WO2018203553A1 - Method for improving quality of differentiated cells - Google Patents

Abstract

Good-quality differentiated cells are acquired. The method for improving the quality of differentiated cells is carried out in the steps for inducing the differentiation of stem cells, and includes a step for adding, to the culture medium, (a) a polynucleotide that contains the base sequence of SEQ ID NO: 1 or a base sequence provided by the execution on the aforementioned base sequence of the deletion, substitution, insertion, or addition of 1 to 3 bases or (b) a vector that encodes the polynucleotide according to (a).

Description

Method for improving the quality of differentiated cells

The present invention relates to a method for improving quality of differentiated cells, 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 and drug efficacy / side effect evaluation. As a typical iPS cell production technique, the 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 iPS cell production technique, Patent Document 2 describes that iPS cells were produced by introducing miR-520d-5p into cells.

International Publication No. 2007/069666 Patent No.5099571

As described in Examples below, iPS cells have many mutations in the genome. This can be a cause of canceration of differentiated cells obtained by differentiating iPS cells. That is, differentiated cells obtained from iPS cells by conventional methods have room for improvement in terms of quality.

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

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

That is, according to one aspect of the present invention, in the step of inducing differentiation of stem cells, (a) the base sequence of SEQ ID NO: 1 or 1 to 3 bases relative to the base sequence is deleted or substituted in the medium. Provided is a method for improving the quality of differentiated cells, which comprises the step of adding a polynucleotide containing the inserted or added nucleotide sequence, or (b) a vector encoding the polynucleotide of (a). By using this method, differentiated cells with good quality can be obtained.

Further, according to one aspect of the present invention, in the step of inducing differentiation of a stem cell, (a) the nucleotide sequence of SEQ ID NO: 1 or 1 to 3 bases 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 the added base sequence, or (b) a vector encoding the polynucleotide of (a). By using this method, differentiated cells with good quality can be obtained.

Further, according to one aspect of the present invention, in the step of inducing differentiation of stem cells, (a) the base sequence of SEQ ID NO: 1 or 1 to 3 bases relative to the base sequence is deleted or substituted in the medium. There is provided a method for producing differentiated cells, which comprises the step of adding a polynucleotide comprising the inserted or added nucleotide sequence, or (b) a vector encoding the polynucleotide of (a). By using this method, differentiated cells with good quality can be obtained.

Further, according to one aspect of the present invention, in the step of inducing differentiation of a stem cell, (a) the nucleotide sequence of SEQ ID NO: 1 or 1 to 3 bases of the nucleotide sequence is deleted, substituted, inserted, or There is provided a method for producing differentiated cells, comprising a step of using a medium containing a polynucleotide containing the added base sequence, or (b) a vector encoding the polynucleotide of (a). By using this method, differentiated cells with good quality can be obtained.

Further, according to one aspect of the present invention, (a) a base sequence of SEQ ID NO: 1 or a polynucleotide comprising a base sequence in which 1 to 3 bases are deleted, substituted, inserted or added to the base sequence There is provided a composition for improving the quality of differentiated cells, comprising a nucleotide or (b) a vector encoding the polynucleotide of (a). By using this composition for improving quality, differentiated cells with 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 the second day after differentiation induction. FIG. 2 is a photograph of cells on day 14 after differentiation induction. FIG. 3 is a photograph of cells on day 20 after differentiation induction. FIG. 4 is a diagram showing a comparison result of the frequency at the site where single base conversion occurs frequently in the entire genome. FIG. 5 is a graph showing the rate of mutation reduction due to 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, in order to avoid the repetition complexity about the same content, description is abbreviate | omitted suitably.

One embodiment of the present invention is a novel method for improving the quality of differentiated cells. In this method, for example, in the step of inducing differentiation of stem cells, (a) the base sequence of SEQ ID NO: 1 or 1 to 3 bases deleted, substituted, inserted or added to the base sequence is added to the medium. And a method for improving the quality of differentiated cells, which comprises the step of adding a polynucleotide comprising the prepared nucleotide sequence, or (b) a vector encoding the polynucleotide of (a). According to this method, as demonstrated in Examples described later, it is possible to reduce the mutation of differentiated cells. Therefore, according to this method, a differentiated cell with good quality can be obtained. Alternatively, differentiated cells with improved quality can be obtained.

In the quality improvement method, 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 is deleted, substituted, inserted or added to the nucleotide sequence. And a method for improving the quality of differentiated cells, comprising a step of using a medium containing a polynucleotide containing the nucleotide sequence or (b) a vector encoding the polynucleotide of (a). According to this method, as demonstrated in Examples described later, it is possible to reduce the mutation of differentiated cells. Therefore, according to this method, a differentiated cell with good quality can be obtained. Alternatively, differentiated cells with improved quality can be obtained.

One embodiment of the present invention is a novel differentiated cell production method. In this method, for example, in the step of inducing differentiation of stem cells, (a) the base sequence of SEQ ID NO: 1 or 1 to 3 bases deleted, substituted, inserted or added to the base sequence is added to the medium. A method for producing a differentiated cell, comprising the step of adding a polynucleotide comprising the prepared nucleotide sequence, or (b) a vector encoding the polynucleotide of (a). According to this method, it is possible to obtain differentiated cells with reduced mutation, as demonstrated in the examples described later. Therefore, according to this method, a differentiated cell with good quality can be obtained. Alternatively, differentiated cells with improved quality can be obtained.

The method for producing a differentiated cell includes, for example, in the step of inducing differentiation of a stem cell: (a) the nucleotide sequence of SEQ ID NO: 1 or 1 to 3 bases deleted, substituted, inserted, or inserted into the nucleotide sequence; A method for producing differentiated cells may be included, including a step of using a medium containing a polynucleotide containing the added base sequence, or (b) a vector encoding the polynucleotide of (a). According to this method, it is possible to obtain differentiated cells with reduced mutation, as demonstrated in the examples described later. Therefore, according to this method, a differentiated cell with good quality can be obtained. Alternatively, differentiated cells with improved quality can be obtained.

One embodiment of the present invention is a novel composition for improving the quality of differentiated cells. This composition for improving quality includes, for example, (a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence in which 1 to 3 nucleotides are 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). By adding this quality-improving composition to the medium during differentiation induction, it is possible to reduce the mutation of differentiated cells, as demonstrated in the examples described later. For this reason, differentiated cells with good quality 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 miR-520d-5p mature miRNA (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-inhibiting RNA molecule. Translation-inhibiting RNA molecules include RNA molecules that have the effect of suppressing translation from gene to protein. Translation inhibitory RNA molecules include, for example, miRNA-related molecules or RNAi molecules. Confirmation of the translation inhibitory effect can be performed by quantifying the expression level of the RNA strand by real-time RT-PCR. Alternatively, it can also be performed by methods such as analysis of RNA strand expression level by Northern blot, analysis of protein amount by Western blot, and observation of phenotype. Moreover, the plasmid which produces | generates the translation suppression RNA molecule with respect to a specific gene can be purchased from a trust company (for example, Takara Bio Inc. etc.), for example.

Further, the polynucleotide includes 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 polynucleotide that forms a salt, a polynucleotide that forms a solvate, and a polynucleotide that binds to a chemical substance. Chemical modification includes, for example, methylation. The chemical substance includes a cell uptake promoting substance (for example, PEG or a derivative thereof), a labeling tag (for example, a fluorescent labeling tag), or a linker (for example, a nucleotide linker). The nucleotide is a concept including, for example, a non-chemically modified or chemically modified RNA base or DNA base. 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. The polynucleotide includes single-stranded or double-stranded polynucleotides. A polynucleotide includes a 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). Further, T and U (uracil) may be interchanged with each other according to the application. Bases in the base sequence represented in this way 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 within the range of any two of them. When the polynucleotide is present in the form of base pairs as in a plasmid vector, the number of base pairs is, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, It may be 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 5000, 7544, 8000, 9000, or 10000 bp, and may be within the range of any two of them. . The polynucleotide can be synthesized, for example, using a DNA / RNA synthesizer. In addition, it can also be purchased from a DNA base or RNA base synthesis contract company (for example, Invitrogen, Takara Bio, etc.). Vector synthesis can also be outsourced to each manufacturer.

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

In one embodiment of the present invention, the “miRNA-related molecule” includes, 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 miRNA binds only partially and causes translational inhibition even with an incomplete complementary strand containing a mismatch.

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

The translation-inhibiting RNA molecule may contain 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, and may be within the range of any two of them. Further, when the translation-inhibiting RNA molecule is double-stranded, mismatch RNA may exist between each RNA strand. The number may be, for example, 1, 2, 3, 4, 5, or 10 or less, and may be in the range of any two of them. The translation-inhibiting RNA molecule may contain a hairpin loop. The number of bases in the hairpin loop may be, for example, 10, 8, 6, 5, 4, or 3 bases, and any two of them It may be within the range of values. In addition, 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-inhibiting 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 within the range of any two of them. This number is preferably 15 or more or 100 or less from the viewpoint of improving the quality of differentiated cells.

In one embodiment of the present invention, as long as the base sequences represented by SEQ ID NOs: 1 to 5 have a desired effect, (a) one or a plurality of base sequences is deleted or substituted in any base sequence, One or more base sequences selected from the group consisting of inserted or added base sequences, and (b) base sequences having 90% or more homology to any of the base sequences 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 any value or less. From the viewpoint of enhancing the quality of differentiated cells, 3 or less is preferable, and 2 or less is more preferable.

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 the range of any two of them. Also good. From the viewpoint of enhancing the quality of differentiated cells, it is preferably 95% or more, more preferably 98% or more, and most preferably 100%. The above-mentioned “homology” may be calculated according to a method known in the art, based on the ratio of the number of bases homologous in the base sequence between two or more bases. 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 bases. Methods for alignment, percentage calculation, comparison methods, and related 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 of the base sequence of the comparison source) × 100.

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

In one embodiment of the present invention, the “vector” refers to 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 plasmids (eg, pSH19), bacteriophages such as lambda phage, 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 contain components necessary for the expression of a DNA strand, such as a promoter, a replication origin, or an antibiotic resistance gene. The vector may be a so-called expression vector. A vector encoding a specific base sequence may contain a base sequence complementary to the base sequence. The complementary base sequences 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 artificial pluripotent stem cell may be, for example, a cell obtained by introducing Oct3 / 4, Klf4, Sox2, or c-Myc into a cell (eg, a somatic cell). Or (a) a nucleotide sequence of SEQ ID NO: 1, or a polynucleotide comprising a nucleotide sequence in which 1 to 3 bases are deleted, substituted, inserted or added to the nucleotide sequence, or (b) the above ( It may be a cell obtained by introducing a vector encoding the polynucleotide of a) into a cell (for example, a somatic cell).

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-inducing agent. Alternatively, the step of inducing differentiation includes, for example, a state in which cells are cultured in a medium containing a differentiation-inducing agent or a state. Alternatively, the step of inducing differentiation includes, for example, from the start of differentiation induction to the point at which the transformation from the stem cell group in the medium to the differentiated cell is substantially completed. The differentiation induction start may be, for example, when the stem cell group in the medium is in contact with the differentiation inducer. The initiation of differentiation induction may be when the stem cells are introduced into the medium containing the differentiation inducer or when the differentiation inducer is introduced into the medium containing the stem cells. The substantial completion may be, for example, when cells are cultured in a medium that does not contain a differentiation-inducing agent. The substantial completion of the above may be, for example, a state in which the proportion of differentiated cells among the cells present in the medium is 99, 99.5, or 100%, and is in a state that is greater than any of these values. Or may be in a state within the range of any two of them. The substantial completion of the above may be, for example, a state where the proportion of pluripotent stem cells is 1, 0.5, or 0% of the cells present in the medium, It may be, or it may be in a state within the range of any two of them. The presence of differentiated cells may be confirmed, for example, by observing cell morphology with a microscope. The presence of pluripotent stem cells may be confirmed, for example, by observing 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 differentiated cells may be confirmed by using a protein marker specific to differentiated cells as an indicator. Alternatively, the presence of pluripotent stem cells may be confirmed by using a protein marker unique 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 stemming. Alternatively, the step of inducing differentiation may be during differentiation induction. Alternatively, the step of inducing differentiation is, for example, when the differentiation induction start is defined as day 0, from which 0.1, 0.5, 1, 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 20, 20, 21, 22, 23, 24, 25, or any of those two values. From the viewpoint of further suppressing the differentiation of differentiated cells, the time of introducing miR-520d-5p into the cells is preferably a transient phase of differentiation induction. 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, either It may be between two values. From the viewpoint of further suppressing differentiation cell differentiation, the time when miR-520d-5p is introduced into the cell is particularly preferably 14, 17, or 20 days.

Cell differentiation induction includes, for example, STEMdiff ^™ 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.). Examples of differentiation inducers include 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 further differentiation ability, and include, for example, somatic stem cells (for example, including mesenchymal stem cells and 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 intestinal cells, colon 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 precursors thereof Contains cells. The quality of differentiated cells can be improved by using the above-mentioned polynucleotide (a) or the vector (b) when using these kits or differentiation inducers or 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 the medium or stem cells. The number of times of addition may be, for example, 1, 2, 3, 4, or 5 times, and may be within the range of any two of them.

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

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

The method includes, 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 1 to 3 bases deleted or substituted with respect to the nucleotide sequence; A step of adding a polynucleotide containing the inserted or added base sequence, or (b) a vector encoding the polynucleotide of (a) may be included. The differentiation induction medium includes a medium containing a differentiation inducer. The medium includes, for example, a liquid medium or a solid medium.

The above method includes, for example, (a) the base sequence of SEQ ID NO: 1 or a base sequence in which 1 to 3 bases are deleted, substituted, inserted or added to the base sequence in a cell or a group of cells. A step of contacting, introducing, or infecting the polynucleotide or (b) the vector encoding the polynucleotide of (a) may be included. The introduction includes, for example, a step of infecting a cell with a vector encoding the 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, differentiated cell precursor cells, or differentiated cells. The medium may contain, for example, pluripotent stem cells, somatic stem cells, differentiated cell precursor cells, or differentiated cells. The method may include a step of collecting differentiated cells. In one embodiment of the present invention, the cell comprises a stem cell. The method includes the invitro method.

In one embodiment of the present invention, “quality improvement” includes reducing mutation. When the genomic DNA of wild-type cells and the genomic DNA of differentiated cells derived from iPS cells are compared, if the base sequence is different, it may be determined that the latter base is mutated. The wild-type cell may be a cell excluding cells obtained from iPS cells or a cell excluding cells derived from iPS cells. The genomic DNA (or normal genomic DNA) of a wild-type cell may be, for example, the genomic DNA of human hg19, human hg18, human (1 kg reference), b36.

All publications and publications (patents or patent applications) cited in this specification 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, when “in the range of two values” is specified, the range includes the two values themselves. In the present specification, “A to B” means A or more and B or less.

As mentioned above, although embodiment of this invention was described, these are illustrations of this invention and various structures other than the above are also employable. Moreover, it is also possible to adopt a combination of the configurations described in the above embodiments.

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

Example 1 Differentiation Experiment A differentiation experiment from iPS cells to MSC (mesenchymal stem cells) was performed using 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 feeder cells (SL10: RCHEFC001) were cultured in the iPS cell 253G1 strain (riken cell bank, Resource NO. RBRC-HPS0002, Resource name: 253G1, Lot No .: 024) has been moved. The culture solution was brought to a concentration of 5 ng / ml bFGF to grow iPS cells. One day later, the next step was performed using a cell group mixed with feeder cells.

(1) Matrigel was diluted 30 times with 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 added to 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 so as to be 10 μM.
(5) 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) Gently pipet off and place in a 15 ml tube containing 2 ml of ReproXF.
(8) 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 placed 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) Washed once with 1 ml of D-PBS Without Ca ²⁺ and Mg ²⁺ .
(3) Medium was changed with 3 ml of STEMdiff ^TM -ACF Mesenchymal induction Medium.
(4) Incubate 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) Washed once with 1 ml of D-PBS Without Ca ²⁺ and Mg ²⁺ .
(3) Medium changed to 2 ml MesenCult ^™ -ACF Basal Medium.
(4) Incubated at 37 ° C for 24 hours.
(5) MesenCult ^™ -ACF Basal Medium was placed at room temperature.
(6) Medium change 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, wrapped with parafilm, and overnight at 4 ° C.

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 PBS Without Ca ²⁺ and Mg ²⁺ .
(3) 1 ml of Accutase was added and incubated at 37 ° C. for 8-10 minutes.
(4) Gently pipet off and place 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 coated plate 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 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) Pipette slowly and peel off. MesenCult ^™ -ACF Basal Medium was added and subcultured.

7) Day 12-20
(1) The medium was changed by half according to the number of cells. Photos of the cells on day 14 and day 20 are shown in FIGS. 2 and 3, respectively.
(2) After a medium change on Day 14, 17, and 20, a lentiviral vector (pMIRNA1-miR-520d-5p / GFP, System Biosciences) encoding miR-520d-5p was added. This lentiviral vector is a vector having a base sequence encoding miR-520d-5p and its complementary strand. At this time, infection was introduced into cultured cells at a rate of 1 copy per cell using a lentiviral vector. The base sequence of the mature miRNA of miR-520d-5p is shown in SEQ ID NO: 1.

8) Day 21-
(1) When 80% confluent, DNA was extracted and NGS (Next Generation Sequence) was performed.

<Example 2> Evaluation of mutations Based on the results of NGS, the frequencies of the single-base conversion sites in the entire genome were compared. The results are shown in FIG. Compared with the normal genome base sequence (reference), the frequency of single base mutation (ALT1) of iPS cells was generally higher (blue: bottom, iPSC). On the other hand, the frequency of miR-520d-5p was reduced and improved with the introduction of miR-520d-5p, approaching the normal MSC mutation level (black: Chubu, MSC induced from iPSC with 520d-5p). The evaluation was performed based on the color area ratio.

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

In addition, although the increase of the mutation is slightly seen in FIG. 5, since most of them coincide with the mutation site of MSC and the frequency, it is considered that the mutation occurs during differentiation into MSC (FIG. 6). ). That is, the increased mutation is considered to be due to normal differentiation induction.

The above results indicate that the quality of differentiated cells can be improved by the introduction of miR-520d-5p.

In the above, this invention was demonstrated based on the Example. It is to be understood by those skilled in the art that this embodiment is merely an example, and that various modifications are possible and that such modifications are 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 nucleotides are 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) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence in which 1 to 3 nucleotides are 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 a vector encoding the polynucleotide of (a). 4. The method according to claim 3, wherein the differentiated cell is a cell differentiated from a pluripotent stem cell. 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 nucleotides are deleted, substituted, inserted or added to the nucleotide sequence. A method for producing a differentiated cell, 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) a polynucleotide comprising the nucleotide sequence of SEQ ID NO: 1, or a nucleotide sequence in which 1 to 3 nucleotides are deleted, substituted, inserted or added to the nucleotide sequence; Or (b) a method for producing differentiated cells, comprising a step of using a medium containing a vector encoding the polynucleotide of (a). The production method according to claim 7, wherein the differentiated cells are cells differentiated from pluripotent stem cells. (a) a polynucleotide comprising the base sequence of SEQ ID NO: 1 or a base sequence in which 1 to 3 bases are deleted, substituted, inserted or added to the base sequence, or (b) the above (a) A composition for improving the quality of differentiated cells, comprising a vector encoding the polynucleotide. 10. The quality improving composition according to claim 9, wherein the differentiated cells are cells differentiated from pluripotent stem cells.

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