WO2009119105A1 - PROCÉDÉ DE PRÉPARATION IN VITRO DE PLAQUETTE GPIBα+GPV+GPVI+ - Google Patents

PROCÉDÉ DE PRÉPARATION IN VITRO DE PLAQUETTE GPIBα+GPV+GPVI+ Download PDF

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WO2009119105A1
WO2009119105A1 PCT/JP2009/001383 JP2009001383W WO2009119105A1 WO 2009119105 A1 WO2009119105 A1 WO 2009119105A1 JP 2009001383 W JP2009001383 W JP 2009001383W WO 2009119105 A1 WO2009119105 A1 WO 2009119105A1
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cells
protein
positive
adam10
adam17
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PCT/JP2009/001383
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English (en)
Japanese (ja)
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中内啓光
江藤浩之
錦井秀和
高山直也
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国立大学法人東京大学
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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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0644—Platelets; Megakaryocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/19—Platelets; Megacaryocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/02—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from embryonic cells

Definitions

  • the present invention relates to a method for preparing platelets in vitro. More particularly, the present invention relates to a method for preparing platelets that retain in vivo function in vitro.
  • hematopoietic progenitor cells For the treatment of blood-related diseases such as leukemia, it is extremely important to stably amplify and supply the amount of blood cells necessary for the treatment. Efficient amplification of stem cells or hematopoietic progenitor cells has been attempted.
  • megakaryocytes are cells that produce platelet precursors and platelets and occupy an important position in therapeutic applications.
  • platelets since platelets are essential cells for blood coagulation (hemostasis), the demand for platelets in leukemia, bone marrow transplantation, anticancer therapy, etc. is extremely high.
  • platelets were collected by donating blood from donors, as well as methods of administering TPO, methods of differentiating megakaryocytes from umbilical cord blood or bone marrow cells. Furthermore, recently, a method of amplifying hematopoietic stem cells or hematopoietic progenitor cells in vitro and preparing platelets from these progenitor cells has also been attempted. For example, self-replicating from mouse ES cells to lymphocytes has been attempted.
  • a method of establishing a hematopoietic stem cell line capable of differentiating (Patent Document 1), a primate ES cell induced by in vitro differentiation, and the resulting cell transplanted into a fetus in a sheep uterus
  • Patent Document 2 A method for obtaining primate hematopoietic cells from the above (Patent Document 2), or a method for simply and stably amplifying CD34 positive / CD38 negative cells maintaining the undifferentiated nature of hematopoietic stem cells in vitro (Patent Document 3) ) Etc. have been reported.
  • Non-patent Document 3 the binding between the platelet receptor GPVI and collagen is also important for blood coagulation, but GPVI is cleaved by ADAM10 and cannot bind to collagen. It is thought to be lost (Non-Patent Document 3).
  • Non-patent Documents 4 and 5 reports that indirectly suggest that platelet production cannot be induced in an in vitro culture system in a state where the activity of ADAM10 and / or ADAM17 is inhibited.
  • Non-Patent Document 6 reports that a signal via ADAM10 rather inhibits the induction of megakaryocytes, and simply suppressing the function of ADAM10 and / or ADAM17, and obtaining platelets that stably maintain the function This is not possible.
  • a methodology for obtaining functionally stable platelets in vitro has not been established.
  • the present inventors provide a preparation method for maintaining the blood coagulation function of platelets for a long period of time in preparing platelets in vitro.
  • ADAM10 and ADAM17 As described above, it is necessary to inhibit the activity of ADAM10 and / or ADAM17 in order to maintain the hemostasis (blood coagulation) function of platelets.
  • ADAM10 and ADAM17 since the activity of ADAM10 and ADAM17 is necessary for the induction of platelets, it was necessary to clarify at which point of the platelet induction process the activity of ADAM10 and ADAM17 should be suppressed. Accordingly, the present inventors have studied in detail the timing at which the activities of ADAM10 and ADAM17 should be suppressed in the process of inducing platelets from ES cells, and completed the present invention.
  • the present inventors also added a p38 MAP kinase inhibitor that indirectly inhibits activation of these metalloprotease activities. It was found that the cleavage and removal of GPIb ⁇ , GPV and GPVI on the platelet surface can be suppressed and the hemostasis (blood coagulation) function of platelets can be maintained.
  • a first aspect of the present invention is “a method for preparing platelets comprising the following (a) to (c): (A) Megakaryocyte progenitor cells that are human-derived c-Kit negative / CD34 positive / integrin ⁇ IIb (CD41) positive / GPI ⁇ negative cells or c-Kit negative / CD34 positive / integrin ⁇ IIb (CD41) positive / GPI ⁇ weakly positive cells Culturing the cells on feeder cells under conditions suitable for inducing differentiation of megakaryocytes, (B) a step of inducing functional loss or loss of ADAM10 protein and / or ADAM17 protein present in megakaryocyte progenitor cells after culturing in step (a), (C) a step of further culturing the megakaryocyte progenitor cells after inducing reduced or lost function of ADAM10 protein and / or ADAM17 protein in step (b) to release platelets
  • the second aspect of the present invention is as follows: “The culture in the step (a) is carried out for 6 to 10 days until GPIb ⁇ -positive multinucleated cells appear, and then the function is reduced or lost in the step (b). The method according to (1) above, which is induced. (3) According to a third aspect of the present invention, “the c-Kit negative / CD34 positive / integrin ⁇ IIb (CD41) positive / GPI ⁇ negative cell or c-Kit negative / CD34 positive / integrin ⁇ IIb (CD41) positive / GPI ⁇ weak” The method according to (1) or (2) above, wherein the positive cells are prepared from a net-like structure obtained by culturing human-derived ES cells or iPS cells.
  • the fourth aspect of the present invention is as follows: “Human-derived ES cells or iPS cells are cultured for 20 to 23 days until GPI ⁇ -positive multinucleated cells appear; The method according to (3) above, which induces loss.
  • a fifth aspect of the present invention is as follows: “The culture in the step (a) is performed in the presence of TPO, SCF and / or Heparin, any of the above (1) to (4) Is the method described in the above.
  • a sixth aspect of the present invention is “a method for preparing platelets comprising the following (a) to (c): (A) Mice-derived megakaryocyte progenitor cells that are c-Kit negative / integrin ⁇ IIb (CD41) positive / GPI ⁇ negative cells or c-Kit negative / integrin ⁇ IIb (CD41) positive / GPI ⁇ weakly positive cells on feeder cells, Culturing under conditions suitable for inducing differentiation of megakaryocytes, (B) a step of inducing functional loss or loss of ADAM10 protein and / or ADAM17 protein present in megakaryocyte progenitor cells after culturing in step (a), (C) a step of further culturing the megakaryocyte progenitor cells after inducing reduced or lost function of ADAM10 protein and / or ADAM17 protein in step (b) to release platelets ”.
  • the seventh aspect of the present invention is as follows: “After the culture in the step (a) is carried out for 18 to 24 hours until GPIb ⁇ -positive multinucleated cells appear, the function in the step (b) is reduced or lost. The method according to (6) above, which is induced.
  • the eighth aspect of the present invention is that the c-Kit negative / integrin ⁇ IIb (CD41) positive / GPI ⁇ negative cell or c-Kit negative / integrin ⁇ IIb (CD41) positive / GPI ⁇ weak positive cell is derived from a mouse.
  • the ninth aspect of the present invention is as follows: “Mouse-derived ES cells or iPS cells are cultured for 9 to 11 days until GPI ⁇ -positive multinucleated cells appear; The method according to (8) above, which induces loss. (10) According to a tenth aspect of the present invention, “the culture in the step (a) is performed in the presence of TPO, IL-6 and / or IL-11” 9) ”.
  • the ADAM10 protein and / or ADAM17 protein is reduced in function or lost by introducing a mutation or deletion into the ADAM10 gene and / or ADAM17 gene.
  • the twelfth aspect of the present invention is characterized in that “the ADAM10 protein and / or the ADAM17 protein can be reduced in function or lost by destroying the entire ADAM10 gene and / or ADAM17 gene. The method according to any one of (1) to (10) above.
  • the thirteenth aspect of the present invention is characterized in that “the functional decrease or loss of ADAM10 protein and / or ADAM17 protein is achieved by addition of an inhibitor of ADAM10 protein and / or ADAM17 protein.
  • “the inhibitor is selected from the group consisting of GM6001, TAPI-1, TAPI-2, TAPI-3, or a p38 MAP kinase inhibitor, 13).
  • any one of the above (1) to (14) characterized in that it induces a decrease in function or loss of function of the ADAM10 protein and / or ADAM17 protein present in the feeder cell.
  • the ADAM10 protein and / or the ADAM17 protein is reduced in function or lost by introducing a mutation or deletion into the ADAM10 gene and / or the ADAM17 gene.
  • the method according to (15) above which is characterized in that (17)
  • the seventeenth aspect of the present invention is characterized in that “the functional decrease or loss of the ADAM10 protein and / or ADAM17 protein is achieved by disrupting the entire ADAM10 gene and / or ADAM17 gene. The method described in (15) above.
  • the eighteenth aspect of the present invention is characterized in that “the functional decrease or loss of the ADAM10 protein and / or ADAM17 protein is achieved by addition of an inhibitor of the ADAM10 protein and / or ADAM17 protein.
  • the method according to (15) above “the inhibitor is selected from the group consisting of GM6001, TAPI-1, TAPI-2, TAPI-3, or a p38 MAP kinase inhibitor ( 18) ".
  • a twentieth aspect of the present invention is “platelets prepared by any one of the methods (1) to (19)”.
  • a twenty-first aspect of the present invention is the “platelet preparation containing platelets according to (20) above”.
  • a twenty-second aspect of the present invention is “a kit for preparing GPIb ⁇ + GPV + GPVI + platelets containing an activity inhibitor or antagonist of ADAM10 protein and / or ADAM17 protein”.
  • the ADAM10 protein and / or ADAM17 protein activity inhibitor is selected from the group consisting of GM6001, TAPI-1, TAPI-2, TAPI-3, or p38MAP kinase inhibitor.” Or a kit according to (22) above.
  • the function of platelets prepared in an in vitro culture system can be stably maintained.
  • the method of the present invention exhibits an indispensable effect for maintaining the function of platelets.
  • GM6001 or TAPI-1 was added 10 days after culturing mouse ES cells. The results are shown as the mean value ⁇ standard error of four different experiments.
  • A On day 10 after culturing mouse ES cells, 1% DMSO (vehicle) or 100 ⁇ M GM6001 dissolved in 1% DMSO was added to the culture solution. The results of flow cytometry on day 12 after culturing mouse ES cells are shown.
  • ESC-MKs shows the results of mature megakaryocytes (GM6001 not added)
  • ESPs show the results of ES cell-derived platelets (hereinafter referred to as ESPs) (vehicle: GM6001 not added, GM-6001: GM6001 added).
  • (B) The total number of ⁇ IIb + ESPs per plate (6-well plate) after 12 days of culture is shown. When inhibitors were added on the 10th day of culture, the total number of platelets produced was not affected.
  • (C) When 1 ⁇ M or 10 ⁇ M TAPI-1 or 100 ⁇ M GM6001 is added after 10 days of culture, it is a graph showing the ratio of GPIb ⁇ + cells in ⁇ IIb + ESPs after 12 days of culture. It was shown that GPIba-positive platelets, which are functional indicators, are retained by the addition of GM6001.
  • (D) shows the effect of GM6001 on GPV or GPVI surface-expressing cells in ⁇ IIb + ESPs after 12 days of culture.
  • mice platelets that have passed through the time after blood collection are used as controls. It has also been confirmed that the hemostatic thrombus function is lower than that in the GM6001 treatment group. It is a result which shows that the lifetime in the living body of ESPs which performed GM6001 process is extended. (A) It is the figure which showed the flow of experiment typically.
  • (A) Flow cytometry results of washed human platelets (Fresh human plates) and human platelets (In vitro aged platelets) incubated at 37 ° C. for 24 hours in the presence or absence of GM6001 are shown.
  • (B) Cell spreading function by washed human platelets (Fresh human plates) and human platelets (In vitro aged platelets) incubated at 37 ° C. for 24 hours in the presence (GM6001) or absence (vehicle) of GM6001. The comparison result is shown.
  • (C) The area of cells expanded by human platelets incubated at 37 ° C. for 24 hours in the presence (GM6001) or absence (vehicle) of washed human platelets (fresh human platelets) The result is shown as 100%.
  • GM6001 The inhibitory effect on the cleavage of CD42b (GPIb ⁇ ) on the surface of platelets derived from human iPS cells and human ES cells by a matrix metalloproteinase inhibitor (GM6001) was examined. On day 22 after culturing iPS cells (TkDN4-M) and ES cells (KhES-3), GM6001 was added at a final concentration of 0 ⁇ M, 20 ⁇ M, 50 ⁇ M (TkDn4-M), or 0 ⁇ M, 100 ⁇ M (KhES) 2 days before platelet collection. -3), and platelets were collected and measured with a flow cytometer.
  • the present invention provides platelets that stably maintain the thrombus formation function (or blood coagulation function) by appropriately adjusting the activity of ADAM10 and / or ADAM17 in the process of preparing platelets from ES cells or iPS cells.
  • ADAM10 or ADAM17
  • ADAM10 protein or When referring to an ADAM17 protein gene, it is described as an ADAM10 gene (or ADAM17 gene).
  • ADAM10 protein and ADAM17 protein are maintained until a hematopoietic progenitor cell population consisting of c-Kit positive / integrin ⁇ IIb (CD41) positive cells is induced, and thereafter platelets are induced. It is necessary to inhibit the activity of ADAM10 protein and ADAM17 protein at a specific time.
  • mouse-derived c-Kit negative / integrin ⁇ IIb (CD41) positive / GPI ⁇ weak positive cell hereinafter, c-Kit ⁇ / ⁇ IIb (CD41) + / GPI ⁇ ⁇ cell.
  • c-Kit negative / integrin ⁇ IIb (CD41) positive / GPI ⁇ negative cells hereinafter also referred to as c-Kit ⁇ / ⁇ IIb (CD41) + / GPI ⁇ ⁇ cells
  • “Positive” means a state in which a cell surface marker (eg, c-Kit, integrin ⁇ IIb (CD41), GPI ⁇ , etc.) is expressed on the cell surface and the expression can be confirmed by an antibody or the like (previous) “Weakly positive” means that expression of a cell surface marker can be confirmed with an antibody or the like, but the expression level is low (for example, when measured with a flow cytometer, expression of 1 log or less) “A negative expression state” indicates that the cell surface marker is not expressed on the cell surface and the expression cannot be confirmed by an antibody or the like.
  • a cell surface marker eg, c-Kit, integrin ⁇ IIb (CD41), GPI ⁇ , etc.
  • mouse-derived c-Kit ⁇ / ⁇ IIb (CD41) + / GPI ⁇ ⁇ cell or “mouse-derived c-Kit ⁇ / ⁇ IIb (CD41) + / GPI ⁇ ⁇ cell” is derived from mouse-derived ES cells or iPS cells.
  • a differentiation promoting factor such as TPO (for example, about 1 to 100 ng / ml, preferably about 10 to 30 ng / ml) is added from the differentiated c-Kit + / ⁇ IIb (CD41) + cells, for example, on feeder cells. Can be guided.
  • the culture period required for inducing c-Kit + / ⁇ IIb (CD41) + cells from mouse-derived ES cells or iPS cells varies depending on the culture method or cell type, and is, for example, about 6 days (or 5 to 5 to About 7 days).
  • the culture period is not particularly limited, for example, it is 1 to 5 days, preferably 2 to 3 days (the culture period from mouse ES cells or iPS cells is about 8 to 9 days). is there.
  • c-Kit ⁇ / ⁇ IIb (CD41) + / GPI ⁇ ⁇ cells or c-Kit ⁇ / ⁇ IIb (CD41) + / GPI ⁇ ⁇ cells are identified by the absence of c-kit on the cell surface and the presence of integrin ⁇ IIb.
  • the cells can be obtained by using an antibody or the like, for example, using a cell sorter system. It should be noted that c-Kit + / ⁇ IIb (CD41) + cells can be induced by any method that can be selected by those skilled in the art, and the cells can be identified as described above.
  • TPO about 1 to 100 ng / ml
  • IL-6 about 1 to 100 ng / ml
  • IL-11 about 1 to 100 ng / ml
  • the appearance of GPI ⁇ -positive multinucleated cells is confirmed after the start of the culture. Thereafter, for example, about 18 to 24 hours later (from mouse ES cells or iPS cells, about 10 days of culture (eg, about 9 to 11 days).
  • human-derived c-Kit negative / CD34 positive / integrin ⁇ IIb (CD41) positive / GPI ⁇ weak positive cells hereinafter c-Kit ⁇ / CD34 + / ⁇ IIb (CD41) + / GPI ⁇ ⁇ cells
  • c-Kit negative / CD34 positive / integrin ⁇ IIb (CD41) positive / GPI ⁇ negative cells hereinafter also referred to as c-Kit ⁇ / CD34 + / ⁇ IIb (CD41) + / GPI ⁇ ⁇ cells
  • “Positive” means a state in which cell surface markers (for example, CD34, integrin ⁇ IIb (CD41), GPI ⁇ , etc.) are expressed on the cell surface and the expression can be confirmed by an antibody or the like (see above). Synonymous), “weakly positive” means that the expression of a cell surface marker can be confirmed by an antibody or the like, but the expression level is low (for example, an expression of 1 log or less when measured with a flow cytometer or the like) “Negative” means that the cell surface marker is not expressed on the cell surface, and the expression cannot be confirmed by an antibody or the like.
  • cell surface markers for example, CD34, integrin ⁇ IIb (CD41), GPI ⁇ , etc.
  • human-derived c-Kit negative / CD34 positive / integrin ⁇ IIb (CD41) positive / GPI ⁇ weakly positive cells or c-Kit negative / CD34 positive / integrin ⁇ IIb (CD41) positive / GPI ⁇ negative cells are human-derived ES cells or It can be prepared from a net-like structure prepared from iPS cells (ES-sac, described later).
  • TPO about 1 to 100 ng / ml
  • SCF about 10 to 200 ng
  • Heparin about 10 to 50 U / ml
  • timing to start the suppression for example, after confirming the appearance of GPI ⁇ -positive multinucleated cells, for example, c-Kit negative / CD34 positive / Integrin ⁇ IIb (CD41) positive / GPI ⁇ weakly positive cells or c-Kit negative / CD34 positive / integrin ⁇ IIb (CD41) positive / GPI ⁇ negative cells after culturing for 6 to 10 days, preferably 7 to After about 9 days of culturing or after culturing from human-derived ES cells or iPS cells, about 22 days (for example, about 20 to 23 days).
  • the method of the present invention can be applied to the preparation of platelets of any animal species, and includes, but is not limited to, cells derived from, for example, humans, mice, rats, dogs, cats, cows, horses, sheep, etc. Can be used, preferably cells derived from mice, rats, and humans, more preferably cells derived from mice or humans, and particularly preferably cells derived from humans.
  • the starting cells for inducing platelets of the invention can be “ES cells”, “iPS cells” and the like.
  • the source of “ES cells” or “iPS cells” may be any animal, but is not limited to, for example, cells derived from humans, mice, rats, dogs, cats, cows, horses, sheep, etc. Even if it exists, it is preferably a cell derived from mouse, rat or human, more preferably a cell derived from mouse or human, and particularly preferably a cell derived from human.
  • feeder When inducing platelets in an in vitro culture system, when culturing ES cells or iPS cells, or cells in the differentiation stage during differentiation from these cells into platelets (particularly in the process of inducing mature megakaryocytes), feeder Preferably co-cultured with cells. Any cell can be used as the “feeder cell” as long as it contributes to differentiation induction of ES cells or iPS cells.
  • mouse embryo fibroblasts preferably 10T1 / 2 cells Strains, OP9 cells and the like can be used.
  • human bone marrow-derived mesenchymal stem cells cells obtained by direct preparation from human bone marrow and cultured
  • they are derived from human ES cells or iPS cells. Megakaryocyte maturation and platelet production are possible.
  • extracellular matrix such as Matrigel
  • the efficiency is reduced, but platelets can be induced.
  • feeder cells it is preferable to suppress the proliferation of cells by irradiating with radiation.
  • any method that can be easily selected by those skilled in the art may be used.
  • an embryoid body differentiated mesodermal undifferentiated cells in the middle of reaching platelets.
  • ES-sac net-like structure
  • ES-sac The method for forming a net-like structure (ES-sac) is outlined below.
  • megakaryocytes derived from ES-sac are cultured under the conditions in which TPO / SCF / Heparin is added around day 14 to day 16 after the culture of ES cells or iPS cells. Re-roll and maintain culture.
  • an inhibitor of ADAM10 or ADAM17 activity such as GM6001 is added on the 20th to 23rd day after the start of culture (6th to 10th day after re-wrinkling of ES-sac).
  • the culture conditions suitable for preparing the net-like structure vary depending on the ES cell or iPS cell to be used.
  • IMDM IMDM supplemented with FBS at a final concentration of 15%
  • those appropriately added with growth factors and supplements can be used.
  • VEGF should be added at about 0 to 100 ng / ml and about 0 to 300 ng / ml, more preferably about 20 ng / ml and about 200 ng / ml.
  • the culture environment varies depending on the type of ES cells or iPS cells to be used. For example, conditions of 5% CO 2 , 36 to 38 ° C., preferably 37 ° C. can be used.
  • the culture period until the net-like structure is formed varies depending on the type of ES cell or iPS cell, but its presence can be confirmed about 14 to 16 days after seeding on the feeder cell.
  • the formed net-like structure has a follicular structure in which hematopoietic progenitor cells are present in a concentrated state. Hematopoietic progenitor cells present inside the net-like structure can be separated by passing through physical means such as a sterilized sieving instrument (eg, a cell strainer).
  • This hematopoietic progenitor cell is further cultured, and c-Kit negative / CD34 positive / integrin ⁇ IIb (CD41) positive / GPI ⁇ weak positive cell or c-Kit negative / CD34 positive / integrin ⁇ IIb (CD41) positive / GPI ⁇ negative cell, Platelets can be prepared.
  • ADAM10 protein which is the gene product, is a membrane protein having a disintegrin domain in structure and having a metalloprotease activity, and is considered to be involved in cell differentiation through cleavage of the extracellular domain of various membrane proteins. Yes.
  • ADAM10 protein is defined based on the characteristics of the primary sequence of amino acids, it is not limited. For example, it is a protein consisting of an amino acid sequence identical or substantially identical to the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4. .
  • the “protein consisting of substantially the same amino acid sequence” means about 60% or more, preferably about 70% or more, more preferably about 80%, with the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, most preferably about 99% amino acid sequence, and a protein having metalloprotease activity.
  • amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 As a protein consisting of an amino acid sequence substantially the same as the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4, one or several of the amino acid sequences represented by SEQ ID NO: 2 or SEQ ID NO: 4 (preferably, A protein having an amino acid sequence in which about 1 to 30, more preferably about 1 to 10, and more preferably 1 to 5 amino acids are deleted, substituted, or added, and has a metalloprotease activity. .
  • ADAM17 (or TACE) protein was identified as a membrane protein that cleaves TNF- ⁇ involved in cell differentiation and cell death.
  • ADAM17 protein has a disintegrin domain structurally and has metalloprotease activity.
  • the ADAM17 protein is defined from the characteristics on the primary sequence of amino acids, it is not limited, but for example, it is the same or substantially the same as the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12.
  • the “protein consisting of substantially the same amino acid sequence” means about 60% or more, preferably about 70% or more, of the amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12. More preferably, about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, A protein having an amino acid sequence having 95%, 96%, 97%, 98%, most preferably about 99% amino acid identity and having metalloprotease activity.
  • amino acid sequence represented by SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 12 SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 or SEQ ID NO: 1 or several (preferably about 1 to 30, more preferably about 1 to 10, more preferably 1 to 5) amino acids in the amino acid sequence represented by 12 have been deleted, substituted or added
  • a protein consisting of an amino acid sequence and having metalloprotease activity.
  • the gene product is produced by introducing a mutation (substitution, insertion or deletion, or modification or modification) into the gene.
  • a mutation substitution, insertion or deletion, or modification or modification
  • Examples thereof include a method of inhibiting, and a method of directly inhibiting the activity (for example, metalloprotease activity) of the protein that is the gene product.
  • a method for introducing mutation substitution, insertion or deletion, or modification or modification
  • a method of destroying the entire ADAM10 gene and ADAM17 gene by homologous recombination using an appropriate gene targeting vector A method for introducing a mutation into a region important for the activity of the gene product using a Cre-loX system or the like, a method for introducing an antisense nucleotide or an antisense oligonucleotide, etc., and suppressing transcription of an ADAM10 gene or an ADAM17 gene (For example, an RNAi method using a lentiviral vector).
  • RNAi method when ADAM10 or ADAM17 functions are lost or lowered by the RNAi method, a method of inducing inhibition of ADAM10 or ADAM17 gene expression by siRNA at a desired timing can be suitably performed. For example, by appropriately introducing an siRNA expression gene linked to an inducible promoter that functions in response to a drug, a nutritional factor, or the like into an ES cell or iPS cell and activating the promoter at a desired timing, ADAM10 and ADAM17 gene expression can be suppressed at the time.
  • ADAM10 protein or ADAM17 protein in addition to a method using an inhibitor for the metalloprotease activity of the gene product, the activity of ADAM10 protein or ADAM17 protein is inhibited.
  • a method using an antagonist may be considered.
  • the inhibitor for metalloprotease activity include, but are not limited to, GM6001, TAPI-1, TAPI-2, TAPI-3, and the like.
  • inhibitors that indirectly inhibit these activations it is also possible to use inhibitors that indirectly inhibit these activations.
  • p38 MAP kinase inhibitor for example, product numbers SB202190, SB203580 (Calbiochem) and the like can be used, but not limited thereto, any of those that inhibit the kinase activity of p38MAP kinase can be used. These compounds can also be used.
  • the object of the present invention can be achieved by inhibiting the activity of a factor that functions in the activation pathway of ADAM10 or ADAM17, such as 38MAP kinase.
  • a factor that functions in the activation pathway of ADAM10 or ADAM17 such as 38MAP kinase.
  • These inhibitors can be used alone or in combination.
  • the optimal concentration of the inhibitor varies depending on the inhibitor used and the culture conditions, but those skilled in the art can easily determine the optimal concentration by preliminary experiments.
  • GM6001, TAPI-1, TAPI -2 or TAPI-3 about 50-200 ⁇ M, preferably about 100 ⁇ M
  • p38 MAP kinase inhibitor for example, about 1-100 ⁇ M, preferably about 1-50 ⁇ M, more preferably 5-20 ⁇ M. Degree.
  • any substance that inhibits the activity of ADAM10 protein or ADAM17 protein can be used, and is not limited.
  • the antagonist specifically binds to ADAM10 protein or ADAM17 protein and exhibits its activity.
  • Examples thereof include an aptamer molecule to be inhibited or an antibody.
  • a method for introducing a mutation (substitution, insertion or deletion, modification or modification) into the ADAM10 gene or ADAM17 gene, an aptamer molecule for inhibiting the metalloprotease activity of the ADAM10 protein or ADAM17 protein, or The method for preparing the antibody is not limited to the method described herein, and can be easily carried out by a method arbitrarily selected by those skilled in the art from methods well known in the art.
  • kits for preparing GPIb ⁇ + GPV + GPVI + platelets include an inhibitor that directly inhibits the protease activity of ADAM10 protein and / or ADAM17 protein (eg, GM6001, TAPI-1, TAPI-2, or TAPI-3), and an inhibitor that indirectly inhibits the activity of the protease.
  • an inhibitor that directly inhibits the protease activity of ADAM10 protein and / or ADAM17 protein eg, GM6001, TAPI-1, TAPI-2, or TAPI-3
  • an inhibitor that indirectly inhibits the activity of the protease e.g., p38 MAP kinase inhibitor
  • antagonists that inhibit the protease activity aptamer molecules that inhibit the activity, antibodies, etc.
  • as well as diluted solutions of inhibitors antibiotics, cell washing reagents, etc.
  • Inhibitors and the like contained in the kit may be in a solution state or a powder state, and the reagents and the like contained in the kit are such that the components keep the activity effectively for a long period of time and are not adsorbed by the material of the container. Supplied in any type of container that is not subject to alteration.
  • a sealed glass ampoule includes a buffer packaged under a neutral and non-reactive gas such as nitrogen gas. Ampoules are composed of glass, polycarbonate, organic polymers such as polystyrene, ceramics, metals, or any other suitable material commonly used to hold reagents.
  • the kit also includes instructions for use.
  • kit Instructions for using the kit are printed on paper or other material and / or electrically or electromagnetically such as floppy disk, CD-ROM, DVD-ROM, Zip disk, video tape, audio tape, etc. It may be supplied as a readable medium. Detailed instructions for use may be actually attached to the kit, or may be posted on a website designated by the manufacturer or distributor of the kit or notified by e-mail or the like.
  • the present invention includes blood products (platelet products) containing platelets prepared by the method of the present invention.
  • blood products containing platelets can be easily performed by those skilled in the art.
  • a solution necessary to preserve the function of platelets eg, ACD-A solution (prepared from sodium citrate / citric acid / glucose)
  • fresh frozen plasma obtained by blood donation
  • the container which stores the formulation containing platelets from the material which activates platelets like glass.
  • Feeder cell-independent ES cells are 10% FBS (JRH BIOSCIENCES, USA), 0.1 mM non-essential amino acids (NEAA; Invitrogen / GIBCO), 0.11 mM 2-mercaptoethanol (GIBCO), 1 mM pyruvate (GIBCO) , 1000 U / ml LIF (leukemia inhibitory factor) (CHEMICON), 2 mM L-glutamine, 100 U penicillin, 0.1 mg / ml streptomycin added GMEM (Glasgow minimum essential medium). The medium was changed every other day and subcultured every two days.
  • mice stromal cell line OP9 cells OP9 cells were subcultured with ⁇ -Minimum Essential Medium ( ⁇ -MEM (Invitrogen / GIBCO)) supplemented with 15% FBS, 2 mM L-glutamine, 100 U penicillin, 0.1 mg / mL streptomycin. The culture medium was changed every day, and in order not to change the character of the cells, cells having a subculture number of 30 or less from the primary culture were used in the experiment. 1-3.
  • ⁇ -MEM ⁇ -Minimum Essential Medium
  • mice Embryoid body formation (1st to 6th day of culture) Subcultured mouse ES cells were treated with 0.25% trypsin-EDTA, 15% FBS, 300 ⁇ g / mL human transferrin (Sigma), 4.5 mM monothioglycerol (Sigma), 50 ⁇ g / mL ascorbic acid ( Sigma), 0.1 mM 2-mercaptoethanol (Invitrogen / GIBCO), Iscove's Modified Dulbecco's Medium (IMDM; Invitrogen / GIBCO) supplemented with 2 mM L-glutamine, 100 U penicillin, 0.1 mg / mL streptomycin And cultured in a Petri dish (Sterilin).
  • IMDM Iscove's Modified Dulbecco's Medium
  • Culture is started at a cell number of 2 ⁇ 10 5 cells per 10 mL of culture solution in a 10 cm Petri dish, and embryoid body formation is attempted. On the fourth day of culture, the culture medium was replaced with a fresh one. Embryoid bodies produced on day 6 of culture were treated with 0.25% trypsin, reacted with anti-CD41 antibody and anti-c-Kit antibody, sorted, and co-cultured with OP9 cells (megakaryocytes). The progenitor cell population is enriched in c-Kit + ⁇ IIb (CD41) + cell population).
  • OP9 co-culture method (6th day after culture) OP-9 cells are prepared 6 days before the culture, and are prepared in a 90-100% confluent state on the day (6th day of ES cell differentiation start) on a 6-well plate.
  • -Kit + ⁇ IIb (CD41) prepared from embryoid bodies against 2 mL of differentiation medium ( ⁇ MEM supplemented with 15% FBS, 0.1 mM 2-mercaptoethanol, 20 ng / ml human TPO (thrombopoietin) (Peprotec)) A cell population of 2 ⁇ 10 4 was added and the culture was continued.
  • ADAM17 (and ADAM10) protein excises the extracellular domain of GPIb ⁇ on human and mouse platelets as platelets It has been reported to lose function. Therefore, GM6001 or TAPI-1 which is an inhibitor of metalloprotease activity of ADAM17 (and ADAM10) protein was added to a culture system for inducing platelets, and the function of induced platelets was examined.
  • FIG. 2D From the above, GPIb ⁇ plays an important role in the thrombus formation (blood coagulation) action of platelets by adding GM6001 or TAPI-1 on the 10th day of culture to inhibit the activity of ADAM17 protein and ADAM10 protein. It was revealed that the expression of GPV and GPVI on the platelet surface was maintained. In addition, it was confirmed that similar results were obtained when a p38 MAP kinase inhibitor (product numbers SB202190, SB203580, Calbiochem) was used (5 to 20 ⁇ M) (data not shown).
  • a p38 MAP kinase inhibitor product numbers SB202190, SB203580, Calbiochem
  • the flow chamber is an apparatus that can observe the time-dependent change of thrombus formation due to two colors of fluorescence with an epifluorescence microscope and quantify the ability to stop and form blood clots by platelets.
  • ESPs that emit red fluorescence from ES cells that express DsRed (red fluorescent label) under the control of the CAG promoter (ES cells were provided by Dr. Hitoshi Niwa, RIKEN, Kobe) (FIG. 3A).
  • the prepared ESPs were mixed with mouse whole blood labeled with platelets with mepacrine (green fluorescent label) at a ratio of 1: 1000.
  • the blood thus prepared was injected into the chamber by a syringe pump (Harvar Appratus) at a constant rate (for example, 1,500 s ⁇ 1 ).
  • the process of thrombus formation by platelets on the collagen surface was visualized by an inverted epifluorescence microscope (DMIRB, Leica) equipped with a video system.
  • DMIRB inverted epifluorescence microscope
  • washed platelets prepared from 10-12 week-old mice serum with 4 ⁇ g / ml PE-labeled anti- ⁇ IIb antibody
  • ESPs vehicle pretreated with 1% DMSO failed to adhere to the collagen-VWF matrix, but the adhesion ability improved when pretreated with GM6001 (FIGS.
  • GM6001 GM6001 added
  • the presence of approximately the same amount of GPIb ⁇ as that of untreated human platelets (FIG. 5A, Fresh human plates) was confirmed.
  • Integrin ⁇ IIb ⁇ 3 causes platelet aggregation when bound to the ligand fibrinogen. If this aggregation state is maintained, a signal is transmitted to the inside of the cell by integrin ⁇ IIb ⁇ 3, the shape of the cell changes, and cell extension is induced. It has been known.
  • KhES cells were dissociated using 0.05% trypsin-EDTA (Sigma), crushed into small colonies using a P-1000 pipette, 15% FBS, 2 mM L-glutamine (Invitrogen), ITS supplement (10 ⁇ g / ml insulin, 5.5 mg / ml transferrin, 5 ng / ml sodium selenite) (Sigma), 50 ⁇ g / ml ascorbic acid (Sigma), 0.45 mM MTG (Sigma), 20 ng / ml VEGF (R & D systems) Seeded on 10T1 / 2 cells in IMDM (IMDM; Invitrogen / GIBCO).
  • the net-like structure containing blood cell-like cells was collected around 14-15 days in culture, and the blood cell and the net-like structure were separated using a 70 ⁇ m cell strainer.
  • Fresh seeded blood cells at 2 ⁇ 3 ⁇ 10 4 / well on 10T1 / 2 cells were irradiated already prepared 6-well plates (one 6 ⁇ 10 5/6-well plate), 15% FBS (JRH BIOSCIENCES, USA), 2 mM L-glutamine (Invitrogen), ITS supplement (10 ⁇ g / ml insulin, 5.5 mg / ml transferrin, 5 ng / ml sodium selenite) (Sigma), 50 ⁇ g / ml ascorbic acid (Sigma), 0 Further culture in IMDM (IMDM; Invitrogen / GIBCO) supplemented with .45 mM MTG (Sigma), 100 ng / ml human TPO (Peprotec), 50 ng / ml SCF and 25
  • Human iPS cells consist of 15% FBS (JRH BIOSCIENCES, USA), 2 mM L-glutamine (Invitrogen), ITS supplements (10 ⁇ g / ml insulin, 5.5 mg / ml human transferrin, 5 ng / ml sodium selenite) (Sigma) 50 ⁇ g / ml ascorbic acid (Sigma), 0.45 mM MTG (Sigma), 20 ng / ml VEGF (R & D systems) in IMDM (IMDM; Invitrogen / GIBCO) seeded on OP9 cells or 10T1 / 2 cells Cultured. After the culturing, a large number of net-like structures containing blood cell-like cells were confirmed around 14 to 17 days.
  • a net-like structure was picked up using a P-1000 pipette under a phase-contrast microscope, and blood cells and net-like structures were separated using a 70 ⁇ m cell strainer.
  • Fresh seeded blood cells at 2 ⁇ 3 ⁇ 10 4 / well on 10T1 / 2 cells were irradiated already prepared 6-well plates (one 6 ⁇ 10 5/6-well plate), 15% FBS (JRH BIOSCIENCES, USA), 2 mM L-glutamine (Invitrogen), ITS supplement (10 ⁇ g / ml insulin, 5.5 mg / ml transferrin, 5 ng / ml sodium selenite) (Sigma), 50 ⁇ g / ml ascorbic acid (Sigma), 0 Further culture in IMDM (IMDM; Invitrogen / GIBCO) supplemented with .45 mM MTG (Sigma), 100 ng / ml human TPO (Peprotec), 50 ng /
  • the present invention makes it possible to stabilize the function of platelets induced as a result of culturing from ES cells and the like over a long period of time in vitro, so that a stable supply of platelets can be achieved and therapeutically beneficial effects can be achieved. Bring.

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Abstract

L’invention concerne un procédé permettant de conserver de manière stable la fonction d’une plaquette induite à partir d’une cellule souche embryonnaire ou d’une cellule souche pluripotente induite. L’invention concerne en particulier un procédé de préparation d’une plaquette comprenant les étapes suivantes (a) à (c) qui consiste à : (a) cultiver une cellule souche de mégacaryocyte, ladite cellule souche de mégacaryocyte étant une cellule humaine c-Kit négative/CD34 positive/intégrine αIIb (CD41) positive/ GPIα négative ou une cellule humaine c-Kit négative/CD34 positive/intégrine αIIb (CD41) positive/ GPIα faiblement positive; (b) induire la réduction ou la perte de la fonction de protéine ADAM10 et/ou de protéine ADAM17 dans la cellule souche de mégacaryocyte après la culture dans l’étape (a); et (c) cultiver davantage la cellule souche de mégacaryocyte obtenue après l’induction de la réduction ou la perte de la fonction de protéine ADAM10 et/ou de protéine ADAM17 dans l’étape (b) pour provoquer la libération d’une plaquette à partir de la cellule.
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EP2277995A1 (fr) * 2008-04-01 2011-01-26 The University of Tokyo Procédé de préparation de plaquette à partir de cellule ips
WO2012036257A1 (fr) 2010-09-17 2012-03-22 国立大学法人東京大学 Composition destinée à maintenir la fonction plaquettaire
WO2014100779A1 (fr) * 2012-12-21 2014-06-26 Advanced Cell Technology, Inc. Procédés de production de plaquettes à partir de cellules souches pluripotentes, et compositions associées
WO2014196624A1 (fr) 2013-06-07 2014-12-11 科研製薬株式会社 Composition pour maintenir la fonction plaquettaire
JP2018519845A (ja) * 2015-07-23 2018-07-26 エタブリスマン フランセ デュ サン Ｃｄ３４＋ｃｄ４１ｄｉｍ巨核球前駆細胞及び血小板前駆細胞を有するｍｋ及び／又はその血小板を製造するためのその使用。
US11566228B2 (en) 2006-04-14 2023-01-31 Astellas Institute For Regenerative Medicine Hemangio-colony forming cells

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US11566228B2 (en) 2006-04-14 2023-01-31 Astellas Institute For Regenerative Medicine Hemangio-colony forming cells
EP2277995A4 (fr) * 2008-04-01 2012-08-22 Univ Tokyo Procédé de préparation de plaquette à partir de cellule ips
US8546141B2 (en) 2008-04-01 2013-10-01 The University Of Tokyo Method for preparation of platelet from iPS cell
EP2277995A1 (fr) * 2008-04-01 2011-01-26 The University of Tokyo Procédé de préparation de plaquette à partir de cellule ips
JP5876828B2 (ja) * 2010-09-17 2016-03-02 国立大学法人 東京大学 血小板の機能を維持するための組成物
WO2012036257A1 (fr) 2010-09-17 2012-03-22 国立大学法人東京大学 Composition destinée à maintenir la fonction plaquettaire
US20130177900A1 (en) * 2010-09-17 2013-07-11 Kaken Pharmaceutical Co., Ltd. Composition for maintaining function of platelets
EP2617812A1 (fr) * 2010-09-17 2013-07-24 The University of Tokyo Composition destinée à maintenir la fonction plaquettaire
EP2617812A4 (fr) * 2010-09-17 2014-10-22 Univ Tokyo Composition destinée à maintenir la fonction plaquettaire
US9034922B2 (en) 2010-09-17 2015-05-19 The University Of Tokyo Composition for maintaining function of platelets
CN105101979B (zh) * 2012-12-21 2021-10-08 安斯泰来再生医药协会 由多能干细胞制备血小板的方法及其组合物
EP3973967A1 (fr) * 2012-12-21 2022-03-30 Astellas Institute for Regenerative Medicine Procédés de production de plaquettes à partir de cellules souches pluripotentes, et compositions associées
CN105101979A (zh) * 2012-12-21 2015-11-25 奥卡塔治疗公司 由多能干细胞制备血小板的方法及其组合物
WO2014100779A1 (fr) * 2012-12-21 2014-06-26 Advanced Cell Technology, Inc. Procédés de production de plaquettes à partir de cellules souches pluripotentes, et compositions associées
US11400118B2 (en) 2012-12-21 2022-08-02 Astellas Institute For Regenerative Medicine Methods for production of platelets from pluripotent stem cells and compositions thereof
US10894065B2 (en) 2012-12-21 2021-01-19 Astellas Institute For Regenerative Medicine Methods for production of platelets from pluripotent stem cells and compositions thereof
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US10426799B2 (en) 2012-12-21 2019-10-01 Astellas Institute For Regenerative Medicine Methods for production of platelets from pluripotent stem cells and compositions thereof
CN105392482B (zh) * 2013-06-07 2017-08-08 科研制药株式会社 用于维持血小板的功能的组合物
WO2014196624A1 (fr) 2013-06-07 2014-12-11 科研製薬株式会社 Composition pour maintenir la fonction plaquettaire
KR20160018517A (ko) 2013-06-07 2016-02-17 가껭세이야꾸가부시기가이샤 혈소판의 기능을 유지하기 위한 조성물
JPWO2014196624A1 (ja) * 2013-06-07 2017-02-23 科研製薬株式会社 血小板の機能を維持するための組成物
CN105392482A (zh) * 2013-06-07 2016-03-09 科研制药株式会社 用于维持血小板的功能的组合物
JP2018519845A (ja) * 2015-07-23 2018-07-26 エタブリスマン フランセ デュ サン Ｃｄ３４＋ｃｄ４１ｄｉｍ巨核球前駆細胞及び血小板前駆細胞を有するｍｋ及び／又はその血小板を製造するためのその使用。
US11236303B2 (en) 2015-07-23 2022-02-01 Institut National De La Sante Et De La Recherche Medicale (Inserm) CD34+CD41DIM megakaryocytes progenitors and uses thereof for producing proplatelet-bearing MKs and/or platelets
JP7068162B2 (ja) 2015-07-23 2022-05-16 エタブリスマン フランセ デュ サン Ｃｄ３４＋ｃｄ４１ｄｉｍ巨核球前駆細胞及び血小板前駆細胞を有するｍｋ及び／又はその血小板を製造するためのその使用。
JP7068162B6 (ja) 2015-07-23 2023-12-20 エタブリスマン フランセ デュ サン Ｃｄ３４＋ｃｄ４１ｄｉｍ巨核球前駆細胞及び血小板前駆細胞を有するｍｋ及び／又はその血小板を製造するためのその使用。

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