Patent Description:
Blood cells are required for the treatment of blood-related diseases and for surgical operations. Among blood cells, platelets, which are cells required for blood coagulation (hemostasis), proplatelets, and megakaryocyte cells, which are cells that produce platelets, are cells where the need is particularly high. Leukemia treatments, bone marrow transplantation, and anticancer therapies create great demand for platelets in particular, and the requirement for a stable platelet supply is thus acute.

To date, methods in which various types of stem cells are differentiated to obtain megakaryocyte cells and these are cultured to release platelets, have been developed as in vitro platelet production methods. Due to the creation of iPS cells, greater attention has been directed in recent years to the usefulness of pluripotent cells as an important source of cell therapies in regenerative medicine. To date, for example, Takayama et al. have succeeded in inducing the differentiation of human ES cells into megakaryocyte cells and platelets (Non-Patent Document <NUM>).

A method for producing mature megakaryocytes comprising the step of culturing immature megakaryocytes in a culture medium containing an ABC transporter inhibitor is disclosed in Patent Document <NUM>.

Thrombopoietin (TPO) is conventionally known to be a platelet growth factor. Prior to the present invention, TPO receptors in the form of c-MPL-dependent drugs were widely studied as candidate substances of platelet growth promoters. However, platelet count may decrease following the appearance of neutralizing antibodies in the bodies of patients administered the c-MPL ligand, human megakaryocyte cell growth and development factor (PEG-r HuMGDF). More recently, although TPO acts to promote increases in megakaryocyte cells and megakaryocyte cell maturation, it has been reported to have no direct effect on platelet release (Non-Patent Documents <NUM> and <NUM>).

Moreover, although the majority of research on megakaryocyte cells has been conducted on mice, megakaryocyte cells may demonstrate different behavior in humans and mice as in congenital amegakaryocytic thrombocytopenia (CAMT) (Non-Patent Document <NUM>). Even if systems using human cells were able to be used, there were problems in terms of stability, safety and ethics depending on the type of cells used.

Known examples of megakaryocyte cell maturation factors include GATA1, FOG1, NFE2 and TUBB1. The inventors of the present invention found that, among these marker genes, TUBB1 is superior as an indicator when searching for platelet production promoters, and produced a reporter cell line derived from human iPS cells that emits fluorescence corresponding to the expression level of TUBB1, thereby leading to completion of the present invention.

Namely, the invention of the present application includes the inventions indicated below.

According to the screening method of the present invention, drugs or genes (siRNA) having an effect on megakaryocyte cell maturation or platelet production that have conventionally been difficult to evaluate can be evaluated both comprehensively and easily. In addition, the present invention is far superior to the prior art in that it enables platelet production promoters to be efficiently screened while relying only on TUBB1 , as mentioned above, the inventors of the present invention found that, among the marker genes GATA1, FOG1, NFE2 and TUBB1, TUBB1 is superior as an indicator when searching for platelet production promoters.

The present invention provides an in vitro method for screening for platelet production promoters, the method comprising: a step of contacting megakaryocytes or progenitor cells thereof with a candidate substance; a step of culturing the megakaryocytes or progenitor cells thereof in the presence of the candidate substance; a step of measuring expression of TUBB1 in the cultured mature megakaryocyte cells following said step of culturing the megakaryocytes or progenitor cells thereof in the presence of the candidate substance, and; a step of selecting as a platelet production promoter a candidate substance that significantly increases the expression of TUBB1 in the mature megakaryocyte cells as compared to the expression of GATA1, FOG1 and/or NFE2; wherein the platelet production promoter is a drug that promotes production of platelets and increases the amount of platelets produced per unit time.

Candidate substances of such drugs can be selected from known compound libraries accessible by a person with ordinary skill in the art. In addition, the candidate substances are not limited to a compound, salt thereof or derivative thereof, but rather may be an antibody or antibody fragment thereof, an antisense oligonucleotide, ribozyme or molecule that causes RNA interference (to be referred to as "RNAi") provided it brings about an increase in the expression of TUBB1 in megakaryocyte cells or progenitor cells thereof.

Microtubules are composed of heterodimers of α-tubulin and β-tubulin. β-tubulin has seven isotypes in humans. TUBB1 is a gene that encodes β1-tubulin, which is a type I isotype that is a member of the family of β-tubulin proteins. Since TUBB1 is a gene that is specifically expressed in megakaryocyte cells and platelets in the same manner as GATA1, FOG1 and NFE2, it is known to be a megakaryocyte cell maturation factor. In the present invention, a candidate substance that significantly increases expression of TUBB1 is evaluated as a platelet production promoter. Although it is possible to combine the use of other megakaryocyte cell maturation factors as indicators, the use of TUBB1 alone as an indicator for screening for platelet production promoters makes it possible to screen for platelet production promoters efficiently.

In the case of using in the present description, "increased expression" or "promotion of platelet production" refers to a state in which production has been increased by <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more, <NUM>% or more or <NUM>% or more in comparison with a control that can be suitably evaluated by a person with ordinary skill in the art. Substances that prominently increase expression of TUBB1 in comparison with a positive control can be evaluated as having a high level of platelet increasing action. A known platelet production promoter such as an AhR antagonist, and preferably StemRegenin1 (SR1) (Selleckchem), can be used as a positive control.

Changes in expression of TUBB1 can be confirmed by contacting a candidate substance with megakaryocyte cells or progenitor cells thereof. The platelet production promoter can be contacted with megakaryocyte cells or progenitor cells thereof in a medium. The expression level of a reporter gene inserted at the gene locus of TUBB1 may be evaluated instead of determining the expression level of TUBB1. A gene that encodes a fluorescent protein such as VENUS can be used as a reporter gene.

Insertion of a reporter gene can be carried out by a person with ordinary skill in the art using a known technology such as a genome editing technology in the manner of the CRISPR/Cas9 system (<NPL>), TALEN (transcription activator-like effector nuclease) (<NPL>), or ZFN (zinc finger nuclease) (<NPL>).

The step for selecting a platelet production promoter can be carried out repeatedly. For example, secondary screening or additional screening may be carried out following primary screening corresponding to the number of libraries of compounds subjected to screening or the number of ultimately required candidate substances. In addition, cutoff values at that time can be set by a person with ordinary skill in the art.

The reporter cell line which can be used in the present invention consists of megakaryocyte cells or progenitor cells thereof in which a reporter gene has been inserted into the gene locus of TUBB1. The megakaryocyte cells are preferably human-derived immortalized megakaryocyte cells.

In this Description, "megakaryocyte cells" are characteristically the largest cells present in the bone marrow in an organism and characteristically release platelets. They are also characterized by being positive for the CD41a, CD42a, and CD42b cell surface markers, and may also additionally express a marker selected from the group consisting of CD9, CD61, CD62p, CD42c, CD42d, CD49f, CD51, CD110, CD123, CD131, and CD203c. A "megakaryocyte cell", when multinucleated (polyploidization), has 16X to 32X genomes relative to that in an ordinary cell, and in this Description the term "megakaryocyte cells" by itself includes, insofar as the characteristic features indicated above are present, both multinucleated megakaryocyte cells and pre-multinucleated megakaryocyte cells. "Pre-multinucleated megakaryocyte cells" is also synonymous with "immature megakaryocyte cells" and "growth-phase megakaryocyte cells".

Immortalized megakaryocyte cells can be obtained by various known methods. A nonlimiting example of a megakaryocyte cell production method is the method described in <CIT>. Using this method, immortalized megakaryocyte cells that can propagate indefinitely can be obtained by the overexpression of an oncogene and a polycomb gene in a "cell less differentiated than a megakaryocyte cell". In addition, immortalized megakaryocyte cells can also be obtained according to the method described in <CIT> by inducing the overexpression of an apoptosis suppression gene in a "cell less differentiated than a megakaryocyte cell", i.e., a megakaryocyte progenitor cell (also referred to in this Description simply as a "progenitor cell"). By terminaing the overexpression of gene, these immortalized megakaryocyte cells undergo the development of multinucleation and release platelets.

A combination of the methods described in the aforementioned documents may be used to obtain the megakaryocyte cells. In this case, overexpression of an oncogene, overexpression of a polycomb gene, and overexpression of an apoptosis suppression gene may be carried out simultaneously or sequentially. For example, multinucleated megakaryocyte cells may be obtained by bringing about the overexpression of an oncogene and polycomb gene; inhibiting this overexpression; then bringing about the overexpression of an apoptosis suppression gene; and inhibiting this overexpression. In addition, multinucleated megakaryocyte cells may also be obtained by bringing about overexpression of the oncogene, overexpression of the polycomb gene, and overexpression of the apoptosis suppression gene all at the same time and then terminating these overexpressions at the same time. Multinucleated megakaryocyte cells may also be obtained by first inducing overexpression of the oncogene and polycomb gene and then inducing overexpression of the apoptosis suppression gene and terminating these overexpressions at the same time.

In this Description, a "cell less differentiated than a megakaryocyte cell" and a "megakaryocyte progenitor cell" denote a cell that has the capacity to differentiate into a megakaryocyte and that resides in various stages of differentiation from a hematopoietic stem cell lineage to a megakaryocyte cell. Nonlimiting examples of cells less differentiated than megakaryocytes are hematopoietic stem cells, hematopoietic progenitor cells, CD34-positive cells, and megakaryocyte-erythroid progenitor cells (MEP). Described herein are cells that may be obtained by isolation from the bone marrow, umbilical cord blood, or peripheral blood, or may be obtained by inducing differentiation from pluripotent stem cells, e.g., iPS cells, and so forth, which are even less differentiated cells.

In this Description, "oncogene" refers to a gene that induces the malignant transformation of a cell within an organism, and examples are MYC family genes (for example, c-MYC, N-MYC, L-MYC), SRC family genes, RAS family genes, RAF family genes, and protein kinase family genes such as c-Kit, PDGFR, and Abl.

In this Description, a "polycomb gene" is known as a gene that functions to circumvent cell senescence by negatively regulating the CDKN2a (INK4a/ARF) gene (<NPL>; <NPL>; <NPL>). Nonlimiting examples of polycomb genes are BMI1, Mel18, Ring1a/b, Phc1/<NUM>/<NUM>, Cbx2/<NUM>/<NUM>/<NUM>/<NUM>, Ezh2, Eed, Suz12, HADC, and Dnmt1/3a/3b.

In this Description, "apoptosis suppression gene" refers to a gene that has the function of suppressing the apoptosis of a cell, and can be exemplified by the BCL2 gene, BCL-xL gene, Survivin gene, and MCL1 gene.

Overexpression of genes and termination of the overexpression can be performed by the methods described in <CIT>, <CIT>, <CIT>, and <NPL>, and by other known methods and by methods based on the preceding.

Common conditions may be used for the megakaryocyte cell culture conditions in all of the embodiments of the present invention. For example, the temperature may be approximately <NUM> to approximately <NUM>, approximately <NUM> to approximately <NUM>, or approximately <NUM> to approximately <NUM>, while <NUM>% CO<NUM> and/or <NUM>% O<NUM> may be used. Static culture or shake culture may be used. There are also no particular limitations on the shaking rate in the case of shake culture, and, for example, <NUM> rpm to <NUM> rpm, <NUM> rpm to <NUM> rpm, and so forth can be used.

By culturing megakaryocyte cells as described in the preceding in the platelet production method, the megakaryocyte cells undergo maturation and platelets are produced from their cytoplasm. This maturation of the megakaryocyte cells refers to multinucleation of the megakaryocyte cells and the release of platelets.

The functionality of these platelets can be evaluated through measurements carried out in accordance with known methods. For example, the amount of activated platelets can be measured using the PAC-<NUM> antibody, which is an antibody that binds specifically to the integrin αIIBβ3 (glycoprotein IIb/IIIa, CD41a/CD61 complex) activation marker that is present on the membrane of activated platelets. The amount of activated platelets may also be measured using antibody to detect CD62b (P-selectin), which is likewise an activation marker for platelets. The amount of platelets can be measured, for example, using flow cytometry and carrying out gating with antibody to the activation-independent platelet markers CD61 or CD41, followed by detection of binding by PAC-<NUM> antibody or anti-CD62P antibody to the platelets. These processes may be carried out in the presence of adenosine diphosphate (ADP).

Platelet functionality may also be evaluated by examining whether binding with fibrinogen in the presence of ADP occurs. The activation of integrin as required in the initial stage of thrombus formation is produced by the binding of fibrinogen by platelets. In addition, platelet functionality may also be evaluated by a method in which the thrombus formation capacity is visualized and observed in vivo, as shown in <FIG> in <CIT>.

There are no particular limitations on the culture medium for the cultivation of the megakaryocyte cells, and known media suitable for the production of platelets from megakaryocyte cells, or media based thereon, may be used as appropriate. For example, a medium used for the culture of animal cells can be prepared to function as a basal medium. The basal medium can be exemplified by IMDM medium, medium <NUM> medium, Eagle's minimum essential medium (EMEM) medium, α-MEM medium, Dulbecco's modified Eagle's medium (DMEM) medium, Ham's F12 medium, RPMI <NUM> medium, Fischer's medium, Neurobasal Medium (Life Technologies Corporation), and mixed media from the preceding.

The culture medium may contain serum or plasma or may be serum-free. As necessary, the medium may also contain one or more substances such as, for example, albumin, insulin, transferrin, selenium, fatty acid, trace elements, <NUM>-mercaptoethanol, thiol glycerol, monothioglycerol (MTG), lipids, amino acids (for example, L-glutamine), ascorbic acid, heparin, nonessential amino acids, vitamins, growth factors, low molecular weight compounds, antibiotics, antioxidants, pyruvic acid, buffers, inorganic salts, and cytokines. The cytokines are proteins that promote blood cell differentiation and can be exemplified by vascular endothelial growth factor (VEGF), thrombopoietin (TPO), various TPO-like agents, stem cell factor (SCF), insulin-transferrin-selenite (ITS) supplement, and ADAM inhibitor. A preferred medium for the present invention is IMDM medium containing serum, insulin, transferrin, serine, thiolglycerol, ascorbic acid, and TPO. It may also contain SCF and may also contain heparin. The co-use of TPO and SCF is preferred. There are no particular limitations on the concentration of each, but the TPO can be approximately <NUM> ng/mL to approximately <NUM> ng/mL or approximately <NUM> ng/mL to approximately <NUM> ng/mL; the SCF can be approximately <NUM> ng/mL to approximately <NUM> ng/mL or approximately <NUM> ng/mL; and the heparin can be approximately <NUM> U/mL to approximately <NUM> U/mL or approximately <NUM> U/mL. A phorbol ester (for example, phorbol <NUM>-myristate <NUM>-acetate, PMA) may also be added.

Human serum is preferred when serum is used. In addition, human plasma and so forth may be used instead of serum. Using the method, platelets equivalent to those obtained using serum can be obtained even with the use of these components.

When a drug-responsive gene expression induction system, e.g., the Tet-on (registered trademark) or Tet-off (registered trademark) system, is used for overexpression of genes or the termination thereof, in the overexpression process the corresponding drug, for example, tetracycline or doxycycline, may be incorporated in the culture medium, and overexpression may be inhibited by its removal from the culture medium.

The step for culturing the megakaryocyte cells may be carried out in the present invention without feeder cells. As demonstrated in the following examples, the method according to the present invention can provide functional platelets even by culture in the absence of feeder cells.

In this Description, "feeder cells" refer to cells that are co-cultured with the target cells in order to adjust to the environment required for the culture of the cells (target cells) that are to be propagated or differentiated. Insofar as they are cells that can be distinguished from the target cells, the feeder cells may contain cells originating from the same species or may contain cells of heterologous origin. The feeder cells may be cells that have been treated with antibiotics or gamma radiation to be nonpropagating, or may be cells not thusly treated.

An immortalized megakaryocyte line (MKCL SeV2) was produced in accordance with the method described by <NPL> by the introduction of Bcl-xL, c-Myc, and Bmi1 into iPS cells (SeV2: cells produced, in accordance with the method of <CIT>, by the introduction of c-MYC, OCT3/<NUM>, SOX2, and KLF4 into neonate human fibroblasts using a sendai virus vector). iPS cells (MK iPS #<NUM>) were produced from the immortalized megakaryocyte line (MKCL SeV2) according to the method described by <NPL>. The obtained iPS cells (MK iPS #<NUM>) were cultured in accordance with the method described by <NPL> using StemFit (registered trademark) AK03 (Ajinomoto) and laminin <NUM> (iMatrix <NUM> (Nippi)).

The cultured iPS cells (MK iPS #<NUM>) were dissociated using TrypLE (registered trademark) Select, and <NUM> × <NUM><NUM> cells were mixed with <NUM>µg template vector (<FIG>), <NUM>µg guide vector (<FIG>. <NUM>µg cas9 vector (pHL-EF1a-SphcCas9-iC-A, received from Dr. Hotta, Center for iPS Cell Research and Application, Kyoto University), and the vectors were introduced into the cells by electroporation using a Human Stem Cell Nucleofector (registered trademark) Kit <NUM> (Lonza) and Nucleofector. After electroporation, suspension was carried out in StemFit (registered trademark) AK03 followed by seeding to a laminin <NUM>-coated <NUM>-cm dish and culture under conditions of <NUM> and <NUM>% CO<NUM>. After <NUM> days, puromycin was added to the culture medium at <NUM> ng/mL and subculturing was performed. After <NUM> days, the colonies that had formed were picked up and DNA was extracted from each of the obtained colonies using a QIAamp DNA Mini Kit (QIAGEN), and homologous recombination was confirmed by genotyping PCR using primers (TUBB1 insert check Fw and Rv, <NUM>-<NUM> insert check Fw and <NUM>-<NUM> insert check Rv, and <NUM>-<NUM> insert check Fw and <NUM>-<NUM> insert check Rv; the sequences are given in Table <NUM>). The homozygous homologous recombinant iPS cell line was expansion cultured and established as MK iPS#<NUM>-<NUM>.

The obtained MK iPS#<NUM>-<NUM> was then mixed with <NUM>µg cre expression vector (pCXW-Cre-Puro, received from Dr. Hotta, Center for iPS Cell Research and Application, Kyoto University), and the vector was introduced into the cells by electroporation using a Human Stem Cell Nucleofector (registered trademark) Kit <NUM> (Lonza) and Nucleofector. After electroporation, suspension was carried out in StemFit (registered trademark) AK03 followed by seeding to a laminin <NUM>-coated <NUM>-cm dish and culture under conditions of <NUM> and <NUM>% CO<NUM>. After <NUM> days, multiple colonies were picked up and were divided in two: one half was cultured under the addition of puromycin at <NUM> ng/mL to the culture medium and the other half was stored. For stored cells deriving from the same colony for which cell death had been confirmed for the line, the removal of the puromycin resistance cassette was checked by genotyping PCR using primers (TUBB1 insert check Fw and Rv), and the resulting iPS cell line was expansion cultured to establish MK iPS#<NUM>-<NUM> cre2.

The induction of hematopoietic progenitor cells (HPC) was performed from the iPS cells (MK iPS#<NUM>-<NUM> cre2) via iPS-sac. In more detail, the iPS cells were released from the culture dish using a cell scraper, and about one-twentieth of the cells were seeded as colony clusters onto mitomycin C (MMC)-treated C3H10T1/<NUM> (available from Riken). The MMC-treated C3H10T1/<NUM> was prepared by seeding <NUM> × <NUM><NUM> cells/dish to a <NUM>-cm dish on the day prior to seeding with the iPS cells. After seeding, the culture was started (day <NUM>) in Eagle's basal medium (EBM) supplemented with <NUM> ng/mL VEGF, in an environment of <NUM>% O<NUM>, <NUM>% CO<NUM>, and <NUM>. Medium exchange with the same culture medium was performed at a frequency of twice per week.

The cells were physically released on day <NUM> using a cell scraper and the tip of a pipet, and cells of uniform size were recovered by passage through a <NUM>-micrometer cell strainer.

On day <NUM>, the cells were recovered and seeded into a <NUM>-well dish at <NUM> × <NUM><NUM> cells/well on the MMC-treated C3H10T1/<NUM> cells. EBM containing <NUM> ng/ml of SCF, <NUM> ng/ml of TPO and <NUM>µg/ml of doxycycline was used for the medium. On day <NUM>, the cells were recovered by pipetting and seeded into a <NUM> dish at <NUM> × <NUM><NUM> cells/dish on MMC-treated C3H10T1/<NUM> cells. The cells were recovered on day <NUM> and seeded into a <NUM> dish at <NUM> × <NUM><NUM> cells/dish to prepare the reporter immortalized megakaryocyte cell line (MKCL #<NUM>-<NUM> cre2).

Continuing, the megakaryocyte cells were cultured in differentiation medium not containing doxycycline to induce the release of platelets. More specifically, the reporter immortalized megakaryocyte cell line (MKCL #<NUM>-<NUM> cre2) obtained according to the method described above was cultured for <NUM> days in differentiation medium containing <NUM> StemRegenin <NUM> (SR1) (Selleckchem), <NUM> Y-<NUM> (Wako), <NUM> ng/ml of TPO (R&D) and <NUM> ng/ml of SCF (R&D). After suspending the cultured cells, the cells were recovered from the culture supernatant and analyzed by FACS after staining with anti-CD41a antibody and anti-CD42b antibody. As a result, expression intensity of CD41a and CD42b was enhanced (megakaryocyte cell maturation was induced) by culturing for <NUM> days (<FIG>) and platelets positive for CD41a and CD42b were confirmed to have been produced from the megakaryocyte cell line as a result of culturing for <NUM> days (<FIG>).

After culturing the immortalized megakaryocyte line obtained according to the method described above for <NUM> days on 10T1/2R feeder cells in differentiation medium containing <NUM> ng/ml of TPO and <NUM> ng/ml of SCF, the cells were stained with anti-CD41a antibody and anti-CD42b antibody and sorted into a VENUS high group and VENUS low group based on the expression level of VENUS as determined by FACS.

A cell group cultured for <NUM> days in differentiation medium containing <NUM> ng/ml of TPO and <NUM> ng/ml of SCF or differentiation medium containing <NUM> SR1, <NUM> Y-<NUM>, <NUM> ng/ml of TPO and <NUM> ng/ml of SCF was used as a control (designated as an SR1+rocki group and plain group, respectively).

When the expression levels of TUBB1 and VENUS were measured by qPCR for the VENUS high group (high level of VENUS fluorescence intensity), VENUS low group (low level of VENUS fluorescence intensity), SR1+rocki group (Y-<NUM>) and plain group, the expression levels of TUBB1 and VENUS were confirmed to be proportional (<FIG> and <FIG>).

On the basis of the above, the expression level of TUBB1 was confirmed to be able to be used interchangeably with the expression level of VENUS in the immortalized megakaryocyte cell line derived from MK iPS #<NUM>-<NUM> cre2. Moreover, when the expression level of TUBB1 was compared with that of other megakaryocyte cell maturation factors, TUBB1 was clearly determined to be particularly superior as an indicator when searching for platelet production promoters. More specifically, as a result of measuring and comparing the expression levels of GATA1, FOG1, NFE2 and VENUS after culturing for <NUM> days in differentiation medium containing SR1 or DMSO from which doxycycline had been removed, the expression level of TUBB1 demonstrated the most favorable sensitivity (<FIG>).

Continuing, the immortalized megakaryocyte cell line was cultured on 10T1/2R feeder cells in differentiation medium containing <NUM> ng/ml of TPO and <NUM> ng/ml of SCF or differentiation medium containing <NUM> SR1, <NUM> Y-<NUM>, <NUM> ng/ml of TPO and <NUM> ng/ml of SCF, followed by suspending the cells and harvesting the supernatant on day <NUM>, day <NUM>, day <NUM>, day <NUM>, day <NUM> and day <NUM> of culturing and analyzing the cells by FACS after staining with anti-CD41a antibody and anti-CD42b antibody. As a result, the expression level of VENUS was confirmed to rise and the number of platelets produced was confirmed to increase due to the addition of SR1 (<FIG> and <FIG>).

Primary screening consisted of seeding the immortalized megakaryocyte cell line obtained according to the method described above into a <NUM>-well dish followed by culturing by adding <NUM> Y-<NUM>, <NUM> ng/ml of TPO, <NUM> ng/ml of SCF and <NUM> of each compound obtained from a compound library provided by Professor Ohta of the Center for iPS Cell Research and Application (CiRA), Kyoto University (total of <NUM> compounds, including duplicate compounds). The cells were also cultured in differentiation medium containing <NUM> SR1, <NUM> Y-<NUM>, <NUM> ng/ml of TPO and <NUM> ng/ml of SCF (positive control) or differentiation medium containing <NUM>% DMSO, <NUM> Y-<NUM>, <NUM> ng/ml of TPO and <NUM> ng/ml of SCF (negative control). On day <NUM> of culturing, the fluorescence intensity of VENUS in each compound group was measured with Array Scan VTI (Thermo Scientific), and the value of fluorescence intensity of each compound was corrected according to the fluorescence intensity of the positive control (assigned a value of <NUM>%) and the fluorescence intensity of the negative control (assigned a value of <NUM>%) (<FIG>). Those drugs for which this value was <NUM>% or higher were selected as primary candidate compounds. The primary candidate compounds consisted of <NUM> types of compounds, and included, for example, TCS-<NUM> (<NPL> Santa Cruz Biotechnology, Inc. ) and C59 (<NPL>).

Secondary screening consisted of seeding the immortalized megakaryocyte cell line obtained according to the method described above into a <NUM>-well dish and culturing after adding <NUM> Y-<NUM>, <NUM> ng/ml of TPO, <NUM> ng/ml of SCF and the primary candidate compounds at concentrations of <NUM>, <NUM>, <NUM> and <NUM> each. The cells were also cultured in differentiation medium containing <NUM> SR1, <NUM> Y-<NUM>, <NUM> ng/ml of TPO and <NUM> ng/ml of SCF (positive control) or differentiation medium containing <NUM>% DMSO, <NUM> Y-<NUM>, <NUM> ng/ml of TPO and <NUM> ng/ml of SCF (negative control). On day <NUM> of culturing, the fluorescence intensity of VENUS in each compound group was measured with Array Scan VTI (Thermo Scientific), and the value of fluorescence intensity of each compound was corrected according to the fluorescence intensity of the positive control (assigned a value of <NUM>%) and the fluorescence intensity of the negative control (assigned a value of <NUM>%). Those drugs for which this value was <NUM>% or higher and for which concentration dependency was observed were selected as secondary candidate compounds. The secondary candidate compounds consisted of <NUM> types of compounds, and included, for example, TCS-<NUM> (<NPL>, Santa Cruz Biotechnology, Inc. ) and C59 (<NPL>).

Continuing, the effect of promoting platelet production was confirmed for <NUM> compounds from among the secondary candidate compounds excluding duplicate compounds and compounds that were unable to be used commercially. The above-mentioned immortalized megakaryocyte cell line was cultured in a <NUM>-well dish in differentiation medium containing each of the compounds at concentrations of <NUM>, <NUM>, <NUM> and <NUM>, <NUM> Y-<NUM>, <NUM> ng/ml of TPO and <NUM> ng/ml of SCF, differentiation medium containing <NUM> SR1, <NUM> Y-<NUM>, <NUM> ng/ml of TPO and <NUM> ng/ml of SCF (positive control), or differentiation medium containing <NUM> % DMSO, <NUM> Y-<NUM>, <NUM> ng/ml of TPO and <NUM> ng/ml of SCF (negative control), followed by suspending the cells and harvesting the supernatant on day <NUM> of culturing and analyzing by FACS after staining with anti-CD41a antibody and anti-CD42b antibody. The relative number of platelets was calculated by correcting the number of platelets in the case of using each candidate compound with the number of platelets of the positive control (assigned a value of <NUM>%) and the number of platelets of the negative control (assigned a value of <NUM>%) (<FIG>).

On the basis of the above results, compounds were discovered that have the potential to induce platelet production more potently than SR1 in excess of <NUM>%. C59 in particular was suggested to be a potent platelet inducer.

After culturing iPS cells (TkDA3-<NUM>), the colonies were detached with a cell scraper and cultured in differentiation medium on 10T1/<NUM> feeder cells following the addition of <NUM>µg/ml of VEGF in accordance with <NPL>. Fourteen days later, the cells were recovered using a cell scraper and cultured for <NUM> days in the absence of feeder cells following the addition of <NUM> U/mL of heparin, <NUM> Y-<NUM>, <NUM> ng/ml of TPO and <NUM> ng/ml of SCF, and further adding <NUM>% DMSO, <NUM> SR1, <NUM> C59 (Albiochem) or <NUM> TCS-<NUM> (Santa Cruz Biotechnology, Inc. Culturing was also carried out in the same manner using feeder cells. The resulting cells were suspended and the supernatant was harvested followed by analyzing by FACS after staining with anti-CD41a antibody and anti-CD42b antibody (<FIG>). As a result, C59 and TCS-<NUM> were confirmed to have the ability to produce platelets that was equal to or greater than that of SR1 in the absence of feeder cells.

Claim 1:
An in vitro method for screening for platelet production promoters, the method comprising:
a step of contacting megakaryocytes or progenitor cells thereof with a candidate substance,
a step of culturing the megakaryocytes or progenitor cells thereof in the presence of the candidate substance,
a step of measuring expression of TUBB1 in cultured mature megakaryocyte cells following said step of culturing the megakaryocytes or progenitor cells thereof in the presence of the candidate substance, and
a step for selecting, as a platelet production promoter, a candidate substance that significantly increases expression of TUBB1 in the mature megakaryocyte cells as compared to the expression of GATA1, FOG1, and/or NFE2;
wherein the platelet production promoter is a drug that promotes production of platelets and increases the amount of platelets produced per unit time.