WO2006080314A1 - Méthode d'élimination de cellules leucémiques au sein d'une population de cellules de testicule et kit de réactifs employé dans ladite méthode - Google Patents

Méthode d'élimination de cellules leucémiques au sein d'une population de cellules de testicule et kit de réactifs employé dans ladite méthode Download PDF

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WO2006080314A1
WO2006080314A1 PCT/JP2006/301063 JP2006301063W WO2006080314A1 WO 2006080314 A1 WO2006080314 A1 WO 2006080314A1 JP 2006301063 W JP2006301063 W JP 2006301063W WO 2006080314 A1 WO2006080314 A1 WO 2006080314A1
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Prior art keywords
antibody
cells
cell
sperm
fraction
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Japanese (ja)
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Akira Tsujimura
Kazutoshi Fujita
Kiyomi Matsumiya
Akihiko Okuyama
Hiroshi Ohta
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University of Osaka NUC
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Osaka University NUC
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0081—Purging biological preparations of unwanted cells
    • C12N5/0093—Purging against cancer cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
    • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis

Definitions

  • the present invention relates to a method for completely removing leukemia cells from a cell population of male germ cells obtained with leukemia infiltrating testicular force.
  • the present invention also relates to a reagent kit suitably used for carrying out the above method.
  • the present invention relates to non-human male germ cells from which leukemia cells have been completely removed by the above method, and uses thereof.
  • cancer cells may be mixed with sperm cells and spermatogonia collected before treatment. Even if it is completely cured, there is a risk that the cancer will recur due to subsequent infertility treatment (intratubal transplantation). Thus, the conventionally proposed infertility treatment has a problem of contamination with cancer cells, which is a barrier to clinical application.
  • the purpose of the present invention is to obtain highly purified spermatogonia cells that are completely mixed with cancer cells (leukemia cells) as transplanted sperm cells used for the treatment of male infertility.
  • An object is to provide a method for completely removing leukemia cells from a cell population containing leukemia cells and spermatogonia.
  • Another object of the present invention is to provide a reagent that can be suitably used for carrying out the method.
  • an object of the present invention is to provide a non-human vertebrate sperm cell obtained by the above method and its use.
  • spermatogonial stem cell fraction a cell fraction containing spermatogonial stem cells (hereinafter also referred to as "spermatogonial stem cell fraction"). From the resulting spermatogonial stem cell fraction, cell sorting does not express to spermatogonia cells and is unreactive to both antibodies to surface markers and to surface markers expressed in leukemia cells. White blood by obtaining cell fraction We have found that leukemia cells can be completely isolated or removed from a population of test male germ cells containing diseased cells.
  • the present inventors have confirmed that male germ cells from which leukemia cells have been separated and removed by the above method have the ability to form sperm having normal fertility, and the male sex cells obtained by the method have been confirmed. I was convinced that germ cells could be used effectively to treat male infertility caused by chemotherapy.
  • the present invention has been completed based on strong knowledge.
  • the present invention includes the following embodiments:
  • Item 1 A method of separating or removing leukemia cells from a test male germ cell population containing leukemia cells, wherein the fraction contains spermatogonial stem cells from the test male germ cell population (spermatogonial stem cell fraction) Next, in the fraction, do not express in the spermatogonia !, and obtain a cell fraction that is non-reactive with both the antibody against the surface marker and the antibody against the surface marker expressed on the leukemia cell. Method.
  • Item 2 Surface marker power not expressed in spermatogonia MHC-class I, j8 2-myoglobulin, and Sea-Hari group force At least one selected and a surface marker expressed in leukemia cells Power CD2, CD7, CD10, CD13, CD3, CD14, CD15, CD19, CD20, CD21, CD22, CD33, CD34, CD38, CD45, CD54, CD56, CD65, HLA-DR, HLA-DQ, HLA-DP and Item 2. The method according to Item 1, wherein a group force consisting of TCR is also selected.
  • Item 3 A method performed by flow cytometry, wherein the antibody is directly or indirectly fluorescently labeled as an antibody against a surface marker that is not expressed on spermatogonia and an antibody against a surface marker that is expressed on leukemia cells.
  • Item 3. The method according to Item 1 or 2, wherein an antibody is used to obtain a fraction of cells that have not reacted with the antibody by fluorescence-active fluorescence cell sorting.
  • Item 4 The method according to any one of Items 1 to 3, wherein the test male germ cell population containing leukemia cells is a population of germ cells obtained from testicular strength of a male vertebrate affected with leukemia.
  • Item 5 A reagent kit for separating or removing leukemic cells from a test male germ cell population containing leukemia cells, and at least as a fluorescently active antibody for cell sorting, it is not expressed in spermatogonia.
  • Antibodies to surface markers and expressed on leukemia cells A reagent kit comprising an antibody against a surface marker.
  • Item 6 Surface marker power not expressed in spermatogonia MHC-class I, j8 2-myoglobulin, and Sea-harness group power At least one surface marker expressed in leukemia cells Power CD2, CD3, CD7, CD10, CD13, CD14, CD15, CD19, CD20, CD21, CD22, CD33, CD34, CD38, CD45, CD54, CD56, CD65, HLA-DR, HLA-DQ, HLA-DP and Item 6.
  • Item 7 A non-human male male obtained by separating and removing leukemia cells from a test male germ cell population containing leukemia cells derived from a non-human male vertebrate by the method of any one of Items 1 to 4. Germ cells.
  • the non-human male germ cell according to Item 8. is transferred into the testes of a donor or similar recipient male vertebrate, and the male vertebrate is mated with a female vertebrate of that species.
  • Item 9 The method according to Item 8, wherein the male vertebrate is an azoospermia non-human male vertebrate.
  • Item 10 Mating is natural mating between a male vertebrate and a female vertebrate of that species, artificial insemination of a female vertebrate by a male vertebrate sperm, in vitro of a female vertebrate egg by a male vertebrate sperm Item 10.
  • Item 11 A method for restoring fertility of an azoospermic non-human male vertebrate, wherein the non-human male germ cell described in Item 7 is treated with an azoospermia donor or a non-human recipient of the same species. Transferring the testis of a male vertebrate.
  • leukemia malignant tumors
  • treated boys have male infertility at reproductive age. It causes great emotional distress to the patient, and urgent measures are urgently needed.
  • leukemia cells can be completely removed from the sperm cell population obtained from leukemia infiltrating testis, so that the sperm cells obtained by the method are used to treat male infertility caused by chemotherapy for boys. Can be suitably used.
  • the method for isolating or removing leukemia cells of the present invention is a method capable of completely isolating or removing leukemia cells from a test male germ cell population containing leukemia cells.
  • a fraction containing a spermatogonial stem cell is obtained from a test male germ cell population containing leukemia cells, and then further expressed from the fraction into a spermatogonia cell. This can be achieved by obtaining a cell fraction that is non-reactive with both the antibody against and the antibody against the surface marker expressed on leukemia cells.
  • the cell population (test cell population) targeted by the method of the present invention is a male germ cell in which leukemia cells are mixed. Usually, the test cell population can also acquire the testicular strength of male vertebrates affected by leukemia.
  • male germ cells include sperm cells and their progenitor cells.
  • Sperm progenitor cells include spermatogonia (including spermatogonial stem cells), spermatocytes, and sperm cells. Preferred are spermatogonia cells (including spermatogonial stem cells) or male germ cells containing at least this.
  • the target vertebrates include humans, non-human primates, dogs, cats, pigs, mice, rats, gerbils, mice, musters, usagis, marine mammals, pachyderms, horses, Mammals such as hidge, pig, and eagle; and birds including ducks, geese, turkeys, and birds.
  • test cell population can be obtained according to methods known in the art. For example, as will be described in detail in Examples described later, the test cell population can also acquire seminiferous tubule strength that can also obtain testicular strength of subjects (including test humans and non-human animals). Specifically, the seminiferous tubule obtained from testis tissue is cut and incubated in a solution containing an enzyme such as spleen trypsin, collagenase type I, or spleen deoxyribonuclease type I (eg, modified DMEM medium). A test cell population (germ cell population) can be obtained by releasing non-damaged cells through an enzymatic reaction.
  • an enzyme such as spleen trypsin, collagenase type I, or spleen deoxyribonuclease type I
  • the seminiferous tubules are prepared by peeling off the testis or a part of the tissue strength of the testis collected from the subject, or the collected testis tissue is collagenase or deoxyribo. It can be prepared from seminiferous tubule tissue obtained by treatment with an enzyme such as nuclease (DNAse).
  • DNAse nuclease
  • cell sorting and recovery techniques known in the art (for example, “The Handbook of Experimental Immunology, 1— 4 ⁇ : A. Radbrucn editors' “Flow Cytometry and ell sorting (Spring Verlag, 1992): and Olweus, J et al., 1997, Proc. Natl. Acad. Sci., USA 94: 1255 1-12556”) Can be used without restriction.
  • flow cytometry particularly flow cytometry with fluorescence active cell sorting is used. That is, the method of the present invention preferably includes the step of subjecting the test cell population prepared by the above method to flow cytometry.
  • the method of the present invention using flow cytometry can be carried out by performing the following steps (a) and (b):
  • test cell population is divided into antibodies against surface markers that are not expressed on spermatogonia (hereinafter “anti-non-progenitor cells-antibody” t) and antibodies against surface markers that are expressed on leukemia cells (hereinafter “pile”). -"Leukemia cells-antibodies”), and
  • MHC-class I labeling step
  • ⁇ 2-myoglobulin and Sca- 1
  • MHC-class J8 2-myoglobulin is a surface marker that is expressed on leukemia cells, but is not limited to CD2, CD3, CD7, CD10, CD13, CD14, CD15, CD19, CD20, CD21, CD22.
  • CD45 is preferred.
  • a combination of a surface marker not expressed in spermatogonia and a surface marker expressed in leukemia cells a combination of MHC-class I and CD45, or a combination of 13 2-myoglobulin and CD45 can be preferably exemplified. .
  • Antibodies used in the present invention are polyclonal antibodies, monoclonal antibodies, chimeric antibodies of any isotype (IgA, IgG, IgE, IgD, IgM) as long as they are antibodies against the various surface markers described above.
  • Humanized antibodies eg, Fab fragments containing antigen binding sites, F (ab ') fragments, Fab' fragments, Fv fragments such as scFv, isolated heavy chains (H chains) and light chains (
  • the various surface markers are all known and can be obtained commercially, for example, from BD Biosciences PharMingen .
  • the above step (a) (labeling step) consists of (0) an antibody against a surface marker not expressed in spermatogonia (stake-non-sperm cell-antibody) and an antibody against a surface marker expressed in GO leukemia cells (stake- In combination with leukemia cells-antibodies) and reacting the test cell population with both of these antibodies.
  • Anti-MHC-class I antibody / 3 antibody against 2-myoglobulin (anti- ⁇ 2-myoglobulin antibody), and antibody against Sca-1 (anti-Sca-1 antibody)
  • These can be used, but can also be used in combination of two or more, preferably an anti-MHC-class I antibody or an anti-j8 2-myoglobulin antibody.
  • Anti-leukemic cells-antibodies include the above-mentioned CD2, CD3, CD7, CD10, CD13, CD15, CD14, CD19, CD20, CD21, CD22, CD33, CD34, CD38, CD45, CD54, CD56, CD65, HLA Antibodies against -DR, HLA-DQ, HLA-DP or TCR (anti-CD2 antibody, anti-CD3 antibody, anti-CD7 antibody, anti-CD10 antibody, anti-CD13 antibody, anti-CD14 antibody, anti-CD15 antibody Anti-CD19 antibody, anti-CD20 antibody, anti-CD21 antibody, anti-CD22 antibody, anti-CD33 antibody, anti-CD 34 antibody, anti-CD38 antibody, anti-CD45 antibody, anti-CD54 antibody, anti-CD56 antibody, anti-CD65 antibody, anti-HLA-DR antibody, anti-HLA-DQ antibody, anti-HLA-DP antibody or The ability to use any one of anti-TCR antibodies) It is also possible to use a combination of two
  • These antibodies are preferably labeled with a fluorescent dye in order to perform fluorescence active cell sorting.
  • a fluorescent dye those known in the art as fluorescent dyes for flow cytometry can be arbitrarily used.
  • fluorescein isothiocyanate FITC
  • PE phycoerythrin
  • PE-Cy5 PE-Cyanin5
  • PE-Cy5.5 PE-Cyanin5.5
  • PE-Cy5 PE-) Cyanin7
  • rhodamine isothiocyanate Texas Red, ECD (PE-Texas Red-x), Arophycosanine (APC), APC-Cy7 (APC-Cyanin 7, PharRed), Per—CP (Peridinin Chlorophyll Protein), and PerCP-Cy5.5 (Per-CP-Cyanin5.5).
  • the labeling can be performed by covalently binding the fluorescent dye to the antibody, and methods known in the art can be used for strong binding (for example, E. Har low & D. Lane, Antibodies: A Laboratory Manual (Old Spring Harbor Laboratory Presess, Cold Spring Harbor, New York, 1988)).
  • an antibody pre-labeled with a fluorescent dye for example, “fluorescent-labeled antibody for flow cytometry”
  • the antibody of the present invention is preferably labeled with a fluorescent dye, but it does not limit the use of other labels such as a chemiluminescent label or a bioluminescent label.
  • the above antibody may be labeled with a fluorescent dye, or may be one that can be labeled with a fluorescent dye.
  • a strong antibody include an antibody treated with piotin or streptavidin.
  • the antibody can be indirectly labeled with a fluorescent dye through a piotine avidin reaction by reacting with a fluorescent dye-conjugated streptavidin or piotin.
  • antibodies not labeled with fluorescent dyes Can be indirectly labeled with a fluorescent dye by reacting with the secondary antibody labeled with the fluorescent dye.
  • the reaction between the test cell population and the antibody consists of an antigen (surface marker) that is expressed on the cell surface and the antibody against it.
  • an antigen surface marker
  • a buffered saline solution such as a phosphate buffered saline solution that is isotonic or equivalent to the test cell can be used as a reaction solvent, although not particularly limited as long as this is the case.
  • step (b) flow cytometry step.
  • the flow cytometry process consists of b-1) gating the spermatogonial stem cell fraction (gating process), and b-2) gating the spermatogonial stem cell fraction.
  • gating process gating process
  • b-2 gating the spermatogonial stem cell fraction.
  • ”And“ stake-leukemia cell-antibody ”fractions that are non-reactive [stake-non-progenitor cell-antibody (1) Z anti-leukemia cell-antibody () fraction] It consists of at least two process powers (sorting process).
  • FSC Forward scatter
  • SSC Side s catter
  • a two-parameter histogram [linear 'scale (constant scale): X-axis and Y-axis are the measurement parameters FSC and SSC, respectively: X-axis : 0-1000, Y-axis: 0-1000] X-axis 50-1000, Y-axis force ⁇ -600, preferably X-axis 100-1000, Y-axis 0-500, more preferably X-axis 200-1000, Y-axis 0-400 Can be set as an area wear.
  • the sorting process is based on the results of the reaction with stake-non-spermatogonia cell-antibody and stake-leukemia cell-antibody obtained by flow cytometry for the spermatogonial stem cell fraction identified in the gating process. , “Stake-non-sperm cell-antibody” and “anti-leukemia cell-antibody”
  • This step can be preferably performed using fluorescence activated cell sorting (FACS).
  • FACS fluorescence activated cell sorting
  • an antibody directly or indirectly labeled with a fluorescent dye is preferably used as the “stake-non-sperm cell-antibody” and “stake-leukemia cell-antibody”.
  • Fluorescence activated cell sorting is, for example, “Flow cytometry, actual battle protocol”, Hirokawa, Chemistry and Biological Experiment Line, MG Ormerod (Editing; “Flow ytometry ana Sorting (2nd Edition)” , Myron R. Melamed et al, Wiley-Liss, Inc. NY, 1990; "Flow cytometry one-technique and practice-" 2nd edition Supervised by Kazuo Ota, Sakai Shobo, 1988, etc. be able to.
  • FACS Fluorescence activated cell sorting
  • Fluorescence-activated cell sorting is specifically performed by the luminescence (fluorescence) of fluorescent dyes used for the labeling of “stake-non-sperm cell-antibody” and “stake-leukemia cell-antibody”. This is performed by at least two-dimensional analysis using the luminescence (fluorescence) of the fluorescent dye used for labeling as a parameter.
  • FACS Fluorescence-activated cell sorting
  • the classification into four fractions is based on the luminescence (fluorescence) of the spermatogonial stem cell fraction generated by the reaction with “stake-non-sperm cell-antibody” and “anti-leukemia cell-antibody”! Done.
  • cell sorting of 1) "stake-non-sperm cell-antibody (-) Z anti-leukemia cell-antibody (-) fraction refers to cells belonging to the fraction according to the conventional method of FACS. This can be done by separating electrostatically into individual liquid droplets that can be deflected into the tube.
  • the method of the present invention using flow cytometry may be any one having a to above (a) step (labeling step) and (b) as E (Flow Cytometry step), further, (a In step (labeling step), the test cell population is reacted with a dead cell stain, and in step (b) (flow cytometry step), the test cell labeled with the dead cell stain is removed. Even if it has a work.
  • the operation of reacting the test cell population with the dead cell stain can be performed under the condition that the cells are alive and the dead cells are stained with the stain, and so far, the conditions are not particularly questioned. Nah ...
  • the operation of removing the test cells labeled with the dead cell stain is preferably performed after (b) the gating step (b-1), for example, simultaneously with, and before or after the sorting step (b-2). Can do.
  • the sorting step (b-2) “stake-non-spermatogonia cell-antibody (-) z stake-non-sperm cell-antibody (-) fraction” and dead cells
  • sorting can be performed by sorting the fraction (dead cell stain ( ⁇ ) fraction) that is not labeled with a dead cell stain.
  • the dead cell stain is not particularly limited as long as it is a dye that specifically stains dead cells.
  • a dye that specifically stains dead cells For example, Trypan Blue, Erythrosin B, Propidium iodide (PI), and
  • fluorescent dyes such as 7-amino-actinomycin D can be used.
  • ( ⁇ ) Reagent kit The present invention also provides a reagent kit used to isolate or remove leukemic cells from a test male germ cell population containing leukemia cells.
  • the reagent kit contains at least (1) an antibody against a surface marker that is not expressed in spermatogonia and (2) an antibody against a surface marker that is expressed in leukemia cells in the same or separate container.
  • the antibody (1) include an antibody against MHC-class I (anti-MHC-class I antibody), an antibody against ⁇ 2-microglobulin (anti- ⁇ 2-microglobulin antibody), and Sca.
  • Antibody against -1 anti-Sca-1 antibody.
  • Anti-MHC-class I antibody or anti-j8 2-microglobulin antibody is preferred.
  • Anti-CD45 antibody is preferable.
  • the combination of antibody (1) (stake-non-sperm cell-antibody) and antibody (2) (anti-leukemia cell-antibody) is a combination of anti-MHC-class I antibody and anti-CD45 antibody. Or a combination of anti- ⁇ 2-myoglobulin antibody and anti-CD45 antibody.
  • antibodies (1) and (2) may be any isotype (IgA, IgG, IgE, etc.) as long as they are antibodies that specifically bind to the target antigen (cell marker).
  • IgD, IgM polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies, antibody fragments (e.g., Fab fragments containing antigen binding sites, F (ab ') fragments, Fab' fragments, Fv such as scFv)
  • H chain isolated heavy chain
  • L chain light chain
  • antibodies (1) and (2) are not limited, but may be antibodies bound to a labeling agent (labeled antibodies).
  • the antibody label is not particularly limited as long as it is a detectable label.
  • non-radioactive isotopes, chemiluminescent labels, bioluminescent labels, and fluorescent labels can be listed.
  • fluorescent labeled antibody fluorescent labeled antibody
  • fluorescent dye those known in the art as fluorescent dyes for flow cytometry can be arbitrarily used.
  • fluorescein soyocyanate FITC
  • PE phycoerythrin
  • PE-Cy5 PE-Cyanin5
  • PE—Cy5.5 PE—Cyanin5.5
  • PE—Cy5 PE — Cyanin7
  • Rhodamine isothiocyanate Texas Red
  • ECD PE-Texas Red-x
  • Alophycosanine APC— Cy7 (APC— Cyanin 7, PharRed)
  • Per— CP Peridinin Chlorophylllin
  • PerCP-Cy5.5 Per-CP-Cyanin5.5
  • Labeling can be performed by covalently binding the above-described fluorescent dye to the aforementioned antibody using a known method (for example, E. Harlow & Dane, "Antibodies: A Laboratory Manual” ( Cold Spring Harbor Laboratory Press, old bpnng Harbor, New York, 198 8)) 0
  • a commercially available fluorescent dye-labeled antibody eg, “fluorescent-labeled antibody for flow cytometry”. You can also.
  • these antibodies (1) and (2) may not be fluorescent dye-labeled antibodies but may be those that can be labeled with a labeling agent such as a fluorescent dye.
  • a strong antibody include an antibody treated with piotin or streptavidin.
  • the antibody can be indirectly labeled with a labeling agent (fluorescent dye, etc.) via a piotine-avidin reaction by reacting with the streptavidin or piotin bound with a labeling agent such as a fluorescent dye. it can. Therefore, in this case, the reagent kit of the present invention preferably contains streptavidin or piotin bound with a labeling agent such as a fluorescent dye as another reagent component.
  • a secondary antibody that is labeled with a labeling agent such as a fluorescent dye as another reagent component in the reagent kit (each of the above antibodies and An antibody that specifically binds).
  • a labeling agent such as a fluorescent dye
  • the antibody (1) or (2) can be indirectly labeled with a labeling agent (fluorescent dye or the like) by reacting with the secondary antibody.
  • the reagent kit of the present invention has at least the antibody (1) and the antibody (2). Any other reagent may be contained in the same or separately separated container. Examples of other reagents that can be used include streptavidin or piotin bound with a labeling agent such as the above-described fluorescent dye, or a secondary antibody labeled with a labeling agent such as a fluorescent dye.
  • reagents include dead cell stains (eg, Trypan Blue, Erythrosin B, Propiaium iodide (PI), Ethidium bromiae (EB), i-amino-actinomycin D, etc.), reaction solvents (eg, phosphate Buffered saline such as buffered saline) and control reagents.
  • dead cell stains eg, Trypan Blue, Erythrosin B, Propiaium iodide (PI), Ethidium bromiae (EB), i-amino-actinomycin D, etc.
  • reaction solvents eg, phosphate Buffered saline such as buffered saline
  • control reagent include vertebrate sperm cells or mimics thereof. Examples of the mimics include beads having a sperm cell surface marker on the surface thereof.
  • the present invention also provides a non-human leukemia cell obtained by separating and removing leukemia cells from a test male reproductive cell population containing leukemia cells derived from a non-human male vertebrate by the method described in (I) above. Relates to the male germ cell population.
  • vertebrates include non-human primates, dogs, cats, pigs, mice, rats, gerbils, wild animals, mustards, usagis, marine mammals, pachyderms, horses, hidges, pigs, and Non-human vertebrates such as mammals such as duck; and birds including ducks, geese, turkeys, and birds.
  • it is a non-human vertebrate that is generally used in biological tests (preclinical tests, nonclinical tests) for confirming the efficacy and safety of drugs.
  • the non-human male germ cell population of the present invention is a collection of non-human sperm cells obtained by the method described in (I) above, wherein leukemia cells have been completely removed, and at least normal reproductive cells.
  • spermatogonia cells spermatogonial stem cells
  • the number of sperm cells constituting the sperm cell population is not particularly limited as long as it contains spermatogonia (spermatogonial stem cells) having the ability to form sperm having normal fertility.
  • the non-human male germ cell population can be used to produce normal offspring of allogeneic non-human vertebrates. Specifically, the production of such offspring begins by using the above-mentioned non-human male germ cell population (sperm cell population) as a donor or similar recipient male. This can be done by transferring into the vertebrate testis, maturing it into sperm, and mating the male vertebrate with a female vertebrate of the same species.
  • the sperm cell population can be transferred into the testis under conditions that allow the sperm cells to remain in the seminiferous tubule of the testis, and thus support Leydig cells or Sertoli cells in the seminiferous tubule. Under the action of cells, they mature into self-motile haploid cells (male gametes, sperm) that can fertilize eggs.
  • the sperm cell population can be transferred into the testis by direct injection, usually using a micropipette.
  • support cells such as Leydig cells or Sertoli cells, which are involved in spermatogonial stem cell sorting, may be transferred.
  • the cell concentration to be transferred is not particularly limited as long as the effect of the present invention can be obtained, but a range of 1 ⁇ 10 5 to 10 ⁇ 10 5 cells / 10 ⁇ ⁇ can be exemplified.
  • the male vertebrate to be transplanted with the sperm cell population may be a donor male vertebrate derived from the sperm cell population or the same type of recipient male vertebrate.
  • male vertebrates to which the sperm cell population is transplanted are preferably azoospermic male vertebrates that have lost or reduced spermatogenic ability.
  • Is an azoospermic male vertebrate administered for example, to a male vertebrate with a cytotoxic alkyl agent (eg, busulfan, chlorambucil, cyclophosphamide, melphalan, ethylethanesulfonic acid, etc.) (chemical treatment)
  • a cytotoxic alkyl agent eg, busulfan, chlorambucil, cyclophosphamide, melphalan, ethylethanesulfonic acid, etc.
  • chemical treatment such as ⁇ irradiation.
  • the male vertebrate is mated with a female vertebrate of the same species, so that the male and female gametes combine to produce fertilization. After a certain gestation period, these vertebrate offspring are born.
  • Male and female gametes can be joined (mating) by natural mating (natural mating) or by in vitro or in vivo artificial mating means. Examples of powerful artificial mating means include, but are not limited to, artificial artificial sperm, artificial insemination, in vitro fertilization, intracytoplasmic sperm injection, subtropical sperm, or partial stratification.
  • the offspring thus born are normal without the onset of leukemia and can be equipped with normal reproductive functions to produce their subsequent generations (breeding).
  • the method of the present invention is suitable for chemical treatment of animals with malignant diseases other than humans (leukemia animals).
  • leukemia animals can be used to correct normal male infertility to restore normal reproductive function and obtain normal offspring, but also to treat human infertility, such as in preclinical studies It can also be used effectively for predictive evaluation of effects.
  • the non-human male germ cell population (sperm cell population) obtained by the method (I) can be effectively used to restore the fertility of allogeneic non-human vertebrates.
  • Such a method can be performed by transferring the non-human male germ cell population (sperm cell population) into the testis of an azoospermia donor or the same recipient non-human vertebrate. This method is suitably used for the treatment of non-human vertebrates having spermatogenic disorders.
  • the therapeutic effect on non-human vertebrates by the method of the present invention suggests the possibility of reproductive application of the method of the present invention to humans. Therefore, the method of the present invention can be effectively used for predicting and evaluating the therapeutic effects on human male infertility, such as in preclinical studies.
  • cell surface markers used to separate leukemia cells and sperm cells by flow cytometry fluorescence-activated cell sorting satisfy at least two conditions. Need to be. First, 100% pure sperm cells can be obtained without contamination with leukemia cells, and secondly, the obtained sperm cells contain spermatogonia that can form sperm after transplantation. Therefore, as a cell surface marker, using a CD4 5 and MHC class I [using MHC class I heavy chain (H-2K b / H- 2D b) ], it was examined their effectiveness.
  • C1498 cell line leukemia cell line of mouse of C57BL / 6 origin
  • sperm cells isolated from normal C57BL / 6 mouse testicular force were used as sperm cells.
  • sperm cells were prepared as follows. First, the testicular force was also removed from the white membrane, and the seminiferous tubule was buffered to pH 7.3 with 20 mM Hepes. modified Eagle's medium) and incubated at 37 ° C for 15 minutes with manual shaking for 5 minutes. The resulting seminiferous tubule was washed twice with calcium-free phosphate buffered saline (PBS) and then washed with 0.25% trypsin and DNase 1 (100 g / ml) (Sigma). Incubated in PBS containing 15 minutes at 37 ° C with shaking at 5 minute intervals. Add 1/2 volume of DMEM containing 10% fetal bovine serum (FBS), and filter the resulting cell suspension through a nylon mesh with a 30 m pore size. Was removed. The cell suspension thus obtained was used as sperm cells.
  • PBS calcium-free phosphate buffered saline
  • trypsin and DNase 1 100 g / m
  • each sperm cells described above were suspended in PBS (PBS / FBS) containing 0.5% FBS, for 20 minutes on ice, the anti-H- 2K b / H- 2D b having attached Piochin antibody (28- 8- 6; BD Biosciences Phar Mingen, San Diego (CA)) (hereinafter, "Piochin bond - anti H- 2K b / H- 2D b antibody" hereinafter) were cultured with co. Next, after washing twice with a large amount of PBS / FBS, the cells were fluorescently labeled for fluorescent activity cell sorting (FACS).
  • FACS fluorescent activity cell sorting
  • streptavidin conjugated with PE (phycoerythrin) (BD Biosciences PharMingen) (hereinafter referred to as “PE-bound streptavidin”) and anti-CD45 antibody conjugated with FITC (Fluorescein isothiocianate) (30- Fl l; BD Biosciences PharMingen) (hereinafter referred to as “FITC-conjugated anti-CD45 antibody”) for 20 minutes.
  • the first antibody Pieriotin binding-Pile H-2K b / H-2D b antibody
  • the second reagent PE binding-Streptavidin, FITC binding-Pile CD45 antibody
  • Cell sorting was performed using a FACS Ventage (BD Biosciences, San Jose) equipped with a 488 nm argon laser (250 mW).
  • Argon ion laser was used to excite the fluorescent dyes FITC, PE, and PI, and the fluorescence generated from FITC was collected and detected with a 530 nm filter, and the fluorescence generated from PE and PI was collected with a 575 nm filter. .
  • Each cell fluorescently labeled in (3) was subjected to flow cytometry.
  • FSC forward scattered light
  • SSC side-scattered light
  • G 1 gate the clusters in the FSC 1 " 811 and SSC lQW cell population area (G 1) ( Figure la, b), and for the resulting gate region cells (G1 fraction), then anti H-2K b / H_2D b antibody and anti-CD45 antibody is an antibody against the surface marker, as well as examined for labeling with PI.
  • Fig. 1 c and d show the analyzed histograms (quadrant regions) on the Y-axis, FITC fluorescence used for labeling the anti-CD45 antibody on the X-axis, and the analyzed region (quadrant region).
  • anti-H-2K b / H-2D b negative fraction for antibody and anti-CD45 antibodies [anti-H-2K b / H-2D b anti (-) / anti-CD45 antibody (-) fraction Min] (G2) did not contain any C1498 cells (Fig. Lc, d). Furthermore, the cells of the fraction (G2) were negative for PI as a dead cell stain.
  • the fraction positive for anti-H-2K b / H-2D b antibody and negative for anti-CD45 antibody contained 0.3% of C1498 cells.
  • the cells contained in the “anti-H-2K b / H-2D b antibody ( ⁇ ) / anti-CD45 ( ⁇ ) antibody fraction” (G3) are sperm cells. It was confirmed by showing a positive reaction to (specific marker for) (Tanaka, H. et al., Int J Androl 20, 361-366 (1997)).
  • sperm cells and leukemia cells can be rigorously used by combining two antibodies (antibody against CD45 and MHC class I heavy chain (H-2K b / H-2D b )). (Fig. 1).
  • anti-CD45 antibodies antibodies against CD45 [hereinafter referred to as “anti-CD45 antibodies”), antibodies against HLA-A, B, and C corresponding to human MHC class I [hereinafter antiantibodies] The effectiveness of HLA-A, B, and C antibodies) was investigated.
  • a Jurkat cell line (human leukemia cell line) was used as a human leukemia cell.
  • Immunostaining was performed according to the method of Kubota et al. In the same manner as in Example 1 (3). Specifically, the above human leukemia cells were suspended in PBS containing 0.5% FBS (PBS / FBS), and anti-HLA-A, B, C bound with PE (phycoerythrin) was used for flow cytometry.
  • PBS / FBS 0.5% FBS
  • PE phytoerythrin
  • Flow cytometry was performed in the same manner as in Example 1 (4). Specifically, each cell fluorescently labeled in (3) was subjected to flow cytometry.
  • Fig. 2a shows a two-parameter histogram obtained for human leukemia cells using forward-scattered light (FSC) and side-scattered light (SSC) as parameters.
  • FSC forward-scattered light
  • SSC side-scattered light
  • G1 gate clusters in the FSC high and SSC lQW cell population areas
  • G1 fraction gate region cells
  • the presence or absence of labeling with certain anti-HLA-A, B, C and anti-CD45 antibodies and PI was examined in the same manner as in Example 1.
  • Fig. 2b shows the analyzed histogram (quadrant region) on the Y-axis and the fluorescence of FITC used to label the anti-CD45 antibody on the X-axis.
  • 99.6% of cells in the gate region (FSC high and SSC lQW cell population area) (G1) responded to both anti-HLA-A, B, C and anti-CD45 antibodies (positive reaction) ).
  • the cell line human leukemia cells
  • the cells of the fraction (G2) were negative for PI as a dead cell stain.
  • a leukemia-affected mouse (leukemia mouse) was used (hereinafter also referred to as “donor mouse”).
  • the leukemia mice are C57BL / 6 mice (3 weeks old, obtained from Shizuoka Experimental Animal Center) or acrosin / eGFP (Acr3- EGFP) and pCX-EGFP (C57BL / 6 Recombinant mouse (6 weeks old) recombined to express GFP (Green Fluorescent Protein) by having TgN (acro / act-EGFP) OsbC3-N01-FJ002) (hereinafter referred to as “GFP mouse”) (Ohta, H., et al., Dev Growth Differ 42, 105-112 (2000); Nakanishi, T.
  • C1498 cells a mouse leukemia cell line of C57BL / 6 origin, obtained from ATCC
  • the optimal dose of C1498 cells was determined in advance in a preliminary test!
  • recipient male vertebrate As a recipient male vertebrate, a male mouse having lost spermatogenic ability was used (hereinafter referred to as “recipient 'mouse”).
  • the recipient mice were C57BL / 6 mice (4 weeks old, obtained from Shizuoka Experimental Animal Center), busulfan (40 mg / kg, Sigma) Chemical, an aralkylating agent used to treat chronic myeloid leukemia. (St. Louis, Inc.) was administered to eliminate the ability to form sperm.
  • Derna 1 'mice C1BL-injected C57BL / 6 mice, C1498-injected GFP mice prepared in (1) and testicular strength excised were also obtained.
  • normal C57BL / 6 mouse force testes were excised and sperm cells were obtained.
  • Dulbecco's modified eagle containing collagenase 'Type IV (lmg / m 1) (Sigma's Chemical), with the white membrane removed from each testis and the seminiferous tubule buffered to pH 7.3 with 20 mM Hepes.
  • the medium was placed in a medium (DMEM: Dulbecco's modified Eagle's medium) and cultured at 37 ° C for 15 minutes with hand shaking for 5 minutes.
  • DMEM Dulbecco's modified Eagle's medium
  • the resulting seminiferous tubules were washed twice with calcium-free phosphate buffered saline (PBS) and then washed with PBS containing 0.25% trypsin and DNase 1 (100 g / ml) (Sigma). Incubated at 37 ° C for 15 minutes with shaking at 5 minute intervals. Add DMEM containing 1/2 volume of 10% fetal calf serum (FBS), and filter the resulting cell suspension through a nylon mesh with a 30 m pore size. The sperm cells were prepared by removing the cells.
  • PBS calcium-free phosphate buffered saline
  • FBS fetal calf serum
  • GFP sperm cells derived from GFP mice injected with C 1498 are referred to as “GFP sperm cells”, and sperm cells derived from C57BL / 6 mice injected with C 1498 and normal C57BL / 6 mice are referred to as “non-GFP fine cells”. To distinguish between the two.
  • Example 2 In the same manner as in Example 1 (3), the sperm cells obtained in (2) were immunostained according to the method of Kubota et al.
  • each of the above sperm cells was suspended in PBS (PBS / FBS) containing 0.5% FBS, and bound with piotin for 20 minutes on ice.
  • PBS PBS / FBS
  • anti H-2K b / H-2D b antibody anti H-2K b / H-2D b antibody; and cultured with (28-8-6 BD Biosciences PharMingen, San Diego (CA)).
  • non-GFP sperm cells for fluorescence activity sorting (FACS), PE-conjugated-streptavidin (BD Biosciences PharMingen) and FITC-conjugated-anti-CD45 Incubated with antibody (30-Fll; BD Biosciences PharMingen) for 20 minutes.
  • FACS fluorescence activity sorting
  • PE-conjugated-streptavidin BD Biosciences PharMingen
  • FITC-conjugated-anti-CD45 Incubated with antibody (30-Fll; BD Biosciences PharMingen) for 20 minutes.
  • GFP sperm cells were cultured for 20 minutes with anti-CD45 antibody (30-Fll; BD Biosciences PharMingen) conjugated with PE-bound streptavidin and PE-Cy5.
  • the first antibody (Piotin binding-Pile H-2K b / H-2D b antibody) and the second reagent (PE binding streptavidin, FITC binding-anti-CD45 antibody or PE-Cy5 binding-anti-CD45 antibody) All were used at a concentration of 5 / zg / ml.
  • these sperm cells are finally washed and then resuspended in PBS / FBS containing 1 ⁇ g / ml of propidium iodide (PI: propidium iodideX Sigma Chemical), a dead cell stain. (4 ⁇ 10 6 cells / ml), stored on ice in a dark room until subjected to fluorescence activity sorting (FACS).
  • PI propidium iodideX Sigma Chemical
  • non-GFP sperm cells derived from C57BL / 6 mice (leukemia mice) injected with C1498 were subjected to flow cytometry in the same manner as in Example 1 (4).
  • the laser was used to excite FITC, PE, and PI, and the fluorescence generated from FITC was collected and detected with a 530 ⁇ m filter, and the fluorescence generated from PE and PI was collected with a 575 nm filter.
  • a two-parameter histogram of this non-GFP sperm cell by forward-scattered light (FSC) and side-scattered light (SSC) is shown in FIG.
  • FSC forward-scattered light
  • SSC side-scattered light
  • the G1 fraction of non-GFP sperm cells derived from C57BL / 6 mice shows the response to anti-H-2K b / H-2D b antibody, the response to anti-CD45 antibody and the response to PI in a three-parameter histogram The results are shown in Fig. 3b.
  • the cell population area (G3) (positive fraction) that reacts with anti-H-2K b / H-2 D b antibody and anti-CD45 antibody is gated and the cell fraction [anti-H-2K b / H- 2D b antibody (+) / anti-CD45 antibody (+)] (G3 fraction) was sorted as “leukemia cell fraction” (FIG. 3b).
  • the cells were sorted into a 5 ml tube containing 2 ml DMEM (DMEM / FBS) containing 10% FBS.
  • An FSC threshold was set to exclude cell debris.
  • the cells of the obtained G2 fraction and G3 fraction corresponded to 51.4% and 2.5% of the total non-GFP cells, respectively.
  • the sperm cell fraction (G2) and leukemia cell fraction (G3) cells sorted from non-GFP sperm cells of C57BL / 6 mice (leukemia mice) injected with C1498 were each 10 0 / zl PBS / Suspended in FCS.
  • the concentration of the sperm cell fraction was 2 ⁇ 10 6 cells / ml and the concentration of the leukemic cell fraction was approximately 1 ⁇ 10 5 cells / ml.
  • a suspension of each cell was injected intraperitoneally into the recipient mouse.
  • 12 mice injected with sperm cell fraction (G2) cells were All survived for 300 days without developing leukemia.
  • mice injected with leukemia cell fraction (G3) cells all showed typical signs of end-stage leukemia with hemorrhagic ascites within 40 days and died (Figure 3c). This revealed that leukemia cells were mixed in the sperm cell fraction (G2)! / ,!
  • GFP sperm cells obtained from C 1498-injected GFP mice were subjected to flow cytometry according to the method described in (4).
  • the laser was used to excite PE, PE-Cy5 and PI, and the fluorescence generated from PE and PI was collected and detected with a 575 nm filter, and the fluorescence generated from PE-Cy5 was collected with a 682 nm filter.
  • the ratio of viable cells contained in the obtained sperm cell fraction (G2) was confirmed to be 95% or more by measuring trypan blue exclusion ability.
  • the cells of the washed sperm cell fraction (G2) were suspended in the injection medium to a concentration of 1 X 10 8 cells / ml, and this was used as a donor cell suspension.
  • the donor cell suspension thus obtained was administered to the testes of recipient mice at a ratio of approximately 101 per testis. Specifically, sperm formation ability by busulfan treatment
  • the sperm cell fraction (G2) obtained by cell sorting by the method of the present invention contains a sufficient number of spermatogonia having spermatogenic activity, and leukemic cells are It was confirmed that it was mixed.
  • the above recipient's testicular strength of the mouse The sperm obtained was able to talk about the power of normal reproductive ability.
  • a recipient transplanted with the above donor cell suspension obtained by cell sorting. From the testes of mice, spermatozoa were obtained 8 weeks after transplantation. Specifically, under a fluorescent stereomicroscope, the seminiferous tubes emitting green fluorescence were collected from the recipient's testis, cut with scissors, and suspended in Hepes-CZB medium (GFP semen suspension ).
  • Recipient ' contains approximately 2 ⁇ l of GFP semen suspension collected from mouse testis, containing 12% (w / v) polypyrrole pyrrolidone (PVP; Mr 360 000; Wako Pure Chemical Industries) Mixed with 1 drop of Hepes-CZB medium.
  • PVP polypyrrole pyrrolidone
  • the sperm head was separated from the tail
  • the oocytes thus obtained were cultured in CZB medium in air at 37 ° C. and 5% CO. 40 (95.2) of the 42 embryos formed
  • the two-cell embryo was transferred to the oviduct of 0.5-dpc pseudopregnant ICR female mice, resulting in a total of 12 mice (28.6%). Since hemizygous GFP transgenic mice were used, about half of the resulting pups (5) showed green fluorescence derived from GFP under excitation light. This indicates that fertilized spermatozoa are derived from transplanted GFP sperm cells. Because of this, the sperm cell fraction (G2) obtained by the recipient's testicular force cell sorting (G2) force The derived sperm has a normal reproductive ability, that is, included in the sperm cell fraction (G2). It has been confirmed that the sperm cells have sufficient spermatogenic ability.
  • testicular seminiferous tubules contain functional Sertoli cells after chemotherapy [Ohta, H., etal, Int J Androl 24, 15-23 (2001); Ohta, H., et al., Devel opment 127, 2125-2131 (2000)], by immature transplantation of spermatogonia and spermatocytes into the seminiferous tubule, immature sperm cells can be matured to sperm.
  • the remaining problem is to develop a first problem, a method that completely separates cancer cells from testicular sperm cells and prevents recurrence after transplantation. As shown in the above example, I That is, by cell sorting by flow cytometry,
  • the fraction (G1) is obtained, and this fractional force fluorescence activity cell sorting (FACS) is used to obtain anti-H-2K b / H- that does not react with anti-H-2K b / H-2D b antibody and anti-CD45 antibody.
  • FACS fractional force fluorescence activity cell sorting
  • 2D b antibody (-) / anti-CD45 antibody (-) fraction (G2) sperm cells with testicular strength and spermatogonia activity in leukemic mice are obtained completely separated from leukemia cells. Succeeded. By using these sperm cells as transplanted cells, the fertility of male mice that had been treated with busulfan (an alkylating agent used for the treatment of chronic myeloid leukemia) and lost spermatogenic activity was It succeeded in restoring without causing.
  • busulfan an alkylating agent used for the treatment of chronic myeloid leukemia
  • the recipient's mouse transplanted with the obtained sperm cells did not suffer from leukemia, formed normal sperm, and its normal sperm power was born, which is associated with anticancer treatment of childhood leukemia This shows that autotransplantation of sperm cells obtained by separating and removing leukemia cells by the method of the present invention is effective for the treatment of male infertility.
  • Figures a and c show the results of flow cytometry of leukemia cells (C1498 cells), and figures b and d show the flow of sperm cells (Germ cells) derived from normal C57BL / 6 mice. The results of cytometry are shown.
  • Figures a and b show two parameter histograms for forward scatten FSC and side scatter (SSC) obtained for leukemia cells (C 1498 cells) and sperm cells, respectively.
  • SSC side scatter
  • Figure c shows the FSC high and SSC 1 TM cell population fractions (G1 2 is a histogram showing the response of the (fraction) to anti-CD45 antibody, anti-H-2K b / H-2D b antibody, and PI (propidium iode).
  • Figure d shows the anti-CD45 antibody and anti-H-2K b / H_2D b antibody of the FSC high and SSC tow cell population fractions (G1 fraction) collected from the above two-parameter histogram for sperm cells. , And a histogram showing the response to PI (4 divisions).
  • Figures a and b show the results of flow cytometry of human leukemia cells (Jurkat cell line).
  • Figure a shows a two-parameter histogram for forward scatter (FSC) and side scatter (SSC) obtained for human leukemia cells.
  • Figure b shows the anti-CD45 antibody, anti-HLA-A, B, and FSC 1 " 8 * 1 and SS C lQW cell population fractions (G1 fraction) gated on human leukemia cells. It is a histogram which shows the reaction with respect to C antibody and PI (4 divisions).
  • FIG. 3 shows the results of flow cytometry performed in Example 3 (4).
  • Figure a is a two-parameter histogram of forward scatter (FSC) and side scatter (SSC) of mouse sperm cells (non-GFP sperm cells) injected with C1498.
  • FSC forward scatter
  • SSC side scatter
  • Response of anti-CD45 antibody, anti-H-2K b / H-2D b antibody, and PI (propidium iode) of FSChigh and SSClow cell population fractions (G1 fraction) gated with the above two-parameter histogram force (4 divisions).
  • H-2Kb / H-2Db (-) and CD45 (-) fractions are sperm cells
  • H-2Kb / H-2Db (+) and CD45 (+) fractions are Including leukemia cells.
  • Figure c shows the survival rate of the recipient mice after intraperitoneal injection of the G2 fraction (sperm cell fraction) (broken line) and G3 fraction (leukemia cells) (solid line) separated by FACS into the recipient mouse. (Survival (%)) is the figure which showed with time (days).
  • FIG. 5 shows the results of observing the testes of recipient mice after transplantation of sperm cells of leukemia GFP mice into the seminiferous tubules of azoospermia treated with busulfan.
  • Figures a and b show the results of staining testis sections of the recipient mice 2 weeks after transplanting non-cell sorted sperm cells with hematoxylone and eosin.
  • Figure c is the testis force of leukemia GFP mice Cell transplanted sperm cell fraction (G2 fraction) 8 weeks after transplanting recipient's mouse testis image
  • Figure d shows fluorescence detection It is an image.
  • Fig. E shows an image of the seminiferous tubule taken from the testes of the recipient's mouse
  • F shows an image obtained by fluorescence detection.
  • Transplanted donor sperm cells green are observed to accumulate in the recipient seminiferous tubule.
  • Figure g shows sperm cells isolated from recipient mouse testes.
  • Figures h and i are images of hematoxylon-stained ( Figure h) sections of recipient mouse testis transplanted with the sorted sperm cell fraction, and fluorescence-detected images ( Figure i). Note that the scale bar size in each figure is lmm for figures a, c, d, e, and f, 10 ⁇ for figure b, 20 m for figure g, and 100 m for figures h and i. In fc.
  • Fig. 6 shows the testicular strength of recipient mice transplanted with GFP sperm cells (donor sperm cells) obtained by cell sorting in Example 3 (6). The normal offspring (pups) born by injecting and mating are shown.
  • Figure b shows the result of observation under fluorescence. This shows that spermatozoa derived from transplanted donor sperm cells are normal.

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Abstract

La présente invention concerne une méthode d'élimination totale des cellules leucémiques au sein d'une population de cellules comprenant des cellules leucémiques et des spermatogonies. La présente invention concerne également un réactif pouvant être employé pour mener à bien ladite méthode. La présente invention concerne en outre des cellules séminales de vertébrés non humains pouvant être obtenues à l'aide de ladite méthode, ainsi que l'emploi desdites cellules. Dans ladite méthode, un prélèvement contenant des cellules souches spermatogoniales (fraction de cellules souches spermatogoniales) est obtenu à partir de cellules reproductrices mâles à analyser contenant les cellules leucémiques. Puis, une fraction de cellules, qui ne réagit ni à un anticorps anti-marqueur de surface non exprimé par les spermatogonies, ni à un autre anticorps anti-marqueur de surface exprimé par les cellules leucémiques, est obtenue à partir de la fraction ci-dessus.
PCT/JP2006/301063 2005-01-26 2006-01-24 Méthode d'élimination de cellules leucémiques au sein d'une population de cellules de testicule et kit de réactifs employé dans ladite méthode Ceased WO2006080314A1 (fr)

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CN101550406B (zh) * 2008-04-03 2016-02-10 北京大学 制备多潜能干细胞的方法,试剂盒及用途
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US12259311B2 (en) 2018-06-13 2025-03-25 Thinkcyte K.K. Methods and systems for cytometry
US12235202B2 (en) 2019-12-27 2025-02-25 Thinkcyte K.K. Flow cytometer performance evaluation method and standard particle suspension
US12298221B2 (en) 2020-04-01 2025-05-13 Thinkcyte K.K. Observation device
US12339217B2 (en) 2020-04-01 2025-06-24 Thinkcyte K.K. Flow cytometer

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