stem cells (Yomogida K, Yagura Y, Tadokoro Y, Nishimune Y. Dramatic expansion of germinal stem cells by ectopically expressed human glial cell line-derived neurotrophic factor in mouse Sertoli cells. Biol Reprod 2003 ; 69: 1303-1307). These observations suggested that colony morphology was dependent on the amount of GDNF, spermatogonial stem cells normally proliferated with chain formation, and formed a mass when stem cell proliferation was strongly stimulated. In Example 1, GDNF was used at the same concentration throughout the experiment, so the results indicate that the MEF feeder cells either enhance the effect of GDNF or stimulate some other molecule that stimulates spermatogonial stem cell proliferation. It suggests that it produces a positive effect by secreting. This is supported by the observation that GS cell growth on laminin is slower than that when using the MEF system. Thus, cell-cell interactions between stem cells and MEFs provided additional growth stimuli that may have manifested as changes in colony morphology.

Another unexpected feature of feeder-free culture was a change in surface marker expression. In particular, the expression of SSEA-1 was unexpected. SSEA-1 is expressed on early embryos and PGCs, but its expression disappears after midgestation (Cooke JE, Godin I, French-Constant C, Heasman J, Wylie C. Culture and manipulation of primordial germ cells. In: Wassarman, PM, DePamphilis, ML (eds.), Guide to techniques in mouse development, San Diego: Academic Press; 1993: 37-58). At the same time, however, GS cells on laminin are markers of undifferentiated spermatogonia in the postnatal testis (Yoshida S, Takakura A, Ohbo K, Abe K, Wakabayashi J, Yamamoto M, Suda T, Nabeshima Y. Neurogenin3 Dev Biol 2004; 269: 447-458) ngn3 was also expressed. delineates the earliest stages of spermatogenesis in the mouse testis. The proportion of cells expressing c-kit also decreased in feeder-free culture. Therefore, the pattern of gene expression in GS cells is affected by the culture environment. It is unclear why GS cells begin to express such embryonic markers in feeder-free culture. Although this cannot exclude the possibility that it reflects an abnormal response of spermatogonial stem cells to a non-physiological environment, there is a rare population of spermatogonial stem cells that express SSEA-1 in vivo. obtain. Stem cells are extremely not only there just ⁽¹⁰⁴ testis cells 2 to 3 only) (Meistrich ML, van Beek MEAB Spermatogonial stem cells In:... Desjardins, C, Ewing, LL (eds), Cell and Molecular Biology of the Testis, New York: Oxford University Press; 1993: 266-295, Tegelenbosch RAJ, de Rooij DG.A quantitative study of spermatogonial multiplication and stem cell renewal in the C3H / 101 F1 hybrid mouse.Mutation Res 1993; 290: 193-200), such a rare population may escape detection using normal immunohistological methods in testis sections. Recent studies also suggest heterogeneity of spermatogonial stem cell populations (Tadokoro Y, Yomogida K, Ohta H, Tohada A, Nishimune Y. Homeostatic regulation of germinal stem cell proliferation by the GDNF / FSH pathway. Mech Dev 2002 ; 113: 29-39). Growth of such populations is inhibited in vivo by somatic cells and also by MEF feeders in vitro, but culture under feeder-free conditions may have changed its relative frequency in the cell population. Alternatively, perhaps GS cells have the ability to turn into cells with more primitive features. It is well known that PGC can be transformed into ES-like cells (EG cells) when cultured under special conditions. EG cells have been derived exclusively from 8.5 to 12.5 days (dpc) PGC after sexual intercourse (Matsui Y, Zsebo K, Hogan BLM. Derivation of pluripotential embryonic stem cells from murine primordial germ cells in culture. Cell 1992; 70: 841-847., Resnick JL, Bixler LS, Cheng L, Donovan PJ. Long-term proliferation of mouse primordial germ cells in culture. Nature 1992; 359: 550-551). May hold the characteristics of. GS cells are different from EG cells in that they are derived from postnatal male germocytes and are destined for the germline. However, as the results of Example 1 show, modification of the culture conditions can induce stem cell properties in GS cells and may allow the cells to acquire more primitive features.

  Despite the different colony morphology and surface marker expression, GS cells on laminin retain the ability to produce spermatogenic colonies and offspring upon transplantation into infertile mice. If the ability of self-renewal is defined as a characteristic of stem cells, the results indicate that cultured cells have stem cell activity as spermatogonial stem cells. Recent studies have reported the behavior of embryonic germline cells in mature seminiferous tubules (Ohta H, Wakayama T, Nishimune Y. Commitment of fetal male germ cells to spermatogonial stem cells during mouse embryonic development. Biol Reprod 2004; 1286-1291): According to this, PGCs from 12.5 dpc embryos cannot produce spermatogenic colonies, but male germ cells after 14.5 dpc collect tubules and spermatogenic colonies Can be produced. Based on these results, PGC may be destined for spermatogonia between 12.5 and 14.5 dpc. In Example 1, SSEA-1 expression suggested fetal traits of GS cells, but spermatogenesis occurred efficiently in transplant recipients, indicating that GS cells on laminin were not affected by SSEA-1 expression. , Functionally equivalent to spermatogonial stem cells. In summary, the results of Example 1 show that self-renewal of spermatogonial stem cells does not require direct contact with somatic stromal cells, and cytokines (growth cofactors) and laminin are at least some of the Nische functions. It proves that it can replace the situation.

  Laminin could replace MEF in serum-containing cultures, but it was difficult to efficiently grow GS cells on laminin under serum-free conditions. It is unclear what factor in serum promotes GS cell adhesion to laminin. GS cells were able to adhere to MEF in the same serum-free medium, but adhesion to laminin was reduced: therefore, MEF probably expresses other molecules that mediate GS cell attachment or serum Can substitute for MEF by providing the molecule or an alternative molecule. MEF cells secrete a variety of extracellular matrix molecules, including laminin, type IV collagen and fibronectin (Xu C, Inokuma MS, Denham J, Golds K, Kundu P, Gold JD, Carpenter MK. Feeder). -Free growth of undifferentiated human embryonic stem cells. Nat Biotechnol 2001; 19: 971-974). Although the composition of the basement membrane is significantly different in different tissues, only laminin may not provide the best microenvironment for spermatogonial stem cells. For example, genetic studies in Drosphila have demonstrated an important role for E-cadherin in keeping differentiated or stem cells tethered to the niche (Yamashita YM., Leanne Jones D., Fuller MT. Orientation of asymmetric stem cell division by the APC tumor suppressor and centrosome. Science 2003; 301: 1547-1550). The cadherin molecule is also expressed on PGC and spermatogonia in mice (Bendel-Stenzel MR, Gomperts M, Anderson R, Heasman J, Wylie C. The role of cadherins during primordial germ cell migration and early gonad formation in the mouse. Mech Dev 2000; 91: 143-152, Wu JC, Gregory CW, DePhilip RM.Expression of E-cadherin in immature rat and mouse testis and in rat Sertoli cell cultures.Biol Reprod 1993; 49: 1353-1361), The molecule may play a similar role in mammalian spermatogonial stem cell biology. Adhesion molecules deliver important signals for stem cells (Gonzalez-Reyes A. Stem cells, niches and cadherins: a view from Drosophila. J Cell Sci 2003; 116: 949-954). It is important to identify other candidate molecules that are included, which can help to further refine the growth methods of the present invention.

  The present invention provides a first step to reproduce the self-renewal division of spermatogonial stem cells under defined conditions in vitro, but allows for a more detailed analysis of self-renewal division and its involvement in the niche In addition, it is desirable to develop a more improved culture system. For example, it may be important to establish culture conditions that lead to specific types of self-renewal, such as asymmetric division and self-renewal division (Meistrich ML, van Beek MEAB. Spermatogonial stem cells. In: Desjardins, C, Ewing , LL (eds.), Cell and Molecular Biology of the Testis, New York: Oxford University Press; 1993: 266-295). In this sense, the growth method of the present invention is extremely useful because the serum-free culture system and feeder-free culture system allow a more definitive test for evaluating the influence of individual factors on stem cells. The identification of decisive factors increases our knowledge of how spermatogonial stem cell self-renewal division is controlled and enables us to develop more efficient techniques for male germline modification. Will bring.

(Reference Example 1)
[1] Experimental methods and materials (1) Animals Testis cells are DBA / 2 mice, or transgenic mouse strain C57BL6 / Tg14 (act-EGFP-Osby01) crossed to DBA / 2 background (hereinafter referred to as Green mice). From newborn pups (0 days of age), pups (5 days of age) or adults (4-5 weeks of age), which may be called, were collected by two-stage enzymatic degradation and used for culture.
The cultured cells were transplanted into W mice (4 weeks old, manufactured by Japan SLC). In experiments using W recipients, 50 μg of anti-CD4 antibody (GK1.5) was administered intraperitoneally at 0, 2, 4 days after transplantation to induce tolerance to allogeneic donor cells. All animal experiment protocols were approved by the Kyoto University Animal Protection and Use System Committee.

(2) Culture conditions StemPro-34 SFM (manufactured by Invitrogen) medium supplemented with the same additives as in Example 1 was used as the basal culture medium. GDNF, EGF, bFGF, LIF and fetal calf serum were the same as in Example 1. GDNF and fetal bovine serum were always added to the medium at concentrations of 10 ng / ml and 1 (v / v)%, respectively, unless otherwise noted. EGF, bFGF and LIF may Except where otherwise specified, each, 20 ng / ml, were added to the appropriate medium at a concentration of ^{10ng / ml, 10 3 units /} ml.
The cells were maintained at 37 ° C. in air containing 5% carbon dioxide.

(3) Collection of spermatogonial stem cells Testicular cells were collected by two-stage enzymatic degradation using collagenase (type IV, Sigma) and trypsin (Invitrogen).
Specifically, the mouse testis was removed, the white membrane was removed in PBS, and incubated for 15 minutes at 37 ° C in a Hanks solution containing 1 mg / ml collagenase (type I) to loosen the seminiferous tubule. It was. After removing stromal cells separated by washing twice with PBS, the cells were incubated in a 0.25% trypsin solution containing 1.4 mg / ml DNase at 37 ° C. for 15 minutes with appropriate shaking. Was broken apart. PBS was added to inactivate trypsin and then pipetting was performed to obtain a cell suspension.

When a newborn testis was used, the cell suspension was used as it was for culturing.
On the other hand, when a testicle of a pup or adult is used, the cell suspension is passed through a 20-30 μm nylon mesh to remove undigested cell mass, and centrifuged at 600 × g for 5 minutes to collect testicular cells. From the testis cells, spermatogonial stem cells were concentrated as CD9-positive cells by a magnetic bead method using an anti-CD9 antibody.

  The collected spermatogonial stem cells were dispersed in the medium and transferred onto gelatin-coated plates or mouse embryo fibroblasts (MEF) inactivated with mitomycin C (first culture).

  After overnight culture from the start of the first culture, the cultured cells in the first culture, preferably the suspended cultured cells (including not only spermatogonial stem cells), were subcultured to another cell culture container ( Second culture). Although the passaged cells differed depending on the culture conditions, colonies formed on the bottom of the cell culture vessel usually within one week after the passage.

  Five to 14 days after the start of the second culture, the cells were dispersed by trypsin treatment or the like, resuspended in the medium, and further subcultured to a new culture plate (third culture). Similarly, by repeating the passage, the flat somatic cells disappeared. After the second or third passage, the cells were cultured in the presence of feeder cells. Cells were passaged every 2-5 days at a dilution of 1-1 / 4 (preferably a dilution of 1-1 / 2 at the beginning of the culture). By 3-4 weeks, the cells settled in a relatively stable state. Thereafter, the cells were passaged at 1 / 4-1 / 10 dilution every 2-5 days.

  In some experiments, spermatogonial stem cells (GS cells) obtained by culture were cultured in a medium containing only GDNF as a growth cofactor and not containing LIF, EGF, and bFGF.

(4) Antibody Staining In order to confirm the properties of cells cultured under the above conditions, flow cytometry for examining expression of molecular markers on spermatogenic cells known in the art was performed as follows.
As the primary antibody, in addition to the antibody used in Example 1, rat anti-mouse CD9 antibody (BD Bioscience) and rat anti-TDA antigen (EE2) (provided by Dr. Nishimune, Osaka University) were used. As the secondary antibody, the same antibody as in Example 1 was used. Cell staining and analysis were performed as in Example 1.

(5) Transplantation of cultured spermatogonial stem cells About 2 μl of donor cell suspension containing spermatogonial stem cells obtained by the above culture method was introduced into the testes of W mice through an export tube. In each recipient's testis, the infusion accounted for 75-80% of the tubules. Adult recipient mice were anesthetized with Avertin Inxon (640 mg / kg).
To count colonies, the recipient mice were analyzed by removing the recipient testes and observing fluorescence under ultraviolet light 7-8 weeks after donor cell transplantation. Donor cells were clearly identified because host testis cells do not have intrinsic fluorescence. A population of cells was defined as a colony if it occupied the entire perimeter of the seminiferous tubule and had a length of at least 0.1 mm.

(6) Histological analysis Testes of newborn (0 day old), offspring (5 day old) and adult (4-5 week old) mice were collected, and tissue sections were prepared by a conventional method. The sections were stained with nuclear fast red and observed under a microscope.

[2] Results (1) Proliferation of spermatogonial stem cells in vitro The spermatogonial stem cells derived from newborn mice (0-day-old) do not proliferate even when cultured with feeder cells in GDNF, EGF and bFGF-free medium. Although it was not confirmed and a colony was not formed (see the upper left of FIG. 4), significant growth was observed in the medium containing GDNF, EGF, bFGF and LIF, and a colony was formed (see the upper right of FIG. 4).
On the other hand, spermatogonial stem cells derived from offspring (5-day-old) mice were observed to proliferate significantly when using any of GDNF, EGF and bFGF-containing LIF-free medium and GDNF, EGF, bFGF and LIF-containing medium, Colonies were formed (FIG. 4, lower left and lower right).
In addition, spermatogonial stem cells derived from adult (4 to 5 week-old) mice can use any of GDNF, EGF and bFGF-containing LIF-free medium and GDNF, EGF, bFGF and LIF-containing medium, as in the case of pup mice. Significant growth was observed and colonies formed (data not shown).

The state of proliferation of the cultured cells (ie, spermatogonial stem cells) on the feeder cells in this experiment is shown in the graph of FIG. In this graph, the horizontal axis indicates the number of days elapsed from the start of culture, and the vertical axis indicates the relative number of cells. As shown in FIG. 5, spermatogonial stem cells derived from pups were confirmed to maintain cell growth on a log scale for about 4 months even when GDNF, EGF and bFGF-containing LIF-free medium was used. It was. Cell number four months from the start of the culture was grown to about 10 ⁹ times. Similar growth was confirmed when media containing GDNF, EGF, bFGF and LIF were used (data not shown).

  On the other hand, spermatogonial stem cells derived from newborn mice were confirmed to maintain cell growth on a log scale for 4 months when GDNF, EGF, bFGF and LIF-containing medium was used. Little cell growth was observed when EGF and bFGF containing LIF-free medium was used (data not shown).

  In addition, spermatogonial stem cells derived from newborn mice were cultured in a medium containing GDNF, EGF, bFGF and LIF for 5 to 15 days, and then the cultured cells were collected, and the cells were collected in a medium containing no LIF containing GDNF, EGF and bFGF. When the cells were co-cultured with feeder cells, cell growth continued for a long period of time in the same manner as spermatogonial stem cells derived from pups, and colonies formed (data not shown).

  Furthermore, the spermatogonial stem cells derived from the newborn mouse (0 day old) prepared above were transformed into a medium containing 15% FCS-containing GDNF-containing EGF, bFGF and LIF after 4 months from the start of the culture. Subsequently, proliferation was continuously maintained by co-culturing with feeder cells (FIG. 6). The number of spermatogonial stem cells was 5.97 ± 0.53 times (n = 3) 5 days after medium change, 38.90 ± 8.44 times (n = 3) 11 days (all values are averages) Value ± indicated by standard error).

From the above results, it was suggested that the following two types exist in the differentiation stage of spermatogonial stem cells.
(A) A differentiation stage (growth stage (A)) requiring glial cell-derived neurotrophic factor (GDNF) and leukemia inhibitory factor (LIF) as growth factors for in vitro growth.
(B) A differentiation stage (differentiation stage (B)) capable of growing in a medium containing substantially only glial cell-derived neurotrophic factor (GDNF) as a growth auxiliary factor for spermatogonial stem cells.

It was also revealed that spermatogonial stem cells of newborn animals are in the differentiation stage (A), and spermatogonial stem cells of offspring and adult animals are in the differentiation stage (B).
Furthermore, the spermatogonial stem cells in the differentiation stage (A) are differentiated into the differentiation stage (B) by culturing in a medium containing glial cell-derived neurotrophic factor (GDNF) and leukemia inhibitory factor (LIF). Was suggested.

(2) Properties of cultured cells Spermatogonia stem cells of pupal green mice (Green Mouse) are grown in GDNF, EGF and bFGF containing LIF-free medium in the presence of feeder cells, and the surface characteristics of the cells are determined by flow cytometry. Analyzed. The mouse GFP gene is expressed everywhere including spermatogonial stem cells. Therefore, cultured cells containing GFP can be distinguished from feeder cells by observation under UV irradiation.
The results are shown in FIG. Each figure shown in FIG. 7 shows the results of analysis using molecular markers such as α6-integrin, β1-integrin, EpCAM, EE2, c-kit, SSEA-1, CD9, and Forssman antigen in order from (a). is there. In addition, the white column enclosed by the black solid line is the result in the case of the control immunoglobulin, and the black column is the result in the case of each molecular marker (antibody).
The cultured cells were positive for α6-integrin, β1-integrin, EpCAM, EE2, and CD9.
Although many cells were negative for c-kit, weak expression was confirmed, suggesting that some colonies were differentiated.
The cultured cells were negative for SSEA-1 and Forssman antigen.
From these results, it was found that the majority of the proliferated cells had the properties of undifferentiated spermatogonia.

(3) Determination of stem cell activity by spermatogonia transplantation technique Based on the result of (2) above, spermatogonia transplantation was performed to confirm whether the cultured cells were really spermatogonial stem cells.
Green mouse spermatogonial stem cells were cultured for 29-58 days in LIF-free medium containing GDNF, EGF and bFGF in the presence of feeder cells, and the harvested cells were transferred to W mouse seminiferous tubules. Transplanted. Colonies in the transplantation experiments were confirmed by fluorescence under UV irradiation 7-8 weeks after transplantation.
The results are shown in FIG. Analysis of the W recipient testis obtained by this experiment demonstrated that vast colony formation was performed by cultured cells filled with numerous seminiferous tubules having apparently normal spermatogenesis (FIG. 8). As a result, it was confirmed that the cultured cells were spermatogonial stem cells.

(4) Histological analysis FIG. 9 shows the results of histological analysis of each mouse of a newborn (0 day old), offspring (5 day old), and adult (4-5 week old).
Although neonatal spermatogonial stem cells are present in the lumen of the seminiferous tubule (FIG. 9d), in 5-day-old pups, the spermatogonial stem cells are in contact with the basement membrane of the seminiferous tubule (FIG. 9e), In adults, it was observed that spermatogonial stem cells were firmly adhered to the basement membrane of the seminiferous tubule (FIG. 9f).
From the above, it was suggested that it is possible to determine in which differentiation stage the spermatogonial stem cell is in the above-described differentiation stage using the histological feature as an index.

By using the proliferation method of the present invention, it is possible to efficiently proliferate spermatogonial stem cells even in the absence of feeder cells and serum, and to eliminate the influence of undetermined factors. It is possible to proliferate spermatogonial stem cells in a more stable and simple manner while maintaining the ability as spermatogonial stem cells. Moreover, in the clinical application of spermatogonial stem cells, the risk of infectious diseases derived from different animals can be avoided. Therefore, the propagation method of the present invention includes the production of genetically modified animals (gene-transfected animals, gene-deficient animals, etc.), the development of treatment methods and treatment agents for male infertility, research and drugs for gene therapy at the germ cell level It is useful for the development of
Furthermore, since the proliferation method of the present invention does not include the influence of feeder cells and serum-derived uncertain factors, it enables a more definitive test for evaluating factors that control the proliferation and differentiation of spermatogonial stem cells, It provides a new opportunity to understand the regulatory mechanisms governing spermatogonial stem cells.

It is a figure which shows the in vitro growth of a GS cell. A is a photograph showing the morphology of cultured cells. The upper left panel shows GS cells on MEF in serum-containing medium, the upper center panel shows GS cells on MEF and serum-free medium, and the upper right panel shows GS cells on laminin. The lower left panel shows GS cells on laminin and round colonies are observed. The lower center panel shows GS cells on laminin and germline chain formation is observed. The lower right panel shows GS cells on laminin, and confluent GS cells are observed in the culture wells. B is a graph showing the proliferation of GS cells in a serum-free or feeder-free condition. The scale bar corresponds to 100 μm. It is a figure which shows the characteristic characterization of a cultured cell. A is a graph showing the results of testing the properties of cultured cells by flow cytometry using each molecular marker. The vertical axis represents the number of cells (cells), and the horizontal axis represents the relative fluorescence intensity. The white column shows a histogram when cells are stained without using a primary antibody (negative control), and the black column shows a histogram when cells are stained with a primary antibody. The upper (serum (−)) corresponds to serum-free culture, and the lower (feeder (−)) corresponds to feeder-free culture. In serum-free culture, only EGFP positive cells were gated to analyze GS cells. B is a diagram showing RT-PCR analysis. Specific primers were used to amplify cDNA from GS cells under serum-free (serum (−)) or feeder-free (feeder (−)) conditions. “MEF” corresponds to culture in the presence of serum and feeder. It is a photograph which shows spermatogenesis from a cultured cell and production of offspring. A shows a recipient testis that received EGFP-expressing donor GS cells from a serum-free culture. B shows the histological appearance of W mouse testis (non-transplanted control) and the absence of differentiated germ cells is observed. C shows the histological appearance of W mouse testis that received donor GS cells from serum-free culture, with normal appearance of spermatogenesis and extended spermatids. D shows recipient testis that received GFP-expressing donor GS cells from feeder-free culture. E shows the histological aspect of the recipient testis showing normal spermatogenesis. F shows F2 progeny due to GS cells from feeder-free culture. Green fluorescence under UV light establishes the presence of the donor transgene (arrow). No fluorescence is observed in control littermates (arrowheads). The scale bar corresponds to 1 mm (A, D), 100 μm (B, C), and 50 μm (E), respectively. In an Example, it is a figure which shows the result of having observed the development of the spermatogonial stem cell expansion colony from the testis cell of a mouse | mouth with the microscope. Upper left figure is culture of newborn mouse testis cells in LIF (-) medium, upper right figure is culture of newborn mouse testis cells in LIF (+) medium, lower left figure is LIF (-) of 5-day-old mouse testis cells Culture on medium, lower right figure shows culture of 5-day-old mouse testis cells in LIF (+) medium. The scale bar corresponds to 50 μm. It is a graph which shows the proliferation of the spermatogonial stem cell cultured in the Example, Specifically, the number of the spermatogonial stem cell which can be distinguished from a feeder cell by GFP label | marker is measured. The vertical axis represents the relative number of cells (times) when the number of cells at the start of culture is 1, and the horizontal axis represents the number of days after the start of culture. It is a graph which shows the proliferation of the spermatogonial stem cell in a 15% FCS containing GDNF containing EGF, bFGF, and LIF free medium. The vertical axis shows the relative number of cells (average value ± standard error) when the number of cells at the time of medium change is 1, and the horizontal axis shows the number of days after the medium change. (A)-(h) is a graph which shows the result of having tested the property of the cultured cell by flow cytometry in each Example using each molecular marker. The vertical axis represents the number of cells (cells), and the horizontal axis represents the relative fluorescence intensity. The white column shows a histogram when cells are stained without using a primary antibody (negative control), and the black column shows a histogram when cells are stained with a primary antibody. Each graph shown in (a) to (h) shows α6-integrin, β1-integrin, EpCAM, EE2, c-kit, SSEA-1, CD9, and Forssman antigen in order from (a) as molecular markers. This is the case. The percentage of cells in the gate relative to the total number of cells is (a) 95.91%, (b) 98.12%, (c) 96.75%, (d) 99.89%, (e) 12.74%, (f), respectively. 0.06%, (g) 99.84%, (h) 0.80%. It is a figure which shows the sperm formation from the spermatogonial stem cell after a transplant in an Example. Figure 5 shows GFP-labeled spermatogonial stem cell-derived spermatogenic colonies that fluoresce under UV irradiation. It is a figure which shows the macroscopic (upper stage (a)-(c)) and histological (lower stage (d)-(f)) external appearance of a testis. Newborn (0 day old), offspring (5 day old) and adult (4-5 week old) testis are shown on the left, center and right, respectively. As the newborn matures into adults, there are more stromal cells in the testis. Arrows in the lower inset indicate germ cells (spermatogonial stem cells). The upper scale bar corresponds to 2 mm and the lower scale bar corresponds to 20 μm.

Claims (39)

  A method of proliferating spermatogonial stem cells, comprising culturing spermatogonial stem cells in the absence of feeder cells in a state adhered to an insoluble carrier.   The method according to claim 1, wherein the spermatogonial stem cells adhere to an insoluble carrier via an extracellular matrix.   The method of claim 2, wherein the extracellular matrix is laminin.   The method according to claim 1, wherein the spermatogonial stem cells are cultured in a medium containing a GDNF receptor agonist compound.   The method of claim 4, wherein the medium further comprises LIF.   The method according to claim 4, wherein the medium further comprises at least one of EGF and bFGF.   The method according to claim 1, wherein the spermatogonial stem cells are cultured in a medium containing serum.   The method according to claim 1, wherein the spermatogonial stem cells are cultured in a medium not containing a conditioned medium of feeder cells.   2. The method of claim 1, wherein the expanded spermatogonial stem cells have an embryonic phenotype.   10. The method of claim 9, wherein the embryonic phenotype is positive expression of SSEA-1.   A method for growing spermatogonial stem cells, comprising culturing spermatogonial stem cells in a serum-free medium.   12. The method of claim 11, wherein the medium comprises a GDNF receptor agonist compound.   The method of claim 12, wherein the medium further comprises LIF.   The method according to claim 12, wherein the medium further comprises at least one of EGF and bFGF.   The method of claim 11, wherein the medium comprises B27.   The method according to claim 11, comprising culturing spermatogonial stem cells in the presence of feeder cells.   The method according to claim 1 or 11, wherein the spermatogonial stem cells to be cultured are in a differentiation stage capable of growing in a medium containing substantially only a GDNF receptor agonist compound as a growth cofactor for spermatogonial stem cells.   The method according to claim 17, wherein the spermatogonial stem cells to be cultured are in a differentiation stage in contact with the basement membrane of the seminiferous tubule.   For spermatogonial stem cells to be cultured, spermatogonial stem cells in a differentiation stage requiring GDNF receptor agonist compound and LIF as growth factors for in vitro growth are cultured in a medium containing GDNF receptor agonist compound and LIF. The method according to claim 17, which is obtained by   The method according to claim 17, wherein the spermatogonial stem cells are cultured in a medium containing a GDNF receptor agonist compound.   21. The method of claim 20, wherein the medium does not contain LIF.   21. The growth method according to claim 20, wherein the culture medium contains substantially only a GDNF receptor agonist compound as a growth cofactor for spermatogonial stem cells.   A spermatogonial stem cell grown in vitro using the method according to claim 1 or 11.   An infertility therapeutic agent comprising the spermatogonial stem cell according to claim 23. A method for producing a non-human animal that forms sperm derived from transplanted spermatogonial stem cells, comprising the following steps:
a) proliferating spermatogonial stem cells by culturing spermatogonial stem cells in the absence of feeder cells in a state of being attached to an insoluble carrier;
b) A step of transplanting the spermatogonial stem cells grown in a) into the seminiferous tubule of an infertile non-human animal to obtain a non-human animal that has caused spermatogenesis derived from the spermatogonial stem cell. A method for producing sperm comprising the following steps:
a) proliferating spermatogonial stem cells by culturing spermatogonial stem cells in the absence of feeder cells in a state of being attached to an insoluble carrier;
b) transplanting the spermatogonial stem cells grown in a) into the seminiferous tubule of an infertile non-human animal to obtain a non-human animal that has caused spermatogenesis derived from the spermatogonial stem cell;
c) A step of obtaining sperm from the non-human animal. A method for producing an embryo derived from a spermatogonial stem cell, comprising the following steps:
a) proliferating spermatogonial stem cells by culturing spermatogonial stem cells in the absence of feeder cells in a state of being attached to an insoluble carrier;
b) transplanting the spermatogonial stem cells grown in a) into the seminiferous tubule of an infertile non-human animal to obtain a non-human animal that has caused spermatogenesis derived from the spermatogonial stem cell;
c) obtaining sperm from said non-human animal;
d) A step of fertilizing the sperm into an egg to obtain an embryo. A method for producing non-human progeny derived from spermatogonial stem cells, comprising the following steps:
a) proliferating spermatogonial stem cells by culturing spermatogonial stem cells in the absence of feeder cells in a state of being attached to an insoluble carrier;
b) transplanting the spermatogonial stem cells grown in a) into the seminiferous tubule of an infertile non-human animal to obtain a non-human animal that has caused spermatogenesis derived from the spermatogonial stem cell;
c) obtaining sperm from said non-human animal;
d) fertilizing the sperm into an egg to obtain an embryo;
e) transferring the embryo into the uterus or fallopian tube of the host female animal to obtain non-human offspring. A method for producing non-human progeny derived from spermatogonial stem cells, comprising the following steps:
a) proliferating spermatogonial stem cells by culturing spermatogonial stem cells in the absence of feeder cells in a state of being attached to an insoluble carrier;
b) transplanting the spermatogonial stem cells grown in a) into the seminiferous tubule of an infertile non-human animal to obtain a non-human animal that has caused spermatogenesis derived from the spermatogonial stem cell;
c) A step of naturally mating the non-human animal with a female to obtain non-human offspring. A method for producing a non-human animal that forms sperm derived from transplanted spermatogonial stem cells, comprising the following steps:
a) proliferating spermatogonial stem cells by culturing spermatogonial stem cells in a serum-free medium;
b) A step of transplanting the spermatogonial stem cells grown in a) into the seminiferous tubule of an infertile non-human animal to obtain a non-human animal that has caused spermatogenesis derived from the spermatogonial stem cell. A method for producing sperm comprising the following steps:
a) proliferating spermatogonial stem cells by culturing spermatogonial stem cells in a serum-free medium;
b) transplanting the spermatogonial stem cells grown in a) into the seminiferous tubule of an infertile non-human animal to obtain a non-human animal that has caused spermatogenesis derived from the spermatogonial stem cell;
c) A step of obtaining sperm from the non-human animal. A method for producing an embryo derived from a spermatogonial stem cell, comprising the following steps:
a) proliferating spermatogonial stem cells by culturing spermatogonial stem cells in a serum-free medium;
b) transplanting the spermatogonial stem cells grown in a) into the seminiferous tubule of an infertile non-human animal to obtain a non-human animal that has caused spermatogenesis derived from the spermatogonial stem cell;
c) obtaining sperm from said non-human animal;
d) A step of fertilizing the sperm into an egg to obtain an embryo. A method for producing non-human progeny derived from spermatogonial stem cells, comprising the following steps:
a) proliferating spermatogonial stem cells by culturing spermatogonial stem cells in a serum-free medium;
b) transplanting the spermatogonial stem cells grown in a) into the seminiferous tubule of an infertile non-human animal to obtain a non-human animal that has caused spermatogenesis derived from the spermatogonial stem cell;
c) obtaining sperm from said non-human animal;
d) fertilizing the sperm into an egg to obtain an embryo;
e) transferring the embryo into the uterus or fallopian tube of the host female animal to obtain non-human offspring. A method for producing non-human progeny derived from spermatogonial stem cells, comprising the following steps:
a) proliferating spermatogonial stem cells by culturing spermatogonial stem cells in a serum-free medium;
b) transplanting the spermatogonial stem cells grown in a) into the seminiferous tubule of an infertile non-human animal to obtain a non-human animal that has caused spermatogenesis derived from the spermatogonial stem cell;
c) A step of naturally mating the non-human animal with a female to obtain non-human offspring.   An agent for culturing spermatogonial stem cells in the absence of feeder cells in an adherent state to an insoluble carrier via an extracellular matrix, the agent comprising an extracellular matrix and an insoluble carrier.   36. The agent of claim 35, wherein the extracellular matrix is laminin.   36. The agent of claim 35, wherein the extracellular matrix is bound to the surface of an insoluble carrier.   A method for producing spermatogonial stem cells, comprising culturing spermatogonial stem cells in the absence of feeder cells in a state of being adhered to an insoluble carrier.   A method for producing spermatogonial stem cells, comprising culturing spermatogonial stem cells in a serum-free medium.