METHOD OF PRODUCTION OF POLYPLOIDIZED MEGAKYOCYTES AND METHOD OF PRODUCTION OF PLATELETS

专利摘要:
method of producing polyploidized megakaryocytes, composition of blood cells, method of producing a platelet, platelet and blood product. the present invention relates to a method of promoting megakaryocyte polyploidization and, therefore, megakaryocyte production. highly polyploid, a method of efficiently producing platelets from polyploidized megakaryocytes and the like. The present invention provides a method of producing polyploidized megakaryocytes comprising a step of forcing expression of an apoptosis suppressor gene in megakaryocytes prior to polyploidization and culturing the resulting cells.
公开号:BR112013029334B1
申请号:R112013029334-9
申请日:2012-05-11
公开日:2022-01-18
发明作者:Hiromitsu Nakauchi；Koji Eto；Naoya Takayama；Sou Nakamura
申请人:The University Of Tokyo；
IPC主号:

专利说明:

technical field
[0001] The present invention relates to a method for efficiently polyploidizing megakaryocytes prior to polyploidization, a method for producing platelets from such megakaryocytes, and the like. Prior Art
[0002] A large number of blood cells are needed for the treatment of blood-related diseases or surgical treatments. Among blood cells, a platelet which is an indispensable cell for blood clotting and hemostasis is a particularly important blood cell. The demand for platelets is high in cases of leukemia, bone marrow transplantation, anti-cancer treatment and the like, so the need for their stable supply is high. Platelets have so far been stably supplied by a method of collecting blood from donors, a method of administering a drug with a TPO-like structure (mimetic), a method of differentiating megakaryocytes from umbilical cord blood, or bone marrow cells, or similar methods. Recently, a technology for in vitro induction and differentiation of pluripotent stem cells, such as ES cells or iPS cells, has been developed to prepare blood cells such as platelets.
[0003] The present inventors have established a technology for inducing the differentiation of megakaryocytes and platelets from human ES cells and have demonstrated the effectiveness of ES cells as a source of platelets (Patent Reference 1 and Non-Patent Reference 1). Furthermore, the present inventors have established a method for the preparation of megakaryocytes and platelets from iPS cells and enabled the elimination of the human leukocyte antigen (HLA) compatibility problem that inevitably occurred in the transfusion of platelets derived from ES cells (Reference Patent 2).
[0004] Furthermore, with the aim of overcoming the problem of the quantity of platelets and the like prepared from stem cells, the present inventors found a method of establishing and, thus, preparing a lineage of immortalized megakaryocyte progenitor cells from stem cells and then developed an important technology for the in vitro preparation of a large number of platelets and the like (Patent Reference 3).
[0005] In vivo megakaryocytes form pseudopodial arrays called proplatelets (platelet progenitors), fractionate their cytoplasm and release platelets. Polyploidization of megakaryocytes is thought to occur by endomitosis until they release platelets. Megakaryocyte endomitosis is multipolar mitosis unaccompanied by cleavage furrow formation and shaft extension, and is caused by abnormal mitosis and mitosis. As a result of endomitosis, cells containing several segmented nuclei are formed. Polyploidization of megakaryocytes is induced by the repetition of such endomitosis.
[0006] Many study results have been reported to date on the polyploidization of megakaryocytes. Lodier, et al. elucidated (Non-Patent Reference 1) that, in megakaryocyte endomitosis, the location of myosin II from non-muscle cells in a contractile ring was not recognized despite the formation of the cleavage groove and the occurrence of defects in the formation of the contractile ring and extension of the contractile ring. axle. Such abnormalities in the contractile ring or axis extension have been shown to become more accentuated by inhibition of RhoA and ROCK activities (Non-Patent Reference 2). RhoA accumulates in the cleavage groove and promotes the activation of some effector factors including Rho kinase (ROCK), citron kinase, LIM kinase and mDia/formins. These results suggest that by inhibiting the activities of factors such as RhoA and ROCK involved in the formation of a contractile ring, endomitosis of megakaryocytes is stimulated. There is also a report that when a Rho signal positioned downstream of the alpha2/beta1 integrin is enhanced, the formation of immature megakaryocyte proplatelets prior to polyploidization is inhibited.
[0007] All of trans-retinoic acid (ATRA), a transcription factor, and valproic acid known as a histone deacetylase inhibitor are reported to be involved in megakaryocyte differentiation. Schweinfurth, et al. found that treatment of immature megakaryocytes with trans-retinoic acid or valproic acid promotes polyploidization (Non-Patent Reference 3). Furthermore, it is reported that polyploidization of megakaryocytes is promoted when p53, a knockdown-reduced cancer suppressor gene product (Non-Patent Reference 4).
[0008] It has also been shown that, as an influence on a process of megakaryocyte differentiation, the cultivation of immature megakaryocytes at 39 °C, a temperature higher than the temperature of conventional culture, promotes the induction of mature polyploidized megakaryocytes and the formation of proplatelets (Reference Non-Patent 5). List of Citations
[0009] Patent References Patent Reference 1: WO2008/041370 Patent Reference 2: WO2009/122747 Patent Reference 3: WO2011/034073
[0010] Non-Patent References Non-Patent Reference 1: Takayama, et al., Blood, 111: 5298-5306 2008 Non-Patent Reference 2: Lordier, et al., Blood, 112: 3164-3174 2009 Non-Patent Reference 3: Schweinfurth, et. al., Platelets, 21: 648-657 2010 Non-Patent Reference 4: Fuhrken, et al., J. Biol. Chem., 283: 2008 1558915600 Non-Patent Reference 5: Proulx et al., Biotechnol. Bioeng., 88: 675680 2004 Summary of the Invention
[0011] Problems to be solved by the Invention
[0012] Once it has been verified that the amount of functional platelets (platelets with in vivo activities such as hemostatic action and characterized as CD42b+) available from megakaryocytes, whose “polyploidization” has not occurred sufficiently and is too small to develop clinical application, the The present inventors have considered that polyploidization of megakaryocytes must be promoted in order to efficiently produce functional platelets in vitro.
[0013] It is therefore an object of the present invention to provide a method of promoting polyploidization of megakaryocytes and then preparing more polyploidized megakaryocytes, a method of effective platelet production from polyploidized megakaryocytes, and the like. Means to Solve the Problem
[0014] In view of the above problems, the present inventors tried to promote the polyploidization of megakaryocytes which were prepared from pluripotent stem cells (ES cells, iPS cells and the like) and whose polyploidization did not sufficiently occur. First, the present inventors performed the test with the immortalized megakaryocytic progenitor cell line (see Patent Reference 3) prepared from self-grown pluripotent stem cells. This immortalized megakaryocytic progenitor cell lineage is a lineage that is transmitted with increased proliferative potential and is established (immortalized) by inducing the expression of an oncogene such as MYC or a gene such as BMI1 in megakaryocytic progenitor cells derived from pluripotent stem cells.
[0015] In order to promote polyploidization with this immortalized megakaryocytic progenitor cell line, the present inventors were able to efficiently promote polyploidization by forcing expression of an apoptosis suppressor gene when suppression of expression of an oncogene and a polycomb gene is performed.
[0016] The present inventors have also confirmed that, like the forced expression of an apoptosis suppressor gene, inhibition of the expression or function of a p53 gene product further increases the efficiency of polyploidization. They further confirmed that subjecting the megakaryocytic progenitor cell line to treatment with a ROCK inhibitor (Rho-associated coil-forming kinase/Rho-associated kinase) or a HADC inhibitor, cultivation at 39°C and the like is also effective to induce polyploidization. Furthermore, they found that treatment with a functional inhibitor of the actomyosin complex (actin-myosin complex) promotes marked polyploidization.
[0017] It has been found that highly polyploidized megakaryocytes produced by the present invention contain megakaryocytes of 4N or 8N or more in a greater proportion than known, and at the same time, contain such cells in a much greater proportion than that of megakaryocytes. mature produced in vivo.
[0018] Furthermore, the present inventors have found that in sufficiently polyploidized mature megakaryocytes, the number of platelets produced from a megakaryocyte shows a dramatic increase by suppressing the forced expression of an apoptosis suppressor gene. Furthermore, they confirmed that the efficiency of platelet production can be further increased by culturing in a medium added with a ROCK inhibitor. After studying the best conditions for the cultivation period, the cultivation temperature and other parameters, they concluded the present invention.
[0019] The present invention relates to:[1] a method of producing polyploidized megakaryocytes, including a step forcibly expressing an apoptosis suppressor gene in megakaryocytes prior to polyploidization and cultivation of the cells;[2] the method, as described in [1], wherein the apoptosis suppressor gene is a BCL-XL gene;[3] the method described above in [1] or [2], wherein in the cultivation step, the expression or function of a p53 gene product is inhibited;[4] the method described above in any one of [1] to [3], wherein, in the cultivation step, megakaryocytes prior to polyploidization are subjected to at least one of the following from ( a) a (c): (a) treatment with an inhibitor of actomyosin complex function; (b) treatment with a ROCK inhibitor; and (c) treatment with an HDAC inhibitor;[5] the method as described above in [4], wherein the ROCK inhibitor is Y27632; the HDAC inhibitor is valproic acid; and the inhibitor of the complex function of actomyosin is blebistatin;[6] the method described above in any one of [1] to [5], wherein the culturing step is carried out at a temperature greater than 37 °C;[7] the method described above in any one of [1] to [6], wherein megakaryocytes prior to polyploidization are obtained by a step of forced expression of an oncogene and any one of the following genes (i) to (iii) in the cells in any stage of differentiation of hematopoietic progenitor cells into megakaryocytes prior to polyploidization; a p16 gene expression suppressor gene or a p19 gene; (ii) an Ink4a/Arf gene suppressor gene expression; and (iii) a polycomb gene; and the cultivation and proliferation of the cells;[8] the method described above in [7], wherein a c-MYC gene is used as the oncogene and BMI1 is used as any one of genes (i) to (iii);[ 9] the method described above in [7] or [8], wherein the hematopoietic progenitor cells are derived from cells selected from the group consisting of iPS cells, ES cells, hematopoietic stem cells derived from umbilical cord blood, bone marrow blood or peripheral blood, and hematopoietic stem cells;[10] a composition of blood cells containing the polyploidized megakaryocytes produced by the method described above in any one of [1] to [9];[11] a method of producing a platelet, including : a step of obtaining polyploidized megakaryocytes using the method described above in any one of [1] to [9] and culturing the cells; and a step of collecting a platelet from the culture of polyploidized megakaryocytes;[12] the method described above in [11], in which the step of culturing the polyploidized megakaryocytes is carried out during the suppression of expression of the apoptosis suppressor gene that was forcibly expressed or after the apoptosis suppressor gene is removed from the cells;[13] the method described above in [11] or [12], wherein the step of culturing the polyploidized megakaryocytes is carried out in the absence of serum and/or in the absence of a feeder cell;[14] the method described above in any one of [11] to [13], wherein the step of culturing the polyploidized megakaryocytes is carried out from one day to 15 days;[15] the method described in any one of [11] to [14], in which the step of culturing the polyploidized megakaryocytes is carried out at 37 °C;[16] the method described above in any one of [11] to [15], where in the step of culturing the polyploidized megakaryocytes, a ROCK inhibitor and/or an inhibitor of a-complex function ctomiosin is added to the medium;[17] the method described above in [16], wherein the ROCK inhibitor is Y27632 and the inhibitor of actomyosin complex function is blebstatin;[18] a platelet produced by the method described in any of [ 11] to [17]; and[19] a blood product containing the platelets described above in [18]. Effect of the Invention
[0020] The present invention makes it possible to artificially promote the polyploidization of megakaryocytes. In particular, the present invention is also effective for promoting polyploidization of megakaryocytes prepared in vitro as previously reported, and the invention makes it possible to provide megakaryocytes (e.g., a population of megakaryocytes with megakaryocytes 4N or at a greater ratio), whose level of polyploidization has advanced further than available megakaryocytes in vivo.
[0021] Furthermore, the present invention makes it possible to considerably increase the number of platelets produced by polyploidized megakaryocyte.
[0022] It becomes possible to drastically decrease the time required for platelet production from stem cells and then carry out mass production of platelets through megakaryocyte induction before stem cell polyploidization, megakaryocyte proliferation before of polyploidization using, for example, the method described in Patent Reference 3 and polyploidizing the megakaryocytes prior to polyploidization to produce platelets in accordance with the method of the present invention. Platelets obtained as described above are CD42b positive and contribute largely to clinical application. Brief Description of Drawings
[0023] [FIGURE 1A] shows the schematic of a test performed to study the cytokine dependence of iMKPC-type I proliferation.
[0024] [FIGURE 1B] shows a change in cell number when iMKPC-type I was cultured in medium in which SCF and TPO (S+T), SCF and EPO (S+E), SCF (S), TPO (T ) and EPO (E) were added, respectively, according to the schedule indicated in FIGURE 1A.
[0025] [FIGURE 1C] shows flow cytometry histograms of the expression of an iMKPC-type I surface marker on Day 8 of the schedule shown in FIGURE 1A.
[0026] [FIGURE 1D] shows the results of forcing the expression of c-MYC and BMI1 with CD34 positive cells derived from umbilical cord blood and promoting megakaryocyte proliferation prior to polyploidization.
[0027] [FIGURE 2A] shows the results of the study of the influence of cytokines on the proliferation of iMKPC-type II.
[0028] [FIGURE 2B] shows the results of studying the expression of CD41a and CD42b in iMKPC-type II.
[0029] [FIGURE 3] Shows microscopic observations of polyploidization of iMKPC-type II when forced expression of BCL-XL and suppression of expression of a p53 gene were performed, and of polyploidization when blebistatin was added to the medium.
[0030] [FIGURE 4] Shows the microscopic observations in the study of the influence of BCL-XL on the proliferation of iMKPC-type II.
[0031] [FIGURE 5] The influence of a ROCK inhibitor on the polyploidization of megakaryocytes. After MYC/BMI1 expression in megakaryocytes was suppressed (by culturing in the presence of doxycycline and in the absence of estradiol), a ROCK inhibitor (Y27632) (10 μM) was added. After 7 days of cultivation, the degree of polyploidization was studied. A shows the flow cytometry histograms of cells (vehicle) to which ROCK inhibitor was not added and cells (ROCK i) to which inhibitor was added, where these cells were stained with Hoechst, a nuclear dye, and then CD41a, a megakaryocyte marker was stained with an anti-CD41a antibody. B is a graph showing the results of A.
[0032] [FIGURE 6] Results of the study of the expression level of a gene involved in the maturation of megakaryocytes in culture at 39 °C. After MYC/BMI1 expression in megakaryocytes, the resulting cells were cultured at 39°C for 5 days. The expression level of a group of genes (GATA1 (A), PF4 (B), NFE2 (C) and β-tubulin (D)), essential for the maturation of megakaryocytes, was studied by quantitative PCR (q-PCR ). The expression level shown in these graphs is a reason for the expression level of GAPDH.
[0033] [FIGURE 7] Results of the study of the influence of BCL-XL, one of the apoptosis suppressor genes, on the polyploidization of megakaryocytes. The degree of polyploidization was studied after suppressing MYC/BMI1 expression in megakaryocytes, inducing BCL-XL expression in the presence of a ROCK inhibitor (10 μM) and culturing the resulting cells for 7 days. A shows flow cytometry histograms of each of the expressed MYC/BMI1 cells (left graph), of cells treated with a ROCK inhibitor after suppression of MYC/BMI1 expression (middle graph), and of cells subjected to expression of BCL-XL plus suppression of MYC/BMI1 expression and treatment with a ROCK inhibitor (graph on the right), in which these cells were stained with a nuclear dye, Hoechst and then CD41a, a megakaryocyte marker, was stained with an anti-CD41a antibody. B is a graph showing the results of A. C includes micrographs of cells that have a 2N, 4N, 8N, and 8N nucleus or greater.
[0034] [FIGURE 8] Growth curve of cells expressed BCL-XL. It shows the results of a change in the number of cells expressed with BCL-XL (CD41+) (■) and the number of unexpressed cells (CD41a+) (▲) as a function of days of culture during suppression of MYC/ BMI1 in megakaryocytes in the presence of a ROCK inhibitor (10 μM).
[0035] [FIGURE 9] Influence of p53 knockdown on polyploidization. The degree of polyploidization of CD41a+ cells was studied by suppressing MYC/BMI1 expression in megakaryocytes, inducing BCL-XL expression in the presence of a ROCK inhibitor (10 μM), knockdown knockdown the p53 gene, and culturing the resulting cells for 7 days at 39°C. A shows flow cytometry histograms of each of the control cells (control) in which there was no p53 knockdown and of cells (SiP53) in which there was p53 knockdown, where these cells were stained with a Hoechst nuclear dye and then , CD41a, a megakaryocyte marker, was stained with an anti-CD41a antibody. B is a graph showing the results of A.
[0036] [FIGURE 10] Influence of valproic acid treatment on polyploidization. The degree of polyploidization of CD41a+ cells was studied after the suppression of MYC/BMI1 expression in megakaryocytes, inducing the expression of BCL-XL in the presence of a ROCK inhibitor (10 μM), knockdown reducing a p53 gene, treating the resulting cells with valproic acid (0.5 mM) and culturing at 39°C for 7 days. A shows flow cytometry histograms of each of cells (Si P53) not treated with valproic acid and of cells (SiP53 VLP) treated with valproic acid, in which these cells were stained with a Hoechst nuclear dye, and then CD41a, a megakaryocyte marker was stained with an anti-CD41a antibody. B is a graph showing the results of A.
[0037] [FIGURE 11] Influence of a myosin heavy chain IIA/B ATPase inhibitor (inhibitor of actomyosin complex function) on megakaryocyte polyploidization. The degree of polymerization was studied after suppressing MYC/BMI1 expression in megakaryocytes (by culturing in the presence of doxycycline and in the absence of estradiol), adding blebstatin (10 μg/mL), an inhibitor of IIA/B ATPase of the heavy chain of myosin and culturing for 7 days. The flow cytometry shows histograms of cells (-), to which blebstatin was not added, and cells (+), to which blebstatin (10 μg/mL) was added, where these cells were stained with a Hoechst nuclear dye. and then CD41a, a megakaryocyte marker, was stained with an anti-CD41a antibody. B is a graph showing the results of A.
[0038] [FIGURE 12] Influence, on the polyploidization of megakaryocytes, of treatment with blebstatin used in combination with other treatments. The degree of polyploidization of CD41a+ cells was studied after suppression of MYC/BMI1 expression in megakaryocytes, induction of BCL-XL expression in the presence of Y27632 (10 μM) and valproic acid (0.5 mM), knockdown of a gene p53, addition of blebistatin (10 μg/ml) and cultivation at 39 °C for 7 days. A shows flow cytometry histograms of each of the blebstatin-untreated (-) cells and blebstatin-treated (+) cells, where these cells were stained with a nuclear dye, Hoechst, and then CD41a, a megakaryocyte marker. was stained with an anti-CD41a antibody. B is a graph showing the results of A.
[0039] [FIGURE 13] Growth curve of cells subjected to treatment with blebstatin in combination with the other treatments. A change in the number of the following cells was plotted (A) as a function of days of culture: cells (CD41a+) treated with blebstatin (CD41a+) (▲) and cells (CD41a+) not treated with blebstatin (■), each after MYC/BMI1 expression in megakaryocytes was suppressed, BCL-XL was expressed in the presence of Y27632 (10 μM) and valproic acid (0.5 mM), and a p53 gene was knockdown reduced. Micrographs of these cells are shown in B.
[0040] [FIGURE 14] Shows the results of studying CD41a and CD42b expression on megakaryocytes and platelets in both cases where BL-XL expression was suppressed and where it was not suppressed during a platelet release stage.
[0041] [FIGURE 15] Shows the cell counts measured at the time of activation/deactivation of BCL-XL expression, based on the results of FIGURE 14. A shows the number of CD42b-positive platelets, B shows the number of megakaryocytes CD41a positive/CD42b positive and C shows the number of CD41a-positive megakaryocytes.
[0042] [FIGURE 16] Shows the results of the study of the influence of the culture temperatures established at 35 °C, 37 °C and 39 °C on the number of platelets in both cases, where the expression of BCL-XL was suppressed and where it was not suppressed during a platelet release stage.
[0043] [FIGURE 17] Shows the results of the study of the influence of the presence or absence of a serum, feeder cells and blebstatin on the number of platelets.
[0044] [FIGURE 18] Shows the results of the study of the influence of a serum, feeder cells and blebstatin on the CD42b platelet ratio.
[0045] [FIGURE 19] Shows an example of preferable cultivation conditions in the polyploidization (MCB expansion) step of megakaryocytes and during a platelet release (platelet production) stage.
[0046] [FIGURE 20] Shows an increase in CD42b platelet ratio by suppressing BCL-XL expression and a further increase in CD42b platelet ratio by removing serum and feeder cells from the medium and adding blebistatin.
[0047] [FIGURE 21] shows the results of the study of the influence of a functional inhibitory antibody HIP1 against CD42b on the agglutination effect of peripheral platelet ristocetin.
[0048] [FIGURE 22] shows the results of the study of the influence of a functional HIP1 inhibitory antibody against CD42b on thrombus formation in vivo.
[0049] [FIGURE 23] shows the results of transplantation, respectively, to NOG mice, of platelets derived from iPS cells produced during the addition of KP-457 (S-45457), an inhibitor of ADAM17, and thus increasing the level expression of GPIba (CD42b) and platelets derived from iPS cells produced without addition of an ADAM17 inhibitor and measurement of platelet number, which contributed to thrombus formation.
[0050] [FIGURE 24] shows the results of transplantation of human peripheral platelets artificially deteriorated by the addition of 100 μm of CCCP, a platelet damaging agent in the presence of KP-457, platelets to which CCCP were added in the absence of KP- 457 and fresh platelets, respectively, and measuring the number of platelets that contributed to thrombus formation. Method for Carrying Out the InventionMethod for producing polyploidized megakaryocytes
[0051] The present invention provides a method of promoting polyploidization of megakaryocytes, thereby preparing the polyploidized megakaryocytes.
[0052] A method of producing polyploidized megakaryocytes according to the present invention includes a step of forcing the expression of an apoptosis suppressor gene in megakaryocytes prior to polyploidization and cultivation of the cells.
[0053] The term "megakaryocytes prior to polyploidization", as used in the present invention, is not particularly limited and may refer to megakaryocytes that are available from cord blood or bone marrow cells and whose polyploidization has not occurred satisfactorily. , or megakaryocytes that have been inductively differentiated from ES cells, iPS cells, hematopoietic stem cells derived from umbilical cord blood, bone marrow blood or peripheral blood, progenitor cells or the like, and whose polyploidization has not sufficiently occurred.
[0054] Furthermore, the term "megakaryocytes prior to polyploidization" as used herein encompasses cells that are characterized, for example, as CD41a positive/CD42a positive/CD42b positive.
[0055] The term "polyploidized megakaryocytes" or "polyploidized megakaryocytes" means cells or a population of cells in which the number of nuclei has relatively increased compared to "pre-polyploided megakaryocytes". For example, when megakaryocytes to which the method of the present invention is to be applied have a 2N nucleus, cells having a 4N nucleus or more correspond to "polyploidized megakaryocytes" or "polyploidized megakaryocytes". Even in megakaryocytes before polyploidization, the number of nuclei is not limited to one. In a cell population, the number of nuclei in the whole cell population shows a significant increase after a predetermined period, the cell population before the predetermined period can be called "megakaryocytes before polyploidization", and the cell population after a predetermined period. can be called "polyploidized megakaryocytes".
[0056] The present invention can also be applied to megakaryocytes prior to polyploidization that have been inductively differentiated from pluripotent stem cells (such as ES cells and iPS cells), hematopoietic stem cells derived from umbilical cord blood, bone marrow blood or peripheral blood, and progenitor cells. For example, megakaryocytes available from a lattice structure (which may also be called ES-sac or iPS-sac) prepared from ES cells or iPS cells are preferred. Here, the "lattice structure" prepared from ES cells or iPS cells means a steric sac-like structure (with an internal space) like the structure derived from ES cells or iPS cells. It is made up of a population of endothelial and similar cells and contains the same hematopoietic progenitor cells (see Patent Reference 1, Patent Reference 2 and Non-Patent Reference 2).
[0057] No particular limitation is imposed on ES cells to be used in the present invention and it is possible to use those finally established as an ES cell line by culturing fertilized eggs in the blastocyst stage together with feeder cells, isolating the proliferating cells derived from them. of the inner cell mass into individual cells, and repeating the subculture.
[0058] When iPS cells are used, cells of any origin can be used as they have acquired pluripotent differentiation similar to ES cells by the introduction of various types of transcription factor (which will henceforth be called "pluripotent differentiation factor" genes ) capable of providing somatic cells (eg, fibroblasts or blood cells) with pluripotent differentiation. As pluripotent differentiating factors, many factors have already been reported. Examples include, but are not limited to, the Oct family (e.g. Oct3/4), SOX family (e.g. SOX2, SOX1, SOX3, SOX15 and SOX17), Klf family (e.g. Klf4 and Klf2), MYC family (e.g. c-MYC, N-MYC and L-MYC), NANOG and LIN28.
[0059] The present inventors have reported that by forcing the expression of an oncogene such as MYC and a gene such as BMI1 in megakaryocytes prior to polyploidization (including so-called "megakaryocyte progenitor cells" in Patent Reference 3) derived from pluripotent stem cells , the resulting megakaryocytes have improved proliferative capacity (Patent Reference 3, JEM, 207: 2817-2830 2010).
[0060] The megakaryocytes before polyploidization, obtained using the above-mentioned method, are suitable for use in the method of the present invention.
[0061] In the method of producing polyploidized megakaryocytes according to the present invention, such as megakaryocytes before polyploidization, those obtained by a step of, at any stage of differentiation from hematopoietic progenitor cells to megakaryocytes before proliferation, forcing the expression of an oncogene and any one of the following genes (i) to (iii): a gene which suppresses the expression of a p16 gene or a p19 gene; (ii) a gene which suppresses the expression of an Ink4a/Arf gene; and (iii) a polycomb gene culturing and proliferating the resulting cells.
[0062] Examples of oncogene include MYC family gene, Src family gene, Ras family gene, Raf family gene and protein kinase family genes like c-Kit, PDGFR and Abl. Examples of genes (i) to (iii) include BMI1, Mel18, Ring1a/b, Phc1/2/3, Cbx2/4/6/7/8, Ezh2, Eed, Suz12, HADC and Dnmt1/3a/3b, with a BMI1 gene being particularly preferred. Control of the expression of the oncogene and the polycomb gene can be carried out by persons skilled in the art in a conventional manner. For example, the method described in detail in Patent Reference 3 and the like can be used. The oncogene and any one of genes (i) to (iii) can be introduced into cells at any stage from hematopoietic progenitor cells to megakaryocytes prior to polyploidization. However, this is not limited as the expression of these genes is induced in megakaryocytes prior to polyploidization to be used in the present invention.
[0063] The oncogene and genes (i) to (iii) (e.g. a BMI1 gene) to be used in the present invention include not only genes with an already known cDNA sequence, but also homologs identified using the prior art based on known cDNA sequence homology.
[0064] For example, among the genes of the MYC family, the c-MYC gene is a gene that has a nucleic acid sequence of SEQ ID No: 1. Homologs of the c-MYC gene are genes that have a cDNA sequence substantially equal to the nucleic acid sequence of SEQ ID No: 1. The cDNA that has a sequence substantially the same as the nucleic acid sequence of SEQ ID No: 1 is DNA that has about 60% or greater sequence identity, preferably about of 70% or more sequence identity, more preferably about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% sequence identity, more preferably about 99% sequence identity with a DNA having a sequence of SEQ ID No: 1, or a DNA capable of hybridizing with a DNA having a sequence complementary to the nucleic acid sequence of SEQ ID No: 1 under stringent conditions, wherein a protein encoded by such DNA contributes to cell amplification cells at a stage of differentiation as megakaryocytes before polyploidization.
[0065] The BMI1 gene is a gene that has a nucleic acid sequence of, for example, SEQ ID NO: 2. A BMI1 gene homolog is a gene that has a cDNA sequence substantially the same as the nucleic acid sequence of, for example, SEQ ID No: 2. The cDNA, which has a sequence substantially the same as the nucleic acid sequence of SEQ ID No: 2, is DNA that has about 60% or more sequence identity, preferably about 70% or more sequence identity, more preferably about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97% or 98% sequence identity, more preferably about 99% sequence identity with a DNA having a sequence of SEQ ID NO: 2 or a DNA capable of being hybridized to a DNA having a sequence complementary to the nucleic acid sequence of SEQ ID No: 2 under stringent conditions, wherein a protein encoded by such DNA suppresses oncogene-induced senescence in cells cells in which the oncogene, such as the MYC family gene, was expressed, thus promoting cell amplification.
[0066] The aforementioned oncogene and genes (i) to (iii) are necessary for cell proliferation, but they can inhibit the promotion of polyploidization or platelet release so that the expression of these genes can be suppressed before a step of polyploidization. Suppressing the expression of these genes in cells facilitates the release of functional platelets (Patent Reference 3).
[0067] The term "apoptosis suppressor gene" as used herein is not particularly limited in that it is a gene that suppresses apoptosis. Examples of this include a BCL2 gene, a BCL-XL gene, Survivin and MCL1.
[0068] The present inventors have found that when forced expression of the oncogene and any one of genes (i) to (iii) is suppressed, death of proliferated megakaryocytes prior to polyploidization can be induced. As shown later in the Examples, suppression of the expression of the oncogene and any one of genes (i) to (iii) in megakaryocytes prior to polyploidization and the forced expression of an apoptosis suppressor gene in the cells promotes polyploidization of the megakaryocytes, resulting in the efficient platelet production from megakaryocytes prior to polyploidization.
[0069] As shown later in the Examples, megakaryocytes continue long-term proliferation by forcing the expression of an apoptosis suppressor gene.
[0070] Apoptosis suppressor genes such as the BCL-XL gene and the BCL2 gene to be used in the present invention not only include genes whose cDNA sequence has already been published, but also homologs identified by the prior art, based on homology with the known cDNA sequence. For example, a BCL-XL gene, one of the apoptosis suppressor genes, is a gene that has a nucleic acid sequence of SEQ ID No: 3. A BCL-XL gene homolog is a gene that has a cDNA sequence substantially equal to the nucleic acid sequence of SEQ ID NO: 3. The cDNA that has a sequence substantially the same as the nucleic acid sequence of SEQ ID NO: 3 is DNA that has about 60% or greater sequence identity, preferably about of 70% or more sequence identity, more preferably about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% or 98% sequence identity, more preferably 99% sequence identity with a DNA that has a sequence of SEQ ID No: 3 or a DNA capable of be hybridized to a DNA having a sequence complementary to the nucleic acid sequence of SEQ ID No: 3 under stringent conditions, wherein a protein encoded by such DNA is effective to suppress apoptosis.
[0071] The term "stringent conditions", as used herein, means hybridization conditions readily determined by those skilled in the art and are empirical experimental conditions that typically depend on probe length, washing temperature and salt concentration. Typically, a temperature for proper annealing becomes higher when a longer probe is used, and becomes lower when a shorter probe is used. The formation of hybrids generally depends on the annealing ability of a complementary strand placed in an environment where the temperature is slightly lower than its melting point.
[0072] Under low stringency conditions, for example in a filter wash stage after hybridization, a filter is washed in 0.1xSSC, 0.1% SDS solution under temperature conditions from 37°C to 42°C. °C Under high stringency conditions, eg in the washing step, a filter is washed in 5 x SSC, 0.1% SDS solution at 65°C. Polynucleotides with high homology can be obtained by making stringent conditions more stringent.
[0073] In order to force the expression of genes as oncogenes, genes (i) to (iii) and the apoptosis suppressor gene in cells, any method well known to those skilled in the art can be employed. For example, the gene can be introduced into cells using a gene delivery system with a lentivirus or a retrovirus and then expressed. When gene expression is performed using a viral gene delivery vector, a target gene can be expressed by functionally linking the gene downstream of a suitable promoter, inserting the resulting gene into the gene delivery vector, and then introducing it into cells. . Here, the term "functionally linked" means that a target gene is linked to a promoter to achieve the desired expression of the target gene. In embodiments of the present invention, for example, the target gene may be constantly expressed using a CMV promoter, an EF1 promoter or the like. Alternatively, an appropriate promoter (inducible promoter) can be placed under the control of an element with activity controlled by a trans factor, e.g. a drug response element as a tetracycline response element and a target gene can be expressed inductively by the drug. realization of such control as drug addition Since such a gene expression system using a drug can effect the desired expression control of the oncogene or genes (i) to (iii), a suitable system can be easily selected by the skilled person in the art. A commercially available kit can be used to carry out such expression. The oncogene and genes (i) to (iii), which are the target genes or in the expression control, can be entered in the respective vectors or in a vector.
[0074] Suppression of the expression of the oncogene or any of the genes (i) to (iii) in megakaryocytes can be achieved, for example, by removing the drug or the like and thus releasing the induction of expression using the inductive expression system above mentioned. Alternatively, the oncogene or any one of genes (i) to (iii), which has been introduced, can be removed using a Cre/lox system or the like to suppress expression of these genes. A commercially available kit or the like can be used, as needed, to suppress-regulate the expression of the oncogene or gene (i) to (iii).
[0075] A method of producing polyploidized megakaryocytes according to the present invention includes a step of forcing the expression of an apoptosis suppressor gene in cells and, in parallel, inhibiting the expression or function of a p53 gene product in the cells. cells. The term "expression" is used as a comprehensive concept of transcription and translation. For example, the term "inhibiting expression" can include the meaning "inhibiting at the transcription level" or "inhibiting at the translation level".
[0076] The p53 gene product is widely known as a tumor suppressor gene and its sequence and similar in various animal species are known.
[0077] A method of inhibiting p53 gene product function in megakaryocytes can be achieved by conventional technology in this technical field. Examples of the method include a method of introducing a mutation (substitution, insertion or deletion, or alteration or modification) into a P53 gene, thereby inhibiting production of the gene product, and a method of directly inhibiting the function of the gene product. Examples of the method of introducing mutation directly (substitution, insertion or deletion, or alteration or modification) into a gene include a method of destroying the entire p53 gene through homologous recombination, using an appropriate gene targeting vector and a method of introducing the mutation into a region important for gene product activity, using a Cre/lox or similar system.
[0078] As a method of inhibiting p53 gene product function, a dominant negative method can be used. The dominant negative method is a method of inducing abundant expression in cells of a p53 protein that has the mutation introduced there to reduce or starve its activity, rendering a portion of the p53 protein inert to the normal p53 protein in cells extremely high, and thus obtaining cells exhibiting the behavior of cells that have lost p53 protein function.
[0079] As the method of suppressing the expression of the p53 gene product, an antisense method, a ribozyme method, an RNAi method or the like can be used.
[0080] The antisense method is a method of suppressing the expression of a gene by using a single-stranded nucleic acid that has a complementary base sequence for a target gene (basically, an mRNA as a transcription product) and having generally a length from 10 bases to 100 bases, preferably from 15 bases to 30 bases. Gene expression is inhibited by introducing an antisense nucleic acid into cells and hybridizing it to the target gene. The antisense nucleic acid is not fully complementary to the target gene, as an expression-inhibiting effect of the target gene can be produced. The antisense nucleic acid can be designed, as needed, by those skilled in the art using known or similar software. The antisense nucleic acid can be any one of DNA, RNA and DNA-RNA chimera, or it can be modified.
[0081] A ribozyme is a nucleic acid molecule that catalytically hydrolyzes a target RNA and is composed of an antisense region having a sequence complementary to the target RNA and a catalytic core region involved in the cleavage reaction. A ribozyme can be engineered, as needed, in a manner known to those skilled in the art. A ribozyme is usually an RNA molecule, but a DNA-RNA chimera molecule can be used instead.
[0082] The RNAi method is a mechanism of suppression of expression of the specific sequence of a gene induced by a double-stranded nucleic acid. The method has high target specificity and, moreover, is highly safe because it uses a gene expression suppression mechanism originally present in vivo.
[0083] Examples of the double-stranded nucleic acid with RNAi effect include siRNA. When siRNA is used for mammalian cells, it is a double-stranded RNA of usually 19 to 30 bases, and preferably about 21 to 25 bases. Double-stranded nucleic acid with an RNAi effect generally has, as one of the strands, a sequence complementary to a portion of a target nucleic acid and, as the other strand, a sequence complementary thereto.
[0084] Double-stranded nucleic acid with an RNAi effect can be designed in a known way, based on the base sequence of a target gene. The double-stranded nucleic acid with an RNAi effect can be any of a double-stranded RNA, a DNA-RNA chimera-like double-stranded nucleic acid, an artificial nucleic acid, and a nucleic acid subjected to various modifications.
[0085] SiRNA, antisense nucleic acid and ribozyme can be expressed in cells by introducing into cells vectors (eg lentivirus vectors) containing nucleic acids encoding them, respectively. Like siRNA, DNAs encoding two strands, respectively, may be used, or DNA encoding a single-stranded nucleic acid obtained by linking two strands of a double-stranded nucleic acid via a loop may be used. In the latter case, single-stranded RNA obtained by intracellular transcription has a hairpin-like structure because the complementary portion of the RNA is hybridized to the molecules. This RNA is called shRNA (short hairpin RNA). When ShRNA is exported to the cytoplasm, the loop portion is cleaved by an enzyme (Dicer) to be a double-stranded RNA, producing an RNAi effect.
[0086] As another method of inhibiting p53 gene product function in megakaryocytes, a method of directly or indirectly inhibiting p53 gene product function, a method of inhibiting p53 phosphorylation and thereby indirectly inhibiting the activation of p53 p53, or a similar method may be employed.
[0087] As described later in the Examples, megakaryocytes prior to forced expression of an apoptosis suppressor gene and inhibition of the expression or function of the p53 gene product, continue proliferation, which includes megakaryocytes, whose cytokine dependence is SCF, and platelets released from them are not CD42b positive. When forced expression of an apoptosis suppressor gene and inhibition of the expression or function of the p53 gene product are performed, megakaryocytes are partially polyploidized while continuing to proliferate and release many CD42 positive platelets. At this stage, the cytokine dependence of the megakaryocytes changes from SCF to TOP and proliferation and maturation proceed in parallel with each other. (a) A method of producing polyploidized megakaryocytes according to the present invention includes at least one of a step treatment of cells, in which an apoptosis suppressor gene was forcibly expressed during cell culture, with (a) an inhibitor of actomyosin complex function, (b) a ROCK inhibitor, and (c) an HDAC inhibitor. By the above-mentioned treatment, more stable proliferation and polyploidization proceeds.
[0088] The term "actomyosin complex", as used herein, means a complex between actin and myosin II and its constituents, for example, a contractile ring that will appear at the time of cytokinesis. In the actomyosin complex, myosin II functions as a motor protein by interacting with actin and is involved in contractile ring contraction and the like. The "inhibitor of actomyosin complex function" in the present invention can inhibit function by any mechanism. It includes, for example, those that inhibit the formation of an actomyosin complex and thus inhibit the function of the actomyosin complex; those that inhibit myosin heavy chain (MHC) ATPase IIA/IIB and thereby inhibit the function of the actomyosin complex; and those that inhibit myosin light chain (MlCK) and thus inhibit the function of the actomyosin complex. Myosin heavy chain ATPase IIA/B is a molecule that plays an important role in the contraction of a contractile ring, while kinases and the myosin light chain phosphorylates L2, between the myosin light chains, and induces a gliding motion between actin and myosin.
[0089] It has been reported to date that a ROCK inhibitor suppresses megakaryocyte endomitosis and promotes polyploidization. The IIA/B myosin heavy chain ATPase or myosin light chain kinase, which controls the formation or function of an actomyosin complex, functions downstream of a ROCK signal and, more directly, controls the contraction of a contractile ring. through the formation or regulation of the function of an actomyosin complex. It is therefore assumed that the inhibitor of actomyosin complex function suppresses endomitosis of megakaryocytes more efficiently and promotes more polyploidization compared to the ROCK inhibitor.
Examples of the actomyosin complex function inhibitor usable in the present invention include blebstatin (Science, 299:1743-17472003), an inhibitor of myosin heavy chain ATPase IIA/B, and ML7, a light chain kinase inhibitor of myosin. Such as the myosin heavy chain IIA/B ATPase inhibitor or myosin light chain kinase inhibitor, nucleic acids (e.g., shRNA) or antibodies that inhibit myosin heavy chain IIA/B ATPase activity or the myosin light chain kinase can also be used.
[0091] It should be noted that the term "treatment", as used in the present invention, means an operation carried out to produce the effect of an inhibitor or the like on target cells, for example, the addition of a suitable amount of an inhibitor or the like. to a cell culture medium to incorporate it into cells. In some cases, an operation that promotes the incorporation of the inhibitors into cells may be used together.
[0092] A method of producing megakaryocytes according to the present invention includes a step of treating cells in which an apoptosis suppressor gene has been forcibly expressed with a ROCK inhibitor during cultivation thereof.
[0093] Examples of the inhibitor ROCK (Rho-associated loop-forming kinase/Rho-associated kinase) include [(R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)- cyclohexane carboxamide 2HCLH 2 O] (Y27632). In some cases, antibodies or nucleic acids (eg, shRNA) that inhibit Rho kinase activity can also be used as an inhibitor of ROCK.
[0094] A method of producing megakaryocytes according to the present invention includes a step of treating cells in which an apoptosis suppressor gene has been forcibly expressed with an HDAC inhibitor during cell cultivation.
[0095] The HDAC inhibitor has an action of inhibiting the activity of histone deacetylase (HDAC). Many HDAC inhibitors are known to date. Examples thereof include valproic acid, trichostatin A, SAHA (suberoylanilide hydroxamic acid) and APHA (aroyl-pyrrolyl-hydroxyamide). In particular, valproic acid and trichostatin A are preferably used. When a drug to be used is provided in a salt form, the inhibitor may be used in a salt form.
[0096] The optimal concentration when cells are treated with an inhibitor of actomyosin complex function, ROCK inhibitor, HDAC inhibitor or the like can be determined in advance by those skilled in the art based on the results of a preliminary test. The treatment time, method or the like may also be selected as needed by those skilled in the art. For example, when cells are treated with blebstatin, a myosin heavy chain ATPase II inhibitor can be added in an amount of about 2 to 15 μg/mL or about 5 to 10 μg/mL to a culture and cultured, for example, preferably for about 5 to 10 days, particularly preferably for about 6 to 7 days. Y27632, a ROCK inhibitor, can be added in an amount of 5 to 15 μM or 8 to 12 μM, preferably about 10 μM, while valproic acid, an HDAC inhibitor, can be added in an amount of about 0.1 to 1 mM or from about 0.2 to 0.7 mM, preferably from about 0.5 mM. The treatment time with Y27632 or valproic acid can be from 10 to 21 days, preferably for about 14 days.
[0097] One method of producing polyploidized megakaryocytes according to the present invention includes placing megakaryocyte cells at a temperature of 37°C or higher, wherein an apoptosis suppressor gene in the cells has been forcibly expressed during cultivation of the cells.
[0098] It has been confirmed that the cultivation of megakaryocytes at the usual temperatures of 37 °C or higher promotes the differentiation of megakaryocytes that have undergone polyploidization. "Temperatures of 37 °C or more" is, for example, from about 37 °C to about 42 °C, preferably from about 37 °C to about 39 °C, as temperatures that do not cause damage to cells are suitable. Although a period of cultivation at temperatures of 37 °C or higher can be determined as needed, when monitoring a series of megakaryocytes that have undergone polyploidization, it is, for example, from 10 days to 28 days, preferably from 14 days to about 21 days.
[0099] No specific limitation is imposed on other cultivation conditions in the step of carrying out the forced expression of an apoptosis suppressor gene in megakaryocytes that have not undergone polyploidization and thus the cultivation of the resulting cells, insofar as the effect of the present invention can be produced, preferably takes place under the conditions. Known growing conditions or conditions equivalent to these can be used. For example, TPO, IL-1, IL-3, IL-4, IL5, IL-6, IL-9, IL-11, EPO, GM-CSF, SCF, G-CSF, Flt3 ligand and heparin can be used singly or in combination of two or more and added to the medium.
[00100] Alternatively, a feeder cell can be used as needed for cultivation. Feeder cells are those that provide helper substances, including adhesion substrates, nutrients, and other factors that are necessary for cell proliferation in culture.
[00101] Polyploidized megakaryocytes obtained using the above-mentioned method efficiently produce functional CD42b-positive platelets. As shown later in the examples, CD42b-positive platelets have a high capacity for thrombus formation both in vivo and in vitro. Furthermore, megakaryocytes that have undergone polyploidization can produce functional platelets even when thawed after cryopreservation.
[00102] The present invention also provides a composition of blood cells with a high content of polyploidized megakaryocytes. The term "blood cell composition" may comprise, as well as "polyploidized megakaryocytes" whose polyploidization has been promoted by the method of the present invention, megakaryocytes prepared using another method, and other blood cells.
[00103] Treatment of megakaryocytes prior to polyploidization by the method of the present invention can promote their differentiation into polyploidized megakaryocytes of 4N or greater. Therefore, application of the method of the invention to a population of differentiated megakaryocytes, for example pluripotent stem cells or the like, makes it possible to obtain a blood cell composition with a high content of polyploidized megakaryocytes of 4N or more. When the megakaryocyte population is treated using the method of the present invention, it is possible to increase the content of polyploidized megakaryocytes with 4N or more to at least 20% or more, 30% or more, preferably 40% or more, 50% or more, more preferably 80% or more (see, for example, FIGURE 11B). Therefore, the present invention makes it possible to prepare a population of megakaryocytes or a population of blood cells with a high existence ratio of polyploidized megakaryocytes.
[00104] Such a composition of blood cells can also be cryopreserved. Therefore, such a composition of blood cells is distributed in the frozen state, and the method of platelet production, which will be described later, can be carried out on the user's side.
[00105] Megakaryocytes and the like, which have been treated to promote polyploidization by the method of the present invention, are also effective to transplant them in vivo and produce functional platelets in vivo by a suitable method.
[00106] Currently, hematopoietic stem cells are transplanted through bone marrow transplantation, umbilical cord blood transplantation or the like. In particular, cord blood transplantation allows for the reduction of bone marrow transplantation problems, such as a shortage of donor numbers and a large burden on donors, so that there are currently more opportunities for blood transplantation than the umbilical cord. Megakaryocytes produced in vivo by umbilical cord blood transplantation, however, did not undergo enough polyploidization and require time to produce a sufficient number of platelets in vivo. When platelet production capacity must be increased rapidly, cord blood transplantation cannot sufficiently satisfy this demand at that time.
[00107] The transplantation of polyploidized megakaryocytes obtained by using the method of the present invention can overcome the problems of bone marrow transplantation, such as a shortage of donor numbers and a heavy donor burden and the problems of umbilical cord blood transplantation , such as the ability to produce platelets in vivo. The method of the present invention is therefore far superior to conventional transplantation methods. Platelet production method
[00108] The platelet production method according to the present invention, on the other hand, produces platelets in vitro from the polyploidized megakaryocytes and the like obtained using the method of the present invention.
[00109] The platelet production method according to the present invention includes a step of culturing the polyploidized megakaryocytes obtained by the aforementioned method and collecting platelets from the cultured product.
[00110] Although no limitations are imposed on the culture conditions, polyploidized megakaryocytes can be cultured for about 7 to 15 days, for example, in the presence of TPO (from about 10 to 200 ng/mL, preferably from about 50 to 100 ng/mL) or in the presence of TPO (from about 10 to 200 ng/mL, preferably from about 50 to 100 ng/mL), SCF (from 10 to 200 ng/mL, preferably from about 50 ng/ml) and heparin (from about 10 to 100 U/ml, preferably from 25 U/ml).
[00111] In one mode of the platelet production method according to the present invention, in the step of culturing polyploidized megakaryocytes, the aforementioned forced expression of an apoptosis gene is suppressed or the aforementioned apoptosis suppressor gene is removed from the polyploidized megakaryocytes.
[00112] Suppression of the expression of an apoptosis suppressor gene can also be achieved, for example, by removing the chemical or the like to allow induction of expression by the aforementioned inductive expression system. Alternatively, the introduced apoptosis suppressor gene can be removed using a Cre/lox system and thus the expression of this gene can be suppressively controlled. A commercially available kit or the like can be used, as needed, to suppressively regulate the expression of an apoptosis suppressor gene.
[00113] As shown later in the Examples, expression of an apoptosis suppressor gene that has been forcibly expressed to promote polyploidization leads to an increase in the efficiency of producing functional CD41a-positive/CD42b-positive platelets.
[00114] Suppression of expression or removal of an apoptosis suppressor gene begins 15 days, preferably 10 days, more preferably 3 to 7 days, even more preferably about 3 days before platelet collection.
[00115] In this step, the expression of not only an exogenous apoptosis suppressor gene, but also an endogenous apoptosis suppressor gene can be suppressed. Inhibition of the expression or function of a p53 gene product can be carried out successively after the present step.
[00116] The temperature of cultivation is not particularly limited insofar as the effect of the present invention can be produced. Cultivation can be carried out from 35°C to 40°C, from 37°C to 39°C being suitable as shown later in the examples.
[00117] In the production method according to the present invention, the step of culturing polyploidized megakaryocytes can be carried out under serum-free conditions and/or feeder cell-free conditions. As demonstrated later in the examples, no major difference was found in the amount of platelet production between culturing in a medium containing a fetal bovine serum and culturing in a serum-free medium. However, the ratio of CD42b positive platelets was higher when the cells were cultured in a serum-free medium or in a feeder cell-free medium. If the platelet production step can be carried out in a serum-free and feeder cell-free medium, the platelets thus obtained can be used clinically without causing the problem of immunogenicity.
[00118] Platelet production without the use of a feeder cell can suppress a production cost and is suitable for mass production because feeder cell adhesion is not required and therefore suspension culture can be performed in a bottle or similar. When the feed cell is not used, a conditioning medium can be used. The conditioned medium is not particularly limited and can be prepared by those skilled in the art in a known manner. For example, it can be obtained, for example, by culturing a feeder cell as needed and then removing the feeder cell from the cultivated product using a filter or the like.
[00119] In one mode of the platelet production method according to the present invention, a ROCK inhibitor and/or inhibitor of actomyosin complex function is added to the medium. ROCK inhibitor and inhibitor of actomyosin complex function similar to that used in the aforementioned method of producing polyploidized megakaryocytes can be used. Examples of the ROCK inhibitor include Y27632. Examples of the inhibitor of actomyosin complex function include blebistatin, an inhibitor of myosin heavy chain ATPase II. ROCK inhibitor can be added alone, ROCK inhibitor and actomyosin complex function inhibitor can be added individually; or they can be added together.
[00120] The ROCK inhibitor and/or the actomyosin complex function inhibitor is preferably added in an amount of 0.1 μM to 30 μM, for example, from 0.5 μM to 25 μM, from 5 μM to 20 μM or similar.
[00121] The culturing period after addition of the ROCK inhibitor and/or inhibitor of actomyosin complex function can be from one day to 15 days. It can be for 3 days, 5 days, 7 days, or something similar. By adding ROCK inhibitor and/or inhibitor of actomyosin complex function, the ratio of CD42b-positive platelets can be further increased.
[00122] The embodiment of the present invention includes a kit to promote megakaryocyte polyploidization and the production of mature megakaryocytes and/or platelets. The kit includes, as well as an expression vector and the like necessary for the induction of intracellular expression of the oncogene, any one of the aforementioned genes (i) to (iii), a BCL-XL gene or the like, and a reagent, a means for o cell culture, a serum, a supplement such as the growth factor (e.g., TPO, EPO, SCF, heparin, IL-6, IL-11 or the like), an antibiotic, and the like. In addition, the kit includes when, for example, cells derived from ES cells or iPS cells are used, an antibody (e.g., antibody against Flk1, CD31, CD34, UEA-I, lecithin or similar) to confirm a marker for the identification of a network structure prepared from these cells. The reagent, antibody and the like included in the kit are supplied in any type of container that allows a representative ingredient to effectively maintain its activity and not cause it to adsorb to the container material or deteriorate. (a) The present kit The invention may additionally include megakaryocytes prior to polyploidization in which the oncogene and any of the aforementioned genes (i) to (iii) have been forcibly expressed.
[00123] The "cells" described in this document are derived from humans and non-human animals (eg mice, rats, cattle, horses, pigs, sheep, monkeys, dogs, cats and birds). While no specific limitations are imposed, human-derived cells are particularly preferred.
[00124] The present invention will be described in more detail below by way of Examples. However, it should be borne in mind that the present invention is not limited to or by Examples.EXAMPLES1. Preparation of megakaryocytes before polyploidization1-1 Preparation of megakaryocytes before polyploidization of ES cells
[00125] In order to study the polyploidization of megakaryocytes, megakaryocytes prior to polyploidization were prepared from ES cells (see Patent Reference 3 for details).
[00126] A human ES cell line [KhES-3] was cultured for 14 days in the presence of 20 ng/ml VEGF to prepare a lattice structure. Hematopoietic progenitor cells removed from the resulting network structure were recovered and seeded into 10T1/2 cells to provide a cell count of 1 x 10 5 /well.
[00127] The hematopoietic progenitor cells thus prepared were infected with a retrovirus vector c-MYC-2A-BMI1-containing pMx tet off c-MYC 2A BMI1 three times every 12 hours at an MOI = 10 (confirmed using Jurkat cells) to induce the expression of c-MYC and BMI1 (Patent Reference 3). The pMx tet off c-MYC 2A BMI1 vector allows the expression of a c-MYC gene and a BMI1 gene in the presence of estradiol, while suppressing the expression of the c-MYC gene and the BMI1 gene in the presence of doxycycline (Dox) and absence of estradiol.
[00128] Simultaneously with the first infection, 2 mM estradiol was added and 12 hours after the last infection, the virus was removed. At that stage, the amount of CD42b-positive platelets released was small, even if expression of the c-MYC gene or BMI1 gene was turned off, suggesting that the cells were immature megakaryocytes. These immature cells can be termed "iMKPC-type I".
[00129] Cytokine dependence of iMKPC type I was studied by seeding 2 x 10 5 iMKPC-type I cells onto 10T1/2 feeder cells and culturing them for 14 days at 37°C under the conditions shown in FIGURE 1A in the presence of of 2 μM β-estradiol, while using the following cytokines: SCF (50 ng/mL), TPO (50 ng/mL) and EPO (6 U/mL). A population in which proliferation was confirmed on Day 4 and Day 11 was counted for cell number, and 2 x 10 5 cells were replated, while the medium of the other populations was changed. Cell numbers were counted on Day 8 and Day 14 and 2 x 10 5 cells were replated. At the same time, on day 8, some of the cells were analyzed with a flow cytometer after staining with a CD41 antibody, a CD42b antibody and a GPA antibody.
[00130] The results are shown in FIGURES 1B and C. As shown in Figure 1B, proliferation of iMKPC type I cells was strongly dependent on SCF. As shown in Figure 1C, cells from either population were nearly all CD41 positive, but in populations without SCF, the CD41+/CD42b- population showed a marked reduction in proliferation. The CD41+/CD42b- population is a population showing good proliferation among iMKPC-type I cells.
[00131] These results, including SCF-dependent proliferation, suggest that iMKPC-type I cells are immature megakaryocytes.1.2 Preparation of megakaryocytes prior to polyploidization from CD34-positive cells derived from umbilical cord blood
[00132] It has been confirmed that megakaryocytes prior to polyploidization can be prepared from CD34 positive cells derived from umbilical cord blood in a similar manner to the use of ES cells or iPS cells.
[00133] More specifically, CD34 positive cells derived from umbilical cord blood were infected three times with pMx-c-MYC virus and DNsam BMI1 (each by retrovirus vector) at MOI = 10 and the number of CD41a positive cells ( megakaryocyte marker) on day 14 and day 21 was counted using FACS. Mock (empty vector) was used as a control.
[00134] The results are shown in FIGURE 1D. In comparison to Mock, proliferation of CD41-positive megakaryocytes was observed from a population in which c-MYC and BMI1 were forcibly expressed. Therefore, it was confirmed that megakaryocytes before polyploidization can be obtained from cells derived from umbilical cord blood in a similar way as described in 1-1. 2. Preparation of polyploidized megakaryocytes from megakaryocytes prior to polyploidization.2-1. Influence of BCL-XL expression on polyploidization
[00135] On day 23 after infection with a retrovirus vector pMx tet off c-MYC 2A BMI1 containing c-MYC-2A-BMI1 in section 1-1, cells were infected once with Lv-BCL-XL-GFP inducible by doxycycline (lentivirus vector) at MOI = 10. The vector was prepared by introducing PCR amplified cDNA from BCL-XL into an Ai-Lv tet into the vector (clontech) treated with EcoRI and BamHI using a cloning by In-fsion advance PCR (clontech). Twenty-four hours after infection, the virus was removed. By removing estradiol and adding doxycillin, the expression of c-MYC and BMI1 was suppressed and, at the same time, the expression of BCL-XL was initiated.2-2. Influence of p53 gene knockdown on polyploidization
[00136] A p53 gene was knocked down by infection with an FG12-sh p53 lentivirus vector in addition to the doxycycline-inducible Lv-BCL-XL-GFP lentivirus vector. The cells thus obtained can be termed "iMKPC-type II" cells.2-3. iMKPC-type II cytokine dependence
[00137] In 10T1/2 feeder cells, 2 x 105 iMKPC-type II cells were seeded, followed by cultivation in the presence of 0.5 μg/mL of Dox at 39 °C for 21 days under cytokine conditions, ie , SCF (50 ng/ml), TPO (50 ng/ml) and EPO (6 U/ml). The results are shown in FIGURE 2A. The iMKPC-type II cell line was confirmed to show proliferation in the absence of cytokine, but proliferation is further promoted by the addition of TPO. Although iMKPC-type I cells are dependent on SCF and TPO, these had no influence on their proliferation, type II cells showed improved proliferation in the presence of TPO, and SCF has no influence on proliferation.2-4. Surface marker analysis of iMKPC-type II
[00138] On day 21, after the addition of cytokines, cells were analyzed with a flow cytometer after staining with a CD41 antibody, a CD42b antibody and a GPA antibody. The results are shown in FIGURE 2B. The population with added TPO showed higher expression of CD41a and CD42b compared to the other populations, which suggests that it is a population that is even more committed to megakaryocytes.2-5. Morphological change and influence of blebstatin
[00139] The results of microscopic observation of iMKPC type I and type II are shown in FIGURE 3. It was observed that by turning off the expression of c-MYC and BMI1, forcing the expression of BCL-XL and knockdown of the p53 gene, the cells become increasingly polyploidized. It was also confirmed that the cells were further polyploidized by the addition of blebstatin (5 μg/mL).
[00140] As shown in Sections 2-3 to 2-5, in the iMKPC-type II stage, megakaryocytes proliferate, the ratio of CD42b positive platelets to released platelets increases, some of the cells have been polyploidized, and cytokine dependence changed to TPO, suggesting that proliferation and maturation occurred in parallel.2-6. Influence of suppression of BCL-XL expression
[00141] In the 10T1/2 feeder cells, 2 x 105 iMKPC type II cells were seeded and the long-term culture was performed at 39 °C in the presence of 0.5 μg/mL of Dox (BCL-XL ON) or in the absence of it (BCL-XL OFF). Every two to five days, the number of cells was counted and 2 x 10 5 cells were replated.
[00142] The results are shown in FIGURE 4. The suppression of BCL-LX by Dox OFF decreased the proliferation rate of iMKPC type II cells and after a long period (after 100 days), the cells lost their ability to proliferate, suggesting that BCL-XL is indispensable for the long-term proliferation of iMKPC type II.2-7 cells. Study of the influence of other treatments on polyploidization2-7-1. Influence of ROCK inhibitor
[00143] After the introduction of the MYC and MBI1 genes, the resulting megakaryocytes (about 105) were cultured for about 30 days in the absence of doxycycline and in the presence of estradiol under culture conditions of 37 °C and in the presence of 5% of CO2 to proliferate until about 1011. Cultivation continued while changing conditions to the presence of doxycycline and absence of estradiol, in order to suppress the expression of the MYC gene and the BMI1 gene in the cell line of megakaryocytes thus proliferated upon addition of a ROCK inhibitor (Y27632; products from Wako Pure Chemicals) to the culture medium to provide a concentration of 10 μM to determine the influence of Y27632 on polyploidization. On day 7 after the culture, the addition of Y27632 to the culture medium was started, the degree of polyploidization was studied using FACS (FIGURE 5). Cells added with ROCK inhibitor showed an increase in the number of 4N cells (upper graph of FIGURE 5 (ROCK I), open square in lower graph) compared to cells added without inhibitor (upper graph of FIGURE 5 ( vehicle), filled square). This revealed that ROCK inhibitor promotes polyploidization of megakaryocytes prior to polyploidization where ES cells were derived, and promotes increased proliferation capacity as a result of expression of a C-MYC gene and a BMI1.2-7-2 gene. Influence of ROCK inhibitor + cultivation under high temperature condition
[00144] It has been reported so far that, as a result of culturing immature megakaryocytes at a temperature, for example, 39 °C, higher than the normal culture temperature, megakaryocyte maturation is promoted, including polyploidization and pro-formation. -platelet (Non-Patent Reference 5). In order to confirm this in megakaryocytes before polyploidization, which were derived from ES cells, an expression level of genes (GATA1, PF4, NFE2 and β-tubulin), known to have enhanced expression with maturation of megakaryocytes, was studied using quantitative PCR.
[00145] Proliferation of megakaryocytes prior to polyploidization was promoted. In order to suppress the expression of the MYC gene and the BMI1 gene in the resulting megakaryocytes before polyploidization, cultivation was carried out for 5 days under unchanged conditions, i.e. in the presence of doxycycline and in the absence of estradiol and at a culture temperature of 39°C. Then, quantitative PCR was performed to measure the expression level of the respective genes (FIGURE 6). As a result, expression levels of genes that serve as an indicator of megakaryocyte maturation were found to be higher in 39 °C culture than in 37 °C culture.2-7-3. Influence of ROCK inhibitor + forced expression of the BCL-XL gene
[00146] MYC/BMI1 expression in megakaryocytes prior to polyploidization was suppressed and, at the same time, a lentivirus vector similar to that used in 2-1 to induce BCL-XL expression was introduced into cells in the presence of doxycycline.
[00147] It was studied whether polyploidization of megakaryocyte progenitor cells was derived from a ROCK inhibitor by the presence or absence of expression of a BCL-XL gene (FIGURE 7).
[00148] The degree of polyploidization was studied (ploidy assay) by suppressing MYC/BMI1 expression in the presence of 10 μM Y27632, during induction of BCL-XL suppression, and culturing the resulting cells for 7 days. It was confirmed that the number of polyploidized cells with 8N or greater was significantly increased in the BCL-XL expressed cell line (a shaded bar in FIGURE 7B) compared to a cell line (a blank bar in FIGURE 7B) in which BCL-XL did not was expressed. Furthermore, it was observed that the number of cells in which BCL-XL was expressed showed a gradual increase (■ in FIGURE 8), while the number of cells in which BCL-XL was expressed decreased (▲ in FIGURE 8).
[00149] This suggests that in order to avoid oncogene dependence on megakaryocytes prior to polyploidization, which acquired high proliferation capacity as a result of forced expression of the oncogene, apoptosis suppressor genes such as BCL-XL gene were effective.
[00150] 2-7-4. Influence of ROCK inhibitor + forced expression of the BCL-XL gene + suppression of the p53 gene
[00151] It was studied whether suppression of p53 expression promoted polyploidization of megakaryocytes prior to polyploidization or not.
[00152] The expression of the p53 gene was suppressed as in 2-2 by lentivirus infection at MOI=10, using an FG12 lentivirus vector into which a promoter, shp53, had been introduced.
[00153] After suppression of MYC/BMI1 expression, forced expression of BCL-XL and suppression of p53 expression in the presence of Y27632, cultivation was performed for 7 days at 39°C. After cultivation, the degree of polyploidization was studied. As a result, it was found that, compared to control cells (black bar in FIGURE 9B) in which there was no p53 knockdown, cells (white bar in FIGURE 9B) in which there was p53 knockdown showed an increase in p53 8N cell number and polyploidization was promoted.2-7-5. Influence of ROCK inhibitor + forced expression of the BCL-XL gene + suppression of the p53 gene + valproic acid
[00154] The influence on polyploidization was studied by further treatment of cells, which were subjected to 2-7-4 treatment with valproic acid. After suppression of MYC/BMI1 expression, forced expression of BCL-XL, treatment with ROCK inhibitor (10 μM) and suppression of p53 expression, valproic acid (final concentration: 0.5 mM) was added to the medium and cultivation was carried out at 39 °C for 7 days. As a result, it was found that cells (blank bar in FIGURE 10B) treated with valproic acid showed promoted polyploidization compared to control cells (black bar in FIGURE 10B), which were not subjected to valproic acid treatment.2-7 -6. Influence of BCL-XL gene forced expression + myosin heavy chain IIA/B ATPase inhibitor and influence of ROCK inhibitor + BCL-XL gene forced expression + p53 gene suppression + valproic acid + myosin heavy chain IIA inhibitor/ B ATPase
[00155] It was studied whether the treatment of megakaryocytes, the polyploidization of which did not proceed sufficiently with blebstatin, ie whether an inhibitor of the myosin heavy chain IIA/B ATPase had an influence on the degree of polyploidization. After suppression of MYC/BMI1 expression, forced expression of BCL-XL and treatment with blebstatin (10 µg/ml), the resulting cells were cultured at 39°C for 7 days. The number of cells 8N or more was found to be greater in cells subjected to blebstatin treatment (white bar in FIGURE 11) than in cells not subjected to blebstatin treatment (black bar in FIGURE 11b), showing promoted polyploidization.
[00156] Next, the degree of polyploidization when blebstatin treatment was used alongside other treatments was studied. The cells were subjected to blebstatin treatment in addition to the treatments mentioned above in Section 2-7-5 and their influence on polyploidization was studied. After suppression of MYC/BMI1 expression, forced expression of BCL-XL, treatment with a ROCK inhibitor (10 μM), suppression of p53 expression and treatment with valproic acid (0.5 mM), treatment with blebstatin (10 μg/mL) was performed and then the resulting cells were cultured at 39 °C for 7 days. It was found that the number of cells 8N or more was greater in cells subjected to treatment with blebstatin (white bar in FIGURE 12) than in control cells not subjected to treatment with blebstatin (black bar in FIGURE 12B), showing that the treatments promoted polyploidization. In addition, after 7 days of culture, cells treated with blebstatin showed a slight deterioration in their ability to proliferate (upper graph in FIGURE 13), but cytoplasmic hypertrophy was observed. Induction for mature megakaryocytes was thus confirmed (bottom graph in FIGURE 13).3. Platelet production from polyploidized megakaryocytes3-1. Influence of suppression of BCL-XL expression on platelet production (1)
[00157] In the 10T1/2 feeder cells, 2 x 105 iMKPC-type II cells obtained in section 2-2 were seeded, followed by cultivation at 39 °C in the presence of 0.5 μg/mL of Dox (BCL-XL ON ) or after removing the Dox from the culture medium (BCL-XL OFF). On days 3-4, megakaryocytes and platelets in the culture medium were analyzed with a flow cytometer after staining with a CD41 antibody and a CD42 antibody.
[00158] The results are shown in FIGURE 14. The population (B) in which BclxL expression was suppressed by Dox OFF was found to be composed mainly of mature megakaryocytes, in which CD42b was expressed (B) compared to the megakaryocytes (A) in which BCL-XL was expressed by Dox ON. It was also found that in relation to platelets released from them, a ratio of platelets in which CD42b required for the expression of the function to be expressed became higher (D) than that of platelets (C) released from megakaryocytes of BCL-XL ON .3-2. Influence of suppression of BCL-XL expression on platelet production (2)
[00159] FIGURE 15 shows the results of measuring the number of cells when BCL-XL expression was turned on or off, based on results similar to those shown in Section 3-1. The number of platelets became markedly higher in the population in which BCL-XL expression was suppressed.
[00160] A shows the number of CD42b-positive platelets, B shows the number of CD41a-positive/CD42b-positive megakaryocytes, and C shows the number of CD41a-positive megakaryocytes.
[00161] No influence of BclxL expression on the number of megakaryocytes in which CD41 was expressed could be found (C), but it was assumed that a ratio of CD42b to CD41+ expression increased due to suppression of BclxL expression and the greater number of mature megakaryocytes.3-3. Influence of culture temperature on platelet production
[00162] In 10T1/2 feeder cells, 2 to 3 x 105 iMKPC type II cells were seeded and cultured for 3 days at culturing temperatures of 35, 37 and 39 °C during the addition of 0.5 μg/mL or not from Dox. Platelets contained in the supernatant were analyzed with a flow cytometer after labeling with a CD41 antibody and a CD42b antibody. With 37°C of Dox+ as 1, the mean and standard deviation of the number of CD41+ CD42b+ platelets from each population are shown in FIGURE 16.
[00163] It was found from the results that the cultivation temperature of 37 °C or 39 °C is adequate. During tests performed afterwards, the cultivation temperature was set at 37 °C.3-4. Influence of feeder cells, use of serum for cultivation and presence or absence of blebstatin on platelet production
[00164] A platelet production efficiency was measured using the following conditions together, as shown in FIGURE 17: use/non-use of feeder cells, use/non-use of conditioned medium, use of serum/ use of serum-free medium and administration/non-administration of blebistatin.
[00165] Conditioned medium was prepared by seeding 8 x 105 MMC-treated 10T1/2 cells in a 10 cm dish, coated with gelatin, by altering the medium with 10 mL of a differentiation medium (EBM) containing SCF (50 ng/mL) and TPO (50 ng/mL) and still containing 15% serum or no serum, the next day (after cell adhesion), recovery of the medium and addition of 10 mL of a new medium ( containing SCF and TPO) 24 hours later, pooling the three-day conditioned medium, and filtering through a 0.22 μm filter to remove 10T1/2 cells. When the resulting medium was used for testing, SCF and TPO were added again.
[00166] In a feeder cell using population, 2 to 3 x 105 cells of iMKPC type II were seeded in 10T1/2 feeder cells, whereas in the non-use population, cells were seeded in the same way in a plate coated with gelatin. In the population using serum medium, a differentiation medium containing 15% serum or conditioned medium was used, while in the blebstatin administration population, 5 μM blebstatin was added to the medium. Cultivation was carried out at 37 °C for 3 days.
[00167] Platelets contained in the medium supernatant were analyzed with a flow cytometer after labeling with a CD41 antibody and a CD42b antibody. With cells obtained by culturing on feeder cells during the addition of 15% serum without addition of blebistatin as cells under normal condition, the mean and standard deviation of a ratio of the number of CD41+ CD42b+ platelets in each population to those in the condition normal are shown in the bar graph in FIGURE 17. No significant difference in the amount of platelet production was found between populations.
[00168] Comparison of CD42b expression level on CD41-positive platelets in each population (a ratio of the average fluorescence intensity of each population to the average fluorescence intensity of the normal condition) is shown in FIGURE 18. Under exempt conditions of serum and feeder cells free, platelets showing a higher expression of CD42b were produced. The influence of blebstatin was not found.
[00169] An example of the optimized conditions is shown in FIGURE 19. A ratio of CD41-positive and CD42b-positive platelets (which may be called "GPIba"), when cultured under the optimized conditions, is shown in FIGURE 20.
[00170] As shown in Figure 20, in the platelets produced from the polyploidized megakaryocytes obtained in the present invention, about 20% were CD41 positive and CD42b positive. When BCL-XL expression was suppressed, the ratio increased to 55%, when culturing was performed by removing the feeder cells and additionally the serum, the ratio increased up to 81%.
[00171] 4. Importance of CD42b expression for platelet function (reference)
[00172] An inhibitory effect of a HIP1 antibody on a ristocetin agglutination reaction (agglutination reaction via vWF and a receptor [heteropentamer composed of GPIba, GPIX and the like] on platelets) was measured using human peripheral blood platelets. The HIP1 antibody was a GPIba function inhibitory antibody.
[00173] After the effect of GPIba was inhibited by suspending 1 x 108 platelets in blood plasma at 50%, adding a HIP1 antibody thereto and pre-culturing at 37°C for 3 seconds, ristocetin (final concentration: 2 mg /mL) was added to induce an agglutination reaction and light transmission was monitored for 7 minutes. The maximum light transmission (showing the agglutination intensity) of each population is shown in the bar graph in FIGURE 21. The HPI1 antibody completely inhibited agglutination due to the association of GPIb/von Willebrand alpha-factor (vWF) at a concentration of 10 μg/mL or greater.
[00174] Next, 100 μg of a control HIP1 or IgG antibody was administered to NOG mice and the platelets stained with TAMRA (red dye) were transplanted. The vascular endothelium was exposed to laser to damage it and induce thrombus formation. The number of human platelets (red) that contributed to thrombus formation was counted.
[00175] The mean and standard deviation of the number of human platelets in the thrombi corrected with one unit of blood vessel length are shown in the bar graph (FIGURE 22). In the group administered with HIP1 antibody, the contribution of human platelets to the thrombus was inhibited.
[00176] It was therefore found that GPIba (CD42b) is a molecule that plays an important role in thrombus formation, both in vivo and in vitro. Industrial Applicability
[00177] The present invention provides a method of promoting polyploidization of megakaryocyte progenitor cells and, furthermore, a method of efficiently inducing platelet release. In particular, the method of the present invention is very effective for the preparation of megakaryocytes or platelets in vitro from various stem cells and contributes greatly to the development of drugs in the medical or blood product fields.

权利要求:
Claims (15)
[0001]
1. Method of producing polyploidized megakaryocytes, characterized in that the method comprises the step of obtaining megakaryocytes by inducing the expression of an oncogene selected from the MYC family of genes, and BMI1, in cells at any stage of differentiation of hematopoietic progenitor cells into megakaryocytes prior to polyploidization, inducing BCL-XL expression in megakaryocytes prior to polyploidization; cultivation and proliferation of the resulting cells.
[0002]
2. Method according to claim 1, characterized in that the c-MYC gene is used as the oncogene
[0003]
3. Method according to claim 1 or 2, characterized in that in the cultivation step, the expression or function of a p53 gene product is inhibited.
[0004]
4. Method according to any one of claims 1 to 3, characterized in that in the cultivation step, the megakaryocytes before polyploidization are subjected to at least one of the following items (a) to (c): (a) treatment with an inhibitor of actomyosin complex function; (b) treatment with a ROCK inhibitor; and (c) treatment with an HDAC inhibitor.
[0005]
5. Method according to claim 4, characterized in that the ROCK inhibitor is Y27632; the HDAC inhibitor is valproic acid; and the inhibitor of actomyosin complex function is blebstatin.
[0006]
6. Method according to any one of claims 1 to 5, characterized in that the cultivation step is carried out at a temperature above 37 °C.
[0007]
7. Method according to any one of claims 1 to 6, characterized in that the hematopoietic progenitor cells are derived from cells selected from the group consisting of iPS cells, ES cells and hematopoietic stem cells.
[0008]
8. Method according to claim 7, characterized in that said hematopoietic stem cells are derived from umbilical cord blood, bone marrow blood or peripheral blood.
[0009]
9. Method according to any one of claims 1 to 6, characterized in that it also comprises the step of suppressing the induced expression of BCL-XL.
[0010]
10. Method of producing a platelet, characterized in that it comprises: obtaining polyploidized megakaryocytes using the method as defined in any one of claims 1 to 9 and culturing the cells; echolet of a platelet from the culture of polyploidized megakaryocytes, wherein the step of culturing the polyploidized megakaryocytes is conducted while suppressing the expression of BCL-XL that has been induced, or after BCL-XL is removed from the cells.
[0011]
11. Method according to claim 10, characterized in that the stage of cultivation of polyploidized megakaryocytes is carried out in the absence of a serum and/or in the absence of a feeder cell.
[0012]
12. Method according to any one of claims 10 or 11, characterized in that the stage of cultivation of polyploidized megakaryocytes is carried out from 1 day to 15 days.
[0013]
13. Method according to any one of claims 10 to 12, characterized in that the step of culturing the polyploidized megakaryocytes is carried out at 37 °C.
[0014]
14. Method according to any one of claims 10 to 12, characterized in that in the step of culturing the polyploidized megakaryocytes, a ROCK inhibitor and/or an inhibitor of the function of the actomyosin complex is added to a medium.
[0015]
15. Method according to claim 14, characterized in that the ROCK inhibitor is Y27632 and the inhibitor of the actomyosin complex function is blebstatin.

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法律状态:
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