Method for producing ips cells and kit for producing ips cells

Abstract

To increase the establishment efficiency of iPS cells in an RNA reprogramming method.SOLUTION: A method for producing iPS cells, comprising culturing, in a ROCK inhibitor-containing medium, blood cells into which mRNA of a reprogramming factor and mRNA encoding p53DD have been introduced.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The embodiments disclosed in the present specification and drawings relate to a method for producing iPS cells and a kit for producing iPS cells. [Background technology]

[0002] Induced pluripotent stem cells (hereinafter referred to as iPS cells) can be generated by introducing a group of genes called reprogramming factors (e.g., Oct3 / 4, Sox2, Klf4, and c-Myc) into somatic cells. Common methods for introducing reprogramming factors into somatic cells include the Sendai virus method and electroporation of episomal vectors. However, iPS cells generated by these methods have the risk of retaining the reprogramming factors, making them unsuitable for clinical application.

[0003] To solve these problems, attempts have been made to generate iPS cells by introducing mRNA for reprogramming factors into somatic cells such as fibroblasts. This method of generating iPS cells using mRNA for reprogramming factors is called "RNA reprogramming." [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] MAPoleganov et al., Efficient Reprogramming of Human Fibroblasts and Blood-Derived Endothelial Progenitor Cells Using Nonmodified RNA for Reprogramming and Immune Evasion, Human Gene Therapy, Vol.26, No.11, p.751-766, 2015 Summary of the Invention [Problem to be solved by the invention]

[0005] One of the problems to be solved by the embodiments disclosed in this specification and drawings is to improve the efficiency of iPS cell establishment in RNA reprogramming methods. However, the problems to be solved by the embodiments disclosed in this specification and drawings are not limited to the above problem. Problems corresponding to the effects of each configuration shown in the embodiments described below can also be positioned as other problems. [Means for solving the problem]

[0006] According to one aspect, there is provided a method for producing iPS cells, comprising culturing blood cells into which mRNA for a reprogramming factor and mRNA encoding p53DD have been introduced, in a medium containing a ROCK inhibitor.

[0007] In another aspect, there is provided a kit for producing iPS cells, comprising mRNA of a reprogramming factor, mRNA encoding p53DD, a transfection reagent, and a ROCK inhibitor. [Brief explanation of the drawings]

[0008] [Figure 1] 1 is a flowchart showing an example of a method for producing iPS cells. [Figure 2] 1 is a micrograph showing the results of iPS cell establishment in Example 1. [Figure 3] 1 is a micrograph showing the results of iPS cell establishment in Example 2. [Figure 4] 1 is a micrograph showing the results of iPS cell establishment in Example 3. [Figure 5] 1 is a micrograph showing the results of iPS cell establishment in Example 4. DETAILED DESCRIPTION OF THE INVENTION

[0009] <1> How iPS cells are generated According to one embodiment, the method for producing iPS cells comprises culturing blood cells into which mRNA for a reprogramming factor and mRNA encoding p53DD have been introduced in a medium containing a ROCK inhibitor. Here, "blood cells into which mRNA for a reprogramming factor and mRNA encoding p53DD have been introduced" refers to blood cells into which a procedure for introducing mRNA has been performed, and it is not necessary for all blood cells to have mRNA introduced.

[0010] According to certain embodiments, the method for producing iPS cells includes introducing mRNA for a reprogramming factor and mRNA encoding p53DD into blood cells, and culturing the blood cells into which the mRNA for the reprogramming factor and mRNA encoding p53DD have been introduced in a medium containing a ROCK inhibitor. For example, the mRNA for the reprogramming factor and the mRNA encoding p53DD can be introduced into blood cells by adding the mRNA to be introduced together with a transfection reagent to the blood cells, and then culturing the blood cells.

[0011] In the above embodiment, the ROCK inhibitor may be added to the blood cells before the addition of mRNA to the blood cells, simultaneously with the addition of mRNA to the blood cells, or after the addition of mRNA to the blood cells. The ROCK inhibitor may be added at multiple times, for example, before the addition of mRNA to the blood cells and simultaneously with the addition of mRNA to the blood cells.

[0012] According to the above embodiment, when blood cells into which mRNA encoding p53DD has been introduced in addition to mRNA for a reprogramming factor are cultured in a medium containing a ROCK inhibitor, the efficiency of establishing iPS cells can be increased.

[0013] In the above-mentioned embodiment, the medium containing the ROCK inhibitor may further contain a TGF-β kinase inhibitor. Alternatively, in the above-mentioned embodiment, the medium containing the ROCK inhibitor may further contain an HDAC inhibitor. Alternatively, in the above-mentioned embodiment, the medium containing the ROCK inhibitor may further contain a TGF-β kinase inhibitor and an HDAC inhibitor.

[0014] Thus, when blood cells into which mRNA encoding p53DD has been introduced in addition to mRNA for reprogramming factors are cultured in a medium containing a TGF-β kinase inhibitor and / or an HDAC inhibitor in addition to a ROCK inhibitor, the efficiency of iPS cell establishment can be further improved.

[0015] The method according to the above embodiment may further comprise introducing the mRNA for the reprogramming factor and the mRNA encoding p53DD into the blood cells into which the mRNA for the reprogramming factor and the mRNA encoding p53DD have been introduced one or more times during culture. That is, in the method according to the above embodiment, the procedure of introducing the mRNA for the reprogramming factor and the mRNA encoding p53DD into the blood cells may be performed a total of two or more times. The number of times mRNA is introduced (i.e., the number of transfections) is not particularly limited, but when performed two or more times, it can be, for example, two to six times, preferably three to five times.

[0016] Thus, in the method according to the above embodiment, if the procedure of introducing the mRNA of the reprogramming factor and the mRNA encoding p53DD into blood cells is performed a total of two or more times, the efficiency of iPS cell establishment can be further improved.

[0017] (Terminology explanation) First, the terms used in this specification will be explained.

[0018] "Blood cells" are, for example, mononuclear cells, generally peripheral blood mononuclear cells (PBMCs). The animal species of blood cells is not particularly limited, but is preferably mammalian, more preferably primate, and even more preferably human. "Blood cells" are preferably CD34-positive blood cells. CD34-positive blood cells are undifferentiated blood cells and can also be called blood cell precursor cells. CD34-positive blood cells can be isolated from peripheral blood mononuclear cells (PBMCs) by, for example, flow cytometry using a CD34 antibody. The use of CD34-positive blood cells can increase the efficiency of iPS cell establishment.

[0019] "Reprogramming factors" refer to a group of genes that can be introduced into somatic cells to induce iPS cells from them. Various gene combinations are known as reprogramming factors, such as a four-factor combination consisting of Oct3 / 4, Sox2, Klf4, and c-Myc, or a six-factor combination consisting of Oct3 / 4, Sox2, Klf4, c-Myc, Lin28, and Nanog.

[0020] "Reprogramming factor mRNA" refers to the mRNA of a group of genes that constitute a reprogramming factor. It is known that reprogramming factor mRNA can also be introduced into somatic cells to induce iPS cells from the somatic cells. The reprogramming factor mRNA is not particularly limited, as long as it can induce iPS cells when introduced into somatic cells. Reprogramming factor mRNA includes, for example, four types: Oct3 / 4 mRNA, Sox2 mRNA, Klf4 mRNA, and c-Myc mRNA. An example of reprogramming factor mRNA is a mixture of Oct3 / 4 mRNA, Sox2 mRNA, Klf4 mRNA, c-Myc mRNA, Lin28 mRNA, and Nanog mRNA.

[0021] mRNA for reprogramming factors is commercially available, for example, StemRNA-3. rdIt is available through the Gen Reprogramming Kit (ReproCell, Inc.). StemRNA-3 rd The Gen Reprogramming Kit (ReproCell Inc.) contains a mixture of mRNA for reprogramming factors, including Oct3 / 4 mRNA, Sox2 mRNA, Klf4 mRNA, c-Myc mRNA, Lin28 mRNA, and Nanog mRNA.

[0022] "p53DD" refers to a dominant-negative mutant of the p53 protein, and preferably refers to a mutant in which the amino acids 14-301 of the mouse p53 protein (consisting of 390 amino acids) have been deleted (Bowman, T., Genes Develop., 10, 826-835 (1996)). p53DD may also be a mutant in which the corresponding amino acids of the human p53 protein have been deleted.

[0023] The "mRNA encoding p53DD" is preferably an mRNA encoding mouse p53DD, the sequence of which is shown below.

[0024] [Table 1]

[0025] "mRNA encoding p53DD" is also referred to herein as "mRNA of p53DD."

[0026] "ROCK inhibitor" refers to a substance that inhibits Rho kinase, such as Y-27632 ((R)-(+)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride; CAS No. 129830-38-2). ROCK inhibitors are known to suppress cell death that can occur when cells are seeded as single cells. ROCK inhibitors can be added to the medium to a final concentration of, for example, 5 to 10 μM, preferably 9 to 10 μM.

[0027] "TGF-β kinase inhibitor" refers to a substance that inhibits transforming growth factor β (TGF-β) kinase, such as A-83-01 (3-(6-methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide; CAS No. 909910-43-6). The TGF-β kinase inhibitor can be added to the medium to a final concentration of, for example, 0.1 to 1 μM, preferably 0.5 μM.

[0028] "HDAC inhibitor" refers to a substance that inhibits histone deacetylase, and examples thereof include sodium butyrate. HDAC inhibitors can be added to the medium to a final concentration of, for example, 10 to 50 μM, preferably 25 μM.

[0029] (An example of how to create iPS cells) An example of a method for producing iPS cells is shown in Figure 1. Below, an example of a method for producing iPS cells will be explained in the order of the steps shown in Figure 1. In this example, a ROCK inhibitor, a TGF-β kinase inhibitor, and an HDAC inhibitor are added to the blood cells simultaneously with the addition of mRNA for the reprogramming factor and mRNA encoding p53DD to the blood cells. In the following description, the mixture of the ROCK inhibitor, TGF-β kinase inhibitor, and HDAC inhibitor is referred to as a "small molecule compound." As described above, these small molecule compounds may be added before or after the addition of mRNA for the reprogramming factor and mRNA encoding p53DD to the blood cells.

[0030] (1) Preparation of blood cells First, blood cells are suspended in a medium to prepare a blood cell suspension. The medium used here is preferably a medium for blood cells, such as StemSpan-AOF (Veritas Corporation).

[0031] (2) Preparation of medium containing mRNA for reprogramming factors, mRNA for p53DD, and small molecules An mRNA-containing medium is prepared by adding mRNA for a reprogramming factor, mRNA for p53DD, a small molecule compound, and a transfection reagent to a base medium. The base medium used here is preferably a mixture of a transfection medium and a medium for blood cells. The transfection reagent refers to a reagent capable of introducing mRNA into cells, and is preferably a lipofection reagent capable of introducing mRNA into cells. Transfection reagents are commercially available, such as Lipofectamine Messenger MAX (Thermo Fisher Scientific, Inc.). The mRNA-containing medium may further contain additives that contribute to improving the efficiency of iPS cell establishment and growth factors.

[0032] (3) Addition of mRNA for reprogramming factors, mRNA for p53DD, and small molecules to blood cells, and then culturing the blood cells. The "blood cell suspension" prepared in (1) is mixed with the "mRNA-containing medium" prepared in (2), and the resulting mixture is seeded into a culture vessel. The culture vessel is placed in a cell culture room and the blood cells are cultured. The day the culture began is designated "culture day 0."

[0033] (4) Addition of mRNA for reprogramming factors, mRNA for p53DD, and small molecules to blood cells, and subculture of blood cells. On the next day (culture day 1), the liquid in the culture vessel is removed and new mRNA-containing medium is added to the culture vessel to replace the medium. Thereafter, the blood cell culture is continued. On the second day of culture, the liquid in the culture vessel is removed and new mRNA-containing medium is added to the culture vessel to perform a medium change. Thereafter, the culture of the blood cells is continued. On the third day of culture, the liquid in the culture vessel is removed and new mRNA-containing medium is added to the culture vessel to replace the medium. Thereafter, the culture of the blood cells is continued.

[0034] The "new mRNA-containing medium" used on days 1 to 3 of culture contains mRNA for reprogramming factors, mRNA for p53DD, and a low-molecular-weight compound in a base medium. The "new mRNA-containing medium" used on days 1 to 3 of culture may have the same composition as the "mRNA-containing medium" prepared in (2), or it may have a different composition. For example, on days 1 and 2 of culture, a medium with the same composition as the "mRNA-containing medium" prepared in (2) may be used, and on day 3 of culture, the base medium of the "mRNA-containing medium" prepared in (2) may be replaced with a "mixed medium of transfection medium, medium for blood cells, and medium for iPS cells."

[0035] In this example, the reprogramming factor mRNA and p53DD mRNA were transfected into the blood cells a total of four times, but as mentioned above, the number of transfections is not particularly limited. Transfections performed twice or more can further increase the efficiency of iPS cell establishment.

[0036] (5) Subculture of blood cells On the fourth day of culture, the liquid in the culture vessel is removed and new medium containing a low molecular weight compound is added to the culture vessel to perform a medium change. Thereafter, the culture of blood cells is continued. On days 5, 7, 9, 11, and 13 of culture, the medium in the culture vessel is replaced with fresh medium containing a small molecule compound. The blood cell culture is continued until day 14 of culture.

[0037] The "new medium containing low-molecular-weight compounds" used on days 4 to 13 of culture contains low-molecular-weight compounds in a base medium. This medium does not contain mRNA for reprogramming factors or mRNA encoding p53DD. The "new medium containing low-molecular-weight compounds" may further contain growth factors. The "new medium containing low-molecular-weight compounds" used on days 4 to 13 of culture may have the same composition as each other, or different compositions. For example, on day 4 of culture, a mixed medium of a medium for blood cells and a medium for iPS cells may be used as the base medium, and on days 5 to 13 of culture, a medium for iPS cells may be used as the base medium.

[0038] (effect) As described above, the efficiency of iPS cell establishment can be improved by culturing blood cells into which mRNA encoding p53DD has been introduced in addition to mRNA for reprogramming factors in a medium containing a ROCK inhibitor.

[0039] In particular, the efficiency of iPS cell establishment can be further improved by culturing blood cells into which mRNA encoding p53DD has been introduced in addition to mRNA for reprogramming factors in a medium containing a TGF-β kinase inhibitor and / or an HDAC inhibitor in addition to a ROCK inhibitor.

[0040] On the other hand, when blood cells transfected with mRNA encoding p53DD in addition to mRNA for reprogramming factors were cultured in a medium that did not contain any of the three types of small molecule compounds, the efficiency of iPS cell establishment was low. Furthermore, when blood cells transfected with only mRNA for reprogramming factors were cultured in a medium that contained all three types of small molecule compounds, the efficiency of iPS cell establishment was also low. Therefore, the above effect is thought to be a synergistic effect obtained by using mRNA encoding p53DD in combination with the small molecule compounds.

[0041] Furthermore, the method according to the embodiment generates iPS cells using mRNA of a reprogramming factor and mRNA encoding p53DD. Therefore, compared with conventional methods that use Sendai virus vectors or episomal vectors to introduce reprogramming factors (DNA), the method according to the embodiment is superior in that there is no risk of the reprogramming factors remaining in the cells, and the generated iPS cells can be used safely in clinical applications.

[0042] Furthermore, the present inventors have demonstrated that iPS cells generated according to the methods of the embodiments have superior quality as iPS cells, as described below. Specifically, iPS cell colonies generated according to the methods of the embodiments, immediately after establishment (i.e., primary culture cells P0), showed higher expression rates of pluripotency markers (undifferentiation markers) and a higher proportion of iPS cells among the cells constituting the colony, compared to iPS cell colonies generated by introducing reprogramming factors (DNA) using Sendai virus vectors or episomal vectors. Furthermore, iPS cells generated according to the methods of the embodiments maintained the expression of pluripotency markers (undifferentiation markers) even after passage. Furthermore, iPS cells generated according to the methods of the embodiments, immediately after establishment (i.e., primary culture cells P0), could be observed as colonies with a more organized morphology than iPS cells generated by introducing reprogramming factors (DNA) using Sendai virus vectors or episomal vectors. Furthermore, iPS cells generated according to the methods of the embodiments were shown to have the ability to differentiate into three germ layers (ectoderm, mesoderm, and endoderm) and into cardiomyocytes.

[0043] <2> Kits for generating iPS cells According to another aspect, a kit for generating iPS cells is provided. The kit according to an embodiment includes mRNA for a reprogramming factor, mRNA encoding p53DD, a transfection reagent, and a ROCK inhibitor. As described above, the "transfection reagent" refers to a reagent capable of introducing mRNA into cells, and is preferably a lipofection reagent capable of introducing mRNA into cells.

[0044] The kit according to the embodiment may further include a TGF-β kinase inhibitor. Alternatively, the kit according to the embodiment may further include an HDAC inhibitor. Alternatively, the kit according to the embodiment may further include a TGF-β kinase inhibitor and an HDAC inhibitor. Furthermore, the kit according to the embodiment may further include a medium for transfection.

[0045] The kit according to the embodiment can be used to carry out the above-mentioned "method for producing iPS cells," thereby achieving the above-mentioned effects.

[0046] Although several embodiments have been described, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other forms, and various omissions, substitutions, modifications, and combinations of embodiments can be made without departing from the spirit of the invention. These embodiments and their modifications are included within the scope and spirit of the invention, as well as within the scope of the invention and its equivalents as defined in the claims.

[0047] With respect to the above embodiment, the following supplementary notes are disclosed as one aspect and optional features of the invention.

[0048] (Appendix 1) A method for producing iPS cells, comprising culturing blood cells into which mRNA for a reprogramming factor and mRNA encoding p53DD have been introduced in a medium containing a ROCK inhibitor.

[0049] (Appendix 2) The method may further comprise introducing the mRNA encoding the reprogramming factor and the mRNA encoding p53DD into a blood cell. That is, the method for producing iPS cells may include introducing mRNA for a reprogramming factor and mRNA encoding p53DD into blood cells, and culturing the blood cells into which the mRNA for the reprogramming factor and mRNA encoding p53DD have been introduced in a medium containing a ROCK inhibitor.

[0050] (Appendix 3) The medium may further contain a TGF-β kinase inhibitor, which may be A-83-01.

[0051] (Appendix 4) The medium may further comprise an HDAC inhibitor, which may be sodium butyrate.

[0052] (Appendix 5) The medium may further comprise a TGF-β kinase inhibitor and an HDAC inhibitor. The TGF-β kinase inhibitor may be A-83-01. The HDAC inhibitor may be sodium butyrate.

[0053] (Appendix 6) The blood cells may be mononuclear cells, peripheral blood mononuclear cells, or CD34-positive blood cells. The blood cells may be mammalian blood cells, primate blood cells, or human blood cells.

[0054] (Appendix 7) The mRNAs of the reprogramming factors may be a mixture consisting of Oct3 / 4 mRNA, Sox2 mRNA, Klf4 mRNA, cMyc mRNA, Lin28 mRNA, and Nanog mRNA. The mRNA encoding p53DD may be an RNA consisting of the base sequence shown in Table 1. The ROCK inhibitor may be Y-27632.

[0055] (Appendix 8) The method may further comprise introducing the mRNA for the reprogramming factor and the mRNA encoding p53DD into the blood cells into which the mRNA for the reprogramming factor and the mRNA encoding p53DD have been introduced, one or more times during the culture. That is, the method may comprise introducing the mRNA of the reprogramming factor and the mRNA encoding p53DD into blood cells two or more times.

[0056] (Appendix 9) mRNA of reprogramming factors, mRNA encoding p53DD, a transfection reagent; ROCK inhibitors and A kit for producing iPS cells, comprising:

[0057] (Appendix 10) The kit may further comprise a TGF-β kinase inhibitor, which may be A-83-01.

[0058] (Appendix 11) The kit may further comprise an HDAC inhibitor, which may be sodium butyrate.

[0059] (Appendix 12) The kit may further comprise a TGF-β kinase inhibitor and an HDAC inhibitor. The TGF-β kinase inhibitor may be A-83-01. The HDAC inhibitor may be sodium butyrate.

[0060] (Appendix 13) The mRNAs of the reprogramming factors may be a mixture consisting of Oct3 / 4 mRNA, Sox2 mRNA, Klf4 mRNA, cMyc mRNA, Lin28 mRNA, and Nanog mRNA. The mRNA encoding p53DD may be an RNA consisting of the base sequence shown in Table 1. The transfection reagent may be a lipofection reagent. The ROCK inhibitor may be Y-27632. [Example]

[0061] The following Examples 1 to 5 were carried out. Example 1: Blood cells transfected with mRNA for reprogramming factors and p53DD mRNA were cultured in a medium containing low molecular weight compounds (a mixture of ROCK inhibitor, TGF-β kinase inhibitor, and HDAC inhibitor). In Example 1, iPS cells were produced according to the above-mentioned "Example of a method for producing iPS cells." Example 2: Blood cells transfected with only mRNA of reprogramming factors were cultured in a medium that did not contain low molecular weight compounds. In Example 2, iPS cells were produced in the same manner as in Example 1, except that neither p53DD mRNA nor a low-molecular-weight compound was added to the blood cells. Example 3: Blood cells transfected with mRNA for reprogramming factors and p53DD mRNA were cultured in a medium that did not contain low-molecular-weight compounds. In Example 3, iPS cells were produced in the same manner as in Example 1, except that no low-molecular-weight compound was added. Example 4: Blood cells transfected with only mRNA for reprogramming factors were cultured in a medium containing low molecular weight compounds (a mixture of ROCK inhibitor, TGF-β kinase inhibitor, and HDAC inhibitor). In Example 4, iPS cells were produced in the same manner as in Example 1, except that p53DD mRNA was not added. Example 5: Blood cells transfected with mRNA for reprogramming factors and p53DD mRNA were cultured in a medium containing a ROCK inhibitor. In Example 5, iPS cells were produced in the same manner as in Example 1, except that a ROCK inhibitor was added instead of a low-molecular-weight compound.

[0062] After the iPS cells were generated, the cells were stained with alkaline phosphatase, a pluripotency marker, and the number of iPS cell colonies generated was counted. Figures 2 to 5 are micrographs showing the results of iPS cell establishment in Examples 1 to 4.

[0063] In Example 1, 10 iPS cell colonies were confirmed, and the iPS cell establishment efficiency was 0.025%. In Example 2, no iPS cell colonies were observed, and the efficiency of iPS cell establishment was 0%. In Example 3, two iPS cell colonies were confirmed, and the iPS cell establishment efficiency was 0.005%. In Example 4, the number of colonies of one iPS cell was confirmed, and the establishment efficiency of iPS cells was 0.0025%. In Example 5, four iPS cell colonies were confirmed, and the iPS cell establishment efficiency was 0.008%.

[0064] The results of Examples 1 to 4 showed that culturing blood cells transfected with p53DD mRNA in addition to mRNA for reprogramming factors in a medium containing a low-molecular-weight compound can increase the efficiency of iPS cell establishment. Furthermore, the results of Examples 1 to 4 showed that the effect obtained in Example 1 is a synergistic effect obtained by using p53DD mRNA in combination with a low-molecular-weight compound.

[0065] The results of Examples 3 and 5 showed that the efficiency of iPS cell establishment can be improved by culturing blood cells into which p53DD mRNA has been introduced in addition to mRNA for reprogramming factors in a medium containing a ROCK inhibitor.

[0066] Immediately after establishment (i.e., primary culture cell P0), the iPS cell colonies produced in Examples 1 and 5 had a higher expression rate of pluripotency markers (undifferentiation markers) and a higher proportion of iPS cells among the cells constituting the colony compared to iPS cells produced by introducing reprogramming factors (DNA) using Sendai virus vectors or episomal vectors. In addition, the iPS cells produced in Examples 1 and 5 maintained the expression of pluripotency markers (undifferentiation markers) even after passage in culture.

[0067] Furthermore, the iPS cells produced in Examples 1 and 5 could be observed immediately after establishment (i.e., primary culture cells P0) as colonies with a regular morphology, specifically, colonies with an overall nearly circular morphology and smooth contours, compared to iPS cells produced by introducing reprogramming factors (DNA) using Sendai virus vectors or episomal vectors.

[0068] Furthermore, when the iPS cells produced in Examples 1 and 5 were differentiated into three germ layers (ectoderm, mesoderm, and endoderm) and cardiomyocytes, they were shown to have the ability to differentiate into the three germ layers and cardiomyocytes.

Claims

1. A method for producing iPS cells, comprising culturing blood cells into which mRNA of a reprogramming factor and mRNA encoding p53DD have been introduced, in a medium containing a ROCK inhibitor.

2. The method of claim 1, further comprising introducing the mRNA encoding a reprogramming factor and the mRNA encoding p53DD into a blood cell.

3. The method of claim 1, wherein the medium further comprises a TGF-β kinase inhibitor.

4. The method of claim 1 , wherein the medium further comprises an HDAC inhibitor.

5. The method of claim 1, wherein the medium further comprises a TGF-β kinase inhibitor and an HDAC inhibitor.

6. The method according to claim 1, wherein the blood cells are CD34-positive blood cells.

7. The method of claim 1, wherein the mRNA of the reprogramming factor is a mixture consisting of Oct3 / 4 mRNA, Sox2 mRNA, Klf4 mRNA, cMyc mRNA, Lin28 mRNA, and Nanog mRNA.

8. The method according to any one of claims 1 to 7, further comprising introducing the mRNA for a reprogramming factor and the mRNA encoding p53DD into the blood cells into which the mRNA for a reprogramming factor and the mRNA encoding p53DD have been introduced one or more times during the culture.

9. mRNA of a reprogramming factor; mRNA encoding p53DD; and a transfection reagent; ROCK inhibitors and A kit for producing iPS cells, comprising:

10. The kit according to claim 9, further comprising a TGF-β kinase inhibitor and / or an HDAC inhibitor.

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