CA2658463A1 - Efficient method for nuclear reprogramming - Google Patents
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Abstract
A method of preparing induced pluripotent stem cells, comprising a nuclear reprogramming step using a nuclear reprogramming factor in the presence of miRNA, wherein said miRNA has a property of providing a higher nuclear reprogramming efficiency in the presence of said miRNA than in the absence thereof.
Description
Description Efficient Method for Nuclear Reprogramming Field of the Invention The present invention relates to an efficient method for preparing induced pluripotent stem cells through reprogramming of differentiated somatic cells.
Background of the Invention Embryonic stem cells (ES cells) are stem cells established from human or mouse early embryos which have a characteristic feature that they can be cultured over a long period of time while maintaining pluripotent ability to differentiate into all kinds of cells existing in living bodies. Human embryonic stem cells are expected for use as resources for cell transplantation therapies for various diseases such as Parkinson's disease, juvenile diabetes, and leukemia, taking advantage of the aforementioned properties. However, transplantation of ES cells has a problem of causing rejection in the same manner as organ transplantation. Moreover, from an ethical viewpoint, there are many dissenting opinions against the use of ES
cells which are established by destroying human embryos.
If dedifferentiation of patients' own differentiated somatic cells could be induced to establish cells having pluripotency and growth ability similar to those of ES
cells (in this specification, these cells are referred to as "induced pluripotent stem cells" or "iPS cells", though they are sometimes called "embryonic stem cell-like cells"
or "ES-like cells"), it is anticipated that such cells could be used as ideal pluripotent cells, free from rejection or ethical difficulties. Recently, it has been reported that such iPS cells can be produced from differentiated cells of mouse or human, which has created a great sensation (International Publication No. W02007/69666; Cell, 126, pp.
1-14, 2006; Cell, 131, pp. 1-12, 2007; Science, 318, pp. 1917-1920, 2007; and Nature, 451, pp. 141-146, 2008).
These methods include a reprogramming step through introduction of a plurality of specific factors (four factors of Oct3/4, Sox2, Klf4, and c-Myc are used in Cell, 126, pp.
1-14, 2006), and the introduction of these factors is mediated by viral vectors such as retrovirus or lentivirus. However, all previously reported nuclear reprogramming methods mediated by the gene introduction involve a problem of low efficiency in which only a small number of induced pluripotent stem cells can be obtained. In particular, there is a problem in that, if reprogramming is carried out in somatic cells through the introduction of three factors (namely, Oct3/4, Sox2, and K1f4), excluding c-Myc that is suspected to cause tumorigensis in tissues and individuals differentiated from resulting induced pluripotent stem cells, then the production efficiency of induced pluripotent stem cells becomes very low.
It is known that various small RNAs are expressed in cells. Examples of small RNA include RNA molecules of about 18-25 nucleotides long which can be cleaved out with a dicer, an RNase specific to double-stranded RNA. Small RNA is mainly classified into siRNA (small interfering RNA) and miRNA (microRNA, hereinafter abbreviated as "miRNA" in the specification). Small RNA is known to function as a guide molecule for finding out the target in a process such as translational suppression, mRNA
degradation, or alteration of chromatin structure, via RNA interference (RNAi)/miRNA molecular mechanism. In addition, small RNA is also known to play an important role in the regulation of developmental process (for example, as reviews, refer to Jikken Igaku (Experimental Medicine), 24, pp. 814-819, 2006; and microRNA Jikken Purotokoru (microRNA Experimental Protocol), pp. 20-35, 2008, YODOSHA CO., LTD.).
With regard to relation between embryonic stem cells (ES cells) and miRNA, ES
cell-specific expression of a microRNA cluster including several types of miRNAs in mouse ES cells has been reported (Developmental cell, 5, pp. 351-358, 2003), and it has also been reported that miRNA-295 suppressed the expression of Rbl2, a member of the Rb tumor suppressor gene family, and increased the expression of methylase to be thereby associated with DNA methylation (Nature Structural & Molecular Biology, 15, pp. 259-267, 2008;
Nature Structural & Molecular Biology, 15, pp. 268-279, 2008). However, it is yet to be known whether or not small RNA plays any role in the nuclear reprogramming of somatic cells.
References:
International Publication No. W02007/69666 Cell, 126, pp. 1-14, 2006 Cell, 131, pp. 1-12, 2007 Science, 318, pp. 1917-1920, 2007 Nature, 451, pp. 141-146, 2008 Developmental cell, 5, pp. 351-358, 2003 Nature Structural & Molecular Biology, 15, pp. 259-267, 2008 Nature Structural & Molecular Biology, 15, pp. 268-279, 2008 Disclosure of the Invention Problems to be Solved by the Invention In the preparation of induced pluripotent stem cells through reprogramming of somatic cell, an object of the present invention is to provide a means for efficiently preparing induced pluripotent stem cells. In particular, the object of the present invention is to provide a means for achieving an efficient preparation of induced pluripotent stem cells without using a suspected tumorigenic factor c-Myc.
Means to Solve the Problems The present inventors have conducted intensive studies to achieve the above objects. As a result, they have now found that induced pluripotent stem cells can be efficiently prepared by introduction of a nuclear reprogramming-inducing gene(s) into somatic cells in the presence of specific miRNA. The present invention was achieved on the basis of the above findings.
The present invention thus provides a method of preparing induced pluripotent stem cells, comprising a step of carrying out a nuclear reprogramming using a nuclear reprogramming factor(s) in the presence of miRNA, wherein said miRNA has a property of providing a higher nuclear reprogramming efficiency in the presence of said miRNA than in the absence of said miRNA.
A preferred embodiment of the present invention provides the aforementioned method wherein: (a) said miRNA is expressed in embryonic stem cells at a higher level than in somatic cells; and (b) said miRNA has a property of providing a higher nuclear reprogramming efficiency in the presence of said miRNA than in the absence of said miRNA.
Another preferred embodiment of the present invention provides: the aforementioned method wherein the nuclear reprogramming factor is either a single substance, or a combination of a plurality of substances, which is/are positive in the screening method of nuclear reprogramming factor described in International Publication No. W02005/80598; the aforementioned method wherein the nuclear reprogramming factor is either a gene product of a single gene, or a combination of gene products of a plurality of genes, which is/are positive in the screening method of nuclear reprogramming factor described in International Publication No.
W02005/80598; the aforementioned method wherein the nuclear reprogramming using the nuclear reprogramming factor is carried out by introduction of the aforementioned gene(s) into somatic cells; the aforementioned method wherein introduction of the aforementioned gene(s) into somatic cells is carried out with a recombinant vector; and the aforementioned method wherein the nuclear reprogramming using the nuclear reprogramming factor is carried out by introduction of gene product(s) of the aforementioned gene(s) into somatic cells.
Yet another preferred embodiment of the present invention provides the aforementioned method wherein: the gene encoding the reprogramming factor consists of one or more genes selected from the group consisting of an Oct family gene, a Klf family gene, a Sox family gene, a Myc family gene, a Lin family gene, and a Nanog gene, preferably a combination of two genes selected from the aforementioned genes except for the Myc family genes, more preferably a combination of three genes, and particularly preferably a combination four or more genes.
More preferable combinations are: (a) a combination of two genes consisting of an Oct family gene and a Sox family gene; (b) a combination of three genes consisting of an Oct family gene, a Klf family gene, and a Sox family gene;
and (c) a combination of four genes consisting of an Oct family gene, a Sox family gene, a Lin family gene, and a Nanog gene. Further, it is also preferable to combine with a TERT
gene and/or a SV40 Large T antigen gene. It may be preferable to omit Klf family genes optionally. The Myc family genes may be included in these combinations if required. However, combinations without the Myc family gene can be suitably used according-to the present invention.
Among these embodiments, particularly preferable combinations are: a combination of two genes consisting of Oct3/4 and Sox2; a combination of three genes consisting of Oct3/4, Klf4, and Sox2; and a combination of four genes consisting of Oct3/4, Sox2, Lin28, and Nanog. It is also preferable to combine with a TERT
gene and/or a SV40 Large T antigen gene therewith. It may be preferable to omit Klf4 optionally. c-Myc may be included in these combinations, if required. However, combinations without c-Myc can be suitably used in the present invention.
Yet another preferred embodiment of the present invention provides: the aforementioned method wherein the somatic cells are those from mammalian including human, preferably somatic cells from human or mouse, and most preferably somatic cells from human; the aforementioned method wherein the somatic cells are human embryonic cells, or adult human-derived somatic cells; and the aforementioned method wherein the somatic cells are somatic cells collected from a patient.
Yet another preferred embodiment of the present invention provides the aforementioned method wherein the miRNA is one or more miRNAs contained in the RNA sequences specified by the registration names on the miRBase database or the accession numbers shown in Table 1 or Table 2 below; the aforementioned method wherein the RNA sequences specified by the registration names on the miRBase database (and the accession numbers) shown in Table 1 or Table 2 below comprise one or more RNAs selected from the group consisting of hsa-miR-372 (MI0000780), hsa-miR-373 (MI0000781), hsa-miR-302b (MI0000772), hsa-miR-302c (MI0000773), hsa-miR-302a (MI0000738), hsa-miR-302d (MI0000774), hsa-miR-367 (MI0000775), hsa-miR-520c (MI0003158), mmu-miR-291a (MI0000389), mmu-miR-294 (MI0000392), and mmu-miR-295 (MI0000393); the aforementioned method wherein the miRNA is miRNAs contained in RNA specified by hsa-miR-302b-367; and the aforementioned method wherein the miRNA is one or more miRNAs contained in one or more RNA sequences selected from the RNA sequences shown in SEQ ID NOS:1 to 12 in the Sequence Listing.
In another aspect, the present invention provides induced pluripotent stem cells that can be obtained by the aforementioned method. In addition, the present invention also provides somatic cells obtained by inducing differentiation from the abovementioned induced pluripotent stem cells.
Further, the present invention provides a stem cell therapy comprising a step of transplanting somatic cells into a patient, wherein the somatic cells are obtained by inducing differentiation from induced pluripotent stem cells that are obtained according to the aforementioned method by using somatic cells isolated and collected from a patient.
In addition, the present invention provides a method for evaluation of physiological effect or toxicity of a compound, a drug, or a toxic agent, with use of various cells obtained by inducing differentiation from induced pluripotent stem cells that are obtained by the aforementioned method.
Further, the present invention provides: a method for preparing induced pluripotent stem cells which uses miRNA expressed in embryonic stem cells at a higher level than in somatic cells, and having a property of providing a higher nuclear reprogramming efficiency in the presence of said miRNA than in the absence thereof;
and a nuclear reprogramming method of somatic cells which uses miRNA expressed in embryonic stem cells at a higher level than in somatic cells, and having a property of providing a higher nuclear reprogramming efficiency in the presence of said miRNA
than in the absence thereof.
In addition, the present invention provides use of miRNA expressed in embryonic stem cells at a higher level than in somatic cells, and having a property of providing a higher nuclear reprogramming efficiency in the presence of said miRNA
than in the absence thereof, for preparation of induced pluripotent stem cells; and use of miRNA expressed in embryonic stem cells at a higher level than in somatic cells, and having a property of providing a higher nuclear reprogramming efficiency in the presence of said miRNA than in the absence of said miRNA, for nuclear reprogramming of somatic cells.
Brief Description of the Drawings Fig. 1 shows the results of confirmation on the production efficiency of induced pluripotent stem cells through induction of nuclear reprogramming in mouse embryonic fibroblasts with a combination of three genes consisting of Oct3/4, Klf4, and Sox2 (this combination is represented as "3fl', and "c-Myc(-)" means that c-Myc was omitted from a combination of four genes consisting of Oct3/4, Klf4, Sox2 and c-Myc, which is highly efficient for nuclear reprogramming), in the presence of each one type of miRNA. 3f+DsRed represents a combination where DsRed (Discosoma sp. red fluorescent protein) as a control was added to the combination of the aforementioned three genes. The table shows the number of colonies assuming that the number of colonies in the case of induction of pluripotent stem cells with 3f+DsRed is 100.
Fig. 2 shows the production efficiency of induced pluripotent stem cells. The top panels show the results when DsRed was added to the combination of three genes consisting of Oct3/4, Klf4, and Sox2 (a combination of three genes in which c-Myc was omitted from the combination of four genes) (Control). Bottom panels show the results of induction of nuclear reprogramming in mouse tail tip fibroblasts (TTFs) with the combination of three genes consisting of Oct3/4, Klf4, and Sox2 in the presence of mmu-miR-295. The number in the figure indicates the number of Nanog GFP
positive colonies/the number of total colonies.
Fig. 3 shows the results of confirmation on the production efficiency of induced pluripotent stem cells through induction of nuclear reprogramming in adult human dermal fibroblasts with the combination of three genes consisting of Oct3/4, Klf4, and Sox2 (Myc(-)3f: a combination of three genes in which c-Myc was omitted from the combination of four genes consisting of Oct3/4, Klf4, Sox2, and c-Myc), or the combination of four genes consisting of Oct3/4, Klf4, Sox2, and c-Myc (Y4f), in the presence of various miRNA.
Best Mode for Carrying Out the Invention The method of the present invention relates to a method for preparing induced pluripotent stem cells, comprising a step of carrying out a nuclear reprogramming using a nuclear reprogramming factor(s) in the presence of miRNA, wherein said miRNA has a property of providing a higher nuclear reprogramming efficiency in the presence of said miRNA than in the absence of said miRNA. In a preferred embodiment of the present invention, (a) said miRNA is expressed in embryonic stem cells at a higher level than in somatic cells; and (b) said miRNA has a property of providing a higher nuclear reprogramming efficiency in the presence of said miRNA
than in the absence of said miRNA.
As for the miRNA, for example, its classification and in vivo functions are described in Jikken Igaku (Experimental Medicine), 24, pp. 814-819, 2006;
microRNA
Jikken Purotokoru (microRNA Experimental Protocol), pp. 20-35, 2008, YODOSHA
CO., LTD. The number of nucleotides of miRNA is for example 18 to 25, and preferably about 19 to 23. At present, a database storing about 1,000 miRNA data is available (for example, miRBase, http://microrna.sanger.ac.uk/sequences/index.shtml), and it is possible for those skilled in the art to obtain any miRNA data therefrom, and to readily extract miRNA expressed in embryonic stem cells at a higher level than in somatic cells.
In addition, it is also possible to readily specify miRNA expressed in embryonic stem cells at a higher level than in somatic cells by confirming the difference in miRNA
expression between embryonic stem cells and somatic cells with use of available techniques for those skilled in the art such as miRNA microarray and real-time PCR
analyses.
The difference in the nuclear reprogramming efficiency between with and without miRNA can be understood by the following manner, as specifically described in Examples of this application: transgenic mice are generated by inserting the sequences encoding Enhanced Green Fluorescent Protein (EGFP) and puromycin resistance gene at a site downstream of the promoter region of the Nanog gene the expression of which is specific to ES cells; then, into embryonic fibroblasts from these transgenic mice are introduced, for example, three genes of Oct3/4, Sox2, and K1f4, and different miRNAs, to induce nuclear reprogramming, followed by confirmation of the production efficiency of induced pluripotent stem cells. The production efficiency can be determined, for example, by counting the number of colonies.
More specifically, the number of colonies can be compared by the following manner:
drug selection is started from day 21 after introduction of the above genes and miRNA; and the number of total colonies and the number of Nanog GFP positive colonies (GFP, the expression of which is induced by Nanog gene locus, is observable under fluorescent microscopy) are counted on day 28. It should be understood, however, that: the confirmation of the nuclear reprogramming efficiency is not limited to the above method; appropriate modification and alteration can be made in the above method; and any appropriate method can be employed by those skilled in the art.
As for the miRNA, it is preferable to use miRNA from the same animal species as the target animal whose somatic cells are to be reprogrammed. Usable miRNAs include wild type miRNAs as well as miRNAs in which one or several nucleotides (for example 1 to 6 nucleotides, preferably 1 to 4 nucleotides, more preferably 1 to 3 nucleotides, yet more preferably 1 or 2 nucleotides, and most preferably 1 nucleotide) are substituted, inserted, and/or deleted, and which are capable of exerting equivalent functions to those of the wild type miRNA in vivo.
Examples of the miRNA preferably used in the method of the present invention can include, but are not limited to, one or more miRNAs included in the following RNA sequences registered on the miRBase database: hsa-miR-372 (MI0000780), hsa-miR-373 (MI0000781), hsa-miR-302b (MI0000772), hsa-miR-302c (MI0000773), hsa-miR-302a (MI0000738), hsa-miR-302d (MI0000774), hsa-miR-367 (MI0000775), hsa-miR-520c (MI0003158), mmu-miR-291a (MI0000389), mmu-miR-294 (MI0000392), and mmu-miR-295 (MI0000393) (Numbers in the brackets respectively indicate miRBase accession numbers. The symbol "hsa-miR-" represents human miRNA, and the symbol "mmu-miR-" represents mouse miRNA.).
In the method of the present invention, miRNA that has been confirmed to improve the nuclear reprogramming efficiency in the above manner can be either solely used or jointly used in combination of two or more types. In addition, a plurality of miRNAs forming a cluster may also be used. For example, hsa-miR-302b-367 which is available as a RNA sequence including a miRNA
cluster, and the like may be used. Examples of RNA sequences for use of these miRNAs are shown in SEQ ID NOS:1 to 12 in the Sequence Listing. SEQ ID NO:1: mmu-mir-294 (MI0000392); SEQ ID NO:2: mmu-mir-295 (MI0000393); SEQ ID NO:3: hsa-mir-372 (MI0000780); SEQ ID NO:4: hsa-mir-373 (MI0000781); SEQ ID NO:5: hsa-mir-302b (MI0000772); SEQ ID NO:6: hsa-mir-302c (MI0000773); SEQ ID NO:7: hsa-mir-302a (MI0000738); SEQ ID NO:8: hsa-mir-302d (MI0000774); SEQ ID NO:9: hsa-mir-367 (MI0000775); SEQ ID NO:10: hsa-mir-520c (MI0003158); and SEQ ID NO:11:
mmu-mir-291a MI00003 89. In addition, RNA represented by SEQ ID NO:12 can also be preferably used. Among these RNA sequences, some RNA sequences may contain a plurality of miRNAs within one sequence. Use of such a RNA sequence may achieve efficient production of iPS cells. Further, a RNA sequence containing a plurality of miRNAs within one sequence and one or more other RNA sequences including one or more miRNAs can also be used in combination.
miRNA is non-coding RNA which is not translated into a protein. miRNA is first transcribed as pri-miRNA from a corresponding gene, then this pri-miRNA
generates pre-miRNA having a characteristic hairpin structure of about 70 nucleotides, and this pre-miRNA is further processed into mature miRNA, which is mediated by Dicer. In the present invention, not only mature miRNA but also pri-miRNA or pre-miRNA can be used as long as the effect of the present invention is not impaired.
In addition, miRNA for use in the present invention may be either natural type or non-natural type.
The production method of miRNA for use in the present invention is not specifically limited, and the production can be achieved, for example, by a chemical synthetic method or a method using genetic recombination technique. When the production is carried out by a method using genetic recombination technique, miRNA
for use in the present invention can be produced through transcription reaction with use of a DNA template and a RNA polymerase obtained by means of gene recombination. Examples of usable RNA polymerase include a T7 RNA polymerase, a T3 RNA polymerase, and a SP6 RNA polymerase.
Alternatively, a recombinant vector capable of expressing miRNA can be produced by insertion of miRNA-encoding DNA into an appropriate vector under the regulation of an expression control sequence (promoter, enhancer or the like).
The type of the vector used herein is not specifically limited, although DNA
vectors are preferred. Examples thereof can include viral vectors and plasmid vectors. The viral vector which can be used includes, but is not limited to, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, and the like. In addition, as to the above plasmids, mammalian expression plasmids well known to those skilled in the art can be employed.
In the nuclear reprogramming step, the means for having the presence of miRNA is not specifically limited, and examples thereof can include a method for directly injecting miRNA into nuclei in somatic cells, and a method for introducing an appropriate recombinant vector capable of expressing miRNA into somatic cells.
However, these methods are not to be considered as limiting.
The method for introducing a recombinant vector into somatic cells is not specifically limited, and can be carried out by a method well known to those skilled in the art. Examples of the employable method can include transient transfection, microinjection, a calcium phosphate precipitation method, liposome-mediated transfection, DEAE dextran-mediated transfection, electroporation, and a method using a gene gun.
As to the means for confirming a nuclear reprogramming factor, for example, the screening method of nuclear reprogramming factor described in International Publication No. W02005/80598 can be used. All the disclosures of the above publication are incorporated herein by reference. Those skilled in the art are able to screen a nuclear reprogramming factor for use in the method of the present invention by referring to the above publication. In addition, the nuclear reprogramming factor can also be confirmed by using a method in which appropriate modification or alteration has been made in the above screening method.
Examples of the combination of genes encoding reprogramming factors are disclosed in International Publication No. W02007/69666. All the disclosures of the above publication are incorporated herein by reference. Those skilled in the art are able to appropriately select a gene that can be preferably used for the method of the present invention by referring to the above publication. In addition, other examples of the combination of genes encoding reprogramming factors are disclosed, for example, in Science, 318, pp. 1917-1920, 2007. Accordingly, those skilled in the art are able to understand the variety of the combination of genes encoding reprogramming factors, and are able to employ an appropriate combination of genes in the method of the present invention, which combination is not disclosed in International Publication No. W02007/69666 or Science, 318, pp. 1917-1920, 2007, by using the screening method of nuclear reprogramming factor described in International Publication No.
W02005/80598.
Examples of the gene encoding a reprogramming factor that can be used for the method of the present invention can include: one or more genes selected from the group consisting of an Oct family gene, a Klf family gene, a Sox family gene, a Myc family gene, a Lin family gene, and a Nanog gene; preferably one or more genes selected from the group consisting of an Oct family gene, a Klf family gene, a Sox family gene, a Lin family gene, and a Nanog gene, and excluding a Myc family gene;
more preferably a combination of two genes; yet more preferably a combination of three genes; and most preferably a combination four genes.
Regarding the Oct family gene, Klf family gene, Sox family gene, and Myc family gene, specific examples of these family genes are described in International Publication No. W02007/69666. Regarding the Lin family gene, those skilled in the art are able to extract the family gene in a similar way.
In addition, reprogramming factors encoded by one or more genes selected from the group consisting of an Oct family gene, a Klf family gene, a Sox family gene, a Myc family gene, a Lin family gene, and a Nanog gene, may be substituted by, for example cytokine, or one or more other low molecular weight compounds in some cases. Examples of such low molecular weight compounds can include low molecular weight compounds having an enhancing action on the expression of one or more genes selected from the group consisting of an Oct family gene, a Klf family gene, a Sox family gene, a Myc family gene, a Lin family gene, and a Nanog gene.
Those skilled in the art are able to readily screen such low molecular weight compounds.
More preferable combinations of genes include, but are not limited to, the following (a) to (c):
(a) a combination of two genes consisting of an Oct family gene and a Sox family gene;
(b) a combination of three genes consisting of an Oct family gene, a Klf family gene, and a Sox family gene; and (c) a combination of four genes consisting of an Oct family gene, a Sox family gene, a Lin family gene, and a Nanog gene.
Any of these genes is commonly present in mammals including human. In order to use the above genes according to the present invention, genes from any mammal (for example, derived from a mammal such as human, mouse, rat, cattle, sheep, horse, and monkey) can be employed. In addition, it is also possible to use a wild type gene product, as well as mutant gene products in which several amino acids (for example 1 to 10 amino acids, preferably 1 to 6 amino acids, more preferably 1 to 4 amino acids, yet more preferably 1 to 3 amino acids, and most preferably 1 or 2 amino acids) have been substituted, inserted, and/or deleted, and which have equivalent functions to those of the wild type gene product. For example, as to the c-Myc gene product, a stable type (T58A) and the like may also be used as well as the wild type. The same principle can be applied to other gene products.
In addition to the above genes, a gene encoding a factor which induces immortalization of cells may also be combined. As disclosed in International Publication No. W02007/69666, for example, one or more genes selected from the group consisting of a TERT gene, and the following genes: SV40 Large T antigen, HPV16 E6, HPV16 E7, and Bmil, can be used solely or in combination where required.
Preferable combinations are as follows, for example:
(e) a combination of four genes consisting of an Oct family gene, a Kif family gene, a Sox family gene, and a TERT gene;
(f) a combination of four genes consisting of an Oct family gene, a Klf family gene, a Sox family gene, and a SV40 Large T antigen gene; and (g) a combination of five genes consisting of an Oct family gene, a Klf family gene, a Sox family gene, a TERT gene, and a SV40 Large T antigen gene.
The Klf family gene may be omitted from the above combinations where required.
Further, in addition to the above genes, one or more genes selected from the group consisting of Fbxl5, ERas, ECAT15-2, Tcll, and (3-catenin may be combined, and/or one or more genes selected from the group consisting of ECAT1, Esgl, Dnmt3L, ECAT8, Gdf3, Soxl5, ECAT15-1, Fth117, Sa114, Rexl, UTFl, Stella, Stat3, and Grb2 may also be combined. These combinations are specifically described in International Publication No.
W02007/69666.
Particularly preferable combinations of genes include, but are not limited to, the following (1) to (5):
(1) a combination of two genes consisting of Oct3/4 and Sox2;
(2) a combination of three genes consisting of Oct3/4, Klf4, and Sox2;
(3) a combination of four genes consisting of Oct3/4, Sox2, Lin28, and Nanog;
(4) a combination of four genes consisting of Oct3/4, Sox2, TERT, and SV40 Large T
antigen gene; and (5) a combination of five genes consisting of Oct3/4, K1f4, Sox2, TERT, and Large T antigen gene.
The factors including the gene products as mentioned above may also be combined with one or more gene products of genes selected from the group consisting of:
Fbxl5, Nanog, ERas, ECAT15-2, Tcll, and 0-catenin. Further, these factors may also be combined with one or more gene products of genes selected from the group consisting of:
ECAT1, Esgl, Dnmt3L, ECAT8, Gdf3, Soxl5, ECAT15-1, Fth117, Sal14, Rexl, UTF1, Stella, Stat3, and Grb2, for example. These gene products are disclosed in International Publication No. W02007/69666. However, gene products that can be included in the nuclear reprogramming factors of the present invention are not limited to the gene products of genes specifically described above. The nuclear reprogramming factors of the present invention can include other gene products which can function as a nuclear reprogramming factor, as well as one ore more factors involving differentiation, development, or proliferation, and factors having other physiological activities. It should be understood that the aforementioned aspect may also be included within the scope of the present invention.
Among these genes, if one or more gene products is/are already expressed in somatic cells to be reprogrammed, such gene products can be excluded from the factors to be introduced. For example, one or more genes besides the already-expressed genes can be introduced into somatic cells by an appropriate gene introduction method, for example, a method using a recombinant vector. Alternatively, among these genes, if one or more gene products is/are introduced into nuclei by a technique such as fusion protein or nuclear microinjection, the other one or more genes can be introduced by an appropriate gene introduction method, for example, a method using a recombinant vector.
In addition, a gene product serving as a nuclear reprogramming factor may be either a protein itself produced from the abovementioned gene, or in the form of a fusion gene product between such a protein and another protein, a peptide, or the like.
For example, a fusion protein having Green Fluorescent Protein (GFP) and a fusion gene product having a peptide such as a histidine tag may also be used. Further, use of a prepared fusion protein having a HIV virus-derived TAT peptide enables the promotion of endocytosis of a nuclear reprogramming factor through cell membrane, and also enables the induction of reprogramming by simply adding such a fusion protein into the medium while avoiding complicated manipulations such as gene introduction. The preparation method of the aforementioned fusion gene product is well known to those skilled in the art, and therefore those skilled in the art are able to readily design and prepare an appropriate fusion gene product according to the purpose.
As used herein, the term "induced pluripotent stem cells" (iPS cells) refers to cells having similar properties to those of ES cells, and more specifically the term includes undifferentiated cells which are reprogrammed from somatic cells and have pluripotency and proliferation potency. However, this term is not to be construed as limiting in any sense, and should be construed to have broadest meaning. The method of preparing induced pluripotent stem cells with use of a nuclear reprogramming factor is described in International Publication No. W02005/80598 (the term "ES-like cell" is used in this publication), and a means for isolating induced pluripotent stem cells is also specifically described therein. In addition, specific examples of the reprogramming factor and specific examples of the method of reprogramming somatic cells with use of such a reprogramming factor are disclosed in International Publication No. W02007/69666.
Accordingly, it is desirable for those skilled in the art to refer to these publications in carrying out the present invention.
The preparation method of induced pluripotent stem cells from somatic cells by the method of the present invention is not specifically limited, and any method can be employed as long as the method enables nuclear reprogramming of somatic cells with a nuclear reprogramming factor in the presence of miRNA in an environment where somatic cells and induced pluripotent stem cells can grow. For example, there may be employed a means in which a vector including a gene which can express a nuclear reprogramming factor is used to introduce such a gene into somatic cells, and at either the same or different timing, a recombinant vector which can express miRNA is introduced into the somatic cells. If such vectors are used, two or more genes may be incorporated into a vector to effect coexpression of respective gene products in somatic cells.
When gene(s) and/or miRNA are introduced into somatic cells with use of a vector which can express the above gene(s), the expression vector may be introduced into somatic cells that have been cultured on feeder cells, or the expression vector may also be introduced into somatic cells alone. The latter methods is sometimes more suitable in order to improve the introduction efficiency of the expression vector. As to the feeder cells, there may be appropriately used feeder cells for use in culture of embryonic stem cells. Examples thereof can include primary culture cells of 14 or 15 day-mouse embryonic fibroblasts and STO cells of fibroblast cell line, which are treated with either radiation or a drug such as mitomycin C.
The culture of somatic cells introduced with a nuclear reprogramming factor under appropriate conditions leads to autonomous nuclear reprogramming, and as a result, induced pluripotent stem cells can be produced from somatic cells. The process for introducing a gene encoding a nuclear reprogramming factor and/or miRNA into somatic cells with use of an express vector to thereby obtain induced pluripotent stem cells can be performed in accordance with, for example, a method using a retrovirus. Examples of such method include methods described in publications such as Cell, 126, pp. 1-14, 2006; Cell, 131, pp. 1-12, 2007; and Science, 318, pp. 1917-1920, 2007. When human induced pluripotent stem cells are produced, it is desirable to set the cell culture density after the introduction of an expression vector so as to become lower than normal cases of culturing animal cells. For example, it is preferable to keep culturing at a density of 1 to 1 x 105 cells/dish, and more preferably about 5 x 104 cells/dish. The medium for use in culture is not specifically limited, and can be appropriately selected by those skilled in the art. For example, it is sometimes preferable to use a medium suitable for human ES cell culture for the production of human induced pluripotent stem cells. The medium selection and culture condition can be referred to the above publications.
Thus produced induced pluripotent stem cells can be identified with various markers specific to undifferentiated cells, and the means therefor is described in the above-described publications specifically in detail. Various media capable of retaining undifferentiation property and pluripotency of ES cells and various media incapable of retaining these properties are known in the art, and appropriate combination of these media enables efficient isolation of induced pluripotent stem cells.
The differentiation ability and proliferation potency of thus isolated induced pluripotent stem cells can be readily determined by those skilled in the art, with use of general means for ES
Background of the Invention Embryonic stem cells (ES cells) are stem cells established from human or mouse early embryos which have a characteristic feature that they can be cultured over a long period of time while maintaining pluripotent ability to differentiate into all kinds of cells existing in living bodies. Human embryonic stem cells are expected for use as resources for cell transplantation therapies for various diseases such as Parkinson's disease, juvenile diabetes, and leukemia, taking advantage of the aforementioned properties. However, transplantation of ES cells has a problem of causing rejection in the same manner as organ transplantation. Moreover, from an ethical viewpoint, there are many dissenting opinions against the use of ES
cells which are established by destroying human embryos.
If dedifferentiation of patients' own differentiated somatic cells could be induced to establish cells having pluripotency and growth ability similar to those of ES
cells (in this specification, these cells are referred to as "induced pluripotent stem cells" or "iPS cells", though they are sometimes called "embryonic stem cell-like cells"
or "ES-like cells"), it is anticipated that such cells could be used as ideal pluripotent cells, free from rejection or ethical difficulties. Recently, it has been reported that such iPS cells can be produced from differentiated cells of mouse or human, which has created a great sensation (International Publication No. W02007/69666; Cell, 126, pp.
1-14, 2006; Cell, 131, pp. 1-12, 2007; Science, 318, pp. 1917-1920, 2007; and Nature, 451, pp. 141-146, 2008).
These methods include a reprogramming step through introduction of a plurality of specific factors (four factors of Oct3/4, Sox2, Klf4, and c-Myc are used in Cell, 126, pp.
1-14, 2006), and the introduction of these factors is mediated by viral vectors such as retrovirus or lentivirus. However, all previously reported nuclear reprogramming methods mediated by the gene introduction involve a problem of low efficiency in which only a small number of induced pluripotent stem cells can be obtained. In particular, there is a problem in that, if reprogramming is carried out in somatic cells through the introduction of three factors (namely, Oct3/4, Sox2, and K1f4), excluding c-Myc that is suspected to cause tumorigensis in tissues and individuals differentiated from resulting induced pluripotent stem cells, then the production efficiency of induced pluripotent stem cells becomes very low.
It is known that various small RNAs are expressed in cells. Examples of small RNA include RNA molecules of about 18-25 nucleotides long which can be cleaved out with a dicer, an RNase specific to double-stranded RNA. Small RNA is mainly classified into siRNA (small interfering RNA) and miRNA (microRNA, hereinafter abbreviated as "miRNA" in the specification). Small RNA is known to function as a guide molecule for finding out the target in a process such as translational suppression, mRNA
degradation, or alteration of chromatin structure, via RNA interference (RNAi)/miRNA molecular mechanism. In addition, small RNA is also known to play an important role in the regulation of developmental process (for example, as reviews, refer to Jikken Igaku (Experimental Medicine), 24, pp. 814-819, 2006; and microRNA Jikken Purotokoru (microRNA Experimental Protocol), pp. 20-35, 2008, YODOSHA CO., LTD.).
With regard to relation between embryonic stem cells (ES cells) and miRNA, ES
cell-specific expression of a microRNA cluster including several types of miRNAs in mouse ES cells has been reported (Developmental cell, 5, pp. 351-358, 2003), and it has also been reported that miRNA-295 suppressed the expression of Rbl2, a member of the Rb tumor suppressor gene family, and increased the expression of methylase to be thereby associated with DNA methylation (Nature Structural & Molecular Biology, 15, pp. 259-267, 2008;
Nature Structural & Molecular Biology, 15, pp. 268-279, 2008). However, it is yet to be known whether or not small RNA plays any role in the nuclear reprogramming of somatic cells.
References:
International Publication No. W02007/69666 Cell, 126, pp. 1-14, 2006 Cell, 131, pp. 1-12, 2007 Science, 318, pp. 1917-1920, 2007 Nature, 451, pp. 141-146, 2008 Developmental cell, 5, pp. 351-358, 2003 Nature Structural & Molecular Biology, 15, pp. 259-267, 2008 Nature Structural & Molecular Biology, 15, pp. 268-279, 2008 Disclosure of the Invention Problems to be Solved by the Invention In the preparation of induced pluripotent stem cells through reprogramming of somatic cell, an object of the present invention is to provide a means for efficiently preparing induced pluripotent stem cells. In particular, the object of the present invention is to provide a means for achieving an efficient preparation of induced pluripotent stem cells without using a suspected tumorigenic factor c-Myc.
Means to Solve the Problems The present inventors have conducted intensive studies to achieve the above objects. As a result, they have now found that induced pluripotent stem cells can be efficiently prepared by introduction of a nuclear reprogramming-inducing gene(s) into somatic cells in the presence of specific miRNA. The present invention was achieved on the basis of the above findings.
The present invention thus provides a method of preparing induced pluripotent stem cells, comprising a step of carrying out a nuclear reprogramming using a nuclear reprogramming factor(s) in the presence of miRNA, wherein said miRNA has a property of providing a higher nuclear reprogramming efficiency in the presence of said miRNA than in the absence of said miRNA.
A preferred embodiment of the present invention provides the aforementioned method wherein: (a) said miRNA is expressed in embryonic stem cells at a higher level than in somatic cells; and (b) said miRNA has a property of providing a higher nuclear reprogramming efficiency in the presence of said miRNA than in the absence of said miRNA.
Another preferred embodiment of the present invention provides: the aforementioned method wherein the nuclear reprogramming factor is either a single substance, or a combination of a plurality of substances, which is/are positive in the screening method of nuclear reprogramming factor described in International Publication No. W02005/80598; the aforementioned method wherein the nuclear reprogramming factor is either a gene product of a single gene, or a combination of gene products of a plurality of genes, which is/are positive in the screening method of nuclear reprogramming factor described in International Publication No.
W02005/80598; the aforementioned method wherein the nuclear reprogramming using the nuclear reprogramming factor is carried out by introduction of the aforementioned gene(s) into somatic cells; the aforementioned method wherein introduction of the aforementioned gene(s) into somatic cells is carried out with a recombinant vector; and the aforementioned method wherein the nuclear reprogramming using the nuclear reprogramming factor is carried out by introduction of gene product(s) of the aforementioned gene(s) into somatic cells.
Yet another preferred embodiment of the present invention provides the aforementioned method wherein: the gene encoding the reprogramming factor consists of one or more genes selected from the group consisting of an Oct family gene, a Klf family gene, a Sox family gene, a Myc family gene, a Lin family gene, and a Nanog gene, preferably a combination of two genes selected from the aforementioned genes except for the Myc family genes, more preferably a combination of three genes, and particularly preferably a combination four or more genes.
More preferable combinations are: (a) a combination of two genes consisting of an Oct family gene and a Sox family gene; (b) a combination of three genes consisting of an Oct family gene, a Klf family gene, and a Sox family gene;
and (c) a combination of four genes consisting of an Oct family gene, a Sox family gene, a Lin family gene, and a Nanog gene. Further, it is also preferable to combine with a TERT
gene and/or a SV40 Large T antigen gene. It may be preferable to omit Klf family genes optionally. The Myc family genes may be included in these combinations if required. However, combinations without the Myc family gene can be suitably used according-to the present invention.
Among these embodiments, particularly preferable combinations are: a combination of two genes consisting of Oct3/4 and Sox2; a combination of three genes consisting of Oct3/4, Klf4, and Sox2; and a combination of four genes consisting of Oct3/4, Sox2, Lin28, and Nanog. It is also preferable to combine with a TERT
gene and/or a SV40 Large T antigen gene therewith. It may be preferable to omit Klf4 optionally. c-Myc may be included in these combinations, if required. However, combinations without c-Myc can be suitably used in the present invention.
Yet another preferred embodiment of the present invention provides: the aforementioned method wherein the somatic cells are those from mammalian including human, preferably somatic cells from human or mouse, and most preferably somatic cells from human; the aforementioned method wherein the somatic cells are human embryonic cells, or adult human-derived somatic cells; and the aforementioned method wherein the somatic cells are somatic cells collected from a patient.
Yet another preferred embodiment of the present invention provides the aforementioned method wherein the miRNA is one or more miRNAs contained in the RNA sequences specified by the registration names on the miRBase database or the accession numbers shown in Table 1 or Table 2 below; the aforementioned method wherein the RNA sequences specified by the registration names on the miRBase database (and the accession numbers) shown in Table 1 or Table 2 below comprise one or more RNAs selected from the group consisting of hsa-miR-372 (MI0000780), hsa-miR-373 (MI0000781), hsa-miR-302b (MI0000772), hsa-miR-302c (MI0000773), hsa-miR-302a (MI0000738), hsa-miR-302d (MI0000774), hsa-miR-367 (MI0000775), hsa-miR-520c (MI0003158), mmu-miR-291a (MI0000389), mmu-miR-294 (MI0000392), and mmu-miR-295 (MI0000393); the aforementioned method wherein the miRNA is miRNAs contained in RNA specified by hsa-miR-302b-367; and the aforementioned method wherein the miRNA is one or more miRNAs contained in one or more RNA sequences selected from the RNA sequences shown in SEQ ID NOS:1 to 12 in the Sequence Listing.
In another aspect, the present invention provides induced pluripotent stem cells that can be obtained by the aforementioned method. In addition, the present invention also provides somatic cells obtained by inducing differentiation from the abovementioned induced pluripotent stem cells.
Further, the present invention provides a stem cell therapy comprising a step of transplanting somatic cells into a patient, wherein the somatic cells are obtained by inducing differentiation from induced pluripotent stem cells that are obtained according to the aforementioned method by using somatic cells isolated and collected from a patient.
In addition, the present invention provides a method for evaluation of physiological effect or toxicity of a compound, a drug, or a toxic agent, with use of various cells obtained by inducing differentiation from induced pluripotent stem cells that are obtained by the aforementioned method.
Further, the present invention provides: a method for preparing induced pluripotent stem cells which uses miRNA expressed in embryonic stem cells at a higher level than in somatic cells, and having a property of providing a higher nuclear reprogramming efficiency in the presence of said miRNA than in the absence thereof;
and a nuclear reprogramming method of somatic cells which uses miRNA expressed in embryonic stem cells at a higher level than in somatic cells, and having a property of providing a higher nuclear reprogramming efficiency in the presence of said miRNA
than in the absence thereof.
In addition, the present invention provides use of miRNA expressed in embryonic stem cells at a higher level than in somatic cells, and having a property of providing a higher nuclear reprogramming efficiency in the presence of said miRNA
than in the absence thereof, for preparation of induced pluripotent stem cells; and use of miRNA expressed in embryonic stem cells at a higher level than in somatic cells, and having a property of providing a higher nuclear reprogramming efficiency in the presence of said miRNA than in the absence of said miRNA, for nuclear reprogramming of somatic cells.
Brief Description of the Drawings Fig. 1 shows the results of confirmation on the production efficiency of induced pluripotent stem cells through induction of nuclear reprogramming in mouse embryonic fibroblasts with a combination of three genes consisting of Oct3/4, Klf4, and Sox2 (this combination is represented as "3fl', and "c-Myc(-)" means that c-Myc was omitted from a combination of four genes consisting of Oct3/4, Klf4, Sox2 and c-Myc, which is highly efficient for nuclear reprogramming), in the presence of each one type of miRNA. 3f+DsRed represents a combination where DsRed (Discosoma sp. red fluorescent protein) as a control was added to the combination of the aforementioned three genes. The table shows the number of colonies assuming that the number of colonies in the case of induction of pluripotent stem cells with 3f+DsRed is 100.
Fig. 2 shows the production efficiency of induced pluripotent stem cells. The top panels show the results when DsRed was added to the combination of three genes consisting of Oct3/4, Klf4, and Sox2 (a combination of three genes in which c-Myc was omitted from the combination of four genes) (Control). Bottom panels show the results of induction of nuclear reprogramming in mouse tail tip fibroblasts (TTFs) with the combination of three genes consisting of Oct3/4, Klf4, and Sox2 in the presence of mmu-miR-295. The number in the figure indicates the number of Nanog GFP
positive colonies/the number of total colonies.
Fig. 3 shows the results of confirmation on the production efficiency of induced pluripotent stem cells through induction of nuclear reprogramming in adult human dermal fibroblasts with the combination of three genes consisting of Oct3/4, Klf4, and Sox2 (Myc(-)3f: a combination of three genes in which c-Myc was omitted from the combination of four genes consisting of Oct3/4, Klf4, Sox2, and c-Myc), or the combination of four genes consisting of Oct3/4, Klf4, Sox2, and c-Myc (Y4f), in the presence of various miRNA.
Best Mode for Carrying Out the Invention The method of the present invention relates to a method for preparing induced pluripotent stem cells, comprising a step of carrying out a nuclear reprogramming using a nuclear reprogramming factor(s) in the presence of miRNA, wherein said miRNA has a property of providing a higher nuclear reprogramming efficiency in the presence of said miRNA than in the absence of said miRNA. In a preferred embodiment of the present invention, (a) said miRNA is expressed in embryonic stem cells at a higher level than in somatic cells; and (b) said miRNA has a property of providing a higher nuclear reprogramming efficiency in the presence of said miRNA
than in the absence of said miRNA.
As for the miRNA, for example, its classification and in vivo functions are described in Jikken Igaku (Experimental Medicine), 24, pp. 814-819, 2006;
microRNA
Jikken Purotokoru (microRNA Experimental Protocol), pp. 20-35, 2008, YODOSHA
CO., LTD. The number of nucleotides of miRNA is for example 18 to 25, and preferably about 19 to 23. At present, a database storing about 1,000 miRNA data is available (for example, miRBase, http://microrna.sanger.ac.uk/sequences/index.shtml), and it is possible for those skilled in the art to obtain any miRNA data therefrom, and to readily extract miRNA expressed in embryonic stem cells at a higher level than in somatic cells.
In addition, it is also possible to readily specify miRNA expressed in embryonic stem cells at a higher level than in somatic cells by confirming the difference in miRNA
expression between embryonic stem cells and somatic cells with use of available techniques for those skilled in the art such as miRNA microarray and real-time PCR
analyses.
The difference in the nuclear reprogramming efficiency between with and without miRNA can be understood by the following manner, as specifically described in Examples of this application: transgenic mice are generated by inserting the sequences encoding Enhanced Green Fluorescent Protein (EGFP) and puromycin resistance gene at a site downstream of the promoter region of the Nanog gene the expression of which is specific to ES cells; then, into embryonic fibroblasts from these transgenic mice are introduced, for example, three genes of Oct3/4, Sox2, and K1f4, and different miRNAs, to induce nuclear reprogramming, followed by confirmation of the production efficiency of induced pluripotent stem cells. The production efficiency can be determined, for example, by counting the number of colonies.
More specifically, the number of colonies can be compared by the following manner:
drug selection is started from day 21 after introduction of the above genes and miRNA; and the number of total colonies and the number of Nanog GFP positive colonies (GFP, the expression of which is induced by Nanog gene locus, is observable under fluorescent microscopy) are counted on day 28. It should be understood, however, that: the confirmation of the nuclear reprogramming efficiency is not limited to the above method; appropriate modification and alteration can be made in the above method; and any appropriate method can be employed by those skilled in the art.
As for the miRNA, it is preferable to use miRNA from the same animal species as the target animal whose somatic cells are to be reprogrammed. Usable miRNAs include wild type miRNAs as well as miRNAs in which one or several nucleotides (for example 1 to 6 nucleotides, preferably 1 to 4 nucleotides, more preferably 1 to 3 nucleotides, yet more preferably 1 or 2 nucleotides, and most preferably 1 nucleotide) are substituted, inserted, and/or deleted, and which are capable of exerting equivalent functions to those of the wild type miRNA in vivo.
Examples of the miRNA preferably used in the method of the present invention can include, but are not limited to, one or more miRNAs included in the following RNA sequences registered on the miRBase database: hsa-miR-372 (MI0000780), hsa-miR-373 (MI0000781), hsa-miR-302b (MI0000772), hsa-miR-302c (MI0000773), hsa-miR-302a (MI0000738), hsa-miR-302d (MI0000774), hsa-miR-367 (MI0000775), hsa-miR-520c (MI0003158), mmu-miR-291a (MI0000389), mmu-miR-294 (MI0000392), and mmu-miR-295 (MI0000393) (Numbers in the brackets respectively indicate miRBase accession numbers. The symbol "hsa-miR-" represents human miRNA, and the symbol "mmu-miR-" represents mouse miRNA.).
In the method of the present invention, miRNA that has been confirmed to improve the nuclear reprogramming efficiency in the above manner can be either solely used or jointly used in combination of two or more types. In addition, a plurality of miRNAs forming a cluster may also be used. For example, hsa-miR-302b-367 which is available as a RNA sequence including a miRNA
cluster, and the like may be used. Examples of RNA sequences for use of these miRNAs are shown in SEQ ID NOS:1 to 12 in the Sequence Listing. SEQ ID NO:1: mmu-mir-294 (MI0000392); SEQ ID NO:2: mmu-mir-295 (MI0000393); SEQ ID NO:3: hsa-mir-372 (MI0000780); SEQ ID NO:4: hsa-mir-373 (MI0000781); SEQ ID NO:5: hsa-mir-302b (MI0000772); SEQ ID NO:6: hsa-mir-302c (MI0000773); SEQ ID NO:7: hsa-mir-302a (MI0000738); SEQ ID NO:8: hsa-mir-302d (MI0000774); SEQ ID NO:9: hsa-mir-367 (MI0000775); SEQ ID NO:10: hsa-mir-520c (MI0003158); and SEQ ID NO:11:
mmu-mir-291a MI00003 89. In addition, RNA represented by SEQ ID NO:12 can also be preferably used. Among these RNA sequences, some RNA sequences may contain a plurality of miRNAs within one sequence. Use of such a RNA sequence may achieve efficient production of iPS cells. Further, a RNA sequence containing a plurality of miRNAs within one sequence and one or more other RNA sequences including one or more miRNAs can also be used in combination.
miRNA is non-coding RNA which is not translated into a protein. miRNA is first transcribed as pri-miRNA from a corresponding gene, then this pri-miRNA
generates pre-miRNA having a characteristic hairpin structure of about 70 nucleotides, and this pre-miRNA is further processed into mature miRNA, which is mediated by Dicer. In the present invention, not only mature miRNA but also pri-miRNA or pre-miRNA can be used as long as the effect of the present invention is not impaired.
In addition, miRNA for use in the present invention may be either natural type or non-natural type.
The production method of miRNA for use in the present invention is not specifically limited, and the production can be achieved, for example, by a chemical synthetic method or a method using genetic recombination technique. When the production is carried out by a method using genetic recombination technique, miRNA
for use in the present invention can be produced through transcription reaction with use of a DNA template and a RNA polymerase obtained by means of gene recombination. Examples of usable RNA polymerase include a T7 RNA polymerase, a T3 RNA polymerase, and a SP6 RNA polymerase.
Alternatively, a recombinant vector capable of expressing miRNA can be produced by insertion of miRNA-encoding DNA into an appropriate vector under the regulation of an expression control sequence (promoter, enhancer or the like).
The type of the vector used herein is not specifically limited, although DNA
vectors are preferred. Examples thereof can include viral vectors and plasmid vectors. The viral vector which can be used includes, but is not limited to, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, and the like. In addition, as to the above plasmids, mammalian expression plasmids well known to those skilled in the art can be employed.
In the nuclear reprogramming step, the means for having the presence of miRNA is not specifically limited, and examples thereof can include a method for directly injecting miRNA into nuclei in somatic cells, and a method for introducing an appropriate recombinant vector capable of expressing miRNA into somatic cells.
However, these methods are not to be considered as limiting.
The method for introducing a recombinant vector into somatic cells is not specifically limited, and can be carried out by a method well known to those skilled in the art. Examples of the employable method can include transient transfection, microinjection, a calcium phosphate precipitation method, liposome-mediated transfection, DEAE dextran-mediated transfection, electroporation, and a method using a gene gun.
As to the means for confirming a nuclear reprogramming factor, for example, the screening method of nuclear reprogramming factor described in International Publication No. W02005/80598 can be used. All the disclosures of the above publication are incorporated herein by reference. Those skilled in the art are able to screen a nuclear reprogramming factor for use in the method of the present invention by referring to the above publication. In addition, the nuclear reprogramming factor can also be confirmed by using a method in which appropriate modification or alteration has been made in the above screening method.
Examples of the combination of genes encoding reprogramming factors are disclosed in International Publication No. W02007/69666. All the disclosures of the above publication are incorporated herein by reference. Those skilled in the art are able to appropriately select a gene that can be preferably used for the method of the present invention by referring to the above publication. In addition, other examples of the combination of genes encoding reprogramming factors are disclosed, for example, in Science, 318, pp. 1917-1920, 2007. Accordingly, those skilled in the art are able to understand the variety of the combination of genes encoding reprogramming factors, and are able to employ an appropriate combination of genes in the method of the present invention, which combination is not disclosed in International Publication No. W02007/69666 or Science, 318, pp. 1917-1920, 2007, by using the screening method of nuclear reprogramming factor described in International Publication No.
W02005/80598.
Examples of the gene encoding a reprogramming factor that can be used for the method of the present invention can include: one or more genes selected from the group consisting of an Oct family gene, a Klf family gene, a Sox family gene, a Myc family gene, a Lin family gene, and a Nanog gene; preferably one or more genes selected from the group consisting of an Oct family gene, a Klf family gene, a Sox family gene, a Lin family gene, and a Nanog gene, and excluding a Myc family gene;
more preferably a combination of two genes; yet more preferably a combination of three genes; and most preferably a combination four genes.
Regarding the Oct family gene, Klf family gene, Sox family gene, and Myc family gene, specific examples of these family genes are described in International Publication No. W02007/69666. Regarding the Lin family gene, those skilled in the art are able to extract the family gene in a similar way.
In addition, reprogramming factors encoded by one or more genes selected from the group consisting of an Oct family gene, a Klf family gene, a Sox family gene, a Myc family gene, a Lin family gene, and a Nanog gene, may be substituted by, for example cytokine, or one or more other low molecular weight compounds in some cases. Examples of such low molecular weight compounds can include low molecular weight compounds having an enhancing action on the expression of one or more genes selected from the group consisting of an Oct family gene, a Klf family gene, a Sox family gene, a Myc family gene, a Lin family gene, and a Nanog gene.
Those skilled in the art are able to readily screen such low molecular weight compounds.
More preferable combinations of genes include, but are not limited to, the following (a) to (c):
(a) a combination of two genes consisting of an Oct family gene and a Sox family gene;
(b) a combination of three genes consisting of an Oct family gene, a Klf family gene, and a Sox family gene; and (c) a combination of four genes consisting of an Oct family gene, a Sox family gene, a Lin family gene, and a Nanog gene.
Any of these genes is commonly present in mammals including human. In order to use the above genes according to the present invention, genes from any mammal (for example, derived from a mammal such as human, mouse, rat, cattle, sheep, horse, and monkey) can be employed. In addition, it is also possible to use a wild type gene product, as well as mutant gene products in which several amino acids (for example 1 to 10 amino acids, preferably 1 to 6 amino acids, more preferably 1 to 4 amino acids, yet more preferably 1 to 3 amino acids, and most preferably 1 or 2 amino acids) have been substituted, inserted, and/or deleted, and which have equivalent functions to those of the wild type gene product. For example, as to the c-Myc gene product, a stable type (T58A) and the like may also be used as well as the wild type. The same principle can be applied to other gene products.
In addition to the above genes, a gene encoding a factor which induces immortalization of cells may also be combined. As disclosed in International Publication No. W02007/69666, for example, one or more genes selected from the group consisting of a TERT gene, and the following genes: SV40 Large T antigen, HPV16 E6, HPV16 E7, and Bmil, can be used solely or in combination where required.
Preferable combinations are as follows, for example:
(e) a combination of four genes consisting of an Oct family gene, a Kif family gene, a Sox family gene, and a TERT gene;
(f) a combination of four genes consisting of an Oct family gene, a Klf family gene, a Sox family gene, and a SV40 Large T antigen gene; and (g) a combination of five genes consisting of an Oct family gene, a Klf family gene, a Sox family gene, a TERT gene, and a SV40 Large T antigen gene.
The Klf family gene may be omitted from the above combinations where required.
Further, in addition to the above genes, one or more genes selected from the group consisting of Fbxl5, ERas, ECAT15-2, Tcll, and (3-catenin may be combined, and/or one or more genes selected from the group consisting of ECAT1, Esgl, Dnmt3L, ECAT8, Gdf3, Soxl5, ECAT15-1, Fth117, Sa114, Rexl, UTFl, Stella, Stat3, and Grb2 may also be combined. These combinations are specifically described in International Publication No.
W02007/69666.
Particularly preferable combinations of genes include, but are not limited to, the following (1) to (5):
(1) a combination of two genes consisting of Oct3/4 and Sox2;
(2) a combination of three genes consisting of Oct3/4, Klf4, and Sox2;
(3) a combination of four genes consisting of Oct3/4, Sox2, Lin28, and Nanog;
(4) a combination of four genes consisting of Oct3/4, Sox2, TERT, and SV40 Large T
antigen gene; and (5) a combination of five genes consisting of Oct3/4, K1f4, Sox2, TERT, and Large T antigen gene.
The factors including the gene products as mentioned above may also be combined with one or more gene products of genes selected from the group consisting of:
Fbxl5, Nanog, ERas, ECAT15-2, Tcll, and 0-catenin. Further, these factors may also be combined with one or more gene products of genes selected from the group consisting of:
ECAT1, Esgl, Dnmt3L, ECAT8, Gdf3, Soxl5, ECAT15-1, Fth117, Sal14, Rexl, UTF1, Stella, Stat3, and Grb2, for example. These gene products are disclosed in International Publication No. W02007/69666. However, gene products that can be included in the nuclear reprogramming factors of the present invention are not limited to the gene products of genes specifically described above. The nuclear reprogramming factors of the present invention can include other gene products which can function as a nuclear reprogramming factor, as well as one ore more factors involving differentiation, development, or proliferation, and factors having other physiological activities. It should be understood that the aforementioned aspect may also be included within the scope of the present invention.
Among these genes, if one or more gene products is/are already expressed in somatic cells to be reprogrammed, such gene products can be excluded from the factors to be introduced. For example, one or more genes besides the already-expressed genes can be introduced into somatic cells by an appropriate gene introduction method, for example, a method using a recombinant vector. Alternatively, among these genes, if one or more gene products is/are introduced into nuclei by a technique such as fusion protein or nuclear microinjection, the other one or more genes can be introduced by an appropriate gene introduction method, for example, a method using a recombinant vector.
In addition, a gene product serving as a nuclear reprogramming factor may be either a protein itself produced from the abovementioned gene, or in the form of a fusion gene product between such a protein and another protein, a peptide, or the like.
For example, a fusion protein having Green Fluorescent Protein (GFP) and a fusion gene product having a peptide such as a histidine tag may also be used. Further, use of a prepared fusion protein having a HIV virus-derived TAT peptide enables the promotion of endocytosis of a nuclear reprogramming factor through cell membrane, and also enables the induction of reprogramming by simply adding such a fusion protein into the medium while avoiding complicated manipulations such as gene introduction. The preparation method of the aforementioned fusion gene product is well known to those skilled in the art, and therefore those skilled in the art are able to readily design and prepare an appropriate fusion gene product according to the purpose.
As used herein, the term "induced pluripotent stem cells" (iPS cells) refers to cells having similar properties to those of ES cells, and more specifically the term includes undifferentiated cells which are reprogrammed from somatic cells and have pluripotency and proliferation potency. However, this term is not to be construed as limiting in any sense, and should be construed to have broadest meaning. The method of preparing induced pluripotent stem cells with use of a nuclear reprogramming factor is described in International Publication No. W02005/80598 (the term "ES-like cell" is used in this publication), and a means for isolating induced pluripotent stem cells is also specifically described therein. In addition, specific examples of the reprogramming factor and specific examples of the method of reprogramming somatic cells with use of such a reprogramming factor are disclosed in International Publication No. W02007/69666.
Accordingly, it is desirable for those skilled in the art to refer to these publications in carrying out the present invention.
The preparation method of induced pluripotent stem cells from somatic cells by the method of the present invention is not specifically limited, and any method can be employed as long as the method enables nuclear reprogramming of somatic cells with a nuclear reprogramming factor in the presence of miRNA in an environment where somatic cells and induced pluripotent stem cells can grow. For example, there may be employed a means in which a vector including a gene which can express a nuclear reprogramming factor is used to introduce such a gene into somatic cells, and at either the same or different timing, a recombinant vector which can express miRNA is introduced into the somatic cells. If such vectors are used, two or more genes may be incorporated into a vector to effect coexpression of respective gene products in somatic cells.
When gene(s) and/or miRNA are introduced into somatic cells with use of a vector which can express the above gene(s), the expression vector may be introduced into somatic cells that have been cultured on feeder cells, or the expression vector may also be introduced into somatic cells alone. The latter methods is sometimes more suitable in order to improve the introduction efficiency of the expression vector. As to the feeder cells, there may be appropriately used feeder cells for use in culture of embryonic stem cells. Examples thereof can include primary culture cells of 14 or 15 day-mouse embryonic fibroblasts and STO cells of fibroblast cell line, which are treated with either radiation or a drug such as mitomycin C.
The culture of somatic cells introduced with a nuclear reprogramming factor under appropriate conditions leads to autonomous nuclear reprogramming, and as a result, induced pluripotent stem cells can be produced from somatic cells. The process for introducing a gene encoding a nuclear reprogramming factor and/or miRNA into somatic cells with use of an express vector to thereby obtain induced pluripotent stem cells can be performed in accordance with, for example, a method using a retrovirus. Examples of such method include methods described in publications such as Cell, 126, pp. 1-14, 2006; Cell, 131, pp. 1-12, 2007; and Science, 318, pp. 1917-1920, 2007. When human induced pluripotent stem cells are produced, it is desirable to set the cell culture density after the introduction of an expression vector so as to become lower than normal cases of culturing animal cells. For example, it is preferable to keep culturing at a density of 1 to 1 x 105 cells/dish, and more preferably about 5 x 104 cells/dish. The medium for use in culture is not specifically limited, and can be appropriately selected by those skilled in the art. For example, it is sometimes preferable to use a medium suitable for human ES cell culture for the production of human induced pluripotent stem cells. The medium selection and culture condition can be referred to the above publications.
Thus produced induced pluripotent stem cells can be identified with various markers specific to undifferentiated cells, and the means therefor is described in the above-described publications specifically in detail. Various media capable of retaining undifferentiation property and pluripotency of ES cells and various media incapable of retaining these properties are known in the art, and appropriate combination of these media enables efficient isolation of induced pluripotent stem cells.
The differentiation ability and proliferation potency of thus isolated induced pluripotent stem cells can be readily determined by those skilled in the art, with use of general means for ES
cells. In addition, colonies of induced pluripotent stem cells can be obtained by growing thus produced induced pluripotent stem cells under appropriate conditions, and the presence of these induced pluripotent stem cells can be determined on the basis of the shape of their colonies. For example, it is known that mouse induced pluripotent stem cells form raised colonies, while human induced pluripotent stem cells form flat colonies. These colony shapes are respectively very similar to those of mouse ES cells and human ES cells, and those skilled in the art are thus able to identify these produced induced pluripotent stem cells with reference to the shape of their colonies.
The type of somatic cell to be reprogrammed by the method of the present invention is not specifically limited, and any somatic cell can be used. For example, somatic cells from any mammal (for example, from a mammal such as human, mouse, rat, cattle, sheep, horse, and monkey) can be employed. Not only embryonic somatic cells but also neonatal somatic cells, matured somatic cell, and tissue stem cells may also be used.
In addition, various somatic cells such as skin cells, liver cells, and gastric mucosa cells can be reprogrammed. For use of induced pluripotent stem cells in therapies against diseases, it is desirable to use somatic cells isolated from the patient. For example, somatic cells involved in a disease and somatic cells associated with a therapy for a disease can be used.
The application of induced pluripotent stem cells produced by the method of the present invention is not specifically limited, and these cells can be used for any tested and studies to be performed with use of ES cells, and for the therapy of diseases with use of ES
cells. For example, induced pluripotent stem cells obtained by the method of the present invention can be induced into desired differentiated cells (such as nerve cells, myocardial cells, and blood cells) by treatment with retinoic acid, a growth factor such as EGF, or glucocorticoid, so that an appropriate tissue can be formed. Stem cell therapies through autologous cell transplantation can be achieved by returning these differentiated cells or tissue obtained in the above manner, into the patient. However, the application of the induced pluripotent stem cells of the present invention is not to be limited to the abovementioned specific aspects.
Examples The present invention will be described more specifically with reference to the following examples. However, the scope of the present invention is not limited to these examples.
The type of somatic cell to be reprogrammed by the method of the present invention is not specifically limited, and any somatic cell can be used. For example, somatic cells from any mammal (for example, from a mammal such as human, mouse, rat, cattle, sheep, horse, and monkey) can be employed. Not only embryonic somatic cells but also neonatal somatic cells, matured somatic cell, and tissue stem cells may also be used.
In addition, various somatic cells such as skin cells, liver cells, and gastric mucosa cells can be reprogrammed. For use of induced pluripotent stem cells in therapies against diseases, it is desirable to use somatic cells isolated from the patient. For example, somatic cells involved in a disease and somatic cells associated with a therapy for a disease can be used.
The application of induced pluripotent stem cells produced by the method of the present invention is not specifically limited, and these cells can be used for any tested and studies to be performed with use of ES cells, and for the therapy of diseases with use of ES
cells. For example, induced pluripotent stem cells obtained by the method of the present invention can be induced into desired differentiated cells (such as nerve cells, myocardial cells, and blood cells) by treatment with retinoic acid, a growth factor such as EGF, or glucocorticoid, so that an appropriate tissue can be formed. Stem cell therapies through autologous cell transplantation can be achieved by returning these differentiated cells or tissue obtained in the above manner, into the patient. However, the application of the induced pluripotent stem cells of the present invention is not to be limited to the abovementioned specific aspects.
Examples The present invention will be described more specifically with reference to the following examples. However, the scope of the present invention is not limited to these examples.
Example 1: Preparation of induced pluripotent stem cells through nuclear reprogramming of mouse embryonic fibroblasts pMXs-based retroviral vectors, which respectively encode each of three genes of mouse-derived Oct3/4, Sox2 and Klf4, control DsRed or each miRNA of 18 types of miRNAs, were transfected into PLAT-E cells using FuGENE 6 reagent (Roche). On the next day, embryonic fibroblasts (Nanog GFP MEF, W02007/69666) from transgenic mice generated by inserting the sequences encoding EGFP gene and puromycin resistance gene at a site downstream of Nanog gene promoter region, were seeded at 1 x 105 cells/well in 6-well plates. On the next day, these cells were infected with retroviruses expressing Oct3/4, Sox2, Klf4, and each type of miRNA
selected from 18 types of miRNAs, at a ratio of 1 ml of virus mixture expressing these three factors to 1 ml of virus solution expressing miRNA, so as to prepare induced pluripotent stem cells through nuclear reprogramming.
Table 1 miRNA number miRNA sequence*) 1 mmu-miR-150(MI0000172) 2 mmu-miR-182(MI0000224) 3 mmu-miR-126(MI0000153) 4 mmu-miR-290-295 mmu-miR-290 (MI0000388) 6 mmu-miR-291a (MI0000389) 7 mmu-miR-292 (MI0000390) 8 mmu-miR-294 (MI0000392) X(9) mmu-miR-295 (MI0000393) mmu-miR-17-92 11 mmu-miR-323 (MI0000592) 12 mmu-miR-130b(MI0000408) 13 mmu-miR-7a-1 (MI0000728) 14 mmu-miR-7a-2 (MI0000729) mmu-miR-205 (MI0000248) 16 mmu-miR-200a (MI0000554) 17 mmu-miR-200c (MI0000694) 18 mmu-miR-mix Numbers in the brackets indicate the accession numbers of miRBase.
From day 3 after infection, the cells were cultured in an ES cell medium containing LIF. On day 4 after infection, the cells were peeled off and the whole amount thereof was reseeded onto feeder cells. Every other day thereafter, the ES
cell medium containing LIF was replaced. From day 21 after infection, drug selection was started with addition of puromycin at a final concentration of 1.5 g/ml.
On day 28, the number of Nanog GFP positive colonies (GFP, the expression of which is induced by Nanog gene locus, can be observed under fluorescent microscopy) was counted. As a control, DsRed was used in place of miRNA. The results are shown in Fig. 1. It was found that mmu-miR-294 and mmu-miR-295 could respectively improve the nuclear reprogramming efficiency when introduced into mouse embryonic fibroblasts together with three factors of Oct3/4, Sox2, and Klf4, and enabled efficient preparation of induced pluripotent stem cells.
Example 2: Preparation of induced pluripotent stem cells through nuclear reprogramming of mouse tail tip fibroblasts pMXs-based retroviral vectors, which respectively encode each of three genes of mouse-derived Oct3/4, Sox2 and K1f4, DsRed (control), or mmu-miR-295, were transfected into PLAT-E cells using FuGENE 6 reagent (Roche). On the next day, tail tip fibroblasts (Nanog GFP tailtip fibroblasts) from transgenic mice generated by inserting the sequences encoding EGFP gene and puromycin resistance gene at a site downstream of Nanog gene promoter region, were seeded at 1 x 105 cells/well in 6-well plates. On the next day, these cells were infected with retroviruses expressing three factors of Oct3/4, Sox2, and Klf4, and either DsRed or mmu-miR-295, at a ratio of 1:1:1:1, so as to prepare induced pluripotent stem cells through nuclear reprogramming.
From day 3 after infection, the cells were cultured in an ES cell medium containing LIF. On day 4 after infection, the cells were peeled off and the whole amount thereof was reseeded onto feeder cells. Every other day thereafter, the ES
cell medium containing LIF was replaced. From day 7, day 21, or day 28 after infection, drug selection was started with addition of puromycin at a final concentration of 1.5 g/ml. On day 39, the number of total colonies and the number of Nanog GFP positive colonies (GFP, the expression of which is induced by Nanog gene locus, can be observed under fluorescent microscopy) were counted. The results are shown in Fig. 2. It was found that mmu-miR-295 could improve the nuclear reprogramming efficiency when introduced into mouse tail tip fibroblasts together with three factors of Oct3/4, Sox2, and Klf4, and could accelerate the reprogramming speed and enabled efficient preparation of induced pluripotent stem cells.
Example 3: Preparation of induced pluripotent stem cells through nuclear reprogramming of adult human dermal fibroblasts pMXs-based retroviral vectors, which encode three genes of human-derived Oct3/4, Sox2 and Klf4, and control DsRed or either each of 23 types of miRNAs or an miRNA cluster, were transfected into PLAT-E cells using FuGENE 6 reagent (Roche).
On the next day, adult human dermal fibroblasts which were generated to express a rodent ecotropic virus receptor Slc7al, were seeded at 3 x 105 cells/well in 6-cm dishes.
On the next day, the cells were infected with retroviruses expressing three genes of Oct3/4, Sox2, Klf4, and various types of miRNAs, at a ratio of 1:1:11, so as to produce induced pluripotent stem cells through nuclear reprogramming.
Table 2 miRNA number miRNA sequence 1 hsa-miR-371 (MI0000779) 2 hsa-miR-372 (MI0000780) 3 hsa-miR-373 (MI0000781) 4 hsa-miR-371-373 hsa-miR-93 (MI0000095) 6 hsa-miR-302a (M 10000738) 7 hsa-miR-302b (MI0000772) 8 hsa-miR-302c (MI0000773) 9 hsa-miR-302d (MI0000774) hsa-miR-367 (MI0000775 ) 11 hsa-miR-302b-367 12 hsa-miR-520a (MI0003149) 13 hsa-miR-520b (MI0003155) 14 hsa-miR-520c (MI0003158) hsa-miR-520d (MI0003164) 16 hsa-miR-520e (MI0003143) 17 mmu-miR-290-295 18 mmu-miR-290 (MI0000388) 19 mmu-miR-291a (MI0000389) 20 mmu-miR-292 (MI0000390) 21 mmu-miR-293 (MI0000391) 22 mmu-miR-294 (MI0000392) 23 mmu-miR-295 (MI0000393) On day 6 after infection, the cells were peeled off and the whole amount of 5 x 105 cells was reseeded onto feeder cells. Every other day thereafter, human ES
cell medium containing bFGF (ReproCELL medium) was replaced. On day 24, day 32, and day 40, the number of total colonies and the number of colonies having morphology of human ES-like cells were counted. As a control, DsRed was used in place of miRNA. The results are shown in Fig. 3. It was found that the number of colonies of induced pluripotent stem cells increased twice or more, as compared to the control, by introduction of three genes in the presence of hsa-miR-372, 373, 302b, 302b-367 (including 302b, 302c, 302a, 302d, and 367), 520c, mmu-miR-291a, 294, or 295.
Industrial Applicability The present invention provides an efficient method for preparing induced pluripotent stem cells. The method of the present invention has higher nuclear reprogramming efficiency as compared to conventional methods. For example, safe induced pluripotent stem cells can be efficiently produced without using c-Myc or gene product thereof Accordingly, the method of the present invention enables efficient production of highly safe induced pluripotent stem cells from patient's own somatic cells.
Cells differentiated from such pluripotent stem cells (for example, myocardial cells, insulin-producing cells, or nerve cells) will be able to be safely utilized in stem cell transplantation therapies for treatment of various diseases, such as heart failure, insulin dependent diabetes mellitus, Parkinson's diseases, and spinal cord injury.
SEQUENCE LISTING
<110> Kyoto University <120> Efficient Method for Nuclear Reprogramming <130> PH-3821-PCT
<140> PCT/JP2008/059586 <141> 2008-05-23 <150> US 60/996,893 <151> 2007-12-10 <160> 12 <170> PatentIn version 3.1 <210> 1 <211> 84 <212> RNA
<213> Mus musculus <400> 1 uuccauauag ccauacucaa aauggaggcc cuaucuaagc uuuuaagugg aaagugcuuc 60 ccuuuugugu guugccaugu ggag 84 <210> 2 <211> 69 <212> RNA
<213> Mus musculus <400> 2 ggugagacuc aaaugugggg cacacuucug gacuguacau agaaagugcu acuacuuuug 60 agucucucc 69 <210> 3 <211> 67 <212> RNA
<213> Homo sapiens <400> 3 gugggccuca aauguggagc acuauucuga uguccaagug gaaagugcug cgacauuuga 60 gcgucac 67 <210> 4 <211> 69 <212> RNA
<213> Homo sapiens <400> 4 gggauacuca aaaugggggc gcuuuccuuu uugucuguac ugggaagugc uucgauuuug 60 ggguguccc 69 <210> 5 <211> 73 <212> RNA
<213> Homo sapiens <400> 5 gcucccuuca acuuuaacau ggaagugcuu ucugugacuu uaaaaguaag ugcuuccaug 60 uuuuaguagg agu 73 <210> 6 <211> 68 <212> RNA
<213> Homo sapiens <400> 6 ccuuugcuuu aacauggggg uaccugcugu gugaaacaaa aguaagugcu uccauguuuc 60 aguggagg 68 <210> 7 <211> 69 <212> RNA
<213> Homo sapiens <400> 7 ccaccacuua aacguggaug uacuugcuuu gaaacuaaag aaguaagugc uuccauguuu 60 uggugaugg 69 <210> 8 <211> 68 <212> RNA
<213> Homo sapiens <400> 8 ccucuacuuu aacauggagg cacuugcugu gacaugacaa aaauaagugc uuccauguuu 60 gagugugg 68 <210> 9 <211> 68 <212> RNA
<213> Homo sapiens <400> 9 ccauuacugu ugcuaauaug caacucuguu gaauauaaau uggaauugca cuuuagcaau 60 ggugaugg 68 <210> 10 <211> 87 <212> RNA
<213> Homo sapiens <400> 10 ucucaggcug ucguccucua gagggaagca cuuucuguug ucugaaagaa aagaaagugc 60 uuccuuuuag aggguuaccg uuugaga 87 <210> 11 <211> 82 <212> RNA
<213> Mus musculus <400> 11 ccuauguagc ggccaucaaa guggaggccc ucucuugagc cugaaugaga aagugcuucc 60 acuuugugug ccacugcaug gg 82 <210> 12 <211> 820 <212> RNA
<213> Homo sapiens <400> 12 guuuucuuuc uccucagcuc uaaauacucu gaaguccaaa gaaguuguau guuggguggg 60 cucccuucaa cuuuaacaug gaagugcuuu cugugacuuu aaaaguaagu gcuuccaugu 120 uuuaguagga gugaauccaa uuuacuucuc caaaauagaa cacgcuaacc ucauuugaag 180 ggauccccuu ugcuuuaaca uggggguacc ugcuguguga aacaaaagua agugcuucca 240 uguuucagug gaggugucuc caagccagca caccuuuugu uacaaaauuu uuuuguuauu 300 guguuuuaag guuacuaagc uuguuacagg uuaaaggauu cuaacuuuuu ccaagacugg 360 gcuccccacc acuuaaacgu ggauguacuu gcuuugaaac uaaagaagua agugcuucca 420 uguuuuggug augguaaguc uuccuuuuac auuuuuauua uuuuuuuaga aaauaacuuu 480 auuguauuga ccgcagcuca uauauuuaag cuuuauuuug uauuuuuaca ucuguuaagg 540 ggcccccucu acuuuaacau ggaggcacuu gcugugacau gacaaaaaua agugcuucca 600 uguuugagug uggugguucc uaccuaauca gcaauugcgu uaacgcccac acugugugca 660 guucuuggcu acaggccauu acuguugcua auaugcaacu cuguugaaua uaaauuggaa 720 uugcacuuua gcaaugguga uggauuguua agccaaugac agaauuuaaa ccacagacuu 780 acuuugauag cacucuuaau gguauaacuu cuucucccau 820
selected from 18 types of miRNAs, at a ratio of 1 ml of virus mixture expressing these three factors to 1 ml of virus solution expressing miRNA, so as to prepare induced pluripotent stem cells through nuclear reprogramming.
Table 1 miRNA number miRNA sequence*) 1 mmu-miR-150(MI0000172) 2 mmu-miR-182(MI0000224) 3 mmu-miR-126(MI0000153) 4 mmu-miR-290-295 mmu-miR-290 (MI0000388) 6 mmu-miR-291a (MI0000389) 7 mmu-miR-292 (MI0000390) 8 mmu-miR-294 (MI0000392) X(9) mmu-miR-295 (MI0000393) mmu-miR-17-92 11 mmu-miR-323 (MI0000592) 12 mmu-miR-130b(MI0000408) 13 mmu-miR-7a-1 (MI0000728) 14 mmu-miR-7a-2 (MI0000729) mmu-miR-205 (MI0000248) 16 mmu-miR-200a (MI0000554) 17 mmu-miR-200c (MI0000694) 18 mmu-miR-mix Numbers in the brackets indicate the accession numbers of miRBase.
From day 3 after infection, the cells were cultured in an ES cell medium containing LIF. On day 4 after infection, the cells were peeled off and the whole amount thereof was reseeded onto feeder cells. Every other day thereafter, the ES
cell medium containing LIF was replaced. From day 21 after infection, drug selection was started with addition of puromycin at a final concentration of 1.5 g/ml.
On day 28, the number of Nanog GFP positive colonies (GFP, the expression of which is induced by Nanog gene locus, can be observed under fluorescent microscopy) was counted. As a control, DsRed was used in place of miRNA. The results are shown in Fig. 1. It was found that mmu-miR-294 and mmu-miR-295 could respectively improve the nuclear reprogramming efficiency when introduced into mouse embryonic fibroblasts together with three factors of Oct3/4, Sox2, and Klf4, and enabled efficient preparation of induced pluripotent stem cells.
Example 2: Preparation of induced pluripotent stem cells through nuclear reprogramming of mouse tail tip fibroblasts pMXs-based retroviral vectors, which respectively encode each of three genes of mouse-derived Oct3/4, Sox2 and K1f4, DsRed (control), or mmu-miR-295, were transfected into PLAT-E cells using FuGENE 6 reagent (Roche). On the next day, tail tip fibroblasts (Nanog GFP tailtip fibroblasts) from transgenic mice generated by inserting the sequences encoding EGFP gene and puromycin resistance gene at a site downstream of Nanog gene promoter region, were seeded at 1 x 105 cells/well in 6-well plates. On the next day, these cells were infected with retroviruses expressing three factors of Oct3/4, Sox2, and Klf4, and either DsRed or mmu-miR-295, at a ratio of 1:1:1:1, so as to prepare induced pluripotent stem cells through nuclear reprogramming.
From day 3 after infection, the cells were cultured in an ES cell medium containing LIF. On day 4 after infection, the cells were peeled off and the whole amount thereof was reseeded onto feeder cells. Every other day thereafter, the ES
cell medium containing LIF was replaced. From day 7, day 21, or day 28 after infection, drug selection was started with addition of puromycin at a final concentration of 1.5 g/ml. On day 39, the number of total colonies and the number of Nanog GFP positive colonies (GFP, the expression of which is induced by Nanog gene locus, can be observed under fluorescent microscopy) were counted. The results are shown in Fig. 2. It was found that mmu-miR-295 could improve the nuclear reprogramming efficiency when introduced into mouse tail tip fibroblasts together with three factors of Oct3/4, Sox2, and Klf4, and could accelerate the reprogramming speed and enabled efficient preparation of induced pluripotent stem cells.
Example 3: Preparation of induced pluripotent stem cells through nuclear reprogramming of adult human dermal fibroblasts pMXs-based retroviral vectors, which encode three genes of human-derived Oct3/4, Sox2 and Klf4, and control DsRed or either each of 23 types of miRNAs or an miRNA cluster, were transfected into PLAT-E cells using FuGENE 6 reagent (Roche).
On the next day, adult human dermal fibroblasts which were generated to express a rodent ecotropic virus receptor Slc7al, were seeded at 3 x 105 cells/well in 6-cm dishes.
On the next day, the cells were infected with retroviruses expressing three genes of Oct3/4, Sox2, Klf4, and various types of miRNAs, at a ratio of 1:1:11, so as to produce induced pluripotent stem cells through nuclear reprogramming.
Table 2 miRNA number miRNA sequence 1 hsa-miR-371 (MI0000779) 2 hsa-miR-372 (MI0000780) 3 hsa-miR-373 (MI0000781) 4 hsa-miR-371-373 hsa-miR-93 (MI0000095) 6 hsa-miR-302a (M 10000738) 7 hsa-miR-302b (MI0000772) 8 hsa-miR-302c (MI0000773) 9 hsa-miR-302d (MI0000774) hsa-miR-367 (MI0000775 ) 11 hsa-miR-302b-367 12 hsa-miR-520a (MI0003149) 13 hsa-miR-520b (MI0003155) 14 hsa-miR-520c (MI0003158) hsa-miR-520d (MI0003164) 16 hsa-miR-520e (MI0003143) 17 mmu-miR-290-295 18 mmu-miR-290 (MI0000388) 19 mmu-miR-291a (MI0000389) 20 mmu-miR-292 (MI0000390) 21 mmu-miR-293 (MI0000391) 22 mmu-miR-294 (MI0000392) 23 mmu-miR-295 (MI0000393) On day 6 after infection, the cells were peeled off and the whole amount of 5 x 105 cells was reseeded onto feeder cells. Every other day thereafter, human ES
cell medium containing bFGF (ReproCELL medium) was replaced. On day 24, day 32, and day 40, the number of total colonies and the number of colonies having morphology of human ES-like cells were counted. As a control, DsRed was used in place of miRNA. The results are shown in Fig. 3. It was found that the number of colonies of induced pluripotent stem cells increased twice or more, as compared to the control, by introduction of three genes in the presence of hsa-miR-372, 373, 302b, 302b-367 (including 302b, 302c, 302a, 302d, and 367), 520c, mmu-miR-291a, 294, or 295.
Industrial Applicability The present invention provides an efficient method for preparing induced pluripotent stem cells. The method of the present invention has higher nuclear reprogramming efficiency as compared to conventional methods. For example, safe induced pluripotent stem cells can be efficiently produced without using c-Myc or gene product thereof Accordingly, the method of the present invention enables efficient production of highly safe induced pluripotent stem cells from patient's own somatic cells.
Cells differentiated from such pluripotent stem cells (for example, myocardial cells, insulin-producing cells, or nerve cells) will be able to be safely utilized in stem cell transplantation therapies for treatment of various diseases, such as heart failure, insulin dependent diabetes mellitus, Parkinson's diseases, and spinal cord injury.
SEQUENCE LISTING
<110> Kyoto University <120> Efficient Method for Nuclear Reprogramming <130> PH-3821-PCT
<140> PCT/JP2008/059586 <141> 2008-05-23 <150> US 60/996,893 <151> 2007-12-10 <160> 12 <170> PatentIn version 3.1 <210> 1 <211> 84 <212> RNA
<213> Mus musculus <400> 1 uuccauauag ccauacucaa aauggaggcc cuaucuaagc uuuuaagugg aaagugcuuc 60 ccuuuugugu guugccaugu ggag 84 <210> 2 <211> 69 <212> RNA
<213> Mus musculus <400> 2 ggugagacuc aaaugugggg cacacuucug gacuguacau agaaagugcu acuacuuuug 60 agucucucc 69 <210> 3 <211> 67 <212> RNA
<213> Homo sapiens <400> 3 gugggccuca aauguggagc acuauucuga uguccaagug gaaagugcug cgacauuuga 60 gcgucac 67 <210> 4 <211> 69 <212> RNA
<213> Homo sapiens <400> 4 gggauacuca aaaugggggc gcuuuccuuu uugucuguac ugggaagugc uucgauuuug 60 ggguguccc 69 <210> 5 <211> 73 <212> RNA
<213> Homo sapiens <400> 5 gcucccuuca acuuuaacau ggaagugcuu ucugugacuu uaaaaguaag ugcuuccaug 60 uuuuaguagg agu 73 <210> 6 <211> 68 <212> RNA
<213> Homo sapiens <400> 6 ccuuugcuuu aacauggggg uaccugcugu gugaaacaaa aguaagugcu uccauguuuc 60 aguggagg 68 <210> 7 <211> 69 <212> RNA
<213> Homo sapiens <400> 7 ccaccacuua aacguggaug uacuugcuuu gaaacuaaag aaguaagugc uuccauguuu 60 uggugaugg 69 <210> 8 <211> 68 <212> RNA
<213> Homo sapiens <400> 8 ccucuacuuu aacauggagg cacuugcugu gacaugacaa aaauaagugc uuccauguuu 60 gagugugg 68 <210> 9 <211> 68 <212> RNA
<213> Homo sapiens <400> 9 ccauuacugu ugcuaauaug caacucuguu gaauauaaau uggaauugca cuuuagcaau 60 ggugaugg 68 <210> 10 <211> 87 <212> RNA
<213> Homo sapiens <400> 10 ucucaggcug ucguccucua gagggaagca cuuucuguug ucugaaagaa aagaaagugc 60 uuccuuuuag aggguuaccg uuugaga 87 <210> 11 <211> 82 <212> RNA
<213> Mus musculus <400> 11 ccuauguagc ggccaucaaa guggaggccc ucucuugagc cugaaugaga aagugcuucc 60 acuuugugug ccacugcaug gg 82 <210> 12 <211> 820 <212> RNA
<213> Homo sapiens <400> 12 guuuucuuuc uccucagcuc uaaauacucu gaaguccaaa gaaguuguau guuggguggg 60 cucccuucaa cuuuaacaug gaagugcuuu cugugacuuu aaaaguaagu gcuuccaugu 120 uuuaguagga gugaauccaa uuuacuucuc caaaauagaa cacgcuaacc ucauuugaag 180 ggauccccuu ugcuuuaaca uggggguacc ugcuguguga aacaaaagua agugcuucca 240 uguuucagug gaggugucuc caagccagca caccuuuugu uacaaaauuu uuuuguuauu 300 guguuuuaag guuacuaagc uuguuacagg uuaaaggauu cuaacuuuuu ccaagacugg 360 gcuccccacc acuuaaacgu ggauguacuu gcuuugaaac uaaagaagua agugcuucca 420 uguuuuggug augguaaguc uuccuuuuac auuuuuauua uuuuuuuaga aaauaacuuu 480 auuguauuga ccgcagcuca uauauuuaag cuuuauuuug uauuuuuaca ucuguuaagg 540 ggcccccucu acuuuaacau ggaggcacuu gcugugacau gacaaaaaua agugcuucca 600 uguuugagug uggugguucc uaccuaauca gcaauugcgu uaacgcccac acugugugca 660 guucuuggcu acaggccauu acuguugcua auaugcaacu cuguugaaua uaaauuggaa 720 uugcacuuua gcaaugguga uggauuguua agccaaugac agaauuuaaa ccacagacuu 780 acuuugauag cacucuuaau gguauaacuu cuucucccau 820
Claims (7)
1. A method of preparing induced pluripotent stem cells, comprising a step of carrying out a nuclear reprogramming using a nuclear reprogramming factor(s) in the presence of miRNA, wherein said miRNA has a property of providing a higher nuclear reprogramming efficiency in the presence of said miRNA than in the absence of said miRNA.
2. The method according to claim 1, wherein said miRNA is expressed in embryonic stem cells at a higher level than in somatic cells
3. The method according to claim 1 or 2, wherein the nuclear reprogramming factor is gene products of three geens consisting of Oct3/4, Klf4 and Sox2 genes.
4. The method according to any one of claims 1-3, wherein the nuclear reprogramming using a reprogramming factor is carried out by introducing the Oct3/4, Klf4 and Sox2 genes into somatic cells.
5. The method according to any one of claims 1-4, wherein the miRNA is one or more miRNAs contained in one or more RNAs selected from the group consisting of hsa-miR-372, hsa-miR-373, hsa-miR-302b, hsa-miR-302c, hsa-miR-302a, hsa-miR-302d, hsa-miR-367, hsa-miR-520c, mmu-miR-291a, mmu-miR-294, and mmu-miR-295, wherein each of the RNA symbols represents a name registered on the miRBase database.
6. The method according to any one of claims 1-4, wherein said miRNA is one or more miRNAs contained in the RNA sequence represented by the name hsa-miR-302b-367 registered on the miRBase database.
7. The method according to any one of claims 1-4, wherein said miRNA is one or more miRNAs contained in one or more RNA sequences selected from the RNA
sequences shown in SEQ IDS:1 to 12 in the Sequence Listing.
sequences shown in SEQ IDS:1 to 12 in the Sequence Listing.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US99689307P | 2007-12-10 | 2007-12-10 | |
| US60/996,893 | 2007-12-10 | ||
| PCT/JP2008/059586 WO2009075119A1 (en) | 2007-12-10 | 2008-05-23 | Effective nucleus initialization method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CA2658463A1 true CA2658463A1 (en) | 2009-06-10 |
| CA2658463C CA2658463C (en) | 2015-07-21 |
Family
ID=40751190
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA2658463A Active CA2658463C (en) | 2007-12-10 | 2008-05-23 | Efficient method for nuclear reprogramming |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA2658463C (en) |
-
2008
- 2008-05-23 CA CA2658463A patent/CA2658463C/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CA2658463C (en) | 2015-07-21 |
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