[0001]The present invention relates to a method for producing a specific differentiated cells, and to cells prepared by the method. In particular, the present invention is a method for producing a differentiated blood cells, and to blood cells produced by the method.
Background technique
[0002]
　For the treatment of a disease, may require a specific cell, it is necessary to secure a sufficient amount of cells to achieve a therapeutic objective. However, it is difficult to obtain a sufficient amount of cells to be subjected to treatment from a living body in vitro, for example, it has been tried a method of preparing differentiated inducing cells of interest from such precursor cells thereof.
　When treating blood-related diseases, or when performing surgical treatment, blood cells to be subjected to treatment is needed. Among blood cells, megakaryocytes platelets, platelet precursors (Proplatelet) even at cells producing platelets are essential cells for blood coagulation (hemostasis) are particularly high demand cell. Especially platelets, leukemia, bone marrow transplantation, many demand in such anticancer treatment, the need for stable supply is high. So far, platelets, other methods of harvesting the blood donation from a donor, have been secured by a method of differentiating methods, megakaryocyte cells from umbilical cord blood or bone marrow cells of administering TPO-like structure similar to (mimetics) Formulation . Furthermore, hematopoietic stem cells or hematopoietic progenitor cells were amplified in vitro, it has been attempted a method of preparing blood cells from precursor cells thereof. For example, a method of establishing a hematopoietic stem cell line from mouse ES cells (Patent Document 1), a method of differentiating the embryonic stem cells of a primate to hematopoietic cells (Patent Document 2), or maintaining the undifferentiated hematopoietic stem cells and a method in which the CD34-positive / CD38-negative cells conveniently and stably amplified in vitro (Patent Document 3) have been reported.
[0003]
　In the case of inducing differentiation of cells, usefulness of pluripotent stem cells is high. Pluripotent stem cells, such as ES cells and iPS cells can be utilized as a source for the production of blood cells such as platelets artificially. In recent years, the establishment of iPS cells, usefulness of pluripotent stem cells has become increasingly spotlight as an important source of cell therapy in regenerative medicine. So far, for example, Takayama et al., Succeeded in inducing differentiation megakaryocyte cells and platelets from human ES cells, as a result, out availability of platelets differentiated from ES cells as a source of platelet transfusions and have (Patent Document 4 and non-Patent Document 1). Furthermore, the inventors to establish a method for preparing megakaryocytes and platelets from iPS cells, transfusion of platelets from ES cells was difficult avoided, leukocyte antigens (human leukocyte antigen; HLA) compatibility problems It was to be solved. Then, conventionally, the platelets have been supplied by donation from a donor, such as by chronic shortage of donors, but was difficult to stably supply a sufficient amount of platelets, in this regard also differentiate platelets from ES cells or iPS cells by inducing, it appeared to be available. However, platelets are prepared from iPS cells or ES cells In the previous method, a smaller amount to be prepared, and, in order not only a method of manufacturing each time a series of steps to ensure the quantitative stability of platelets it would be desirable to improve to efficient methods for.
　A sufficient amount of blood cells, such as such megakaryocytes and platelets, the problem to be solved in order to supply a stable is also true for the supply of other cells.
　The desired cells, even when prepared by inducing differentiation of a cell, for it is not easy to prepare a large amount of precursor cells of the desired cell, sufficient amounts ensure the desired cells terminally differentiated it is difficult at this stage to be.
CITATION
Patent Literature
[0004]
Patent Document 1: JP 2006-141356 Patent Publication
Patent Document 2: JP 2004-350601 Patent Publication
Patent Document 3: JP 2006- 61106 JP
Patent Document 4: WO2008 / 041.37 thousand
Non-Patent Document
[0005]
Non-Patent Document 1: Takayama et al, Blood, 111: 5298-5306 2008
Summary of the Invention
Problems that the Invention is to Solve
[0006]
　The present inventors relates to blood cells, but has established a method for obtaining megakaryocytes and platelets from iPS cells, upon clinical application of this method, to improve the megakaryocytes and platelets in large-scale production possible way It is necessary. Further, rapidly can correspond to the supply needs of platelets, and also to enable a stable supply, it is important in terms of aim future clinical applications.
　In view of the above circumstances, the present invention is for the manufacture of a target cell to induce differentiation of cells, increasing the proliferative capacity of cells in the desired differentiation stage, amplifying the number of cells, target cells from the cell to provide a method for producing a.
　Furthermore, the present invention uses the above methods, to provide the desired differentiated blood cells. In particular, the present invention is a blood cells as a source of mature megakaryocytes and platelets, provides high megakaryocyte progenitors of proliferative, and its manufacturing method.
　Further, the present invention is a method for producing large amounts and stably mature megakaryocytes and platelets from該巨mononuclear progenitor cells, as well as induce mature megakaryocyte cells produced by these methods, and the mature megakaryocyte cell differentiation and an object thereof is to provide a platelet being.
　Further, since the necessity of similarly stable supply and platelets also red blood cells has been pointed out, the present invention aims also to provide a red blood cells produced by the production method and the method of red blood cells.
　Furthermore, the present invention aims to provide a long-term storage methods of megakaryocyte progenitor cells are cells of immature state of mature megakaryocytes are progenitors of platelets.
Means for Solving the Problems
[0007]
　We have 4 genes (OCT3 / 4, SOX2, KLF -4, c-MYC) 3 gene with the exception of iPS cells and c-MYC was established by (OCT3 / 4, SOX2, KLF -4) iPS were established by a comparison of megakaryocytes and platelets production ability of the cells revealed that the direction of iPS cells with 4 genes to produce significantly efficiently megakaryocytes and platelets. Furthermore, the expression of four genes that were introduced during the establishment, the contrast is suppressed in the state of iPS cells, with the megakaryocytes differentiation is induced reactivation of c-MYC gene, megakaryocyte production level It was revealed to be involved in the increase. Additionally, megakaryocyte progenitor cells prior multinucleated forced to express c-MYC gene, it has been found that obtaining a high proliferative capacity.
　In general, when the oncogenes such as c-MYC is overexpressed in cells, the cell cycle is increased, the growth is activated. Cells capturing the growth as a stress, suppressing defense reaction which (oncogene-induced senescence: OIS oncogene induced senescence) is induced, are known to inhibit excessive cellular proliferation. The present inventors have further found a method that focuses on this phenomenon, to produce large quantities of a specific differentiated cell by controlling the OIS of cells in the differentiation stage.
　The present invention is based on the above findings, it has been completed.
[0008]
That is, the present invention relates to the following (1) to (30).
(1) A method for producing a specific cell to induce differentiation of cells,
　in order to expand the cell of the desired differentiation stage, forced expression of oncogenes in the cells of the desired differentiation stage specific cells the method of production.
(2) is induced by forced expression of oncogenes in cells of the desired differentiation stage method for producing a particular cell according to the above (1), characterized in that to suppress the oncogene induced senescence.
(3) the oncogene induced senescence suppression is achieved by expressing the Polycomb gene, a manufacturing method of a particular cell according to the above (1) or (2).
(4) the desired differentiation stage of the cells, the production method of a particular cell of any one of the above characterized in that it is a cell that has been induced to differentiate from ES cells or iPS cells (1) to (3).
(5) into said cells of the desired differentiation stage, cancer gene foreign or introducing oncogenes and Polycomb genes, cancer gene or, characterized in that forced expression of cancer genes and the Polycomb gene method for producing a specific cell of any one of the above (1) to (4).
(6) an oncogene or Polycomb gene of the foreign, and introduced into a precursor cell of a cell of the desired differentiation stage, cancer gene or above, characterized in that the forced expression of cancer genes and the Polycomb gene (5 method for producing a specific cell according to).
(7) the oncogene and / or Polycomb genes, respectively, operably linked to downstream of the inducible promoter, cancer gene or, characterized in that inducibly forcibly expressed cancer gene and the Polycomb gene method for producing a specific cell according to (5) or (6) to.
(8) In order to promote differentiation of the desired differentiation stage of the cell, the desired cancer gene in a cell differentiation stages or above (5), characterized in that inhibiting the expression of oncogenes and Polycomb genes to method for producing a specific cell of any one of (7).
(9) the oncogene or the suppression of the expression of oncogenes and Polycomb genes, respectively, operably linked to inhibiting promoter downstream, characterized in that it is achieved by suppressing the expression of the gene described above ( method for producing a specific cell according to 8).
(10) The method of producing a specific cell of any one of the above said oncogene is characterized by a MYC family genes (1) through (9).
(11) The method of producing a specific cell of any one of above the Polycomb gene characterized in that it is a BMI1 (3) to (10).
(12) wherein a desired progenitor hematopoietic progenitor cells differentiation stage of the cell, the a desired differentiation stage of the cells prior to multinucleated megakaryocyte progenitor cells, the specific cell is a mature megakaryocyte cell method for producing a specific cell of any one of the above (6) to (11), characterized in.
(13) wherein a desired progenitor hematopoietic progenitor cells differentiation stage of the cell, the a desired differentiation stage of the cells prior to multinucleated megakaryocyte progenitor cells, and wherein the specific cell is a platelet method for producing a specific cell of any one of the above (6) to (11) to be.
(14) said hematopoietic progenitor cells, the production method of a particular cell according to the above (12) or (13), characterized in that those present in the net-like structure was prepared from ES cells or iPS cells .
(15) In (12) or (14) mature megakaryocyte cell is a specific cell according to.
(16) platelet is a particular cell according to (13) or (14).
(17) a blood preparation as an active ingredient platelets according to (16).
(18) (15) above or a kit for producing a mature megakaryocyte cells or platelets according to (16).
(19) the desired differentiation stage of hematopoietic cells oncogene is forcibly expressed.
(20) Further polycomb gene was forcibly expressed, hematopoietic cells according to (19).
(21) the blood cells of the desired differentiated stages, blood cells according to (19) or (20), characterized in that the ES cells or iPS cells, which are differentiation-induced cells.
(22) to said desired stage of differentiation hematopoietic intracellular above cancer gene foreign or introducing oncogenes and Polycomb genes, oncogenes or, characterized in that forced expression of oncogenes and Polycomb gene (19) to hematopoietic cells of any one of (21).
(23) an oncogene or Polycomb gene of the foreign, and introduced into a precursor cell of hematopoietic cells of the desired differentiation stage, cancer gene or above, characterized in that the forced expression of cancer genes and the Polycomb gene blood cells according to (22).
(24) the oncogene and / or Polycomb genes, respectively, operably linked to downstream of the inducible promoter, cancer gene or, characterized in that inducibly forcibly expressed cancer gene and the Polycomb gene blood cells according to (22) or (23) to.
(25) hematopoietic cells according to any one of the above said oncogene is characterized by a MYC family genes (19) to (24).
(26) the above (20) which polycomb gene characterized in that it is a BMI1 to (25) blood cells according to any one of.
(27) the precursor cells blood cells in the desired differentiation stage is hematopoietic progenitor cells, above, wherein the blood cells of the desired differentiation stage is megakaryocyte progenitor cells prior multinucleated (23 ) to blood cells according to any one of (26).
(28) said hematopoietic progenitor cells, blood cells according to the above (27), characterized in that those present in the net-like structure was prepared from ES cells or iPS cells.
(29) above (19) to frozen cell composition comprising hematopoietic cells according to any one of (28).
(30) (27) above or a kit for producing the megakaryocyte progenitor cells prior multinucleated a hematopoietic cell according to (28).
Effect of the invention
[0009]
　According to the present invention, it is possible to expand the cell is in the desired differentiation stage, and it is possible to manufacture large quantities of specific cells differentiated from the amplified cells.
[0010]
　Furthermore, when used in the production of hematopoietic cells differentiated present invention, the pluripotent stem cells, for example, it is possible to produce blood cells such as megakaryocytes and platelets stably and in large quantities.
[0011]
　Also, blood cells produced by the present invention can be stored frozen. Thus, as the blood cells, for example, when producing a multinucleated previous megakaryocyte progenitor cells, it is possible to cryopreserve this, it can be supplied mature megakaryocyte cells and platelets megakaryocyte progenitor cells derived from the same source to become.
[0012]
　In particular, according to the method of the present invention, it is possible from the iPS cells are prepared in large quantities cryopreserved possible multinucleated preceding megakaryocyte progenitor cells (progenitor cells mature megakaryocyte cell). As a result, if the multinucleation preceding megakaryocyte progenitor cells in the source to avoid the problems HLA compatibility, and to produce the amount of platelets can be sufficiently corresponding to the repeating transfusion, it is possible to supply.
[0013]
　Furthermore, the present invention, a method of supplying stably is provided red blood cells in vitro.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014]
[1] 4 gene derived iPS cells and 3 genes derived graph comparing the number of megakaryocytes produced from iPS cells in. The vertical axis, CD42b positive megakaryocyte cell count from each cell was 1 to CD42b number of positive megakaryocyte cells of ES cell-derived 22 days after the culture. The horizontal axis, number of days from start of the culture of iPS cells and ES cells. 3-f, the cell lines derived from 3 gene, the 4-f represents the cell lines derived from 4 genes. ES ES cells.
[2] Confirmation of reactivation of a transgene in human iPS cell-derived megakaryocytes. 4 factors derived from iPS cells (TkDA3-2, TkDA3-4 and TkDA3-5) and 3 in factor-derived iPS cells (TkDN4-M), each transgene (OCT3 / 4, SOX2, KLF -4, c-MYC expression of) confirmed the undifferentiated iPS cells and differentiated megakaryocytes. As a control gene introduction, for the human skin fibroblast cells transfected with the gene (HDF), to confirm its expression. endo is that of endogenous genes, Tg indicates that the introduced gene. The undifferentiated iPS cells, was confirmed expression also REX1 and NANOG.
[3] increase in megakaryocyte cell number by c-MYC forced expression in ES cell-derived hematopoietic progenitors cells. Blood progenitor cells from day 15 of the net-like structure after the culture of human ES cells are removed, the gene (OCT3 / 4, SOX2, KLF -4, c-MYC) were each introduced alone, megakaryocytes that are produced the afterbirth the number of cells over time was counted. The vertical axis, CD42b positive megakaryocyte cell count from each cell was 1 to CD42b positive megakaryocyte cell numbers only from the introduced hematopoietic progenitor cells (mock) viral vector. The horizontal axis, number of days from the start of cultivation of the ES cells.
Graph comparing the number of platelets produced from FIG. 4 4 gene derived iPS cells and 3 gene derived from iPS cells. The vertical axis is the number of platelets from the cells with 1 the number of platelets from ES cells 21 days after the culture. The horizontal axis, number of days from start of the culture of iPS cells and ES cells. 3-f, the cell lines derived from 3 gene, the 4-f represents the cell lines derived from 4 genes. ES is that of ES cells.
[5] transfusion experiments to the model mice with platelets from iPS cells. Was prepared immunodeficient mice thrombocytopenia model previously irradiated (A). Platelets produced from TkDA3-4 strain was blood transfusion from the tail vein of immunodeficient mice. B shows the time course of post-transfusion (30 minutes, 2 hours, 24 hours). PB; human peripheral blood
[Figure 6] Confirmation of thrombus formation ability in vivo of human iPS cell-derived platelets. Human iPS cells derived platelets, tetramethylrhodamine ethyl ester; and stained with (TMRE red pigment) was injected from the mouse tail vein mixed with hematoporphyrin (hematoporphyrin). After the laser irradiation of the mesenteric artery, 0 seconds, 6 seconds, 13 seconds, and observed the formation state of the thrombus in the 20 seconds the vessel in time-lapse confocal microscopy. Blood flow: blood flow
showing the overview of FIG. 7 protocol to introduce genes into hematopoietic progenitor cells prepared from ES cells.
[8] After c-MYC gene transfer into hematopoietic progenitor cells prepared from ES cells, we show the results of FACS analysis in 9 days. A a result of FACS analysis. B after c-MYC introducing shows micrographs of 9 day transduced cells. Control, was introduced only MYC virus vector cells.
[9] shows the proliferative capacity of megakaryocyte progenitor cells expressing c-MYC gene. The vertical axis, CD42b-positive cell count. The horizontal axis, days after introducing the c-MYC gene into cells. ■ is, c-MYC control of the result of introducing only viral vectors instead of.
[Figure 10] shows the results of FACS analysis of c-MYC gene and BMI1 gene introduced megakaryocyte progenitor cells. c-MYC / BMI1 (top) shows the FACS analysis of cells transfected with both the c-MYC gene and BMI1 gene, c-MYC alone (see below) are of cells transfected with only the c-MYC gene It shows FACS analysis results.
[11] The c-MYC gene and BMI1 genes are introduced into cells, shows the FACS analysis of cultured day 35 cells. A is a diagram schematically showing the specific functional molecule of megakaryocytes. B shows the results of FACS analysis.
[Figure 12] shows the results of examining the proliferation ability of MYC / BMI1 expressing cells. The vertical axis, the number of cells and the horizontal axis represents the number of days after gene introduction into a cell.
[Figure 13] shows the image observed by an electron microscope of released platelet from c-MYC / BMI1 expressing cells derived from megakaryocyte progenitor cells.
[Figure 14] c-MYC gene and HOXA2 gene, as well, the c-MYC gene and BCLXL gene was introduced into ES cells (KhES) derived hematopoietic progenitor cells, respectively, after 105 days, and, FACS analysis for cells after 27 days We are shown the results of.
[Figure 15] pMX tet off vector according Confirmation of gene regulation system. the pMX tet off vector c-MYC and BMI1And the construct ligated across the 2A expressed 293GPG intracellularly, the results of control of gene expression was examined whether functions. A is a diagram for explaining the constructs and mechanisms of action of the vector. B is a tetracycline and β- estradiol, in addition or non-addition state, the intracellular c-MYC is a result of examining the flow cytometer expression. The horizontal axis of B is, c-MYC is an expression of. "293gpg" shows the results of 293GPG cells of control.
[Figure 16] shows the results of investigation of proliferation potency and differentiation potency of genetic control vector expressing cell lines. A is the various vector c-MYC and BMI1 the results obtained by examining the proliferation ability of cells expressing the. The vertical axis, the number of cells and the horizontal axis represents the number of days after gene introduction into a cell. B is an anti-CD42b (GPIb-alpha) antibodies and anti-CD41a (Integrin alphaIIb / beta3 complex) antibody (top), the cells were stained with anti-Glycophorin-a antibodies and anti-CD41a antibodies (bottom), analyzed on a flow cytometer it is the result. As the upper and lower of B, left, pMX c-MYC and DSAM BMI1 results of cells separately forced to express, right, pMX tet off c-MYC 2A BMI1 the result of cells expressing.
[Figure 17] beta-presence of estradiol, pMX tet off c-MYC2A BMI1 was studied the extent of multinucleated megakaryocyte cell lines that expressed. A is the result of the control of the cell of the vector alone (strain not expressing the gene), B is, c-MYC and BMI1 the result of cells expressing.
[Figure 18] c-MYC and BMI1 relates megakaryocytes derived platelets was forcibly expressed, it shows the results of fibrinogen binding assay. Upper (human platelets) is the result of platelets from human peripheral blood, middle (pMX tet off c-MYC 2A BMI1), in the presence of β- estradiol tet off pMX c-MYC 2A BMI1 of strain-derived platelets result, the lower (pMx Myc Dsam Bmi1) is, pMX c-MYC and DSAM BMI1 in, c-MYC and BMI1 shows the results of platelets from strains forced to express.
[Figure 19] c-MYC and BMI1 shows the results of examining the integrin activation ability of platelets produced from megakaryocytes strain with suppressed expression. Left figure, ADP absence, right figure, were analyzed integrin activation ability by flow cytometer ADP presence (50 [mu] M).
[Figure 20] shows the differentiation pathway to a megakaryocyte from ES cells.
DESCRIPTION OF THE INVENTION
[0015]
　One embodiment of the present invention is a method of producing a specific cell to induce differentiation of cells of the source, the desired differentiation stage of the cells in the middle differentiation into specific cells from the cell as a source , in order to amplify (or to grow), in cells of the desired differentiation stage, forced expression of oncogenes, a manufacturing method of a particular cell.
　Here, the "cells of the source" is a target cell obtained by performing differentiation induction (here, specific cell) be one that corresponds to the progenitor cells, other than terminally differentiated cells retain the ability to differentiate if the cells may be any one, for example, completely even pluripotent stem cells such as undifferentiated or, to some extent differentiate holds but still differentiation ability in progress cells (e.g., such as hematopoietic progenitor cells blood cells) may be used. Further, the "specific cells" or "specific cell" is produced in the present embodiment, full undifferentiated cells (e.g., pluripotent stem cells) if other than the cells, retaining the undifferentiated state to some extent may be a cell, i.e., a cell that appeared complete undifferentiated stage during terminal differentiation stages, is that the full undifferentiated cells other than cells. For example, a "specific cell" or "specific cells" in the present embodiment, if showing an example of a portion of a blood cells, mature megakaryocyte cell, is that such platelets or red blood cells.
[0016]
　Amplified in this embodiment (or proliferation) by the, the "differentiation stage of the cell" is a cell that appeared complete undifferentiated stage during terminal differentiation stages, complete undifferentiated stage of the cell (e.g. , is that of pluripotent stem cells) and terminally differentiated stage of the cell other than the cell. For example, the "differentiation stage of the cell" in the present embodiment, one example of a blood cells are progenitors of mature megakaryocyte, by such megakaryocyte progenitor cells before hematopoietic progenitor cells or multinucleated is there. As cell differentiation stages, for example, it can be used, such as cells derived from pluripotent stem cells such as ES cells or iPS cells.
[0017]
　ES cells used in the present invention is not particularly limited, in general, a fertilized egg blastocyst stage were cultured with feeder cells, and grown cells from the inner cell mass into pieces, Furthermore, repeated operation subcultured, can be finally established as ES cell lines. Thus, ES cells are often obtained from fertilized eggs, other, for example, adipose tissue, chorionic villi, amniotic fluid, placenta, etc. testis cells are obtained from the non-fertilized egg, the characteristics of ES cell-like It has, or may be ES cell-like cells with pluripotent.
[0018]
　Further, iPS cells used in the present invention, somatic cells (e.g., fibroblasts or blood cells, etc.) several transcription factors to confer pluripotency to (hereinafter, wherein the term "pluripotency factors" referred) by introducing the gene, as long as the cell has acquired the ES cells equivalent pluripotent be any cells from. The pluripotent factors, have already been reported many factors including, but not limited to,, Oct family (e.g., Oct3 / 4), SOX family (e.g., SOX2, SOXl, Sox3, etc. SOX15 and SOX17 ), Klf family (e.g., Klf4, Klf2), MYC family (e.g., c-MYC, N-MYC, L-MYC, etc.), and the like NANOG, LIN28. For establishment method of iPS cells, since many documents have been issued, they can be referred to (see, eg, Takahashi et al., Cell 2006,126: 663-676; Okita et al., Nature 2007,448: 313 -317; Wernig et al., Nature 2007,448: 318-324; Maherali et al., Cell Stem Cell 2007,1: 55-70; Park et al., Nature 2007,451: 141-146; Nakagawa et al., Nat Biotechnol 2008,26: 101-106; Wernig et al., Cell Stem Cell 2008,10: 10-12; Yu et al., Science 2007,318: 1917-1920; Takahashi et al., Cell 2 07,131: 861-872; Stadtfeld et al., Science 2008 322: 945-949 that of reference, etc.).
[0019]
　The oncogenes used in the present invention refers to a gene that induces malignant transformation of cells the gene is endogenous, but are not limited to, for example, MYC family gene, SRC family gene, RAS family gene, RAF family gene, c-Kit and PDGFR, such as protein kinase family genes such as Abl may be mentioned.
　In the present invention, forced expression of Polycomb genes described later oncogenes or in cells of the desired differentiated stage, an oncogene or Polycomb gene was introduced into the "cell of a desired differentiation stage", forced expression may be accomplished by, also introducing these genes into precursor in cells of the "cell of a desired differentiation stage", forced to express, to proceed to differentiate while maintaining the expression, "desired differentiated It may be accomplished by maintaining the forced expression status of these genes at the stage of a cell "in the further advance by introducing these genes into precursor in cells of the" cell of a desired differentiation stage "," it may be achieved by inducing a forced expression of these genes when differentiated into cells "in the desired stage of differentiation. For example, when amplifying a multinucleated previous megakaryocyte progenitor cells as a cell of the desired differentiation stage, introducing oncogenes or Polycomb gene to hematopoietic progenitor cells (described later) in the precursor stage, we are forced to express good. If forced expression of the oncogene and the Polycomb gene in cells of the desired differentiation stage, may be introduced into the cell simultaneously with oncogenes and Polycomb gene, it may be introduced at different timings.
[0020]
　Moreover, cancer embodiment of the present invention, induced by the desired as a method for amplifying the cell differentiation stage (or to grow), oncogene forced to express in the cells of the desired differentiation stage It includes suppressing gene induced cellular senescence.
　Oncogene induced senescence (oncogene-induced senescence: OIS) and is that the stress-induced cellular senescence induced by abnormal growth stimulation due oncogenes such as RAS and MYC. When the cancer gene product excessively expressed in a cell, expression of CDKN2a (INK4a / ARF) tumor suppressor gene product such as p16 and p19 encoded by the genetic locus is induced. As a result is induced senescence and apoptosis, proliferation activity of the cells is reduced. Therefore, if avoiding OIS to induce oncogenes are expected to be maintained at a high proliferative cell conditions.
[0021]
　Inhibition of oncogene-induced cellular senescence, for example, can be accomplished by expressing the Polycomb gene into cells oncogene is expressed. Polycomb genes (polycomb group: PcG) controls the CDKN2a (INK4a / ARF) locus negatively functioning to avoid cellular senescence (or, for example, Oguro et al, by "Polycomb group protein complex aging control of stem cells ", regenerative medicine vol.6 No4 pp26-32;.. Jseus et al, Nature Reviews Molecular cell Biology vol.7 pp667-677 2006;.... Proc Natl Acad Sci USA vol.100 pp211-216 2003 see). Therefore, in addition to the expression of oncogenes such as MYC family gene, by expressing the Polycomb gene in a cell, avoiding OIS, it is possible to further enhance the proliferation effect of oncogene product.
　The Polycomb group gene used in the present invention, for example, BMI1, Mel18, Ring1a / b , Phc1 / 2/3, Cbx2 / 4/6/7/8, Ezh2, Eed, Suz12, HADC, Dnmt1 / 3a / , and the like 3b, but particularly preferred polycomb group gene is BMI1 gene.
　Furthermore, suppression of oncogene-induced cellular senescence can also be achieved by expression of HOXA2 gene or BCLXL gene.
[0022]
　If forced expression oncogene, polycomb gene in a cell may be carried out by any method known in the art. For example, a polycomb gene in cancer gene or foreign foreign, e.g., lentiviruses and retroviruses such as using the gene transfer system according to, introduced intracellularly, it may be expressed. When performing gene expression by viral gene transfer vector, the gene operably linked to downstream of an appropriate promoter, which was inserted into a gene transfer vector may be expressed a gene of interest is introduced into cells . Here, coupled to "operably" means that the target gene by the promoter is dominated cis connects the promoter and target gene as desired expression of the gene of interest is achieved. In the practice of this invention, eg, CMV promoter, may be expressed constitutively target gene using, for example, EF1 promoter, or the elements that are activated controlled by the transformer factors such as drug response elements such as tetracycline under dominant, place the appropriate promoter (inducible promoters), for example, it may also be inducibly expressed the gene of interest under the control of such drug addition. Such gene expression system with drugs, in order to achieve a desired regulation of expression of oncogene or Polycomb genes, those skilled in the art can select an appropriate system easily. It may be used to purchase a commercial product kits for such expression systems. Moreover, oncogene is a target gene expression control, Polycomb gene may be inserted into separate vector or, it is more preferable to insert the oncogenes and Polycomb genes into the same vector.
[0023]
　Further, the present embodiment, oncogenes, or even induced to differentiate the cancer gene and the desired differentiation stage of cells Polycomb genes were expressed, includes a method of producing a specific cell of interest. To further induce differentiation of cells of the desired differentiation stage, in addition to culturing the cells differentiation stage culture conditions suitable for the induction of differentiation (medium conditions such culture temperature), if necessary, of said differentiated phase the expression of the oncogene or polycomb genes expressed in a cell may be adjusted suppressively. In this case, oncogene, the inhibition of the expression of Polycomb genes, for example, may be achieved by releasing the induction of gene by removal of the drug or the like expression induced by inducible expression system described above. Alternatively, if the drug or the like does not exist a promoter for performing constitutive expression control, to inhibit promoter performing suppression expression control in drug presence, and operably linked to an oncogene or Polycomb gene it may suppress control the expression of these genes. Additionally, it introduced oncogene, is removed by using, for example, the Cre / Lox system Polycomb gene may suppress control the expression of these genes. To adjust suppressively the expression of oncogenes or Polycomb gene can be appropriately used a commercially available kit or the like.
[0024]
　To the present embodiment, multinucleated previous megakaryocyte progenitor cells was amplified as a cell of the desired differentiation stage (grown), the multinucleated preceding megakaryocyte progenitor cells, as a specific cell, to produce a mature megakaryocyte cell the method is included. Here, oncogene, or cancer gene and Polycomb genes when forcibly expressed in megakaryocyte progenitor cells prior multinucleated, in hematopoietic progenitor cells in the precursor stage of multinucleated preceding megakaryocyte progenitor cells, oncogenes, or it is preferred to express the oncogene and polycomb gene.
　As used herein, "multinucleated preceding megakaryocyte progenitor cell" refers to at CD41a-positive / CD42a-positive / CD42b-positive is a specific marker of megakaryocytic series, mononuclear not cause polyploid body of the nucleus or it is a two-nucleus of the cell. In addition, the "hematopoietic progenitor cell", CD34 + is a hematopoietic cells characterized as cells (CD34 positive cells), for example, ES cells or iPS cell-derived cells, in particular, be prepared from ES cells or iPS cells that the net-like structure cells obtained from (also referred to as ES-sac or iPS-sac) (in particular, cells immediately after isolation from the net-like structure) is preferable. Here, prepared from ES cells or iPS cells as "net-like structure", ES cells or iPS cell-derived three-dimensional saccular (inside those involving space) in the structure, such as endothelial cell populations is formed, is that those containing hematopoietic progenitor cells therein. For more information about the net-like structure, for example, TAKAYAMA et al., BLOOD 2008,111: 5298-5306, see.
[0025]
　Cell culture conditions suitable for preparing the nets like structure from human ES cells or human iPS cells is different depending ES cells or iPS cells used, for example, as a medium to a final concentration of 15% FBS was added using IMDM, it can also be used after adding an appropriate growth factors and supplements in the case of other serum. Furthermore, in order to form a net-like structure efficiently, VEGF and 0 ~ 100 ng / ml, more preferably, it is to add about 20 ng / ml. The culture environment is different depending on the type of ES cells or iPS cells used, for example, 5% CO 2 , 36 ~ 38 ° C., can be preferably used the conditions of 37 ° C.. Culturing period until the net-like structure is formed may vary depending on the type of ES cells or iPS cells, the seeded on feeder cells, it is possible to confirm the existence enough after 14-16 days.
　Net-like structure formed is adapted to the follicular structure, inside, it is present in a state of the hematopoietic progenitor cells are concentrated. Hematopoietic progenitor cells present in the interior of the net-like structure, physical means, for example, by passing through a sterile sieve-like tool (e.g., such as a cell strainer) can be separated. Hematopoietic progenitor cells thus obtained can be used in the present invention.
[0026]
　Oncogene forcibly expressed hematopoietic progenitor intracellularly may be of any of the aforementioned oncogenes, particularly, MYC family gene is preferred, as the MYC family gene, for example, c-MYC, N-MYC and L-MYC. In particular, c-MYC is preferred. Further, Polycomb genes forcibly expressed in hematopoietic progenitor cells, may be any of Polycomb genes described above, particularly, BMI1 genes are preferred.
　Oncogenes such as MYC family gene, and hematopoietic progenitor cells expressing the Polycomb gene such BMI1 gene, SCF (10 ~ 200ng / ml , for example 100ng / ml), TPO (10 ~ 200ng / ml, for example 40 ng / ml ), FL (10 ~ 200ng / ml, for example 100ng / ml), VEGF (10 ~ 200ng / ml, the culture, for example, 40 ng / ml) either or the added condition by combining two or more of these, such as performed, for example, at about 4-7 days after the gene introduction, the megakaryocyte progenitor cells prior to the multinucleated of acquiring a high proliferative capacity. Multinucleated preceding megakaryocyte progenitor cells obtained in this way, cell proliferation is at least about 30 to 50 days, preferably more than about 50 to 60 days, more preferably, it continues more than 60 days, its cell count is about 1.0 × 10 c-MYC gene and cell number during BMI1 gene transfer 4 times or more, preferably, about 1.0 × 10 5 times or more, more preferably, about 1.0 × 10 6 to amplify up to a fold or more (e.g., see FIG. 12).
[0027]
　The present invention is a megakaryocyte progenitor cells prior multinucleated produced by the process of the present invention were cultured in conditions suitable for inducing the differentiation of blood cells, mature megakaryocytes, further includes a method of producing platelets It is. Here, the conditions suitable for inducing the differentiation of blood cells, for example, TPO, IL-1α, IL -3, IL-4, IL-5, IL-6, IL-9, IL-11, EPO, GM -CSF, SCF, G-CSF, Flt3 ligand, mention may be made of any or the added condition by combining two or more of these, such as Heparin. When the differentiation inducing maturation megakaryocytes and platelets, for example, TPO (10 ~ 200ng / mL , preferably about 100ng / mL) in the presence of, or, TPO (10 ~ 200ng / mL , preferably from 100ng / mL approximately), SCF (10 ~ 200ng / mL, preferably 50ng / about mL) and Heparin (10 ~ 100U / mL, preferably in the presence of about 25 U / ml), may be cultured for about 7 to 15 days . The culture environment, in vitro may be a suitable environment for carrying out the differentiation induction of hematopoietic cells, e.g., 5% CO 2 , 36 ~ 38 ° C., preferably carrying out the culturing under conditions of 37 ° C. .
　Oncogenes and Polycomb genes before acquired multinucleated a high proliferative capacity by introducing megakaryocyte progenitor cells, to induce differentiation such as mature megakaryocytes and platelets, as described above, if desired, the oncogene and the expression of polycomb genes may suppress controlled.
[0028]
　Another embodiment of the present invention is a process for preparing red blood cells, in order to amplify the erythroid progenitor cells, oncogenes and HOXA2 gene or BCLXL gene to hematopoietic progenitor intracellular forced to express, in a process for producing red blood cells is there. More specifically, the present embodiment, the oncogene such as MYC family genes, desired in erythroid progenitor intracellularly which is a cell of the differentiation stage is forcibly expressed, the results derived HOXA2 the oncogene induced cell senescence gene or inhibited by expression of BCLXL gene, to amplify the erythroid progenitor cells is a method for producing the red blood cell is a specific cell. This embodiment, the hematopoietic progenitor cells by introducing dozens of hematopoietic transcription factors and anti-apoptotic-associated gene with MYC oncogenes result of screening, based on a finding that HOXA2 or BCLXL is growing erythroid progenitor cells .
　Oncogene forcibly expressed hematopoietic progenitor intracellularly, as described above, it is also possible to use any as long as it is a cancer gene, MYC family gene are preferred, in particular, c-MYC gene is preferred.
　As used herein, "erythroid progenitor cell" is a Glycophorin A (glycophorin A) enucleated before cell positive is erythroid lineage specific molecules.
[0029]
　Oncogenes such as MYC family gene, and HOXA2 gene or BCLXL gene hematopoietic progenitor cells expressing the, SCF (10 ~ 200ng / ml, for example 100ng / ml), TPO (10 ~ 200ng / ml, for example 40 ng / ml) , FL (10 ~ 200ng / ml, for example 100ng / ml), VEGF (10 ~ 200ng / ml, for example 40ng / ml), EPO (1 ~ 100U / ml, for example 6U / ml) or any such of these deeds cultured under conditions added in combination of two or more, e.g., at about 4-7 days after the gene introduction, the high enucleated acquired the ability to grow before the erythroid progenitor cells.
[0030]
　Through the erythroid progenitor cells obtained from MYC family gene and BCLXL gene or HOXA2 gene hematopoietic progenitor cells expressing, the conditions suitable for inducing differentiation of mature red blood cells, for example, TPO, IL-l [alpha], IL -3, two of one or of these, such as IL-4, IL-5, IL-6, IL-9, IL-11, EPO, GM-CSF, SCF, G-CSF, Flt3 ligand, Heparin mention may be made of the added conditions a combination of more. In particular, in the case of red blood cells, EPO (2 ~ 100U / mL , preferably about 10 U / mL) in the presence of, or, EPO (2 ~ 100U / mL , about preferably 10U / mL), SCF (10 ~ 200ng / mL, preferably in the presence of about 50 ng / mL), can be cultured for about 7 to 15 days. The culture environment, in vitro may be a suitable environment for carrying out the differentiation induction of blood cells, for example, 5% CO 2 , 36 ~ 38 ° C., preferably carrying out the culturing under conditions of 37 ° C..
[0031]
　Other embodiments of the present invention include blood cells of desired differentiation stage cancer gene was forcibly expressed. Here, as the oncogene, it can be used oncogene described above, for example, such as MYC family gene may be used, in particular, c-MYC gene is preferred. Further, the "hematopoietic cell differentiation stage", complete a from undifferentiated stage a blood cells that appear during the terminal differentiation stages, other than stage of cells cells and terminal differentiation of complete undifferentiated stage it is of the blood cells it. For example, a "blood cells differentiation stage" of the present embodiment, for example, and the like multinucleated preceding megakaryocyte progenitor cells. As blood cells such differentiation stage, for example, it can be used, such as cells derived from ES cells or iPS cells. In particular, the net-like structure to be prepared from ES cells or iPS cells (ES-sac or also referred to as iPS-sac) blood cells obtained from (especially cells immediately after isolation from the net-like structure) is preferable. Further, the present embodiment, other oncogenes, further blood cells differentiation stage Polycomb genes described above were forced expression are also included. Forced expression of oncogenes and Polycomb genes, as described above, can be implemented using such inducible promoters.
　Forced expression of oncogenes or polycomb gene in cells of the desired differentiation stage, an oncogene or Polycomb genes are introduced into the "blood cells of desired differentiated stage", it is accomplished by forced expression well, also introducing these genes into precursor in cells of the "blood cells of desired differentiated stage", forced to express, to proceed to differentiate while maintaining the expression, "hematopoietic desired differentiation stage it may be achieved by maintaining the forced expression status of these genes in the cells. " Further, the advance by introducing these genes into precursor cells "desired differentiation stage of blood cells", when differentiated into "blood cells of desired differentiated stage", induce forced expression of these genes it may be achieved by. For example, when amplifying a multinucleated previous megakaryocyte progenitor cells as blood cells in the desired differentiation stage, introducing oncogenes or Polycomb gene to hematopoietic progenitor cells in the precursor stage, it may be forced to express .
[0032]
　In this embodiment, for example, blood cells such as multinucleated preceding megakaryocyte progenitor cells, even when thawed after cryopreservation, has a freeze-thaw resistance that maintains cell proliferation and differentiation potential. Therefore, by freezing the blood cells, and dissolved as required, it is possible to produce blood cells induced to differentiate. Thus, by using the present cell, blood cells from ES cells or iPS cells, is necessary to perform the step of beginning a series of operations to produce, for example, platelets eliminated. In other words, the cancer gene of the present invention, or oncogenes and Polycomb genes hematopoietic cells forced to express as a raw material, a large amount of prepared, by previously cryopreserved if necessary, rationalization and efficiency of the manufacturing process is attained, it can be constructed quickly suppliable mechanisms of various blood cells platelets, and the like.
　With blood cells such as multinucleated preceding megakaryocyte progenitor cell of the invention, in the case of manufacturing a frozen cell composition comprises hematopoietic cells such as multinucleated preceding megakaryocyte progenitor cells, and cryopreservation fluid can be composed of, also including additives according Others required may be included in the composition.
The cryopreservation solution, can be utilized, such as frozen solution of DMSO containing. Specifically, cell banker (Nihonzen'yakukogyo Co., Ltd.) and Bambang car (Nippon Genetics Co., Ltd.), TC protector (DS Pharma Biomedical Co., Ltd.), albumin pressure cp-1 (Far East Pharmaceutical Industry Co., Ltd.), etc. is there.
[0033]
　MYC family genes used in the present invention, Polycomb genes (e.g., BMI1 gene), HOXA2 gene and BCLXL gene is already a gene whose cDNA sequence is published, of course, based on homology of these known cDNA sequence homologs identified by the prior art Te are included.
　Of MYC family gene, the homologue of c-MYC gene, the cDNA sequence, for example, is that of the gene of a nucleic acid sequence substantially identical to the sequence shown in SEQ ID NO: 1. The cDNA consisting of the nucleic acid sequence substantially identical to the sequence shown in SEQ ID NO: 1, and DNA consisting of the sequence shown in SEQ ID NO: 1, to about 60%, preferably about 70% or more, more preferably about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, and most preferably DNA consisting of sequences having about 99% identity or hybridize with DNA under stringent conditions having a sequence complementary to the nucleic acid sequence represented by SEQ ID NO: 1 can be a DNA, proteins encoded by these DNA is like multinucleated preceding megakaryocyte progenitor cells are those that contribute to the amplification of cellular differentiation stages.
　Also, a homologue of BMI1 gene used in the present invention, the cDNA sequence, for example, is that of the gene of a nucleic acid sequence substantially identical to the sequence shown in SEQ ID NO: 2. The cDNA consisting of the nucleic acid sequence substantially identical to the sequence shown in SEQ ID NO: 2, and DNA consisting of the sequence shown in SEQ ID NO: 2, about 60%, preferably about 70% or more, more preferably about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, and most preferably DNA consisting of sequences having about 99% identity or hybridize with DNA under stringent conditions having a sequence complementary to the nucleic acid sequence represented by SEQ ID NO: 2 can be a DNA, protein encoded by the DNA is suppressed oncogene induced cell senescence occurring in cells oncogenes such as MYC family gene is expressed, facilitate amplification of the cells Is that of shall.
[0034]
　The HOXA2 gene or BCXL gene used in the present invention, respectively, the cDNA sequences, for example, is that of the gene of a nucleic acid sequence substantially identical to the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4. The cDNA consisting of the nucleic acid sequence substantially identical to the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4, respectively, and DNA consisting of the sequence shown in SEQ ID NO: 3 or SEQ ID NO: 4, about 60% or more, preferably about 70% or more, more preferably about 80%, 81% is 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, and most preferably about 99% of the DNA consisting of the sequence identity with or complementary to the nucleic acid sequence represented by SEQ ID NO: 3 or SEQ ID NO: 4 a DNA capable of hybridizing with the DNA under stringent conditions consisting Do sequences, protein encoded by the DNA is that of having an effect of proliferating erythroid progenitor cells.
[0035]
　Here, the stringent conditions, with the hybridization being readily determined by those skilled in the art conditions, generally probe length, washing temperature, and is an empirical experimental conditions depend on the salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally complementary strand is dependent on the re-annealing capacity in somewhat an environment below their melting point.
　Specifically, for example, as low stringency conditions, in the washing step of the filter after hybridization, a temperature of 37 ℃ ~ 42 ℃, 0.1 × SSC, and washed with 0.1% SDS solution It is raised like that. Further, as a high-stringent conditions, for example, in the washing step, 65 ° C., and the like to be washed in 5 × SSC and 0.1% SDS. By higher stringent conditions, it is possible to obtain a highly homologous polynucleotide.
[0036]
　An embodiment of the present invention, further, desired differentiation stage of cells (e.g., multinucleated preceding megakaryocyte progenitor cells or erythroid progenitors), and finally the specific cells to be produced (e.g., megakaryocytes, platelets or red blood cells), it includes a kit for producing. The said kit oncogene, Polycomb gene, BclxL gene, other such expression vectors, and the like, and reagents necessary to express the HOXA2 gene like in cells, the culture medium for cell culture, serum, supplements such as growth factors (e.g., TPO, EPO, SCF, Heparin, such as IL-6, IL-11), and the like antibiotics. Other, for example, when using the ES cells or iPS cell-derived cells, antibodies for markers confirmation for identifying a net-like structure was prepared from these cells (e.g., Flk1, CD31, CD34, UEA-I lectin It is also included for the antibody) and so on. Reagents included in the kit, such as antibodies, effectively and long lasting constituents activity is not adsorbed by the material of the container, is fed into a vessel of any type that does not undergo alteration.
[0037]
　Furthermore, platelets and red blood cells produced by the present invention, it is possible to stably supplied in the form of a formulation. Platelets produced by the methods of the invention can be released from megakaryocytes fractions were collected culture platelets abundant leukocyte removing filter (e.g., Terumo, available from such Asahi Kasei Medical Co., Ltd.) use like, megakaryocytes, etc., to remove blood cells components other than platelets, it can be prepared. In preparing the blood product can be allowed to contain other components that contribute to the stabilization of platelets or red blood cells. Components that contribute to such stabilization, it is possible to select the methods well known in those skilled in the art.
[0038]
　More specifically, the obtained platelets, for example, be formulated by the following method.
　ACD-A solution: FFP; adjusted by the ratio of (fresh frozen plasma those prepared from whole blood obtained by blood donation, albumin, clotting factors, etc., including everything except blood components) to 1:10, 15-50Gy shaking to save while at 20-24 ℃ after irradiation. ACD-A solution, the entire sodium citrate 22g / citric acid 8 g / Glucose 22g water for injection adjusted to a 1L.
　When using the above method, the concentration of platelets, for example, 1 × 10 9 about platelets / mL is preferred.
　Further, GM6001 (a broad-range hydroxamic acid-based metalloprotease inhibitor) (Calbiochem Corp., La Jolla, CA, USA) the previously added and platelets occurs during frozen storage and room temperature storage capabilities molecule GPIb-V-IX Ya It can prevent inactivation due to cleavage of GPVI. The present inventors have confirmed that this method can prevent inactivation relates mouse ES cell-derived platelets. For reference papers mechanisms for this platelet inactivation using human platelets, Bergmeier, W et al. , Cir Res 95: 677-683,2004 and Gardiner, EE et al. , J Thrombosis and Haemostasis, 5: 1530-1537,2007 see.
　Incidentally, the container for containing a formulation comprising platelets, preferably avoided those materials that activate the platelets as glass.
[0039]
　On the other hand, the formulation of red blood cells, can be performed as follows.
More specifically, the obtained erythrocytes, for example, be formulated by the following method.
MAP solution culture supernatant concentrated erythrocyte solution was concentrated after centrifugation (composition see below) was prepared by adding and stored at 2 ~ 6 ° C. After irradiation 15-50Gy.
　When using the above method, the concentration of red blood cells, for example, 1 × 10 10 approximately erythrocytes / mL is preferred. Obtained erythrocytes as erythrocyte preserving additive solution (MAP solution), for example, D- mannitol (14.57 g), adenine (0.14 g), crystalline sodium dihydrogen phosphate (0.94 g), sodium citrate ( 1.50 g), citric acid (0.20 g), glucose (7.21 g), a sodium chloride (4.97 g), dissolved in water for injection, the total amount can be used after a 1000 ml.
　Other, be appropriately selected from known manner deemed suitable for the formulation of red blood cells is that it is easy to those skilled in the art.
[0040]
　Further the invention includes a frozen composition blood cells of the present invention. In the present compositions, other blood cells, the culture medium required to store the blood lineage cells, DMSO to protect the cells during the buffer and frozen, may be included glycerol, and the like. Other, may include any as long as it is a normal substance required for freezing the cells. Alternatively, when using a commercially available cell freezing reagent may contain a substance contained in the reagent.
[0041]
　From the "cells", as described herein, human and non-human animals (e.g., mice, rats, cows, horses, pigs, sheep, monkey, dog, cat, bird, etc.), and no particular limitation is But. Particularly preferred are cells derived from human.
[0042]
　It will be described in more detail with the following examples, but the present invention is not intended to be limited by the examples.
Example
[0043]
1.4 Gene Comparison of megakaryocyte production efficiency from derived iPS cells and 3 gene derived from iPS cells
　4 genes (OCT3 / 4, SOX2, KLF -4, c-MYC) iPS cells established by (TkDA3-2, TkDA3-4 and TkDA3-5) and 3 genes except c-MYC (OCT3 / 4, SOX2, KLF-4) iPS cells established by (253G1 (Kyoto, donated by Shinya Yamanaka Dr.) and TkDN4-M), and human ES cells; comparing the number of megakaryocyte cells produced from (KhES-3 Kyoto University, from donor Nakatsuji Norio Dr.) (Figure 1). 15 days after the culture from iPS cells and ES cells, hematopoietic progenitor cells from the net-like structures, plated on feeder cells at a final concentration of 15% in IMDM supplemented with FBS TPO (100ng / mL) , the culture is performed in the presence of SCF (50ng / mL) and Heparin (25U / ml). Then over time counting the number of megakaryocyte a CD42b-positive induced (Figure 1). As a result, compared to the 3 gene (without c-MYC) derived iPS cells and human ES cells, 4 genes (c-MYC present) derived iPS cells, the number of megakaryocytes was increased with three strains used .
[0044]
　Then, the gene was introduced when producing iPS cells were examined (OCT3 / 4, SOX2, KLF -4, c-MYC) expression activity in undifferentiated iPS cells, any gene is also expressed by silencing mechanism There was suppressed (Fig. 2A). In contrast, in the culture on day 25 of megakaryocyte cells were differentiation-inducing, re-activation of expression of each transgene was confirmed (Fig. 2B).
　From the above, among the genes introduced to produce iPS cells, reactivation of one of gene expression may be involved in the increase in the number of megakaryocyte cells produced was suggested. Therefore, we verify the cause genes that are involved in the increase in the number of megakaryocytes cell. Human ES cells (unlike iPS cells OCT3 / 4, SOX2, KLF- 4, c-MYC is not introduced exogenously) from hematopoietic progenitor cells alone forced to express each gene by retrovirus, production and counting the number of megakaryocytes cells CD42b-positive, which are. As a result, in the case of introducing c-MYC, as compared with the case of introducing other genes, megakaryocyte number of CD42b-positive produced revealed that increased by about 10-fold (Fig. 3) . From the above, as megakaryocyte induction efficiency is high for reasons of from 4 genes derived iPS cells, reactivation of the expression of c-MYC gene is considered.
　Further, megakaryocyte cells derived from four genes derived iPS cells than megakaryocyte cells derived from ES cells or 3 gene derived from iPS cells, it was confirmed that the higher freeze-thaw after survival. Specifically, human ES cells (KhES-3) or 3 gene derived from human iPS cells (TkDN4-M) derived from freeze-thaw after viability megakaryocyte cells, respectively 56.7% 54.5% When about while remained approximately 50%, freezing and thawing after viability megakaryocyte cells derived from four genes from human iPS cells (TkDA3-4) is found to reach 81.0% and 80% It was. From this, megakaryocyte progenitor cells reactivation occurs oncogenes such as c-MYC gene is more suitable for cryopreservation, considered likely cells supply after thawing.
[0045]
　For even production number of platelets was studied similarly to the megakaryocyte cells. 15 days after the culture from iPS cells and ES cells were seeded hematopoietic progenitor cells from the net-like structure, then the number of platelets induced was over time count, as in the megakaryocyte cell from iPS cells by 4 transgenic production of efficient platelets has been performed (Fig. 4).
　Then, most platelet production ability with high TkDA3-4 strain were transfusion experiments platelets produced in vitro. Previously irradiated to prepare a immunodeficient mice thrombocytopenia model was transfused from the tail vein of platelets from iPS cells (Fig. 5A). 20% at 30 minutes post-transfusion before and after about 10% of platelets chimerism even after 2 hours was observed, was similar to fresh platelets from human peripheral blood (Figure 5B).
[0046]
　Further, to evaluate the thrombus formation ability in vivo of human iPS cell-derived platelets with time-lapse confocal microscopy (Time-lapse confocal microscopy).
　Advance, iPS cell-derived platelets tetramethylrhodamine ethyl ester; and stained with (TMRE red pigment) was injected from the mouse tail vein mixed with hematoporphyrin (hematoporphyrin). By staining the blood flow (other than cellular components) in FITC-dextran (green), visible missing blood components in the blood vessel can be confirmed with cells from the form and size. Laser by reacting the hematoporphyrin and vascular endothelial damage is caused, platelets immobilize and adhere to failure endothelium or endothelial peeling spot, thrombus formation is induced.
　Doing wavelength 488nm to mesenteric small arteries in mice, the laser irradiation of 30 mW, iPS cell-derived platelets stained red after 13 seconds have adhered to the failure endothelium (in FIG. 6, shown as "iPS-derived" with an arrow site). After 20 seconds the thrombus formed in cooperation with platelets (murine platelets) from other hosts, confirmed that caused vascular occlusion capability platelets from iPS cells to form a blood clot in bloody under in vivo it has been demonstrated that there is.
　From the above, is established by the introduction of 4 genes including c-MYC gene, platelets prepared from iPS cells c-MYC gene is reactivated, the same physiological characteristics and platelets from human peripheral blood it was confirmed that holding the.
[0047]
　From the analysis so far, in order to induce megakaryocytes and platelets efficiently from iPS cells, important to maintain the effect in intracellular c-MYC gene expression induction with its c-MYC gene product it became clear it. Therefore, if the inducing megakaryocytes and platelets from iPS cells, to express c-MYC gene in mononuclear megakaryocyte progenitor cells in an undifferentiated megakaryocyte progenitor cells, and the effect of c-MYC gene product to maintain the, inhibiting the oncogene induced senescence (OIS) are expected to be effective. Therefore, in order to suppress the OIS, it expressed simultaneously with the c-MYC gene Polycomb group gene, was studied for its effect.
[0048]
2. c-MYC gene and BMI1 production efficiency of mature megakaryocytes cellular gene from megakaryocyte progenitor cells expressing
　the comparison of the 4 genes established lines and 3 genes established lines of iPS cells megakaryocyte production efficiency, megakaryocyte progenitor cells in, reactivation of c-MYC gene, was found to affect the number of mature megakaryocytes cells are then induced. Therefore, whether the expression of c-MYC gene in ES cell-derived megakaryocytes progenitors are pluripotent stem cells that c-MYC gene is not introduced, to determine its impact on the induction of subsequent megakaryocyte investigated.
　The net-like structure was prepared in the presence of 20 ng / ml VEGF from the human ES cell line (KhES-3) megakaryocyte progenitor cells from the net-like structure (multinucleated ago), the cells on the 10T1 / 2 cells number 1 × 10 5 seeded so that / well, a retroviral vector that holds the c-MYC gene (SEQ ID NO: 1), after seeding, 0 hours, 12 hours, were infected at 24 hours. After 36 hours, change to the medium that does not contain a retrovirus, and the culture was continued. Gene transfer using a retrovirus, using the 6-well plates the culture medium added 2 ~ 3 ml, under conditions of 900 rpm, 90 min, was performed using the spin infection method (Spin infection). Final concentration of 15% FBS in IMDM supplemented with 100 ng / ml SCF, was performed 40ng / ml TPO, 100ng / ml FL, a cultured with medium supplemented with 40 ng / ml VEGF and protamine (Fig. 7).
[0049]
　Was subjected to FACS analysis on day 9 after infection of retroviruses in the cells transfected with c-MYC compared to the control vector, CD41a, it was observed that cells with CD42b is increasing predominantly (Figure 8A ). Further, observation of the cells in cytospin cells that in the control are multinucleated are observed, c-MYC in transduced cells, mononuclear before multinucleated cells was observed (Figure 8B). These results, c-MYC immature megakaryocyte cell mononuclear suggesting that increased by forced expression of. This result, megakaryocytes specifically c-MYC with similar results and transgenic mice was expressed (Alexander et al., Deregulated expression of c-MYC in megakaryocytes of transgenic mice increases megakaryopoiesis and decreases polyploidization. J .Biol.Chem, 1996 Sep 20; 271 ( 38):. 22976-82 see).
[0050]
　Next, observation of the capacity of the cells in a state that expressed c-MYC, it has been observed that proliferation is reduced from 14 days post infection (Figure 9). This phenomenon, c-MYC to aberrant growth signals by overexpression of Oncogene such, cell cycle arrest, cellular senescence, a canceration avoidance mechanism of cells undergoing apoptosis, oncogene-induced senescence (Oncogene- induced senescence: OIS) are called (as described above). Therefore, one of the Polycomb group gene that controls the Ink4a / Arf gene encoding the p16 and p19 is a tumor suppressor gene product negatively, BMI1Was introduced into megakaryocyte progenitors in cells, it was attempted to avoid the OIS. Transgenically method by the aforementioned retrovirus, after c-MYC gene and BMI1 gene (SEQ ID NO: 2) was introduced into and expressed in the cell, was subjected to FACS analysis. As a result, with the lapse of time after the gene introduction, it was possible to obtain a CD41a (a marker of megakaryocytic) positive CD42b-positive cell population growing exponentially stable (Figure 10). When introduced c-MYC gene only in the cells, the 20 days after gene transfer, whereas CD41a-positive CD42b-positive cells is significantly decreased (FIG. 10 under the analysis result), and c-MYC gene BMI1 in case of introducing genes of CD41a positive CD42b positive cells increases with each passing day was confirmed (the analysis results on Figure 10). Differentiated from this result, which is one of the pre-c-MYC gene transfer multinucleated introduced with BMI1 gene megakaryocyte progenitor cells Polycomb gene avoids OIS, into megakaryocyte progenitors while preserving a high proliferative potential it has become clear that the. In order to confirm the characteristics of the megakaryocyte cells obtained, yet whether other megakaryocyte-specific functional molecule CD9 and CD42a are present on the cell surface (see Figure 11A), by FACS analysis investigated. As a result, in cell lines introduced with c-MYC gene and BMI1 gene, it was possible to confirm the presence of CD9 and CD42a (Fig. 11B).
[0051]
　Next, we examined for growth ability of c-MYC / BMI1 expressing cells. The c-MYC gene and BMI1 gene was introduced were multinucleated preceding megakaryocyte progenitor cells, in IMDM supplemented with a final concentration of 15% FBS, 100ng / ml SCF, 40ng / ml TPO, 100ng / ml FL, 40ng / ml were cultured in medium supplemented with VEGF, it was over time by counting the number of cells. As a result, in 49 days after gene transfer, approximately 4 × 10 7 cells of the CD41a-positive cells were obtained (Figure 12). Furthermore, platelets released from megakaryocyte progenitor cells derived from c-MYC / BMI1 expressing cells was observed by an electron microscope, characteristic of platelets, microtubule structure, open canalicular system (Open canalicular system), the platelet granules It was able to be confirmed (Figure 13).
[0052]
3. induction of red blood cells through the erythroid progenitor cells from c-MYC gene transfer hematopoietic progenitor cells
　were then tried the production of red blood cells from erythroid progenitor cells obtained from hematopoietic progenitor cells transduced with c-MYC gene. As with the introduction of c-MYC gene or BMI1 genes described in the above 2, to produce a c-MYC / HOXA2 (SEQ ID NO: 3) expressing cells and c-MYC / BCLXL (SEQ ID NO: 4) expressing cells, the FACS analysis went. As a result, the c-MYC / HOXA2 expressing cells, at 105 days after the gene introduction, the presence of CD71 and GlyA positive cell groups, a marker of red blood cells was observed (FIG. 14 upper right). The presence of GlyA-positive cell population was confirmed in c-MYC / BCLXL expressing cells (Fig. 14 right bottom). This result, hematopoietic progenitor cells transduced in c-MYC gene, by changing the introduction factors combined, it has been found that differentiation into erythrocytes are possible.
[0053]
4. Functional platelet preparation using the expression induction system gene
　megakaryocyte cells, platelets efficiently, for mass prepared is to be effective to increase the number of megakaryocyte progenitor cells, revealed It was. For that purpose, c-MYC family gene, a Polycomb genes co-expressed simultaneously prior multinucleated megakaryocyte progenitor cells, it is necessary to increase the proliferative capacity of the multi nucleation preceding megakaryocyte progenitor cells, the maturation of megakaryocyte cells in order to promote the (multinucleation), in some cases, c-MYC family gene, it is desirable to suppress control the expression of the polycomb gene.
[0054]
　Therefore, by utilizing the pMX tet off system, to adjust inductively expression of c-MYC gene and BMI1 gene to produce platelets was investigated physiological functions of platelets.
4-1. Functional confirmation of gene regulation unit vector
　pMX tet off vector (Jichi Hiroyuki Mano from donor Professor) c-MYC -2A- BMI1 to produce an all-in-one incorporating (all in one) type vector ( "2A] is , a peptide having the self cleavage activity from foot-and-mouth disease virus, by sandwiching the arrangement between a plurality of proteins, is used to efficiently acquire a plurality of proteins from a single promoter ( Hasegawa et al., 2007 Stem Cells)). pMx tet off c-MYC 2A BMI1 vector estradiol presence of, to express c-MYC gene and BMI1 gene. On the other hand, tetracycline presence of, the estradiol absence of, c- suppress the expression of a MYC gene and BMI1 gene.
　pMx tet off to prepare c-MYC 2A BMI1 the vector, it was expressed in 293GPG cells And the status of the regulation of expression of c-MYC gene and BMI1 gene was confirmed by FACS. Figure 15 is a c-MYC protein in the cell, after staining with anti-c-MYC protein antibody, staining with Alexa647 labeled secondary antibody .pMX tet off, and that is the result of FACS analysisc-MYC 2A BMI1 in 293GPG cells incorporating, in the tetracycline presence of, c-MYC is a expression level of the gene does not change the 293GPG control cells (in FIG. 15, the graph indicated by 293gpg and + tetracycline), estradiol present it can be seen that the expression of c-MYC gene is promoted by the formula (Figure 15, + beta-estradiol).
　These results, pMx tet off used herein c-MYC 2A BMI1 was confirmed that it is possible to control the expression of target genes by a vector.
[0055]
4-2. Preparation of megakaryocyte cell lines by gene control vector
　with the gene control vectors described above 4-1, express the c-MYC gene and BMI1 gene in megakaryocyte progenitor cells derived from human ES cell line (KhES-3) then, they were examined for their ability to grow and differentiation potential.
　Only was introduced cell vector (FIG. 16A (a)), pMX c-MYC and DSAM BMI1 were separately forced to express cell lines (FIG. 16A (b)), pMX tet off c-MYC and pMX tet off BMI1 in separately expressed allowed cell lines (FIG. 16A (c)) to, pMX tet off c-MYC 2A BMI1 cells was expressed in strain (Fig. 16A (d)) and pMX tet off BMI1 2A c-MYC (FIG 16A (e was examined cell lines expressed in)). Here, (d) (e) is, across the 2A sequence order of arrangement of the c-MYC gene and BMI1 genes are different constructs.
　These cell lines, CD41a + shown in FIG. 16A growth curves of the cells. Stained each cell line with anti-CD41a antibody and anti-CD42b antibody is megakaryocyte markers were analyzed using a flow cytometer. Tet Off pMX C-MYC 2A BMI1In creating cell lines (FIG. 16A (d)) is, pMX c-MYC and DSAM BMI1 showed similar expression system and were separately forced to express cell lines (FIG. 16A (b)), most of the population megakaryocytes It was expressed ball marker (panel on Figure 16B). Furthermore, pMX tet off c-MYC 2A BMI1 cells created in strain (Fig. 16A (d)) is, pMX tet off c-MYC and pMX tet off BMI1 separately introduced cell lines (FIG. 16A (c)) and tet off pMX BMI1 2A c-MYC cells were created in strain showed higher proliferative capacity than (FIG. 16A (e)).
　Moreover, when stained with anti-Glycophorin-a antibodies and anti-CD41a antibodies, pMX c-MYC and DSAM BMI1 in cell lines that separate forced expression is a common marker megakaryocyte / erythroid CD41a + / Gly-a + against the cell population is present (FIG. 16B, left lower panel), pMX tet off c-MYC 2A BMI1In a cell line that was created was lost Gly-a (FIG. 16B, lower panel on the right). This result, pMX tet off c-MYC 2A BMI1 cell lines created in, pMX c-MYC and DSAM BMI1 than cell lines separately forced to express, cell lines advanced and more differentiation into megakaryocyte system it is shown that it is.
[0056]
4-3. The multinucleated megakaryocytes
　presence of β- estradiol, pMX tet off c-MYC 2A BMI1 examined the extent of multinucleated of vector c-MYC gene and BMI1 gene cell lines forced to express. Human megakaryocytes, usually are multinucleated about 32N (FIG. 17A), pMX tet off c-MYC 2A BMI1 The vector c-MYC gene and BMI1 gene was forcibly expressing cell lines, most multinucleated is not promoted, it was shown to be 2N-4N.
[0057]
4-4. c-MYC gene and BMI1 gene function analysis of megakaryocytic cell line-derived platelets expressing
　was functional assay platelet derived c-MYC gene and megakaryocyte cell lines expressing BMI1 gene.
　Human peripheral blood-derived platelets control, ADP; combine with fibrinogen in the presence of (adenosine diphosphate platelet intracellular factor activating) the initial integrin activation functions required to thrombus formation (inside-out signaling) is shown to be normal (FIG. 18 upper right). On the other hand, pMX tet off c-MYC 2A BMI1 strain (estradiol presence) and pMX c-MYC and DSAM BMI1 both forced expression strain did not bind to fibrinogen also be added ADP (Fig. 18 middle and lower). Therefore, if there remains c-MYC gene and BMI1 gene was forcibly expressed, it was found not to emit platelets with normal function.
　Next, pMX tet off c-MYC 2A BMI1 to c-MYC gene and BMI1 cell lines gene is forced expressed in vector, after releasing the forced expression under the conditions of + tetracycline and -β- estradiol, culture of the fourth day CD41a Tasu / CD42b TasuIntegrin activation ability of platelets were analyzed using a flow cytometer (Fig. 19). As a result, PAC1 antibody (activated integrin αIIbβ3 binding antibody) binds with ADP presence, integrin activation ability (inside-out signaling) was shown to be normal (FIG. 19B).
　From the above results, the platelets produced from megakaryocytes line grown by forced expression of c-MYC gene is with disabilities to function, to release the forced expression of such c-MYC gene megakaryocytic lines it is has been shown to be possible the production of platelets having normal functions.
[0058]
　Expression control of c-MYC gene and BMI1 gene in the megakaryocyte progenitor intracellularly, MYC family gene was used in the same manner as erythroid progenitor cell line established, BclxL gene, can also be used for HOXA2 genes, induction of mature red blood cells It is considered to be made possible.
[0059]
　MYC and BMI1 has been shown that by growing the cells in a common precursor cell is a MEP fraction, or megakaryocyte progenitor cells advanced differentiation than that stage of megakaryocyte-erythroid cells (Figure 20) . Furthermore, c-MYC gene and BMI1 gene was introduced were multinucleated preceding megakaryocyte progenitor cells, since it is possible to cryopreservation, can be prepared from frozen stocks, megakaryocytes, platelets when necessary.
　Similarly, cryopreserved erythroid progenitors strain prepared in MYC gene BCLXL or HOXA2 transgenic be prepared thawed when needed.
　Also, it introduced MYC gene, BMI1 the expression of genes to control the upward or downward, it is possible to a sufficient amount prepared platelets or red blood cells retaining the physiological activity.
Industrial Applicability
[0060]
　The present invention, cell differentiation stages amplifies, provides a method for producing a specific cell which is further differentiated. The method of the present invention for example, by applying the blood cells, it is possible to mass supply of cells of the desired differentiation stage. Accordingly, the present invention is particularly intended to contribute greatly to the development of treatments in the medical field.

claims

[Claim 1]A method for producing a specific cell to induce differentiation of cells,
　in order to expand the cell of the desired differentiation stage, forced expression of oncogenes in the cells of the desired differentiation stage method for producing a specific cell .
[Claim 2]
　Induced by forced expression of oncogenes in cells of the desired differentiation stage method for producing a specific cell of claim 1, characterized in that to suppress the oncogene induced senescence.
[Claim 3]
　The suppression of oncogene-induced senescence is achieved by expressing the Polycomb gene, a manufacturing method of a particular cell according to claim 1 or 2.
[Claim 4]
　The cells of the desired differentiation stage method for producing a specific cell according to any of claims 1 to 3, characterized in that the ES cells or iPS cells, which are differentiation-induced cells.
[Claim 5]
　Into said cells of the desired differentiation stage, cancer gene foreign or introducing oncogenes and Polycomb genes, cancer gene or claim 1, characterized in that forced expression of cancer genes and the Polycomb gene to a manufacturing method of a particular cell of any one of the four.
[Claim 6]
　Oncogenes or Polycomb gene of the foreign, and introduced into a precursor cell of a cell of the desired differentiation stage, cancer gene or, according to claim 5, characterized in that forced expression of cancer genes and the Polycomb gene method for producing a specific cell.
[Claim 7]
　Claims the oncogenes and / or Polycomb genes, respectively, operably linked to downstream of the inducible promoter, cancer gene or, characterized in that inducibly forcibly expressed cancer gene and the Polycomb gene method for producing a specific cell according to claim 5 or 6.
[8.]
In order to promote differentiation of the desired differentiation stage of cells, cancer gene in a cell of the desired differentiation stage, or any of claims 5 to 7, characterized in that inhibiting the expression of oncogenes and Polycomb gene method for producing a specific cell according to.
[Claim 9]
　The oncogene or suppression of the expression of oncogenes and polycomb gene, respectively, operably linked to inhibiting promoter downstream, according to claim 8, characterized in that it is achieved by suppressing the expression of the gene the method for producing specific cells.
[Claim 10]
　Method for producing a specific cell according to any of claims 1 to 9, wherein the oncogene is a MYC family gene.
[Claim 11]
　Method for producing a specific cell according to any one of claims 3 to 10 wherein the Polycomb gene characterized in that it is a BMI1.
[Claim 12]
　Wherein a desired progenitor hematopoietic progenitor cells differentiation stage of the cell, the a desired cell differentiation stage before multinucleated megakaryocyte progenitor cells, and wherein the specific cell is a mature megakaryocyte cell method for producing a specific cell according to any of claims 6 to 11.
[Claim 13]
　Wherein a desired progenitor hematopoietic progenitor cells differentiation stage of the cell, the a desired cell differentiation stage before multinucleated megakaryocyte progenitor cells, claim, wherein the specific cell is a platelet method for producing a specific cell according to 6 or 11 either.
[Claim 14]
　Method for producing a specific cell of claim 12 or 13 wherein the hematopoietic progenitor cells, and characterized in that present in the ES cells or iPS cells net-like structure prepared from.
[Claim 15]
　Mature megakaryocyte cell is a specific cell of claim 12 or 14.
[Claim 16]
　Platelets is a specific cell of claim 13 or 14.
[Claim 17]
　Blood product comprising, as an active ingredient the platelets according to claim 16.
[Claim 18]
　Mature megakaryocyte cells or kit for preparing platelets according to claim 15 or 16.
[Claim 19]
　Desired differentiation stage of hematopoietic cells oncogene is forcibly expressed.
[Claim 20]
　Further Polycomb gene was forcibly expressed, hematopoietic cells of claim 19.
[Claim 21]
　The desired blood cells differentiation stage, blood cells according to claim 19 or 20, characterized in that a cells differentiation-induced from ES cells or iPS cells.
[Claim 22]
　The desired differentiation stage of hematopoietic intracellular oncogene foreign, or by introducing an oncogene and Polycomb genes, oncogenes or 19 through claim, characterized in that forced expression of oncogenes and Polycomb gene blood cells according to any one of 21.
[Claim 23]
　Oncogenes or Polycomb gene of the foreign, and introduced into a precursor cell of hematopoietic cells of the desired differentiation stage, cancer genes or to claim 22, characterized in that forced expression of cancer genes and the Polycomb gene blood cells described.
[Claim 24]
　Claims the oncogenes and / or Polycomb genes, respectively, operably linked to downstream of the inducible promoter, cancer gene or, characterized in that inducibly forcibly expressed cancer gene and the Polycomb gene blood cells according to claim 22 or 23.
[Claim 25]
　Blood cells according to any one of claims 19 to 24, wherein the oncogene is a MYC family gene.
[Claim 26]
　Blood cells according to any one of claims 20 to 25 wherein the Polycomb gene characterized in that it is a BMI1.
[Claim 27]
　Wherein a desired progenitor hematopoietic progenitor cells differentiation stage of blood cells, of claims 23 to 26, wherein the desired differentiation stage of blood cells are megakaryocytes progenitors prior multinucleated blood cells according to any one.
[Claim 28]
　The hematopoietic progenitor cells, blood cells according to claim 27, characterized in that present in the net-like structure was prepared from ES cells or iPS cells.
[Claim 29]
　Frozen cell composition comprising hematopoietic cells according to any one of claims 19 to 28.
[Claim 30]
　Kits for producing a megakaryocyte progenitor cells prior multinucleated a hematopoietic cell of claim 27 or 28.