JPWO2018203553A1 - How to improve the quality of differentiated cells - Google Patents

Abstract

Obtain high quality differentiated cells. In the step of inducing stem cell differentiation, the medium contains (a) the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence in which 1 to 3 bases have been deleted, substituted, inserted, or added to the nucleotide sequence. A method for improving the quality of differentiated cells and the like, including a step of adding a polynucleotide or (b) a vector encoding the polynucleotide of (a), is performed.

Description

  The present invention relates to a method for improving the quality of a differentiated cell, a production method, a composition for improving quality, and the like.

  Artificial pluripotent stem cells (hereinafter sometimes abbreviated as “iPS cells”) are expected to be applied to regenerative medicine, evaluation of drug efficacy / side effects, and the like. As a typical technique for producing iPS cells, a method described in Patent Document 1 can be mentioned. This document describes that pluripotent stem cells were prepared by introducing four genes (Oct3 / 4, Klf4, Sox2, c-Myc) into cells. As another technique for preparing iPS cells, Patent Document 2 describes that iR cells were prepared by introducing miR-520d-5p into cells.

WO 2007/069666 Patent No. 5095971

  As described in the examples below, iPS cells have multiple mutations in the genome. This can be a factor in canceration of differentiated cells obtained by differentiating iPS cells. That is, the differentiated cells obtained from iPS cells by the conventional method have room for improvement in terms of quality.

  The present invention has been made in view of the above circumstances, and has as its object to improve the quality of differentiated cells.

  The present inventors introduced miR-520d-5p into cells during induction of differentiation of iPS cells, as described in Examples below. Surprisingly, the mutations in the differentiated cells were reduced. And based on this result, this invention was completed.

  That is, according to one embodiment of the present invention, in the step of inducing differentiation of stem cells, in the medium, (a) the base sequence of SEQ ID NO: 1, or a deletion of 1 to 3 bases relative to the base sequence, substitution, There is provided a method for improving the quality of differentiated cells, comprising a step of adding a polynucleotide containing an inserted or added nucleotide sequence, or (b) a vector encoding the polynucleotide of (a). If this method is used, high quality differentiated cells can be obtained.

  According to one aspect of the present invention, in the step of inducing stem cell differentiation, (a) the nucleotide sequence of SEQ ID NO: 1, or 1 to 3 nucleotides relative to the nucleotide sequence of the nucleotide sequence is deleted, substituted, inserted, or There is provided a method for improving the quality of differentiated cells, comprising a step of using a medium containing a polynucleotide containing an added base sequence or (b) a vector encoding the polynucleotide of the above (a). If this method is used, high quality differentiated cells can be obtained.

  Further, according to one aspect of the present invention, in the step of inducing differentiation of stem cells, in the medium, (a) the nucleotide sequence of SEQ ID NO: 1, or a deletion of 1 to 3 bases relative to the nucleotide sequence, substitution, There is provided a method for producing a differentiated cell, comprising a step of adding a polynucleotide containing an inserted or added nucleotide sequence, or (b) a vector encoding the polynucleotide of (a). If this method is used, high quality differentiated cells can be obtained.

  According to one aspect of the present invention, in the step of inducing stem cell differentiation, (a) the nucleotide sequence of SEQ ID NO: 1, or 1 to 3 nucleotides relative to the nucleotide sequence of the nucleotide sequence is deleted, substituted, inserted, or There is provided a method for producing a differentiated cell, comprising a step of using a medium containing a polynucleotide containing an added nucleotide sequence or (b) a vector encoding the polynucleotide of the above (a). If this method is used, high quality differentiated cells can be obtained.

  Further, according to one aspect of the present invention, (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1 or a nucleotide sequence comprising 1 to 3 nucleotides deleted, substituted, inserted, or added to the nucleotide sequence. A composition for improving the quality of differentiated cells, comprising a nucleotide or (b) a vector encoding the polynucleotide of (a) is provided. By using this composition for improving quality, differentiated cells having good quality can be obtained.

  According to the present invention, high quality differentiated cells can be obtained.

FIG. 1 is a photograph of cells on day 2 after induction of differentiation. FIG. 2 is a photograph of cells on day 14 after induction of differentiation. FIG. 3 is a photograph of cells on day 20 after induction of differentiation. FIG. 4 is a diagram showing a comparison result of the frequency at a site where single base conversion frequently occurs in the entire genome. FIG. 5 is a diagram showing the rate of mutation reduction by the introduction of miR-520d-5p. FIG. 6 is a diagram showing a comparison result of mutation frequencies.

  Hereinafter, embodiments of the present invention will be described in detail. In addition, the description of the same contents will be appropriately omitted to avoid repetition.

  One embodiment of the present invention is a novel method for improving the quality of differentiated cells. This method is, for example, in the step of inducing differentiation of stem cells, in the medium, (a) the base sequence of SEQ ID NO: 1, or 1 to 3 bases relative to the base sequence deletion, substitution, insertion, or addition And (b) a vector encoding the polynucleotide of the above (a). According to this method, the mutation of the differentiated cells can be reduced as demonstrated in the examples described later. Therefore, according to this method, high-quality differentiated cells can be obtained. Alternatively, differentiated cells with improved quality can be obtained.

  The quality improvement method includes, for example, in the step of inducing differentiation of stem cells, (a) the nucleotide sequence of SEQ ID NO: 1 or 1 to 3 nucleotides are deleted, substituted, inserted, or added to the nucleotide sequence. Or a method for improving the quality of differentiated cells, which comprises a step of using a medium containing a polynucleotide containing the base sequence described above, or (b) a vector encoding the polynucleotide of (a). According to this method, the mutation of the differentiated cells can be reduced as demonstrated in the examples described later. Therefore, according to this method, high-quality differentiated cells can be obtained. Alternatively, differentiated cells with improved quality can be obtained.

  One embodiment of the present invention is a method for producing a novel differentiated cell. This method is, for example, in the step of inducing differentiation of stem cells, in the medium, (a) the base sequence of SEQ ID NO: 1, or 1 to 3 bases relative to the base sequence deletion, substitution, insertion, or addition And (b) a vector encoding the polynucleotide of the above (a). According to this method, a differentiated cell with reduced mutation can be obtained, as demonstrated in Examples described later. Therefore, according to this method, high-quality differentiated cells can be obtained. Alternatively, differentiated cells with improved quality can be obtained.

  The method for producing the differentiated cells, for example, in the step of inducing differentiation of stem cells, (a) the nucleotide sequence of SEQ ID NO: 1, or 1 to 3 bases relative to the nucleotide sequence of the deleted, substituted, inserted, or The method for producing a differentiated cell may include a step of using a medium containing a polynucleotide containing the added nucleotide sequence or (b) a vector encoding the polynucleotide of (a). According to this method, a differentiated cell with reduced mutation can be obtained, as demonstrated in Examples described later. Therefore, according to this method, high-quality differentiated cells can be obtained. Alternatively, differentiated cells with improved quality can be obtained.

  One embodiment of the present invention is a composition for improving the quality of a novel differentiated cell. The composition for improving quality includes, for example, (a) a polysaccharide containing a base sequence of SEQ ID NO: 1 or a base sequence having 1 to 3 bases deleted, substituted, inserted, or added to the base sequence. A composition for improving the quality of differentiated cells, comprising a nucleotide or (b) a vector encoding the polynucleotide of (a). By adding the composition for improving quality to the medium during the induction of differentiation of the stem cells, the mutation of the differentiated cells can be reduced as demonstrated in the Examples described later. Therefore, high-quality differentiated cells can be obtained by using this composition for improving quality. Alternatively, differentiated cells with improved quality can be obtained.

  In one embodiment of the present invention, the “base sequence of SEQ ID NO: 1” includes the base sequence of the mature miRNA of miR-520d-5p (5′-CUACAAAGGGAAGCCCUUUC-3 ′).

  In one embodiment of the present invention, the “polynucleotide” may be, for example, an RNA strand or a DNA strand encoding the RNA strand. This RNA strand may be a translation-suppressing RNA molecule. Translation-suppressing RNA molecules include RNA molecules that have the effect of suppressing the translation of a gene into a protein. Translation-suppressing RNA molecules include, for example, miRNA-related molecules or RNAi molecules. Confirmation of the translation inhibitory effect can be performed by quantifying the expression amount of the RNA chain by real-time RT-PCR. Alternatively, it can also be performed by a method such as analysis of RNA strand expression level by Northern blot, analysis of protein level by Western blot, and observation of phenotype. In addition, a plasmid that produces a translation-suppressing RNA molecule for a specific gene can be purchased, for example, from a contract company (for example, Takara Bio Inc.).

  Polynucleotides also include those in which a plurality of nucleotides or bases or their equivalents are combined. The polynucleotide is a concept including, for example, a chemically modified polynucleotide, a salt-formed polynucleotide, a solvate-formed polynucleotide, and a chemical-binding polynucleotide. Chemical modifications include, for example, methylation. Chemicals include cell uptake promoting substances (eg, PEG or derivatives thereof), labeling tags (eg, fluorescently labeled tags), or linkers (eg, nucleotide linkers). The nucleotide is a concept including, for example, non-chemically modified or chemically modified RNA bases or DNA bases. Equivalents include nucleotide analogs. Nucleotide analogs include unnatural nucleotides. The “RNA strand” includes a form in which two or more non-chemically modified or chemically modified RNA bases or their equivalents are linked. Polynucleotides include single-stranded or double-stranded polynucleotides. The polynucleotide includes the polynucleotide in a state linked to a vector. The base sequence can be represented by, for example, A (adenine), G (guanine), C (cytosine), T (thymine), U (uracil). Also, T and U (uracil) may be interchanged with each other depending on the application. The bases in the base sequence represented in this manner include non-chemically modified or chemically modified A, G, C, T, and U.

  The number of bases of the polynucleotide is, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, or It may be 500 and may be in the range of any two of them. When the polynucleotide is present in base pair form, such as a plasmid vector, the number of base pairs is, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 5000, 7544, 8000, 9000, or 10000 bp, and may be in the range of any two of them. . The polynucleotide can be synthesized, for example, using a DNA / RNA synthesizer. In addition, it can be purchased from a contracted company for DNA base or RNA base synthesis (for example, Invitrogen Corporation or Takara Bio Inc.). Vector synthesis can also be outsourced to each manufacturer.

  The polynucleotide may include the nucleotide sequence of SEQ ID NO: 1. This nucleotide sequence includes the mature miRNA of miR-520d-5p. Further, the polynucleotide may include the base sequence of SEQ ID NO: 2 (5′-UCUCAAGCUGUGAGUCUACAAAGGGAAGCCCUUUCUGUUGUCUAAAAGAAAAGAAAGUGCUUCUCUUUGGUGGGUUACGGUUUGAGA-3 ′). This nucleotide sequence includes the full-length sequence of miR-520d-5p. Further, the polynucleotide may include the base sequence of SEQ ID NO: 3 (5′-AAAGUGCUUCUCUUUGGUG-3 ′). This nucleotide sequence may complementarily bind to the nucleotide sequence of SEQ ID NO: 1. Further, the polynucleotide may include the base sequence of SEQ ID NO: 4 (5′-UCUACAAAGGGAAGCCCUUUCUG-3 ′). This base sequence may function as a guide strand. Further, the polynucleotide may include the base sequence of SEQ ID NO: 5 (5'-AAAGUGCUUCUCUUUGGUGGGU-3 '). This base sequence may function as a passenger strand. These base sequences may have RNA bases at both ends or at one end.

  In one embodiment of the present invention, “miRNA-related molecules” include, for example, miRNA, pri-miRNA, or pre-miRNA. miRNA-related molecules are known to contribute to translational suppression of target RNA strands. It is also known that miRNAs bind only partially and impair translation even with incomplete complementary chains containing mismatches.

  In one embodiment of the present invention, the “RNAi molecule” is an RNA chain having an RNAi action, and examples thereof include siRNA, shRNA, and small RNA having an RNAi action. For the design of RNAi molecules, for example, siDirect 2.0 (Naito et al., BMC Bioinformatics. 2009 Nov 30; 10: 392.) Can be used. In addition, it may be entrusted to a trustee company (for example, Takara Bio Inc.).

  The translation-suppressing RNA molecule may include an overhang consisting of 1 to 5 bases at the 5 ′ end or the 3 ′ end from the viewpoint of increasing the translation suppression efficiency. This number may be, for example, 5, 4, 3, 2, or 1 base, or may be in the range of any two values. When the translation-suppressing RNA molecule is double-stranded, a mismatched RNA may exist between the RNA strands. The number may be, for example, 1, 2, 3, 4, 5, or 10 or less, or may be in the range of any two values. The translation-suppressing RNA molecule may contain a hairpin loop.The number of bases of the hairpin loop may be, for example, 10, 8, 6, 5, 4, or 3 bases, and any two of them may be used. It may be within a range of values. The notation of each base sequence is the 5 'end on the left side and the 3' end on the right side.

  The length of the translation inhibitory RNA molecule is, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 70, 80, 90, 100, It may be 200 or 500, and may be in the range of any two values. This number is preferably 15 or more or 100 or less from the viewpoint of improving the quality of the differentiated cells.

  In one embodiment of the present invention, the nucleotide sequences represented by SEQ ID NOS: 1 to 5 are, as long as they have the desired effect, (a) in any one of the nucleotide sequences, one or more nucleotide sequences are deleted, substituted, Even one or more base sequences selected from the group consisting of an inserted or added base sequence, and (b) a base sequence having 90% or more homology to any base sequence, Good.

  In one embodiment of the present invention, the “plurality” may be, for example, 10, 8, 6, 5, 4, 3, or 2, or may be equal to or less than any of them. From the viewpoint of improving the quality of differentiated cells, the number is preferably 3 or less, more preferably 2 or less.

  In one embodiment of the present invention, “90% or more” may be, for example, 90, 95, 96, 97, 98, 99, or 100% or more, and is within a range of any two values. Is also good. From the viewpoint of improving the quality of differentiated cells, it is preferably 95% or more, more preferably 98% or more, and most preferably 100%. The “homology” may be calculated by calculating the ratio of the number of homologous bases in two or more base sequences according to a method known in the art. Before calculating the ratio, the base sequences of the base sequence groups to be compared are aligned, and a gap is introduced into a part of the base sequence if necessary to maximize the ratio of the same base. Methods for alignment, percentage calculations, comparison methods, and their associated computer programs are well known in the art (eg, BLAST, GENETYX, etc.). Homology (%) may be expressed as a value measured by the default setting of Blastn. Alternatively, the homology (%) may be represented by (the number of homologous bases / the number of bases in the base sequence of the comparison source) × 100.

  For the introduction of the polynucleotide into the cell, for example, infection introduction using a virus vector, calcium phosphate method, lipofection method, electroporation method, microinjection, or the like can be used. In addition, only the introduced cells can be selected using drug resistance, a cell sorter, or the like.

  In one embodiment of the present invention, the “vector” is a virus (eg, lentivirus, adenovirus, retrovirus, or HIV) vector, a plasmid derived from E. coli (eg, pBR322), a plasmid derived from Bacillus subtilis (eg, pUB110). ), Yeast-derived plasmid (eg, pSH19), bacteriophage such as phage λ, psiCHECK-2, pA1-11, pXT1, pRc / CMV, pRc / RSV, pcDNAI / Neo, pSUPER (OligoEngine), BLOCK-it Inducible H1 RNAi Entry Vector (Invitrogen), pRNATin-H1.4 / Lenti (GenScript, corp., NJ, USA) and the like can be used. The vector may include components required for expression of the DNA strand, such as a promoter, an origin of replication, or an antibiotic resistance gene. The vector may be a so-called expression vector. A vector encoding a specific base sequence may include a base sequence complementary to the base sequence. The complementary base sequence may be complementary, for example, between RNA and DNA, between RNA and RNA, or between DNA and DNA.

  In one embodiment of the present invention, “stem cells” include, for example, pluripotent stem cells. The pluripotent stem cells include, for example, induced pluripotent stem cells and ES cells. The induced pluripotent stem cells may be, for example, cells obtained by introducing Oct3 / 4, Klf4, Sox2, or c-Myc into cells (eg, somatic cells). Or, (a) a polynucleotide comprising a base sequence of SEQ ID NO: 1 or a base sequence having 1 to 3 bases deleted, substituted, inserted, or added to the base sequence, or (b) the ( A cell obtained by introducing a vector encoding the polynucleotide of a) into a cell (eg, a somatic cell) may be used.

  In one embodiment of the present invention, the “step of inducing differentiation” includes, for example, a step of culturing cells in a medium containing a differentiation inducer. Alternatively, the step of inducing differentiation includes, for example, while culturing cells in a medium containing a differentiation inducing agent, or in a state. Alternatively, the step of inducing differentiation includes, for example, from the start of the induction of differentiation to the time when the transformation from the stem cell group in the medium to the differentiated cells is substantially completed. The start of the differentiation induction may be, for example, at the time when the group of stem cells in the medium comes into contact with the differentiation inducer. The above-described differentiation induction may be started when the stem cells are introduced into the medium containing the differentiation-inducing agent, or when the differentiation-inducing agent is introduced into the medium containing the stem cells. The above-mentioned substantial completion may be, for example, a time point when the cells are cultured in a medium containing no differentiation inducer. The above-mentioned substantial completion may be, for example, a state in which the percentage of differentiated cells among the cells present in the medium is 99, 99.5, or 100%, and a state in which the ratio is at least one of those values. Or a state within the range of any two of them. Substantially complete, for example, the ratio of pluripotent stem cells may be 1, 0.5, or 0% of the cells present in the medium, and may be less than any of those values. May be present, or may be in the range of any two values. The presence of the differentiated cells may be confirmed, for example, by observing the cell morphology under a microscope. The presence of pluripotent stem cells may be confirmed, for example, by observing the cell morphology under a microscope.Since pluripotent stem cells are known to have a unique cell morphology, those skilled in the art The presence of sex stem cells and differentiated cells can be confirmed. Alternatively, the presence of a differentiated cell may be confirmed by using a protein marker specific to the differentiated cell as an indicator. Alternatively, the presence of pluripotent stem cells may be confirmed by using a protein marker specific to pluripotent stem cells as an index. Alternatively, the step of inducing differentiation includes, for example, a situation where the force acting in the direction of differentiation is stronger than the force acting in the direction of stemification. Alternatively, the step of inducing differentiation may be during the induction of differentiation. Or, the step of inducing differentiation, for example, if the start of differentiation induction is 0 days, 0.1, 0.5, 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 , 20, 21, 22, 23, 24, 25 days, or between any two of these values. From the viewpoint of further suppressing the mutation of the differentiated cells, the transition time of the differentiation induction is preferably at the time of introducing miR-520d-5p into the cells. The transition period may be, for example, between 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 days, any of which. It may be between two values. From the viewpoint of further suppressing the mutation of the differentiated cells, the time when miR-520d-5p is introduced into the cells is particularly preferably 14, 17, or 20 days.

Cell differentiation induction can be performed, for example, by using the STEMdiff ^TM Mesenchymal Progenitor Kit (STEMCELL Technologies Inc.), Cellartis ^(R) iPS Cell to Hepatocyte Differentiation System (TAKARA BIO INC.), CnT-Prime iPS Epithelial Differentiation Medium (Funakoshi Co., Ltd.) .), StemXVivo ^™ cardiomyocyte differentiation induction kit (R & D Systems, Inc.). As differentiation inducers, for example, KY03-I, KY02111, Am580, Ciglitazone, CKI-7 Dihydrochloride, SB431542, Y-27632, DAPT, Dexamethasone, Dorsomorphin, Dorsomorphin Dihydrochloride, DMH1, Forskolin, LY-294002, Purmorphamine, all- trans-Retinoic Acid, Spermine, Trichostatin A, TTNPB, TWS119, LDE225, LDN-193189, or StemRegenin1. Differentiated cells include, for example, cells differentiated from pluripotent stem cells. Cells differentiated from pluripotent stem cells may have additional differentiation potential, and include, for example, somatic stem cells (eg, including mesenchymal stem cells, neural stem cells). Differentiated cells include, for example, mesenchymal stem cells, mesenchymal progenitor cells (MPC), hepatocytes, lung cells, heart cells, esophageal cells, stomach cells, small intestine cells, large intestine cells, pancreatic cells, kidney cells, nerve cells , Neural stem cells, fibroblasts, skin cells, skin epithelial cells, cardiomyocytes, retinal cells, retinal progenitor cells, corneal epithelial cells, osteoblasts, blood cells, hematopoietic stem cells, hematopoietic progenitor cells, bone marrow stem cells, or their precursors Including cells. The quality of differentiated cells can be improved by using the polynucleotide (a) or the vector (b) when using these kits and differentiation inducers or when preparing these differentiated cells.

  In one embodiment of the present invention, “adding” includes adding a target component to a specific component group. The step of adding may include a step of bringing the target component into contact with a medium or stem cells. The number of additions may be, for example, 1, 2, 3, 4, or 5 times, or may be between any two of these values.

  In one embodiment of the present invention, the “composition” may be, for example, a composition containing the active ingredient and one or more pharmacologically acceptable carriers. The composition can be produced, for example, by mixing the active ingredient and the above-mentioned carrier and by any method known in the technical field of pharmaceuticals. The shape of the carrier may be, for example, a solid, semi-solid, or liquid. The composition may be used in vitro or in vivo.

  One embodiment of the present invention, in the step of inducing differentiation of stem cells, in the medium, (a) the base sequence of SEQ ID NO: 1, or a deletion of 1 to 3 bases relative to the base sequence, substitution, insertion, Alternatively, the present invention also includes a method for reducing the mutation of a differentiated cell, comprising a step of adding a polynucleotide containing the added nucleotide sequence or (b) a vector encoding the polynucleotide of (a). In one embodiment of the present invention, in the step of inducing differentiation of stem cells, (a) the nucleotide sequence of SEQ ID NO: 1, or 1 to 3 nucleotides are deleted, substituted, inserted, or added to the nucleotide sequence. A method for reducing mutations in differentiated cells, which comprises a step of using a medium containing the polynucleotide containing the base sequence thus obtained, or (b) a vector encoding the polynucleotide of (a). Further, one embodiment of the present invention, (a) the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence containing 1 to 3 bases deleted, substituted, inserted or added to the base sequence, or Or (b) a vector encoding the polynucleotide of (a), which comprises a composition for reducing mutations in differentiated cells. The reduction may include suppression.

  The method is, for example, a step of culturing stem cells in a differentiation-inducing medium, or (a) the nucleotide sequence of SEQ ID NO: 1 in the differentiation-inducing medium, or deletion of 1 to 3 bases relative to the nucleotide sequence, substitution, The method may include a step of adding a polynucleotide containing the inserted or added nucleotide sequence, or (b) a vector encoding the polynucleotide of (a). The differentiation induction medium includes a medium containing a differentiation inducer. The medium includes, for example, a liquid medium or a solid medium.

  The method includes, for example, (a) the base sequence of SEQ ID NO: 1 or a base sequence in which 1 to 3 bases have been deleted, substituted, inserted, or added to the base sequence of the cell or cell group. The method may include a step of contacting, introducing, or infecting the polynucleotide or (b) a vector encoding the polynucleotide of (a). The above-described introduction includes, for example, a step of infecting a cell with a vector encoding a nucleic acid of interest and expressing the nucleic acid of interest in the cell. The cells may include, for example, pluripotent stem cells, somatic stem cells, precursor cells of differentiated cells, or differentiated cells. The medium may contain, for example, pluripotent stem cells, somatic stem cells, precursor cells of differentiated cells, or differentiated cells. The method may include the step of collecting the differentiated cells. In one embodiment of the invention, the cells include stem cells. The methods include in vitro methods.

  In one embodiment of the invention, "improving quality" includes reducing mutations. When the genomic DNA of a wild-type cell is compared with the genomic DNA of a differentiated cell derived from an iPS cell, if there is a difference in the base sequence, it may be determined that the latter base is mutated. The wild-type cells may be cells excluding cells obtained from iPS cells or cells excluding cells derived from iPS cells. The genomic DNA (or normal genomic DNA) of a wild-type cell may be, for example, human hg19, human hg18, human (1 kg reference), b36 genomic DNA.

  All publications, publications (patents or patent applications) cited herein are incorporated by reference in their entirety.

  In this specification, "or" is used when "at least one or more" of the items listed in the text can be adopted. The same applies to "or". In this specification, the phrase "in the range of two values" includes the two values per se. In the present specification, “A to B” means from A to B.

Although the embodiments of the present invention have been described above, these are merely examples of the present invention, and various configurations other than the above can be adopted. Further, the configurations described in the above embodiments can be combined and adopted.

  Hereinafter, the present invention will be further described with reference to Examples, but the present invention is not limited thereto.

<Example 1> Differentiation experiment
An experiment for differentiating iPS cells into MSCs (mesenchymal stem cells) was performed using the STEMdiff ^™ Mesenchymal Progenitor Kit (STEMCELL Technologies Inc.). The procedure was performed with reference to the protocol attached to the kit. The specific procedure is as follows.

1) Before differentiation induction
A 6-well culture plate was coated with 1 ml of Reprocoat (RCHEOT001: ReproCELL), and cultured in feeder cells (SL10: RCHEFC001), iPS cell 253G1 strain purchased from RIKEN (riken cell bank, Resource NO .: RBRC-HPS0002, Resource name: 253G1, Lot No .: 024). The culture solution was adjusted to a concentration of bFGF of 5 ng / ml, and iPS cells were grown. One day later, the next step was performed using a cell group mixed with feeder cells.

(1) Matrigel was diluted 30-fold with a basal medium, coated, and allowed to stand at room temperature for 3 hours.
(2) 100 ml of MesenCult ^™ -ACF 5X Supplement and 5 ml of 200 mM L-Glutamine were placed in MesenCult ^™ -ACF Basal Medium.
(3) ReproXF, DMEM / F-12 and Gentle Cell Dissociation Reagent were kept at room temperature.
(4) Y-27632 (Dihydrochloride) was added to ReproXF at a concentration of 10 μM.
(5) The plate was washed once with 1 ml of D-PBS Without Ca ²⁺ and Mg ²⁺ .
(6) 1 ml of Gentle Cell Dissociation Reagent was added and incubated at 37 ° C. for 8-10 minutes.
(7) Peeled off slowly by pipetting and placed in a 15 ml tube containing 2 ml of ReproXF.
(8) The mixture was centrifuged at 300 × g for 5 minutes.
(9) After aspirating the supernatant, 2 ml of ReproXF was added.
(10) The whole amount was put on the plate coated in (1) above, and incubated at 37 ° C. for 24 hours.
(11) After 24 hours, the medium was changed with 2 ml of ReproXF kept at room temperature.
(12) Incubated at 37 ° C for 24 hours.

2) Day 0-2
(1) STEMdiff ^™ -ACF Mesenchymal induction Medium was placed at room temperature.
(2) The plate was washed once with 1 ml of D-PBS Without Ca ²⁺ and Mg ²⁺ .
(3) Medium was changed with 3 ml of STEMdiff ^™ -ACF Mesenchymal induction Medium.
(4) Incubated at 37 ° C. for 48 hours. A photograph of the cells on day 2 is shown in FIG.

3) Day 3-4
(1) MesenCult ^™ -ACF Basal Medium was placed at room temperature.
(2) The plate was washed once with 1 ml of D-PBS Without Ca ²⁺ and Mg ²⁺ .
(3) The medium was changed to 2 ml of MesenCult ^™ -ACF Basal Medium.
(4) Incubated at 37 ° C for 24 hours.
(5) The MesenCult ^™ -ACF Basal Medium was placed at room temperature.
(6) The medium was changed with 2 ml of MesenCult ^™ -ACF Basal Medium.
(7) Incubated at 37 ° C for 24 hours.
(8) MesenCult ^™ -ACF Attachment Substrate was diluted 28-fold with PBS Without Ca ²⁺ and Mg ²⁺ , coated with 800 μL, wound with parafilm, and allowed to stand at 4 ° C. overnight.

4) Day 5
(1) MesenCult ^™ -ACF Basal Medium was placed at room temperature, and Y-27632 was added to 10 μM.
(2) Washed once with 2.5 ml of PBS Without Ca ²⁺ and Mg ²⁺ .
(3) 1 ml of Accutase was added and incubated at 37 ° C. for 8-10 minutes.
(4) Peeled off slowly by pipetting and placed in a 15 ml tube containing 2 ml of MesenCult ^™ -ACF Basal Medium.
(5) The mixture was centrifuged at 300 × g for 7 minutes.
(6) After aspirating the supernatant, 3 ml of MesenCult ^™ -ACF Basal Medium was added.
(7) The whole amount was put on the plate coated the day before and incubated at 37 ° C. for 24 hours.

5) Day 6-10
(1) The medium was changed by half according to the number of cells.

6) Day 11
(1) MesenCult ^™ -ACF Basal Medium was placed at room temperature, and Y-27632 was added to 10 μM.
(2) Washed once with 2.5 ml of PBS Without Ca ²⁺ and Mg ²⁺ .
(3) 1 ml of Accutase (cell detachment solution) was added and incubated at 37 ° C. for 8-10 minutes.
(4) Peeled off slowly by pipetting, adding MesenCult ^™ -ACF Basal Medium, and subculturing.

7) Day 12-20
(1) The medium was changed by half according to the number of cells. Photographs of the cells on days 14 and 20 are shown in FIGS. 2 and 3, respectively.
(2) After a medium change on days 14, 17, and 20, a lentivirus vector encoding miR-520d-5p (pMIRNA1-miR-520d-5p / GFP, System Biosciences) was added. This lentivirus vector is a vector having a nucleotide sequence encoding miR-520d-5p and its complementary strand. At this time, the cells were infected with a lentiviral vector so that the cells were infected at a rate of one copy per cell. SEQ ID NO: 1 shows the nucleotide sequence of the mature miRNA of miR-520d-5p.

8) Day 21-
(1) At 80% confluence, DNA was extracted and NGS (next generation sequencing) was performed.

<Example 2> Evaluation of mutation
Based on the results of NGS, we compared the frequency of single nucleotide changes at the most frequently occurring sites in the whole genome. The results are shown in FIG. Compared with the normal genome sequence (reference), the frequency of single nucleotide mutation (ALT1) in iPS cells was generally higher (blue: bottom, iPSC). On the other hand, the frequency of miR-520d-5p was reduced and improved by the introduction of miR-520d-5p, approaching the normal mutation level of MSC (black: middle, MSC induced from iPSC with 520d-5p). The evaluation was performed based on the area ratio of colors.

  Next, based on FIG. 4 as a whole, the rate of mutation reduction due to the introduction of miR-520d-5p was examined. FIG. 5 shows the results. In 60.8% (301733), the mutation found in iPS cells was reduced in cells transfected with miR-520d-5p. This result indicates that by introducing miR-520d-5p into cells during the process of inducing differentiation of iPS cells, mutations can be reduced and the cells can be returned to the wild type. This also indicates that the quality of the differentiated cells has been improved.

  Although a slight increase in mutation is seen in FIG. 5, most of the mutations coincide with the mutation site and frequency of MSC, and are considered to be mutations that occur during differentiation into MSC (FIG. 6). ). That is, the increased mutation is considered to be caused by normal differentiation induction.

  As a result, it was shown that the quality of differentiated cells can be improved by introducing miR-520d-5p.

  The present invention has been described based on the embodiments. This embodiment is merely an example, and it will be understood by those skilled in the art that various modifications are possible and that such modifications are also within the scope of the present invention.

Claims (10)

  In the step of inducing differentiation of stem cells, the medium contains (a) the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence in which 1 to 3 bases have been deleted, substituted, inserted, or added to the nucleotide sequence. A method for improving the quality of differentiated cells, comprising the step of adding a polynucleotide or (b) a vector encoding the polynucleotide of (a).   2. The method according to claim 1, wherein the differentiated cells are cells differentiated from pluripotent stem cells.   In the step of inducing differentiation of stem cells, (a) the nucleotide sequence of SEQ ID NO: 1, or a polynucleotide comprising a nucleotide sequence having 1 to 3 nucleotides deleted, substituted, inserted, or added to the nucleotide sequence, Or (b) a method for improving the quality of differentiated cells, comprising a step of using a medium containing the vector encoding the polynucleotide of (a).   4. The method according to claim 3, wherein the differentiated cells are cells differentiated from pluripotent stem cells.   In the step of inducing differentiation of stem cells, the medium contains (a) the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence in which 1 to 3 bases have been deleted, substituted, inserted, or added to the nucleotide sequence. A method for producing differentiated cells, comprising a step of adding a polynucleotide or (b) a vector encoding the polynucleotide of (a).   6. The production method according to claim 5, wherein the differentiated cells are cells differentiated from pluripotent stem cells.   In the step of inducing differentiation of stem cells, (a) the nucleotide sequence of SEQ ID NO: 1, or a polynucleotide comprising a nucleotide sequence having 1 to 3 nucleotides deleted, substituted, inserted, or added to the nucleotide sequence, Or a method for producing differentiated cells, comprising a step of using a medium containing (b) a vector encoding the polynucleotide of (a).   8. The production method according to claim 7, wherein the differentiated cells are cells differentiated from pluripotent stem cells.   (a) the nucleotide sequence of SEQ ID NO: 1, or a polynucleotide containing a nucleotide sequence having 1-3 nucleotides deleted, substituted, inserted, or added to the nucleotide sequence, or (b) the (a) A composition for improving the quality of differentiated cells, comprising a vector encoding the polynucleotide of   10. The quality improving composition according to claim 9, wherein the differentiated cells are cells differentiated from pluripotent stem cells.