JP7369416B2 - Methods for producing naive pluripotent stem cells, methods for naiveting stem cells, and methods for preserving them - Google Patents

Description

The present invention relates to a method for producing naive pluripotent stem cells from primed pluripotent stem cells, a method for naiveting primed pluripotent stem cells, and a method for preserving naive pluripotent stem cells.

Pluripotent stem cells such as embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) can become all cells that make up the body. Therefore, it is attracting a lot of attention as a material for regenerative medicine.

In addition, because it can induce differentiation of cells such as nerve cells and cardiomyocytes that are difficult to collect from individuals, it is useful for custom-made preparations, drug discovery, and as a sensor for drug efficacy and safety testing.

In order to demonstrate the effectiveness of pluripotent stem cells as described above, it is necessary to induce differentiation of cell types that meet the purpose, and methods for inducing differentiation in in vitro culture have been studied and investigated.

On the other hand, research to date has revealed that the differentiation ability of human pluripotent stem cells is quite limited compared to pluripotent stem cells from mice and rats.

The biggest reason for this is thought to be that human pluripotent stem cells and mouse and rat pluripotent stem cells are at different developmental stages. Currently, pluripotent stem cells from humans, monkeys, rabbits, etc., and pluripotent stem cells from mice and rats are classified as different categories of cells, with the former being considered prime-type stem cells and the latter being naive-type stem cells.

Detailed analysis shows that primed stem cells are in a more differentiated state than naïve stem cells, and that they exhibit heterogeneity in which the properties of individual cells differ slightly, and that some cell lines have specific characteristics. It has been revealed that they exhibit differentiation bias, have unstable chromosomes, and slow proliferation.

Non-Patent Document 1 states that when transplanted into an early embryo, naive stem cells contribute to fetal formation and are incorporated into various tissues including reproductive cells, but primed stem cells are not incorporated into the early embryo. is listed.

Because existing human pluripotent stem cells are primed, they have limited differentiation ability and are not suitable for producing organs through chimeric animal creation. When considering a wide range of medical applications, technology is needed to transform existing human pluripotent stem cells into a naive type, or to create naive human pluripotent stem cells from somatic cells.

To date, several methods have been developed to transform human pluripotent stem cells into naive stem cells, and Non-Patent Document 2 summarizes the naiveization methods for human pluripotent stem cells that have been reported so far. ing.

For example, it has been reported that naive stem cells can be produced by simultaneously inhibiting multiple enzymes involved in differentiation or by forcing the expression of genes that are specifically expressed in naive stem cells.

Leehee Weinberger, Muneef Ayyash, Noa Nevershtern and Jacob H. Hanna, Dynamic stem cell states: naive to primed pluripotency in rodents and humans. NATURE REVIEWS: MOLECULAR CELL BIOLOGY, Vol. 17, No. 3, pp. 155-169 AJ, Rugg-Gunn PJ, Identifying Human Naive Pluripotent Stem Cells - Evaluating State-Specific Reporter Lines and Cell-Surf ace Markers. Bioessays, Vol. 40, No. 5, Electronic version e1700239, Collier Trusler, O. , Huang, Z. , Goodwin, J. , & Laslett, A. Cell surface markers for the identification and study of human naive pluripotent stem cells. Stem Cell Research Vol. 26, pp. 36-43, Vol. 17, No. 3, pp. 155-169

However, the naive stem cells produced by the method described in the above non-patent document have drawbacks such as incomplete methylation modification of the genome, low pluripotency, and low reproducibility as a manufacturing method (non-patent document 2). , development of a simple and highly reproducible method that does not involve genetic recombination is desired.

As a result of extensive research, the present inventors have discovered that by inhibiting the function of β-catenin cofactor histone acetyltransferase EP300 (hereinafter referred to as P300) in pluripotent stem cells with a drug, the differentiation of pluripotent stem cells becomes biased. In addition to discovering that pluripotent stem cells can be induced into naive pluripotent stem cells, post-translational modification of β-catenin is involved in the naivety of pluripotent stem cells, and in particular, the acetyl modification of lysine 49 residue (K49) of β-catenin The present invention was completed by discovering that the oxidation affects naiveization.

Furthermore, the present inventors have discovered that this method can be applied to a method for producing naive pluripotent stem cells from tissue somatic cells or blood cells, and have completed the present invention.

That is, an object of the present invention is to provide a method for easily obtaining naïve pluripotent stem cells from primed pluripotent stem cells or from tissue somatic cells or blood cells in various animal species including humans. That is what it is.

The present invention is a method for producing naive pluripotent stem cells from primed pluripotent stem cells, comprising:
(A) Primed pluripotent stem cells (excluding mouse cells) are treated with a β-catenin-P300 interaction inhibitor, or (B) the higher-order structure of β-catenin in primed pluripotent stem cells is By introducing a gene encoding a mutant β-catenin in which lysine 49 residue is replaced with glutamine or glutamic acid into competent stem cells, the lysine 49 residue is changed to a higher-order structure similar to that of acetylated β-catenin. This method is characterized by

The present invention also provides that the β-catenin-P300 interaction inhibitor in (A) is 2-(4-acetylphenylazo)-2-(3,3-dimethyl-3-,4-dihydro-2H-isoquinoline-1 -ylidene)-acetamide.

The present invention also provides a method for transforming primed pluripotent stem cells (excluding mouse cells) into naïve pluripotent stem cells by treating the primed pluripotent stem cells (excluding mouse cells) with a β-catenin-P300 interaction inhibitor. This is a method for naiveting primed pluripotent stem cells (excluding mouse cells) , which is characterized by:

Furthermore, the present invention stably maintains naïve pluripotent stem cells (excluding mouse cells) by treating them with a β-catenin-P300 interaction inhibitor. This is a method for preserving naïve pluripotent stem cells.

The present invention produces naive stem cells by treating primed pluripotent stem cells, somatic cells, blood cells, etc. with a β-catenin-P300 interaction inhibitor. It does not require any manual handling and has the obvious advantages of being simple and highly reproducible.

Furthermore, unlike conventional methods, the present invention uses protein-protein interaction to change the higher-order structure of β-catenin in primed pluripotent stem cells, somatic cells, blood cells, etc. Since naïve pluripotent stem cells can be produced from a single gene, naïve stem cells can be produced by manipulating a single gene.

Furthermore, since naive pluripotent stem cells can be maintained in a naive state and stably stored for a long period of time, there is an advantage that the naive pluripotent stem cells can be used at any time.

Furthermore, β-catenin has extremely high conservation in amino acid sequence and structure among different animal species, so it has a clear advantage over existing methods in that it can be applied to all animal species.

These are phase contrast micrographs showing the morphology of cells used in Example 1, in which (A) is primed mouse iPS cells, (B) is naive mouse embryonic stem cells, and (C) is β-catenin-P300 interaction inhibition. (D) shows mouse epiblast stem cells cultured without a drug, and (D) shows naive mouse pluripotent stem cells of the present invention. These are phase contrast micrographs showing the morphology of cells used in Example 2, in which (A) is primed human iPS cells, (B) is naive human iPS cells of the present invention, and (C) is β-catenin-P300 reciprocal. Human iPS cells cultured without an effect inhibitor are shown, respectively. These are phase contrast micrographs showing the morphology of cells used in Example 3, in which (A) is primed Amami rabbit iPS cells, (B) is naive Amami rabbit iPS cells according to the present invention, and (C) is β Amami rabbit iPS cells cultured without a catenin-P300 interaction inhibitor are shown. 1 is a diagram showing the results of gene expression by RT-PCR of naive mouse pluripotent stem cells obtained in Example 1. FIG. 1 is a photograph of a chimeric mouse created from the naive mouse pluripotent stem cells obtained in Example 1. FIG. 2 is a diagram showing the evaluation results of naive human iPS cells obtained in Example 2, and (A) shows that the gene expression state of the naive type was prominent in the total RNA sequence analysis of the naive human iPS cells. The results are shown for the gene group that , (B) shows the results of measuring the proportion of methylated cytosine in genomic DNA extracted from the same cells. These are the results of an alkaline phosphatase activity test on the naive Amami rabbit iPS cells used in Example 3, which utilizes the fact that the coloring intensity is specifically enhanced in naive cells, and (A) shows the β-catenin-P300 interaction. These are the results of an alkaline phosphatase activity test on Amami rabbit iPS cells cultured without an inhibitor, and (B) shows the alkaline phosphatase activity test on Amami rabbit iPS cells that were changed into a naive type by the addition of a β-catenin-P300 interaction inhibitor. It is a phase contrast micrograph showing the results of an activity test. 1 shows the results of quantitative RT-PCR gene expression analysis of human iPS cells used in Example 2. These are the results of flow cytometry (FACS) analysis when separating the naive human iPS cells obtained in Example 2. This is a phase contrast micrograph of cells used in Example 5, showing (A) human fibroblasts used as materials, (B) human naive iPS cells created from the same cells, and (C) created in a control experimental group. This is a phase contrast micrograph of human iPS cells.

The present invention can be performed by (A) treating primed pluripotent stem cells with a β-catenin-P300 interaction inhibitor (hereinafter referred to as method A), or (B) increasing β-catenin in primed pluripotent stem cells. Naive pluripotent stem cells are produced from primed pluripotent stem cells by changing the secondary structure to a higher order structure similar to β-catenin in which 49 lysine residues are acetylated (hereinafter referred to as method B). This is a method for producing naïve pluripotent stem cells.

The present invention also provides for (C) treating somatic cells or blood cells of a tissue with a β-catenin-P300 interaction inhibitor, or (D) changing the higher-order structure of β-catenin in the cells at lysine 49 residues. Production of naïve pluripotent stem cells, characterized by producing naive pluripotent stem cells from tissue somatic cells or blood cells by changing the group into a higher-order structure similar to that of acetylated β-catenin It's a method.

First, method A will be explained. In Method A, primed pluripotent stem cells are pluripotent stem cells at various stages of differentiation from cells in the so-called basal state to those corresponding to the post-implantation embryo, or cells in the basal state that have different differentiation markers. Refers to pluripotent stem cells in which at least one has disappeared.

Typical examples of such primed pluripotent stem cells are pluripotent stem cells that have properties similar to post-implantation embryonic cells, lack the ability to form chimeras, and have a colony morphology. refers to one having at least one characteristic, such as being flat and not expressing naive-related genes such as TFCP2L1.

Specific examples of such cells include primed stem cells with so-called pluripotency, such as human iPS cells, human ES cells, and mouse epiblast stem cells. These pluripotent stem cells may be human cells, cells derived from mice, cynomolgus monkeys, rabbits, or other mammals, and in addition to known cells, commercially available cells are also suitable. It can be used for.

For example, iPS cells include cells into which transcription factors such as Oct3/4, Klf4, c-Myc, Tert, Nanog, Oct3/4, Sox2, and Lin28 have been introduced.

iPS cells can be produced by known methods, and as somatic cells used for production, for example, fibroblasts can be suitably used, and blood cells such as monocytes and epithelial cells are also suitably used. can do.

In this case, the most suitable method for producing iPS cells is, for example, a method using Sendai virus, but it can also be carried out by introducing retroviruses or RNA.

Examples of ES cells include human cells, such as KhES-1 cells, KhES-2 cells, and KhES-3 cells, and examples of monkey ES cells include cynomolgus monkey ES cells.

In the present invention, a β-catenin-P300 interaction inhibitor refers to a drug that inhibits the interaction between β-catenin cofactor P300 and β-catenin, and is preferably a drug that promotes dephosphorylation of β-catenin cofactor P300. It can be used for.

Such β-catenin-P300 interaction inhibitors include 2-(4-acetylphenylazo)-2-(3,3-dimethyl-3-,4-dihydro-2H-isoquinolin-1-ylidene)-acetamide (IQ- 1: CEREC Chemicals), 1-(4-methoxyphenyl)-2-methyl-3-nitro-1H-indol-6-ol (ID-8: CEREC Chemicals), and the like.

Among these, IQ-1 is preferred because of its easy availability, low cost, convenience, and low toxicity.

In the present invention, the method of treating primed pluripotent stem cells with a β-catenin-P300 interaction inhibitor (hereinafter sometimes simply referred to as an inhibitor) includes One example is a method of culturing stem cells.

Moreover, the inhibitor can also be added during the production process of iPS cells, ES cells, etc.

In the present invention, the inhibitor may be used at a concentration in the treatment solution of approximately 0.5 to 10 μg/ml, particularly preferably 2 to 4 μg/ml.

At this time, the inhibitor may be added as is, or it may be dissolved in a solvent and added to the medium at a desired concentration. First, it is not particularly limited as long as it dissolves the inhibitor and mixes it with the culture medium.

Examples of such solvents include dimethyl sulfoxide (DMSO) and ethanol. When adding an inhibitor to the culture medium, more favorable results can be obtained by replacing the inhibitor approximately every 12 to 48 hours.

When producing naïve pluripotent stem cells from primed pluripotent stem cells by culturing in the presence of an inhibitor, the culture may be carried out for about two weeks from the start of the culture, and may be continued during the cell culture period. You can go.

Furthermore, the inhibitor may be added to the medium at appropriate intervals, such as every day or every few days, during the culture period, or the medium may be replaced every time.

The treatment temperature with the inhibitor is not particularly limited as long as it is a temperature at which pluripotent stem cells grow, but specifically, it may be 30 to 40°C, and when adding the inhibitor to the culture solution, The temperature is preferably 35 to 38° C., taking into account the suitability of culture. As a culture method when an inhibitor is added to the culture solution, various culture methods used for cell culture can be suitably used.

To give an example of a specific culture method, for example, during the culture of primed pluripotent stem cells, IQ-1 solution containing dimethyl sulfoxide (DMSO) as a solvent is diluted to a dilution rate of about 1/2000. Naive stem cells can be obtained by adding the culture medium and replacing it with a culture medium of the same composition every two days and continuing the culture for about two weeks.

Thus, generation of naive pluripotent stem cells from primed pluripotent stem cells involves recognition of surface antigens specifically possessed by naive cells using known methods (for example, the method described in Non-Patent Document 3). It can be easily identified by labeling with antibodies that can be used to identify the target species.

Specifically, for example, it is sufficient to confirm the expression of CD75, CD77, CD130, etc., or to identify target cells by immunostaining, Western blotting, or flow cytometry using antibodies against intracellular antigens such as KLF2 and TBX3. It can also be confirmed that the cells are naive stem cells.

Further, confirmation may be made by a change in the genome modification state of the cell; for example, it can be confirmed that the cell is a naive pluripotent stem cell by a decrease in the proportion of methylated cytosine contained in the entire genome.

The naive pluripotent stem cells thus obtained are naive pluripotent stem cells that are very close to the so-called basal state, and have various differentiation markers such as POU5F1, SOX2, NANOG, KLF2, KLF4, ZFP42, STELLA, FGF4, and TBX3. It is a stem cell with excellent pluripotency.

Next, method B will be explained. Method B is a method characterized by changing the higher-order structure of β-catenin in primed pluripotent stem cells to a higher-order structure similar to that of β-catenin in which 49 lysine residues are acetylated.

In method B, as the primed pluripotent stem cells, the primed pluripotent stem cells exemplified with respect to method A above can be suitably used.

The higher-order structure of β-catenin changes to a higher-order structure similar to that formed by acetylation of 49 lysine residues of β-catenin due to a mutant β-catenin in which 49 lysine residues are replaced with glutamic acid. The changes are described in the literature (J. Biol. Chem. 2006 sep 8; 281 (36): 26181-7).

In order to change the higher-order structure of β-catenin, a β-catenin protein that has undergone a higher-order structural change similar to the higher-order structural change formed by acetylation of lysine 49 residue in primed pluripotent stem cells is used. What is necessary is to introduce a gene that expresses this.

Examples of genes that express such proteins include genes containing an amino acid sequence encoding β-catenin in which 49 lysine residues of β-catenin are replaced with glutamine or glutamic acid (BC-K49 constitutively acetylated mutant gene). The gene may contain an intron, but is particularly preferably a cDNA encoding the above amino acid sequence.

The above gene can be inserted into a plasmid, vector, etc. commonly used in this field, introduced into primed pluripotent stem cells, and cultured to produce the same effect as β-catenin in which 49 lysine residues are acetylated. can express proteins that have

Such vectors and plasmids may be vectors and plasmids for gene expression in mammals, such as pCDNA3.1, pCAG, pig gy bac transposon vectors, lentivirus vectors, and Sendai virus vectors.

Among these, Sendai virus vector is preferred due to its ease of operation, stability, low toxicity, and safety.

In addition, the method for introducing the recombinant gene into primed pluripotent stem cells may be any known method, and electroportion method, microinjection method, etc. can be used.

Next, the pluripotent stem cells into which the gene has been introduced are cultured in a culture medium, and the target cells are selected and collected using drug resistance, etc., and further cultured to produce the target naive pluripotent stem cells. can be obtained.

The naïve pluripotent stem cells thus obtained, like the naive pluripotent stem cells obtained by method A above, are as close as possible to cells in the basal state, and are pluripotent with various differentiation markers. These are naïve cells that are rich in

The method for naiveting primed pluripotent stem cells of the present invention comprises treating the primed pluripotent stem cells with a β-catenin-P300 interaction inhibitor to transform the primed pluripotent stem cells into naïve pluripotent stem cells. This is a method to change it to

In the present invention, as the primed pluripotent stem cells and the inhibitor, those used in the above-mentioned method for producing naive pluripotent stem cells can be suitably used, and the concentration of the inhibitor, the treatment temperature, and the treatment time also vary. By carrying out the same method as the production method described above, it is possible to make naive pluripotent stem cells as close to the basal state as possible.

Further, the present invention is a method for preserving naive pluripotent stem cells, which maintains naive pluripotent stem cells in the presence of a β-catenin-P300 interaction inhibitor.

In the present invention, as the primed pluripotent stem cells and the inhibitor, those used in the above-mentioned method for producing naive pluripotent stem cells can be suitably used, and in the present invention, maintaining in the presence of the inhibitor means , maintaining naïve pluripotent stem cells in a medium containing an inhibitor, especially if the medium allows the naive pluripotent stem cells to survive without impairing their pluripotency. Not restricted.

Examples of such a medium include physiological saline, a growth medium, a growth medium for stem cells, and a growth medium for stem cells is particularly preferred. The concentration of the inhibitor in the medium may be 0.5 to 10 μg/ml, particularly preferably 2 to 4 μg/ml. The inhibitor may be added as appropriate, or may be replaced with each medium.

By maintaining the temperature at 35 to 38°C, differentiation of naive pluripotent stem cells can be suppressed and the cells can be maintained as naive pluripotent stem cells for a long period of time.

Next, method C will be explained. Method C is a method for producing naive pluripotent stem cells from tissue somatic cells or blood cells by treating them with a β-catenin-P300 interaction inhibitor during reprogramming.

In method C, the tissue somatic cells or blood cells are not particularly limited as long as they can be imparted with pluripotency by introduction of a transcription factor. Such transcription factors include reprogramming translocation factors such as Oct3/4, Klf4, c-Myc, Tert, Nanog, Oct3/4, Sox2, and Lin28.

Examples of somatic cells include fibroblasts, bone marrow mesenchymal stem cells, epithelial cells, and vascular endothelial cells, and examples of blood cells include leukocytes such as lymphocytes and monocytes.

In Method C, the time of cell reprogramming refers to the time when the transcription factor is introduced into somatic cells or blood cells.

Further, as the β-catenin-P300 interaction inhibitor and the treatment conditions using the inhibitor, those employed in the method A above can be preferably used.

Furthermore, method D will be explained. Method D changes the higher-order structure of β-catenin in somatic cells or blood cells of tissues to a higher-order structure similar to β-catenin in which 49 lysine residues are acetylated, and transforms the cells into naive pluripotent stem cells. This is a method of manufacturing.

In Method D, the somatic cells listed in Method C can be suitably used. In addition, in order to change the higher-order structure of β-catenin in the same manner as the higher-order structure change formed by acetylation of 49 lysine residues of β-catenin, the method and genes employed in Method B are preferably used. It can be used for.

EXAMPLES Hereinafter, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto.

Example 1
(1) <Production of naive mouse pluripotent stem cells from mouse epiblast stem cells>
At Kindai University, C57BL/6 mice were mated, and 5.75 days after mating was confirmed, fetal epiblasts collected from the uterus of female mice were transfected on feeder cells made from mouse embryonic fibroblasts (MEFs). The epiblast stem cell KEp1 strain was established by culturing for one week in a pluripotent stem cell culture medium supplemented with basic fibroblast growth factor (bFGF).

The mouse epiblast stem cells were cultured on feeder cells prepared from mouse embryonic fibroblasts (MEFs), and 1 mL of the stem cell culture medium was added to the KEp1 strain that had grown to about 25% of the culture plate (25% confluence). 4 μg of IQ-1 was added to each cell, and cultured for 2 weeks while replacing the stem cell culture medium containing fresh IQ-1 every 24 hours.

The detailed method for establishing the epiplast stem cells is described in the literature (Scientifc Reports. 2015 Sep 30; 5:14722. Doi: 10.1038/srep14722.).

During this time, the cells that reached confluence were detached with a 0.25% trypsin (manufactured by Invitrogen)-0.02% EDTA mixture, a pellet of viable cells was obtained by centrifugation, and after removing all the supernatant, a new IQ- The cells were suspended in a stem cell culture medium supplemented with 1/4 to 1/8 of the amount and cultured on new feeder cells.

Cells that have formed a dome-shaped colony are collected using a glass tube or micropipette, treated with a 0.25% trypsin-0.02% EDTA mixture until individual cells are isolated, and then centrifuged. A pellet of viable cells was obtained, and after removing all the supernatant, the pellet was suspended in fresh IQ-1 supplemented stem cell culture medium and cultured on fresh feeder cells.

Through the above operations, naive mouse pluripotent stem cells were obtained.

The morphological characteristics of the obtained naive mouse pluripotent stem cells were confirmed using a phase contrast microscope (manufactured by Olympus Corporation, model CK30).

The obtained naive mouse pluripotent stem cells exhibited a typical naive morphology of small dome-shaped colonies (Fig. 1, B and D), similar to naive mouse embryonic cells, and had excellent proliferative ability. showed that.

In contrast, the mouse epiblast stem cell KEp1 and the above KEp1 cultured without the β-catenin-P300 interaction inhibitor both had the morphology of primed pluripotent stem cells, with flat and large colonies (Figure 1, A and C). showed that.

Furthermore, when quantitative RT-PCR was performed using the obtained naive mouse pluripotent stem cells as a sample, a significant increase in the expression of KLF4 and ZFP42, which are markers for naive mouse pluripotent stem cells, was observed.

Quantitative RT-PCR was performed using mouse Gapdh and primers specific to the gene sequence prepared in advance. The gene sequence is published at Entrez Gene (http://www.ncbi.nlm.nih.gov/sites/entrez).

For quantitative RT-PCR, cells were treated with TRIzol (Invitrogen), mixed with chloroform, and centrifuged at 15,000 rpm for 10 minutes to separate the aqueous layer containing RNA. The aqueous layer containing RNA was mixed with the same amount of isopropanol and centrifuged again at 15,000 rpm for 10 minutes to obtain an RNA precipitate. The precipitate was washed with ice-cold 80% ethanol, dissolved in RNase-free water, and then used for reverse transcription reaction.

The reverse transcription reaction was performed using Prime Script RT (manufactured by Takara Bio Inc.). The PCR reaction solution was prepared using TB Green Premix Ex Taq II (registered trademark, manufactured by Takara Bio Inc.), and was prepared using RT-PCR quantitative tubes (Microamp Optical 8-Tube Strip, trade name, Applied, manufactured by Biosystems Inc.). ).

The PCR reaction was performed using TAKARA Thermal Cycler Dice Real Time System (manufactured by Takara Bio Inc.), followed by a denaturation reaction at 94°C for 20 seconds, annealing at 94°C for 5 seconds, annealing at 60°C for 30 seconds, and an amplification reaction 40 times. The results were analyzed repeatedly.

Figure 4 shows that the cells produced using the naive mouse pluripotent stem cells obtained in Example 1 were confirmed to have increased expression of genes specific to naive pluripotent stem cells. FIG. 2 is a diagram showing that the cells are type pluripotent stem cells.

(2) Production of a mouse chimeric animal (chimeric mouse) The formation of a chimeric animal was induced by transplanting the naive mouse pluripotent stem cells obtained in (1) into a mouse blastocyst stage embryo. Production of a large amount of fertilized eggs was induced by injecting PMSG and hCG into 4-week-old female mice and mating them with 10-week-old male mice. After 24 hours, the female mouse was euthanized, and the 2-cell stage embryo was removed from the oviduct.

Subsequently, the removed two-cell stage embryo was cultured in KSOM culture solution for 3 days to produce a blastocyst stage embryo. Using a micromanipulator (manufactured by Leica), about 8 to 12 naïve cells produced according to the present invention were injected into the blastocyst cavity of the blastocyst stage embryo. After the injection was completed, the blastocyst stage embryo was transplanted into the uterus of a pseudopregnant ICR female mouse (white mouse). Seventeen days after transplantation, a caesarean section was performed and the chimeric mice were removed.

The obtained mouse had black hair that does not normally appear on the body hair of a white mouse (Figure 5), and the naïve mouse pluripotent stem cells (C57BL/6 line: black mouse) produced according to the present invention appeared. A chimeric mouse obtained by injecting ICR mouse (white mouse) into a blastocyst stage embryo, in which the cells produced have the ability to form tissues, which is unique to naïve pluripotent stem cells. It is clear that it has.

Example 2
<Production of naive human iPS cells from human iPS cells>
The human iPS cell line 409B2 strain (HSP0076), which was created at Kyoto University and received from RIKEN, was cultured on feeder cells made from mouse embryonic fibroblasts (MEF), and approximately 25% of the culture dish ( At the stage when the cells grew to 25% confluence, 4 μg of IQ-1 was added to 1 mL of stem cell culture medium, and the cells were cultured for 2 weeks while replacing with fresh IQ-1-added stem cell culture medium every 24 hours.

In the case of human iPS cells, the incidence of naïve cells is lower than that of mice. Therefore, we introduced a gene that emits green fluorescence (POU5F1ΔPE-GFP: Addgene, Plasmid #52380) into the human iPS cell line 409B2 (HSP0076) in advance, and transferred this gene to mouse embryonic fibroblast cells ( 4 μg of IQ-1 was added to 1 mL of stem cell culture medium to 409B2, which was cultured on feeder cells prepared from MEF and grown to about 25% of the culture dish (25% confluence), and then cultured every 24 hours. The cells were cultured for 2 weeks while replacing the stem cell culture medium with fresh IQ-1 supplemented stem cell culture medium.

During this time, cells that reached confluence were detached using a 0.25% trypsin (Invitrogen)-0.02% EDTA mixture, a pellet of viable cells was obtained by centrifugation, and after removing all the supernatant, a ROCK inhibitor was added. The cells were suspended in a new stem cell culture medium supplemented with Y27632 and IQ-1, and 1/4 to 1/8 of the amount was cultured on new feeder cells.

When the formation of dome-shaped colonies was confirmed, each cell was treated with a 0.25% trypsin-0.02% EDTA mixture until it was unified, and then a pellet of viable cells was obtained by centrifugation. After removing all the supernatant, the cells were suspended in a stem cell culture medium supplemented with HEPES. This was analyzed using FACS-Aria (BD Biosciences), and a fraction positive for green fluorescence was separated to obtain naive human iPS cells according to the method of the present invention.

The obtained naive human iPS cells had a typical naive morphology of small dome-shaped colonies (FIG. 3, B) and exhibited excellent proliferative ability.
In contrast, the human iPS cell line 409B2 and the above-mentioned 409B2 line cultured without the β-catenin-P300 interaction inhibitor both had the morphology of primed pluripotent stem cells with flat and large colonies (Figure 3, A and C )showed that.

RNA was extracted from the obtained naive human iPS cells and total RNA sequence analysis was performed (conducted by Danaform Inc.), which showed a change in gene expression characteristic of naive stem cells (Figure 6, A). Furthermore, when we performed DNA methylation analysis of the whole genome, we obtained results indicating that the content of methylated cytosine was reduced in the naïve stem cells produced according to the present invention (Figure 6, B). .

Example 3
<Production of naive Amami rabbit iPS cells from Amami rabbit iPS cells>
We received a portion of tissue from an adult Amami rabbit that died in an accident from the relevant bureau of the Ministry of the Environment, and isolated Amami rabbit fibroblasts using a standard method. Oct3/4, Klf4, L-Myc, LIN28, Sox2, and a mouse p53 dominant negative mutant were introduced into the same Amami rabbit fibroblast cells using episomal plasmids to generate iPS cells.

Amami Rabbit iPS cells produced by the method described above were cultured on feeder cells, and when they had grown to about 25% of the culture plate (25% confluence), 4 μg of IQ- 1 and cultured for 2 weeks while replacing the stem cell culture medium with fresh IQ-1 added every 24 hours.

During this time, the cells that reached confluence were detached with a 0.25% trypsin (manufactured by Invitrogen)-0.02% EDTA mixture, a pellet of viable cells was obtained by centrifugation, and after removing all the supernatant, a new IQ- The cells were suspended in stem cell culture medium supplemented with 1/4 to 1/8 of the amount and cultured on new feeder cells.

Cells that have formed a dome-shaped colony are collected using a glass tube or micropipette, treated with a 0.25% trypsin-0.02% EDTA mixture until individual cells are isolated, and then centrifuged. A pellet of viable cells was obtained, and after removing all the supernatant, the pellet was suspended in fresh IQ-1 supplemented stem cell culture medium and cultured on fresh feeder cells.

Through the above operations, naive Amami rabbit iPS cells were obtained.

The obtained cells exhibited a typical naïve morphology and exhibited excellent proliferative ability.
The treated cells were subjected to an alkaline phosphatase activity evaluation test, and it was confirmed that the naive Amami rabbit iPS cells produced according to the present invention were strongly positive for alkaline phosphatase activity. Since alkaline phosphatase activity is weakly positive in primed stem cells and strongly positive in naive stem cells, the same results indicate that the present invention is also effective in Amami rabbit cells.

The obtained naive Amami rabbit iPS cells exhibited a typical naive morphology of small dome-shaped colonies (FIG. 3, B) and exhibited excellent proliferative ability.
On the other hand, the Amami rabbit iPS cells and the Amami rabbit iPS cells cultured without the β-catenin-P300 interaction inhibitor both had the morphology of primed pluripotent stem cells (Fig. 3, A and C) were shown.

Furthermore, as shown in Figure 7, the naïve type Amami rabbit iPS cells (B) of the present invention are more stained with alkaline phosphatase activity, which is specifically recognized by the naive type, than the Amami rabbit iPS cells (A). It is clear that the Amami rabbit iPS cells of the present invention are of a naive type.

Example 4
<Production of naive pluripotent stem cells from mouse epiblast stem cells by introducing BC-K49 constantly acetylated mutant gene>
A piggybac transposon and transposase (PBase) containing a cDNA encoding β-catenin (sequence 1) in which 49 lysine residues of β-catenin are replaced with glutamine was electroporated into mouse epiblast stem cell line KEp1 produced at Kindai University. It was introduced by the ration method.

24 hours after the electroporation treatment, cells were cultured in a stem cell culture medium containing hygromycin, and during this period, cells that reached confluence were detached with a mixture of 0.25% trypsin (manufactured by Invitrogen) and 0.02% EDTA, and centrifuged. A viable cell pellet was obtained by the treatment, and after removing all the supernatant, it was suspended in a fresh stem cell culture medium supplemented with hygromycin, and 1/4 to 1/8 of the pellet was cultured on new feeder cells.

The BC-K49 constantly acetylated mutant cDNA and the hygromycin resistance gene are integrated into the same vector, and all cells exhibiting drug resistance express the BC-K49 constantly acetylated mutant cDNA.

After 2 weeks of drug selection, cells that formed colonies were sorted using a glass tube or micropipette, and treated with a 0.25% trypsin-0.02% EDTA mixture until each cell was unified. A pellet of viable cells was obtained by centrifugation, and after removing all the supernatant, the pellet was suspended in a new stem cell culture medium and cultured on new feeder cells. Through the above operations, naive mouse pluripotent stem cells were obtained.

By quantitative RT-PCR analysis of the obtained naive mouse pluripotent stem cells using the method shown in Example 1, it was confirmed that they were positive for naive marker genes such as Klf4 and Zfp42.

Furthermore, as shown in Figure 8, naive mouse pluripotent stem cells clearly have acquired properties characteristic of naive stem cells, as the expression of genes specific to primed stem cells is reduced. be.

Example 5
<Direct production of naive iPS cells from human somatic cells>
Human epidermal fibroblasts were obtained from the RIKEN BioResource Center and cultured in DMEM medium containing 10% fetal bovine serum. Oct3/4, Klf4, L-Myc, LIN28, and Sox2 were introduced into the same cells using a Sendai virus vector. At that time, 4 μg of IQ-1 was added to every 1 mL of the medium, and cultured for 2 weeks while replacing the stem cell culture medium with fresh IQ-1 added every 24 hours.

During this time, the cells that reached confluence were detached with a 0.25% trypsin (manufactured by Invitrogen)-0.02% EDTA mixture, a pellet of viable cells was obtained by centrifugation, and after removing all the supernatant, a new IQ- The cells were suspended in stem cell culture medium supplemented with 1/4 to 1/8 of the amount and cultured on new feeder cells.

Cells that have formed a dome-shaped colony are collected using a glass tube or micropipette, treated with a 0.25% trypsin-0.02% EDTA mixture until individual cells are isolated, and then centrifuged. A pellet of viable cells was obtained, and after removing all the supernatant, the pellet was suspended in fresh IQ-1 supplemented stem cell culture medium and cultured on fresh feeder cells.

Through the above operations, human naive iPS cells were obtained.

The obtained human naive iPS cells exhibited a typical naive morphology of small dome-shaped colonies (FIG. 10, B) and exhibited excellent proliferative ability.
On the other hand, when cultured without a β-catenin-P300 interaction inhibitor, normal primed human iPS cells in the form of flat and large colonies were formed (FIG. 10, C).

Claims (4)

A method for producing naive pluripotent stem cells from primed pluripotent stem cells, the method comprising:
(A) Primed pluripotent stem cells (excluding mouse cells) are treated with a β-catenin-P300 interaction inhibitor, or (B) the higher-order structure of β-catenin in primed pluripotent stem cells is By introducing a gene encoding a mutant β-catenin in which lysine 49 residue is replaced with glutamine or glutamic acid into competent stem cells, the lysine 49 residue is changed to a higher-order structure similar to that of acetylated β-catenin. How to characterize it. The β-catenin-P300 interaction inhibitor in (A) is 2-(4-acetylphenylazo)-2-(3,3-dimethyl-3-,4-dihydro-2H-isoquinolin-1-ylidene)-acetamide. The method according to claim 1, characterized in that: The method is characterized in that the primed pluripotent stem cells (excluding mouse cells) are treated with a β-catenin-P300 interaction inhibitor to transform the primed pluripotent stem cells into naive pluripotent stem cells. Method for naiveting primed pluripotent stem cells (excluding mouse cells). It is characterized by stably maintaining naïve pluripotent stem cells (excluding mouse cells) by treating them with a β-catenin-P300 interaction inhibitor. How to preserve naïve pluripotent stem cells.