JPWO2017175876A1 - How to re-establish stem cells - Google Patents

Abstract

The present invention relates to a method for re-establishing a stem cell having chimera-forming ability by a multi-step process. The method of the present invention comprises the step of co-culturing a first kind of pluripotent stem cell or a multipotent stem cell with a host embryo of a second kind of cell having different culture conditions from the first stem cell. In the step of co-culturing the first type stem cells with the host embryo of the second type cells, the culture medium is a culture medium suitable for the first type stem cells and Using a mixed culture medium suitable for two types of cells and re-establishing chimera-forming stem cells, wherein the re-established chimera-forming stem cells are of the second type A culture medium suitable for cells, a culture medium suitable for the first type of stem cells, or a mixed medium of a culture medium suitable for the first type of stem cells and a culture medium suitable for the second type of cells. But culture Is a function, including that.

Description

  The present invention relates to a method for reestablishing stem cells by a multi-step process.

  Production and supply of organ primordia or organ stem cells from iPS cells (artificial pluripotent stem cells) / ES cells (embryonic stem cells) is necessary for the development of regenerative medicine using iPS cells and the like. For example, in order to transplant human organs / organ primordia regenerated from iPS cells / ES cells to humans, it is considered effective to induce the cells to form target organs in different environments such as pigs and sheep. However, since it is difficult to produce organ primordia / organ stem cells in vitro, it is particularly necessary to produce a heterogeneous chimeric embryo and collect organ primordia or organ stem cells from the chimeric embryo. For this purpose, it is necessary to use high-quality stem cells that are engraftable between different species and maintain chimera-forming ability as starting cells for organ formation.

  In addition, it is useful to produce cloned animals or chimeric animals from iPS cells / ES cells from the viewpoint of storage / regeneration / maintenance of rare animals such as endangered species, pets such as pets, and useful commercial animals. it is conceivable that. In this case, it is necessary to use a high-quality stem cell that is engraftable between different species and maintains the chimera-forming ability as a starting cell.

  Mouse and rat ES cells are generally established as naive pluripotent stem cells, and there are several reports on the production of chimeric animals by the blastocyst complementation method. On the other hand, iPS / ES cells of animals such as monkeys and humans have a prime-type feature in which colonies are flat, and if this is transplanted into a heterologous blastocyst, a chimera can be formed, Even if it can be formed, it is a very small contribution rate.

  Thus, the importance of the method for re-establishing stem cells having the ability to form chimeras is further increased from the viewpoint of increasing the success rate of production of chimeric embryos and chimeric animals.

  Many of the reports so far aimed at obtaining iPS cells / ES cells capable of forming chimeras have established iPS cells / ES cells by means such as gene transfer (Non-patent Document 1). In addition, an example of establishing high-quality iPS cells / ES cells by improving the medium such as addition of environmental factors has been reported (Non-patent Document 2). Although these reported examples are techniques for producing chimera-forming iPS cells / ES cells, the produced iPS cells / ES cells are heterogeneous cell populations, and cells having no chimera-forming ability are mixed. It has not been evaluated enough to deny the possibility of being. These techniques can be said to be techniques for increasing the ratio of cells capable of chimera formation among iPS cells / ES cells.

  Stem cells such as iPS cells and ES cells are generally formed from a cell population having a characteristic with a certain degree of differentiation. For example, it has been reported that cells having totipotency are contained in a very small fraction of ES cells as a whole (Non-patent Document 13).

  At present, cells that maintain the characteristic of “capability of chimera formation” are only a part of all cells obtained at the time of iPS cell / ES cell establishment. In order to obtain starting cells for organ formation and the like, it is necessary to further select and re-establish established iPS cells / ES cells to improve the quality from the viewpoint of maintaining the ability to obtain chimera-forming ability.

  Nagao et al. (Patent Document 1) includes two types including pluripotent cells in which a specific gene is mutated or deleted and the function involved in the gene is lost, and other pluripotent cells other than the pluripotent cell A method for producing a chimeric animal comprising injecting the above-described cells into an animal host embryo is disclosed. Patent Document 1 improves the proliferative ability of other mouse ES cells by co-culturing ES cells established from mouse embryos that cannot form germ cells with other mouse ES cells, for example, ES cells that have been genetically modified. It states that we have found that we can do it.

  However, there is no report at present on a method that enables re-establishment of high-quality iPS / ES cells capable of forming chimeras.

International Publication WO2006 / 009272 Pamphlet PCT / JP2015 / 078699

Hanna, J .; , Et al. , Proc. Natl. Acad. Sci. USA, 107: 9222-9227 (2010) Gafni, O .; , Et al. Nature, 504 (7479): 282-286 (2013). Boroviak, T .; , Et al. , Nature Cell Biology, 16: 513 (2014). Dan, J .; , Et al. , Scientific Rep. 3: 3492 (2013) Suemori H.M. , Et al. Dev Dyn. , 222 (2): 273-9 (2001) Takada T. , Et al. , Cell Transplant. 11 (7): 631-5 (2002) Ban H. , Et al. Proc Natl Acad Sci US, 108 (34): 14234-14239 (2011) Chan Y. , Et al. , Cell Stem Cell, 13: 663-675 (2013) Valamehr B.I. , Et al. , Stem Cell Reports, 2: 366-381 (2014). Wall C.I. , Et al. Proc Natl Acad Sci US, 111 (12): 4484-4489 (2014). Theunissen T. , Et al. , Cell Stem Cell, 15: 1-17 (2014) Takashima Y. et al. , Et al. , Cell 158: 1254-1269 (2014) Macfarlan, T .; S. , Et al. , Nature, 487 (7405): 57-63 (2012).

  In order to transplant human organs / organ organs produced from iPS cells / ES cells into primates such as humans, the step of inducing the formation of target organs in heterogeneous environments such as pigs, sheep, etc. It is valid. For that purpose, it is necessary to use high-quality stem cells that are engraftable between different species and maintain chimera-forming ability as a starting material for organ formation.

  The present invention relates to a method for re-establishing stem cells, re-established stem cells, and utilization of the re-established stem cells.

  Although not necessarily limited, this invention includes the following aspects.

[Aspect 1]
A method for re-establishing a stem cell having the ability to form a chimera by a multi-step process,
A re-establishing step comprising co-culturing a first type of pluripotent stem cell or a multipotent stem cell with a host embryo of a second type of cell having different culture conditions from the first stem cell At least twice, where the culture medium is suitable for the first type of stem cells and the second type of cells in the step of co-culturing the first type of stem cells with the host embryo of the second type of cells. Using a mixed medium of different culture media, and
Re-establishing a stem cell having the ability to form a chimera, wherein the re-established stem cell having the ability to form a chimera is a culture medium suitable for the second type of cell, a culture medium suitable for the first type of stem cell, or The culture medium suitable for the first type of stem cells and the mixed medium of the culture medium suitable for the second type of cells can be cultured in any culture medium.
Said method.

[Aspect 2]
A method for re-establishing a stem cell capable of forming a chimera between different species by a multi-step process,
Performing at least two re-establishment steps comprising co-culturing pluripotent stem cells or multipotent stem cells from a first mammalian species with a host embryo from a second mammalian species; Here, in the step of co-culturing stem cells derived from the first mammalian species with a host embryo derived from the second mammalian species, the culture medium comprises a culture medium suitable for the first mammalian species and a second mammalian species. Using a mixed medium of culture media suitable for the species, and
Re-establishing a stem cell capable of forming a chimera between different species, wherein the stem cell having the ability to form a chimera between different species is a culture medium suitable for the second mammalian species, the first mammalian species Or a culture medium suitable for the first mammalian species and a mixed culture medium suitable for the second mammalian species can be used.
Said method.

[Aspect 3]
The following steps:
(Ia) co-culturing a pluripotent stem cell from a first mammalian species or a multipotent stem cell with a high quality pluripotent stem cell from a second mammalian species Obtaining a population of cells, wherein the culture medium is a culture medium suitable for the first mammalian species; or (ib) Pluripotent stem cells or multipotent from the first mammalian species Combining multipotent stem cells with high quality pluripotent stem cells from a second mammalian species to obtain a population of cells;
(Ii) co-culturing the cell population obtained in step (i) with a host embryo derived from a second mammalian species, wherein a culture medium suitable for the first mammalian species as the culture medium and a second Using a mixed medium of culture media suitable for the mammalian species of
(Iii) separating the inner cell mass from the host embryo co-cultured in step (ii);
(Iv) Step (iii) culturing the resulting inner cell mass, wherein the culture medium is a mixed medium of a culture medium suitable for the first mammalian species and a culture medium suitable for the second mammalian species is there;
(V) co-culturing the cell population of step (iv) with a host embryo derived from a second mammalian species, wherein a culture medium suitable for the first mammalian species as the culture medium and a second mammalian species Using a mixed medium of culture medium suitable for
(Vi) separating the inner cell mass from the host embryo co-cultured in step (v);
(Vii) culturing the inner cell mass obtained in step (vii) and re-establishing stem cells having the ability to form chimera between different species, wherein the culture medium is a culture medium suitable for the second mammalian species A culture medium suitable for the first mammalian species, or the culture medium may be a mixed culture medium suitable for the first mammalian species and a culture medium suitable for the second mammalian species. Good;
Wherein the first mammalian species and the second mammalian species are different species,
A method according to embodiment 2.

[Aspect 4]
In step (ii) and / or step (v) with a host embryo derived from a second mammalian species, after co-culturing in a mixed medium, the medium is a culture medium suitable for the first mammalian species. 4. The method according to aspect 3, further comprising performing co-culture after changing to

[Aspect 5]
The method according to aspect 3 or 4, further comprising repeating steps (v) to (vii) for the stem cells re-established in step (vii).

[Aspect 6]
The method according to any one of aspects 2-5, wherein the first mammalian species is selected from the group consisting of dogs, cats, horses, cows, goats, sheep, monkeys and humans.

[Aspect 7]
The method according to any one of aspects 2-5, wherein the first mammalian species is a species belonging to the order of primates.

[Aspect 8]
The method according to any one of aspects 2-7, wherein the second mammalian species is selected from the group consisting of mice, rats, rabbits and pigs.

[Aspect 9]
The method according to any one of aspects 2-8, wherein the first mammalian species is a monkey and the second mammalian species is a mouse.

[Aspect 10]
The pluripotent stem cells are selected from the group consisting of: ES cells, induced pluripotent stem cells (iPS cells), cloned embryo-derived ES cells (ntES cells), and germ cells (EG cells), and multipotent The stem cell is selected from the group consisting of: trophoblast stem cell (TS cell), epiblast stem cell (EpiS cell), pluripotent germ cell (mGS cell), hematopoietic stem cell, neural stem cell and mesenchymal stem cell The method according to any one of 2-9.

[Aspect 11]
The method according to any one of aspects 2-10, wherein the pluripotent or multipotent stem cell derived from the first mammalian species is an ES cell or an iPS cell.

[Aspect 12]
The method according to any one of aspects 3-11, wherein the high-quality pluripotent stem cells are naïve pluripotent stem cells.

[Aspect 13]
The method according to any one of aspects 1-12, wherein the host embryo is selected from the group consisting of a fertilized egg-derived embryo or an artificially manipulated embryo.

[Aspect 14]
In step (ii) and step (v), the cells of step (i) and step (iv) are microinjected or aggregated into fertilized egg-derived embryos or tetraploid embryos derived from the second mammalian species, respectively. The method according to any one of aspects 2-13, which is carried out by culturing.

[Aspect 15]
A culture medium suitable for the first mammalian species comprises an inhibitor selected from the group consisting of glycogen synthase kinase 3 inhibitor, protein kinase C inhibitor, MEK inhibitor and cyclin dependent kinase inhibitor. A method according to any one of aspects 2-14, characterized in that

[Aspect 16]
A culture medium suitable for the second mammalian species is free of inhibitors selected from the group consisting of glycogen synthase kinase 3 inhibitors, protein kinase C inhibitors, MEK inhibitors and cyclin dependent kinase inhibitors A method according to any one of aspects 2-15, characterized in that

[Aspect 17]
17. The method according to any one of embodiments 2 to 16, wherein the pluripotent stem cell from the first mammalian species is not a human ES cell and the second mammalian species is not a human.

[Aspect 18]
Primate animal stem cells re-established from pluripotent or multipotent stem cells, including:
-Uniformized;
-Chimera formation is possible;
• can form cell aggregates; and • has high affinity for the niche environment of the inner cell mass;
The cell having one or more of the characteristics selected from the group consisting of:

[Aspect 19]
The cell according to aspect 18, re-established by the method according to aspect 1 to 17.

[Aspect 20]
Less than:
Embodiment 19 capable of being cultured in a medium suitable for a second mammalian species; and, having one or more characteristics selected from the group consisting of being easily synchronized with a host embryo derived from a second mammalian species A cell according to 1.

[Aspect 21]
A method for evaluating efficacy or pathophysiology using cells, comprising the following:
(I) Step of obtaining cells: Here, the cells are (A) stem cells having the ability to form chimera re-established by the method according to any one of embodiments 1 to 17, or (B) embodiments 1 to A somatic stem cell, organ progenitor cell, or somatic cell obtained by differentiating a stem cell having the ability to form a chimera re-established by the method according to any one of claims 17, wherein the somatic stem cell, organ Progenitor cells or somatic cells are obtained by any of the following methods (a) to (c):
(A) A chimeric embryo or a chimeric fetus / fetus is produced from a stem cell having the ability to form a chimera re-established by the method according to any one of Embodiments 1 to 17, and a body derived from the chimeric embryo or chimeric fetus / fetus Obtaining sex stem cells, organ progenitor cells, or somatic cells;
(B) The somatic stem cells derived from the chimeric embryo or chimeric fetus / fetus obtained in (a) are differentiated in vitro to obtain organ progenitor cells or somatic cells;
(C) Somatic stem cells, organ progenitor cells, or somatic cells are obtained by differentiating in vitro stem cells having the ability to form chimeras re-established by the method according to any one of aspects 1 to 17;
(Ii) A step of performing drug efficacy evaluation or pathological analysis using the cells obtained in step (i);
Said method.

  The multistage re-establishment method of the present invention has made it possible to efficiently obtain high-quality stem cells having the ability to form chimeras. In particular, the present invention makes it possible to efficiently obtain high-quality stem cells that can form chimeras between different species. The method of the present invention can be widely applied to different stem cells of the same species with different culture conditions.

FIG. 1 shows a schematic diagram of one embodiment of the multi-stage re-establishment method of the present invention. In the process of re-establishing monkey ES cells, the medium is appropriately changed step by step. In particular, the embryo culture medium in the host embryo is optimized.
FIG. 2 is a photomicrograph of monkey ES cells before and after multi-stage re-establishment. Monkey ES cells subcultured in monkey ES medium were observed with a fluorescence microscope (Axio Observer D1 system, Carl Zeiss) using an X10 or X20 objective lens. ES cells after two-stage re-establishment showed green fluorescence and formed colonies of good shape and size.
Upper row: transmission image Middle row: GFP fluorescence image Lower row: superimposed image [Fig. 3] Fig. 3 shows subculture of monkey ES cells re-established in multiple stages in mouse medium, and examined the adaptation to the mouse medium It is a result. Monkey ES cells after two-stage re-establishment cultured using monkey ES medium were subcultured for 5 passages in mouse ES medium. When the obtained cells were observed with a fluorescence microscope, colonies having a good shape and size were maintained.
Upper row: transmission image Middle row: GFP fluorescence image Lower row: superimposed image [FIG. 4] FIG. 4 is a result of examining a HE-stained tissue image of teratoma formed by cell transplantation of monkey ES cells. When the monkey ES cells after multi-stage re-establishment were transplanted into the testicles of NOD-Scid mice, teratomas were formed. An image observed with a microscope after HE staining after fixation is shown.
A. An epithelial tissue (endoderm) (arrowhead) consisting of a luminal structure lined with pseudo-stratified columnar epithelium or single-layer columnar epithelium (arrowhead) is seen around it, fibrous connective tissue (mesoderm) producing collagen (* ) Exists.
B. Neuroepithelial tissue (ectodermal) (arrow) and glial cells (ectodermal) (*) showing a rosette structure can be seen.
C. An epithelial tissue (endoderm) (arrowhead) consisting mainly of a luminal structure lined with a single-layered columnar epithelium is seen, and muscle tissue (mesoderm) (*) exists around it.
D. Neuroepithelial tissue (ectodermal) (arrow) presenting a rosette structure is present. Neuroepithelial tissue (ectodermal) (double arrow) having black-brown melanin pigment is observed.
[FIG. 5] FIG. 5 shows that the teratoma is derived from monkey ES cells that have been re-established in multiple stages. It is the result of detecting the presence of the gene β2 microglobulin (β2MG) by PCR. As a result of agarose gel electrophoresis of the PCR product, a 340 bp long neo-resistant gene PCR product (left figure) and a 133 bp long monkey β2MG gene PCR product (right figure) were detected.
FIG. 6 shows the results of hierarchical clustering of gene expression profiles in each cell before and after re-establishment of monkey ES cells.
[FIG. 7A] FIG. 7A is a graph showing the gene expression levels of 4 endogenous factors of Yamanaka and NANOG before and after re-establishment. FIG. 7A shows the FPKM value of each gene.
[FIG. 7B] FIG. 7B is a diagram showing the gene expression levels of 4 endogenous factors of Yamanaka and NANOG before and after re-establishment. FIG. 7B shows changes in gene expression levels before and after re-establishment as log ₂ values.
[FIG. 7C] FIG. 7C is a graph showing the expression levels of endogenous genes of Yamanaka 4 factor and NANOG before and after re-establishment. FIG. 7C summarizes each FPKM value, log ₂ value, and presence / absence of significance.
[FIG. 8] FIG. 8 shows naive pluripotency, core pluripotency, primed pluripotency, mesoderm / primoderm / p It is the figure which compared before and after re-establishment of the monkey ES cell about the expression of the gene regarding each characteristic of these. Upper: FPKM value of each gene; Lower: log ₂ value of change in gene expression level before and after re-establishment.

  The present invention will be specifically described below, but the present invention is not limited thereto.

  The contents described in PCT / JP2015 / 076699 (filed on October 2, 2015), which is the prior application of the present applicant and the present inventor, are incorporated herein by reference as appropriate.

<Definition>
Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art.

  In the present specification, the “stem cell” is a cell having self-renewal ability and differentiation ability. Here, the self-replicating ability means the ability to replicate cells having the same ability as the self, and means the ability to differentiate into a plurality of cells having functions different from the differentiation ability.

  As used herein, a “pluripotent stem cell” is a stem cell that has the ability to differentiate into all cell types that form an individual. Pluripotent stem cells include embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells), cloned embryo-derived ES cells (ntES cells), embryonic germ cells (EG cells) and the like.

  As used herein, “multipotent stem cell” is a stem cell that has the ability to differentiate into multiple cell types. Pluripotent stem cells include trophoblast stem cells (TS cells), epiblast stem cells (EpiS cells), pluripotent germ cells (mGS cells), hematopoietic stem cells, neural stem cells, mesenchymal stem cells, etc. .

  In the present specification, when “pluripotent stem cells and the like” are described, the expression is understood to include pluripotent stem cells and multipotent stem cells.

  Chimera embryos / chimera animals are produced by transplanting pluripotent stem cells or the like into embryos derived from fertilized eggs of a host (another individual) or artificially engineered embryos (tetraploid embryos, etc.) prepared by developmental engineering. Is done. A chimeric embryo is produced from all or part of a host embryo body in which transplanted pluripotent stem cells and the like are produced. A chimeric animal is produced by transplanted pluripotent stem cells and the like becoming part of an born individual.

In the present specification, a stem cell has “chimera-forming ability” or “capable of chimera formation” means that the stem cell is transplanted into a fertilized egg-derived embryo of a host, an artificially engineered embryo produced by developmental engineering, or the like. Means that the stem cells have the ability to become all or part of the resulting host embryo body, or part of various organs in the born individual. Alternatively, in the present specification, the term “a chimera-forming ability” or “capable of chimera” is used when the stem cell is transplanted into a host fertilized egg-derived embryo or an artificially manipulated embryo. It is also used as a term that means that a stem cell has the ability of a pluripotent stem cell or the like to form a part of an inner cell mass (ICM), that is, the ability to contribute to ICM. However, it does not limit the stem cell, embryo development time, transplant location, and contributing location. Further, as understood from the above description, “chimera-forming ability” does not mean only the ability to form a “individual” chimera.
Here, when the species from which the transplanted stem cells are derived and the species of the host are different, it is particularly said that “chimera can be formed between different species”.

  As used herein, “heterologous” or “different species” refers to different animal species. Unless otherwise stated, “heterologous” or “different species” refers to different animal species at the genus level or higher.

  As used herein, “same species” means belonging to the same animal species. Unless otherwise stated, the range of “same species” includes not only those belonging to the same animal species at the species level but also “same species heterologous” belonging to the same animal species at the genus level.

  In the present specification, the phrase “high quality” of a pluripotent stem cell or the like means that the pluripotent stem cell or the like is “naive type”. A pluripotent stem cell or the like is “naive type” means that a pluripotent stem cell or the like forms a dome-shaped colony, has a chimera-forming ability, and can differentiate into a germ line such as a sperm or egg. It means having one or more, preferably two or more, more preferably all three properties selected from the group consisting of.

  As used herein, “host embryo” means an embryo of a host animal into which a pluripotent stem cell is transplanted when producing a chimeric embryo / chimeric animal.

<Method of re-establishing stem cells through a multi-step process>
The present invention provides a method for re-establishing stem cells having the ability to form chimeras by a multi-step process.

A re-establishing step comprising co-culturing a first type of pluripotent stem cell or a multipotent stem cell with a host embryo of a second type of cell having different culture conditions from the first stem cell At least twice, where the culture medium is suitable for the first type of stem cells and the second type of cells in the step of co-culturing the first type of stem cells with the host embryo of the second type of cells. Using a mixed medium of different culture media, and
Re-establishing a stem cell having the ability to form a chimera, wherein the re-established stem cell having the ability to form a chimera is a culture medium suitable for the second type of cell, a culture medium suitable for the first type of stem cell, or The culture medium suitable for the first type of stem cells and the mixed medium of the culture medium suitable for the second type of cells can be cultured in any culture medium.
Including that.

  In the method of the present invention, the re-establishment step including co-culture with the second-type host embryo is performed at least twice. It is characterized by changing the medium composition in a multistage manner to a culture medium suitable for cells. In at least one stage of the step of co-culturing the first type stem cells with the host embryo of the second type cells, the culture medium is composed of a culture medium suitable for the first type stem cells and a culture medium suitable for the second type cells. Use mixed media. The mixed medium to be used is appropriately optimized depending on the stage of re-establishment. The obtained re-established cells have a chimera-forming ability, and are suitable for the second type of cells, the first type of stem cells, or the first type of stem cells. It is characterized in that it can be cultured in any one of a culture medium and a mixed culture medium suitable for the second type of cells. Preferably, a culture medium suitable for the second type of cells is used.

  In the present invention, the first-type cell and the second-type cell have substantially different cell culture conditions. For example, a case where the species from which the first type cell and the second type cell are derived is different from each other is included.

  Even if it is the same species, for example, embryonic stem cells (ES cells) and induced pluripotent stem cells (iPS cells) of “pluripotent stem cells”, cloned embryo-derived ES cells (ntES cells), embryonic germ cells (EG cells), etc., “multipotent stem cells” trophoblast stem cells (TS cells), epiblast stem cells (EpiS cells), pluripotent germ cells (mGS cells), hematopoietic stem cells, neural stem cells, and Mesenchymal stem cells and the like have different culture conditions depending on the type of each cell. For example, in particular, ES cells and iPS cells, EG cells, TS cells, EpiS cells, and mGS cells have different culture conditions. In such a case, the present invention can be provided.

  Hereinafter, in this specification, the case where the species of the animal from which the first type cell and the second type cell are derived is different is described as an example of the first type cell and the second type cell. .

  The present invention provides a method for re-establishing stem cells having the ability to form chimera between different species by a multi-step process.

The method of the present invention comprises:
Performing at least two re-establishment steps comprising co-culturing pluripotent stem cells or multipotent stem cells from a first mammalian species with a host embryo from a second mammalian species; Here, in the step of co-culturing stem cells derived from the first mammalian species with a host embryo derived from the second mammalian species, the culture medium comprises a culture medium suitable for the first mammalian species and a second mammalian species. Using a mixed medium of culture media suitable for the species and re-establishing a stem cell capable of forming a chimera between different species, wherein the stem cell capable of forming a chimera between different species is A culture medium suitable for a second mammalian species, a culture medium suitable for a first mammalian species, or a culture medium suitable for a first mammalian species and a culture suitable for a second mammalian species Culture medium Can be cultured in any culture medium of
Including that.

  In the method of the present invention, the re-establishment step including co-culture with the second-type host embryo is performed at least twice. The medium used for the re-establishment step is changed from the culture medium suitable for the first mammalian species to the second. It is characterized in that the medium composition is changed in a multistage manner to a culture medium suitable for the mammalian species. During at least one stage of co-culturing stem cells from a first mammalian species with a host embryo from a second mammalian species, the culture medium comprises a culture medium suitable for the first mammalian species and a second mammalian species. Use a mixed culture medium suitable for the species. The mixed medium to be used is appropriately optimized depending on the stage of re-establishment. The obtained re-established cells have the ability to form chimeras between different species, and the culture medium suitable for the second mammalian species, the culture medium suitable for the first mammalian species, or the culture medium It is characterized in that it can be cultured in any one of a culture medium suitable for one mammalian species and a mixed culture medium suitable for a second mammalian species. Preferably, a culture medium suitable for the second mammalian species is used.

Process Steps of the Invention Non-limitingly, the method of the invention comprises, in one aspect, the following steps.
(Ia) co-culturing a pluripotent stem cell from a first mammalian species or a multipotent stem cell with a high quality pluripotent stem cell from a second mammalian species Obtaining a population of cells, wherein the culture medium is a culture medium suitable for the first mammalian species; or (ib) Pluripotent stem cells or multipotent from the first mammalian species Combining multipotent stem cells with high quality pluripotent stem cells from a second mammalian species to obtain a population of cells;
(Ii) co-culturing the cell population obtained in step (i) with a host embryo derived from a second mammalian species, wherein a culture medium suitable for the first mammalian species as the culture medium and a second Using a mixed medium of culture media suitable for the mammalian species of
(Iii) separating the inner cell mass from the host embryo co-cultured in step (ii);
(Iv) Step (iii) culturing the resulting inner cell mass, wherein the culture medium is a mixed medium of a culture medium suitable for the first mammalian species and a culture medium suitable for the second mammalian species is there;
(V) co-culturing the cell population of step (iv) with a host embryo derived from a second mammalian species, wherein a culture medium suitable for the first mammalian species as the culture medium and a second mammalian species Using a mixed medium of culture medium suitable for
(Vi) separating the inner cell mass from the host embryo co-cultured in step (v);
(Vii) culturing the inner cell mass obtained in step (vii) and re-establishing stem cells having the ability to form chimera between different species, wherein the culture medium is a culture medium suitable for the second mammalian species A culture medium suitable for the first mammalian species, or the culture medium may be a mixed culture medium suitable for the first mammalian species and a culture medium suitable for the second mammalian species. Good;
Wherein the first mammalian species and the second mammalian species are different species.

In step (i), a cell comprising a pluripotent or multipotent stem cell derived from a first mammalian species and a high quality pluripotent stem cell derived from a second mammalian species Get a group.
(Ia) co-culturing a pluripotent stem cell from a first mammalian species or a multipotent stem cell with a high quality pluripotent stem cell from a second mammalian species Obtaining a population of cells, wherein the culture medium is a culture medium suitable for the first mammalian species; or (ib) Pluripotent stem cells or multipotent from the first mammalian species A multipotent stem cell is combined with a high quality pluripotent stem cell derived from a second mammalian species to obtain a cell population.

  Preferably, the pluripotent stem cells or multipotent stem cells derived from the first mammalian species are co-cultured with high quality pluripotent stem cells derived from the second mammalian species (step (ia)). Step (ib) comprises combining pluripotent stem cells or multipotent stem cells from a first mammalian species with high quality pluripotent stem cells from a second mammalian species, in particular co-culture. Without being done, it is an embodiment in which it is directly subjected to co-culture with the host embryo in step (ii).

  In the method of the present invention, the type of mammal is not particularly limited. Preferably, the first mammalian species is selected from the group consisting of dogs, cats, horses, cows, goats, sheep, monkeys and humans. Preferably, they are monkeys or humans. Alternatively, preferably, the first mammalian species is a species belonging to the primate.

  "Primates" are also called monkeys in the eyes of the mammals, and the nasal monkeys (original monkeys) including lemurs and loris lower eyes, and other straight-nosed monkeys (true monkeys) ). There are about 220 species of primates (monkeys), including nasal monkeys (true monkeys), including humans, orangutans, gibbons, gibbons, marmosets, capuchins, tarsiers, etc. . Biologically, humans are one of the primates (monkeys), but generally a general term that excludes humans from monkeys is called “monkey”. The same applies to the present specification.

  In the present invention, the medium composition used in the re-establishment process is changed in multiple stages from a culture medium suitable for the first mammalian species to a culture medium suitable for the second mammalian species. It is characterized by. Thus, it is desirable for the second mammalian species to have a culture medium that is substantially different from a culture medium suitable for the first mammalian species. Without limitation, the second mammalian species is preferably selected from the group consisting of mice, rats, rabbits and pigs.

  More preferably, the first mammalian species is a monkey and the second mammalian species is a mouse.

  Preferably, the pluripotent stem cell from the first mammalian species is not a human ES cell and the second mammalian species is not a human.

  The “pluripotent stem cell” is selected from the group consisting of ES cells and induced pluripotent stem cells (iPS cells), cloned embryo-derived ES cells (ntES cells), and germ cells (EG cells). “Multipotent stem cells” include trophoblast stem cells (TS cells), epiblast stem cells (EpiS cells), pluripotent germ cells (mGS cells), hematopoietic stem cells, neural stem cells, and mesenchymal stem cells, Selected from the group consisting of In a preferred embodiment, the “pluripotent or multipotent stem cell” from the first mammalian species is an ES cell or iPS cell.

  “High quality pluripotent stem cells” from the second mammalian species include ES cells, iPS cells, or the inner cell mass of these cells. “High quality pluripotent stem cells” are preferably ES cells. The high-quality pluripotent stem cells are preferably naive pluripotent stem cells.

  After obtaining the cell population in step (i) and before co-culture with the host embryo in step (ii), a cell aggregate is formed from the obtained cell population, and the first mammalian species The cell group containing stem cells derived from derived pluripotent stem cells or multipotent stem cells may be selected. This step is derived from a pluripotent or multipotent stem cell from a first mammalian species that colonizes (especially naïve colony formation) with a high quality pluripotent stem cell from a second mammalian species This is a step of selecting a cell group containing stem cells. Thus, when a stem cell colonies with an ES cell, the stem cell may be expressed as a “stem cell contributing to colony formation”. Selection of cell groups containing stem cells derived from pluripotent stem cells derived from the first mammalian species that colonize (particularly naïve colony formation) with high-quality pluripotent stem cells derived from the second mammalian species, Pluripotent stem cells derived from the first mammalian species can be labeled with an appropriate selection marker before selection, and colonies containing the selection marker can be selected. Alternatively, a high-quality pluripotent stem cell derived from a second mammalian species is labeled with an appropriate selection marker, and colonies that do not contain the selection marker are identified as pluripotent stem cells derived from the first mammalian species. It can also be identified as a cell group containing. Examples of suitable selection markers include: wedge orange (huKO), green fluorescent protein (GFP), Clover, DsRed, mCherry, luciferase, LacZ, neomycin resistance gene, puromycin resistance gene, hygromycin B resistance gene, blasticidin Resistance genes, zeocin resistance genes, DT-A genes, HSV-TK genes, and the like. The identification can be performed by identification by fluorescence, luminescence, staining, or the like, or by drug selection by a drug resistance gene or the like.

  Next, in step (ii) of the above method, the cell population of step (i) is co-cultured with a host embryo derived from a second mammalian species. A chimeric embryo is produced by co-culturing the cell population of step (ii) with a host embryo derived from a second mammalian species. In the co-culture of step (ii), the chimeric embryo is cultured until the initial blastocyst stage where an internal cell mass (ICM) is obtained. In the present invention, in all or part of the step of co-culturing stem cells derived from the first mammalian species with a host embryo derived from the second mammalian species, the culture medium is a culture medium suitable for the first mammalian species. And using a mixed medium of culture medium suitable for the second mammalian species.

  In a preferred embodiment, the co-culture in this step is performed by microinjecting or aggregating the cell population of step (i) into a host embryo derived from a second mammalian species. Microinjection is a method for producing a chimeric embryo by transplanting cells (such as stem cells) into a host embryo. Aggregation is a method of producing a chimeric embryo by using an early embryo up to the morula stage as a host embryo and contacting and assembling stem cells and the like. More preferably, the co-culture of step (ii) is performed by microinjecting the cell population of step (ii) into a host embryo derived from a second mammalian species and co-culturing.

  The host embryo is not particularly limited. Artificially manipulated embryos such as embryos derived from fertilized eggs and tetraploid embryos (tetraploid embryos) are included. In a preferred embodiment, the host embryo is a fertilized egg-derived embryo or a tetraploid embryo early embryo.

  An early embryo means an embryo from a 2-cell stage embryo to a blastocyst stage embryo.

  The tetraploid embryo is an embryo prepared by electrically fusing wild type 2-cell stage blastomeres. When a chimeric embryo is produced by co-culturing with a pluripotent stem cell using a tetraploid embryo, the tetraploid cell cannot contribute to the embryo body itself, but can contribute to extraembryonic tissues such as placenta. Therefore, the embryo body or individual obtained is almost 100% derived from the pluripotent stem cells.

  Preferably, step (ii) of the above method comprises co-culturing the cell population of step (i) by microinjection or aggregation into a fertilized egg-derived embryo or tetraploid embryo derived from a second mammalian species. Done. More preferably, step (ii) of the above method is performed by microinjecting the cell population of step (i) into a fertilized egg-derived embryo or tetraploid embryo derived from a second mammalian species and co-culturing. Is called.

  Next, in step (iii), the inner cell mass is separated from the host embryo co-cultured in step (ii). Separation of the inner cell mass can be performed using a technique known to those skilled in the art. Preferably, the inner cell mass is separated by microscopic surgery or immunosurgery. Microsurgery is an excellent way to remove the inner cell mass while observing the state of the embryo. On the other hand, immunosurgery (Solter, D. and Knowless, BB, Proc. Nat. Acad. Sci. USA, 72 (12): 5099-5102 (1975)) is more mechanically damaged than microscopic surgery. Less, it is excellent for isolating the inner cell mass of blastocysts. Either may be used.

  In step (iv)-(vi), the re-establishment step of step (i) (specifically, step (ia))-step (iii) is repeated to perform the second re-establishment step.

  In the method of the present invention, the re-establishment step including co-culture with the second-type host embryo is performed at least twice. The medium used for the re-establishment step is changed from the culture medium suitable for the first mammalian species to the second. It is characterized in that the medium composition is changed in a multistage manner to a culture medium suitable for the mammalian species. The mixed medium to be used is appropriately optimized depending on the stage of re-establishment.

  Finally, in step (vii), the inner cell mass obtained in step (vii) is cultured, and stem cells having the ability to form chimera between different species are reestablished. Here, the culture medium is a culture medium suitable for the second mammalian species, a culture medium suitable for the first mammalian species, or a culture medium suitable for the first mammalian species and the second culture species. Any of the mixed culture media suitable for the mammalian species. Preferably, it is a culture medium suitable for the second mammalian species.

  Furthermore, steps (v) to (vii) may be repeated for the stem cells re-established in step (vii).

  When the inner cell mass separated from the host embryo in step (iii) and step (vi) is cultured in step (iv) and step (vii), the inner cell mass obtained in step (vi) Cloning of pluripotent stem cells derived from mammalian species or stem cells derived from multipotent stem cells may be performed.

  Cloning of pluripotent stem cells and the like from the inner cell mass can be performed using techniques known to those skilled in the art. That the cloned cell is a pluripotent stem cell derived from the first mammalian species or a stem cell derived from a multipotent stem cell is, for example, labeled with an appropriate selection marker before step (ii). Can be identified. Alternatively, before performing step (ii), the host embryo is labeled with an appropriate selection marker, and cells that do not contain the selection marker are identified as pluripotent or multipotent stem cells derived from the first mammalian species. And can be cloned. Examples of suitable selection markers include: wedge orange (huKO), green fluorescent protein (GFP), Clover, DsRed, mCherry, luciferase, LacZ, neomycin resistance gene, puromycin resistance gene, hygromycin B resistance gene, blasticidin Resistance genes, zeocin resistance genes, DT-A genes, HSV-TK genes, and the like. The identification can be performed by identification by fluorescence, luminescence, staining, or the like, or by drug selection by a drug resistance gene or the like.

  For example, when cloning pluripotent stem cells labeled with wedge orange (huKO) or the like, it can be carried out as follows. Remove the blastocyst together with the blastocyst under a microscope, or remove the part by microscopic surgery. Alternatively, only the inner cell mass is taken out by microscopic surgery or immunosurgery, and transferred and cultured separately on feeder cells in a 4-well plate. From the inner cell mass that has proliferated after seeding, only those in the mosaic state that have been confirmed to have red fluorescence with a fluorescence microscope are visually selected and picked up using the proliferation ability and morphology as indicators. In addition, colonies and cells showing the form of trophoblast-like cells, epithelium-like cells, endoderm-like cells, etc. are removed. The cell mass is dispersed by trypsin treatment, decomposed into several new cell masses, transferred to newly prepared feeder cells, and cultured separately. After sowing, colonies having a good state are selected from newly formed colonies by confirming that they have red fluorescence with a fluorescence microscope. Next, when the cells are dispersed, the cells are further dispersed into a cell cluster having a small number of cells, and the process is repeated several times to obtain colonies containing only pluripotent cells.

  When re-establishing the second stage of step (iv), high-quality pluripotent stem cells derived from the second mammalian species may or may not be added, as in step (i). good. This is because mouse ES cells were established together from mouse ICM through the first-stage re-establishment of steps (i)-(iii) and already in a co-culture state.

Culture medium In the method of the present invention, the re-establishment step including co-culture with the second-type host embryo is performed at least twice. The medium used for the re-establishment step is selected from a culture medium suitable for the first mammalian species. It is characterized by changing the medium composition in a multi-step manner to a culture medium suitable for the second mammalian species. During at least one stage of co-culturing stem cells from a first mammalian species with a host embryo from a second mammalian species, the culture medium comprises a culture medium suitable for the first mammalian species and a second mammalian species. Use a mixed culture medium suitable for the species. The mixed medium to be used is appropriately optimized depending on the stage of re-establishment.

  Conditions suitable for culturing naive pluripotent stem cells or multipotent stem cells derived from the first mammalian species that is the donor species at the beginning of re-establishment, i.e. step (i), in particular Use culture media suitable for mammalian species.

  A person skilled in the art can apply a suitable medium according to the species of the first mammal that is the donor species. For example, when the first mammalian species is a human, it is possible to use primate ES / iPS cell culture medium, RCHEMD001, RCHEMD001A, RCHEMD001B, etc. sold by REPROCELL without limitation. Yes (https://www.reprocell.com/products/research/hes-ipsc/medium). The culture medium for human contains, for example, bFGF.

  For example, when the first mammalian species is a monkey, for example, without limitation, TeSR2 (ST-CELL Technologies) is used in the medium for primate ES / iPS cells sold by REPROCELL. 05860, ST-05880) (http://www.veritastk.co.jp/news.php?id=446) can be used. TeSR2 (ST-05860, ST-05880) is a medium for ES / iPS cells for primates including humans, characterized by feeder cell-free, non-human-derived component-free, and poisonous and deleterious substances-free.

The medium for monkeys and the medium for humans are not particularly distinguished, and a medium that can be used for either one can be used for the other. The culture medium for monkeys and the culture medium for humans may be appropriately added with bFGF or TGFβ, or BMPi, ROCKi, BRAFi, SRCi, activin or the like. For example, a medium having the following composition can be used when the first mammalian species is a monkey or a human.
DMEM / F12 (Invitrogen)
Neurobasal (Invitrogen)
N2 supplement (Invitrogen)
B27 supplement (Invitrogen)
GlutaMAX (Invitrogen)
Non-essential amino acid (NEAA) solution (Invitrogen)
2-mercaptoethanol (2-ME) (Invitrogen)
Penicillin / Streptomycin (Invitrogen)
Bovine serum albumin (BSA) (Sigma)
PD0325901 (Stemgent) MEK1 / 2 inhibitor CHIR99021 (Stemgent) GSK3 inhibitor Forskolin (Sigma) AC (adenylate cyclase) activator Human LIF (hLIF) (Millipore)
Kenpaulone (Sigma)
Cyclin-dependent kinase (CDK) inhibitor Go6983 (Sigma) PKC inhibitor

  In addition, as the culture medium of human or monkey iPS cells, for example, those shown in Table 1 below can be used.

  A person skilled in the art can apply a suitable medium according to the species of the second mammal that is the donor species. For example, when the second kind of mammal is a mouse ES cell or a naive pig iPS cell, a culture solution having the following composition can be used.

Example of mouse ES cell culture composition:
80% (v / v) D-MEM, 20% (v / v) FCS, 1 mM pyruvic acid solution, 0.1 mM 2-mercaptoethanol, 1 × MEM non-essential amino acid solution, 10 ³ U / mL mLIF (Wako) or Medium containing 1xrhLIF (Wako) Example of medium composition of naive porcine iPS cells:
82% (v / v) D-MEM, 15% (v / v) FCS, 0.1 mM 2-mercaptoethanol, 1 × MEM non-essential amino acid solution, 1 × GlutaMAX ^™ -I (GIBCO), 1 × rhLIF (Wako), 10 μM Forskolin Medium containing

  A suitable culture medium for the first mammalian species (eg, primates such as humans, monkeys, etc.) that is ultimately the donor species for organ / organ protoplast formation is the second mammal that provides the host embryo. Compared to a culture medium suitable for various species (pigs, mice, etc.), it has many components and a complicated composition. An example of the composition of the culture medium suitable for the first mammalian species and the example composition of the culture medium suitable for the second mammalian species were compared.

  As shown in Tables 1 and 2, in one aspect, the culture medium suitable for the first mammalian species is preferably glycogen synthase kinase 3 inhibitor, protein kinase C inhibitor, MEK inhibitor and cyclin. Inhibitors selected from the group consisting of dependentase inhibitors. On the other hand, in one embodiment, the culture medium suitable for the second mammalian species is selected from the group consisting of glycogen synthase kinase 3 inhibitor, protein kinase C inhibitor, MEK inhibitor and cyclin dependent kinase inhibitor. Contains no inhibitor.

  The medium composition used in the re-establishment process is changed in a multistage manner from a culture medium suitable for the first mammalian species to a culture medium suitable for the second mammalian species. During at least one stage of co-culturing stem cells from a first mammalian species with a host embryo from a second mammalian species, the culture medium comprises a culture medium suitable for the first mammalian species and a second mammalian species. Use a mixed culture medium suitable for the species. For cocultivation with the host embryo, it is considered that culture using a culture medium suitable for culturing the second mammalian species from which the host embryo is derived is necessary. However, stem cells derived from the first mammalian species are difficult to efficiently co-culture only with a culture medium suitable for culturing the second mammalian species. Therefore, in the present invention, a mixed medium in which a culture medium suitable for the first mammalian species is appropriately added to a culture medium suitable for the second mammalian species is used (optimization of the embryo culture medium). Features. By using a mixed medium, a cell mass containing stem cells derived from the first mammal is efficiently formed in the co-culture in the host embryo derived from the second mammalian species, and the stem cells having the ability to form chimeras among different species are reestablished. Is possible.

  In the first re-establishment, in the step of co-culture in a host embryo derived from a second mammalian species (step (ii)), a culture medium suitable for the first mammalian species is used as the second mammalian species. To a suitable culture medium, it is added, but not limited to, preferably in the range of about 10/1-1/10, more preferably in the range of about 2 / 1-1 / 2.

  In the second re-establishment, in the step of co-culturing in a host embryo derived from a second mammalian species (step (v)), the ratio of adding a culture medium suitable for the first mammalian species may be further increased A culture medium suitable for the first mammalian species is possible relative to a culture medium suitable for the second mammalian species, but is not limited, preferably in the range of about 100: 1-1: 100, Those added in a range of preferably about 10: 1-1: 10 can be used.

  The culture medium is a culture medium suitable for the first mammalian species and a culture medium suitable for the second mammalian species, the presence or absence of serum or serum substitutes, the presence or absence of various inhibitors, the presence or absence of various growth factors, It can be appropriately prepared depending on the presence or absence of other trace components.

  In step (ii) and / or step (v) with a host embryo derived from a second mammalian species, after co-culturing in a mixed medium, the medium is a culture medium suitable for the first mammalian species. Further, co-cultivation may be carried out.

  In step (iii), the inner cell mass separated from the host embryo is cultured (step (iv)). The culture medium in step (iv) differs from step (i) in that it is not a culture medium suitable for the first mammalian species but a culture medium suitable for the first mammalian species and a second mammalian species. The mixed culture medium can be used. In this case, the culture medium suitable for the first mammalian species is non-limiting, preferably in the range of about 1: 3-3: 1, more preferably relative to the culture medium suitable for the second mammalian species. Can be added in the range of about 1: 2-2: 1.

  During the second re-establishment, the culture medium after re-establishment (step (vii)) is a culture medium suitable for the second mammalian species, a culture medium suitable for the first mammalian species, or a culture medium Either a culture medium suitable for the first mammalian species or a mixed medium of culture media suitable for the second mammalian species may be used. Preferably, it is a culture medium suitable for the second mammalian species. That is, the stem cells re-established by the method of the present invention can be adapted to the culture conditions of the recipient. The ratio of the culture medium suitable for the first mammalian species and the culture medium suitable for the second mammalian species in the mixed medium can also be appropriately selected.

<Stem cells re-established from pluripotent or multipotent stem cells>
The invention also provides primate animal stem cells re-established from pluripotent or multipotent stem cells. The stem cells of the present invention are as follows:
-Uniformized;
-Chimera can be formed, especially between different species;
• can form cell aggregates; and • has high affinity for the niche environment of the inner cell mass;
Having one or more features selected from the group consisting of: Preferably, it has 2 or more and 3 or more of the above properties.

  “Uniform” means that the original cell group is re-established by re-establishing it from a group of cells that have multiple properties ranging from a more undifferentiated state to a relatively advanced state. This means that the cell group is made up of cells with more similar properties.

  “A chimera can be formed” and “a chimera can be formed between different species” are as described in the section <Definition>.

  “A cell aggregate can be formed” means that when cultured under the condition of co-culturing different cell groups, it has the ability to form a cell aggregate, or the cells adhere to each other, or the cells are the same. It means that it can exist in the place.

  “High affinity to the niche environment of the inner cell mass” means that the ability of the stem cells to survive in the niche environment of the inner cell mass, that is, the situation in the vicinity of the periphery, is high.

  In the examples of the present invention, the colony shape of the re-established stem cells was clean and showed a large dome shape, and the teratoma formation experiment confirmed the diversity showing trilobal differentiation. Furthermore, when the origin of teratomas was confirmed by PCR, the presence of a marker gene introduced into monkey ES cells (first mammalian ES cells) was confirmed.

The stem cell of the present invention further includes the following:
Can be cultured in a medium suitable for a second mammalian species; and; has one or more characteristics selected from the group consisting of being easily synchronized with a host embryo derived from a second mammalian species. Preferably, it means both of the above two conditions.

  “Can be cultured in a medium suitable for the second mammalian species” means, but is not limited to, for example, five passages when cultured in a medium suitable for the second mammalian species. It means that it has one of the requirements such as being able to survive, maintaining pluripotency as confirmed by, for example, a teratoma experiment, or maintaining the shape of a dome-shaped colony. To do.

  “Easy to synchronize with a host embryo derived from a second mammalian species” means that the re-established stem cell of the present invention is a recipient species (second secondary species) that is different from the donor species (first mammalian species). When co-cultured in a host embryo derived from a mammalian species), it means that the cell cycle of the stem cell of the present invention is easily aligned with the cell cycle of the cell of the host embryo.

  The stem cells having the ability to form chimera between different species re-established by the method of the present invention are not particularly limited, and can be used for the following uses.

  An organ / organ primordium derived from the stem cell can be formed by producing a chimeric animal by the blastocyst complementation method using the stem cell having the chimera-forming ability of the present invention. Such organs / organ discs are useful in regenerative medicine.

  Further, a chimeric embryo or a chimeric fetus / fetus is produced by the blastocyst complementation method using the stem cell having the ability to form a chimera of the present invention, and the somatic stem cell, organ progenitor cell and body derived from the chimeric embryo or chimeric fetus / fetus Sex cells can be obtained. Also, organ progenitor cells and somatic cells can be obtained by differentiating somatic stem cells derived from the chimeric embryo or chimeric fetus / fetus in vitro. Alternatively, somatic stem cells, organ progenitor cells, and somatic cells can be obtained by differentiating stem cells having the ability to form chimeras of the present invention in vitro. These somatic stem cells, organ progenitor cells and somatic cells are useful for use in drug efficacy evaluation and pathological analysis. Since the stem cell having the chimera-forming ability of the present invention is a high-quality stem cell that maintains the chimera-forming ability, it can efficiently produce or differentiate a chimeric embryo / chimera animal using the cell as a starting cell. .

  Alternatively, using the stem cells having the ability to form a chimera of the present invention, an animal to which the stem cells have been transmitted can be produced by the tetraployless cue method. The tetraploid dress cue method utilizes the fact that, by injecting iPS / ES cells or the like into tetraploid embryos, tetraploid cells develop into the placenta and only iPS / ES cells etc. develop into the individual. (Nagy, A., et al., Development, 110, 815-821 (1990)). Such an animal production method is effective for the preservation, reproduction, and / or maintenance of rare animals such as endangered species, pet mammals such as pets, and useful commercial animals.

<Method of evaluating efficacy or pathological analysis using cells>
The present application further provides a medicinal effect using a chimera-forming stem cell obtained by the re-establishment method, or a somatic stem cell, organ progenitor cell or somatic cell obtained by differentiating the chimera-forming stem cell. A method for performing an evaluation or pathological analysis is provided.

The method of the present invention includes the following:
(I) A step of obtaining a cell: wherein the cell is (A) a stem cell having the ability to form a chimera re-established by the method of the present invention, or (B) the cell according to any one of aspects 1 to 17 A somatic stem cell, organ progenitor cell, or somatic cell obtained by differentiating a stem cell having the ability to form a chimera re-established by the method, wherein the somatic stem cell, organ progenitor cell, or somatic cell is It is obtained by any of the following methods (a) to (c):
(A) A chimeric embryo or a chimeric fetus / fetus is produced from a stem cell having the ability to form a chimera re-established by the method according to any one of Embodiments 1 to 17, and a body derived from the chimeric embryo or chimeric fetus / fetus Obtaining sex stem cells, organ progenitor cells, or somatic cells;
(B) The somatic stem cells derived from the chimeric embryo or chimeric fetus / fetus obtained in (a) are differentiated in vitro to obtain organ progenitor cells or somatic cells;
(C) Somatic stem cells, organ progenitor cells, or somatic cells are obtained by differentiating in vitro stem cells having the ability to form chimera re-established by the method according to any one of aspects 1 to 17;
(Ii) A step of performing drug efficacy evaluation or pathological analysis using the cells obtained in step (i);
Said method.

  In the step (i) of the above method, the “stem cell capable of forming a chimera” in (A) is regenerated from a pluripotent stem cell or a multipotent stem cell by the “method for reestablishing a stem cell by a multi-step process”. Can be established.

In step (i) of the above method, the “somatic stem cell, organ progenitor cell or somatic cell obtained by differentiating a stem cell having chimera-forming ability” in (B) After having obtained “stem cells” in the same manner as (A), it can be obtained by performing any of the following methods (a) to (c).
The method of (a) is to produce a chimeric embryo or a chimeric fetus / fetus from a stem cell capable of forming a chimera, and obtain a somatic stem cell, organ progenitor cell, or somatic cell derived from the chimeric fetus / fetus. It is. The method for producing a chimeric embryo or a chimeric fetus / fetus can be carried out by the method described for producing a chimeric embryo / chimera animal in the item “Definitions” above. In order to obtain somatic stem cells, organ progenitor cells, or somatic cells from a chimeric embryo or a chimeric fetus / fetus, the cells are sorted and established. Sorting and establishment of somatic stem cells, organ progenitor cells, or somatic cells from a chimeric embryo or chimeric fetus / fetus can be appropriately performed by methods commonly used by those skilled in the art.
In the method (b), the somatic stem cells derived from the chimeric embryo or chimeric fetus / fetus obtained in (a) are differentiated in vitro to obtain organ progenitor cells or somatic cells. A method for differentiating somatic stem cells in vitro to obtain organ progenitor cells or somatic stem cells can be appropriately performed by methods commonly used by those skilled in the art.
The method (c) is to obtain somatic stem cells, organ progenitor cells, or somatic cells by differentiating stem cells having the ability to form chimera in vitro. A method of obtaining somatic stem cells, organ progenitor cells, or somatic cells by differentiating stem cells having the ability to form chimera in vitro is appropriately performed by a method commonly used by those skilled in the art to differentiate pluripotent stem cells in vitro. be able to.

  The “somatic stem cell, organ progenitor cell, or somatic cell” obtained in step (i) of the above method is related to, for example, the heart, nerve, kidney, liver, pancreas, skeletal muscle, blood cell, etc. Although it is a cell, it is not specifically limited.

  In step (ii) of the above method, drug efficacy evaluation or pathological condition analysis is performed using the cells obtained in step (i). The efficacy evaluation or pathological analysis is not particularly limited as long as it is a test method using cells. This can be appropriately carried out by methods usually used by those skilled in the art.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail based on an Example, this invention is not limited to these Examples. Those skilled in the art can easily modify and change the present invention based on the description of the present specification, and these are included in the technical scope of the present invention.

Example 1 Multi-stage Re-establishment Method Using ES Cells This example describes a multi-stage re-establishment method using ES cells. Monkey ES cells (sESCs) are derived from cynomolgus monkeys (Macaca fascicularis) and are cell lines (CMK6G) (Non-Patent Documents 5 and 6) into which green fluorescent protein (GFP) and neomycin resistance gene (neo) are introduced as marker genes. Furthermore, a multi-stage re-establishment method was carried out using a temperature-sensitive Sendai virus (SeV18 + hNANOG / TS7dF) (Non-patent Document 7) expressing human nanog.

  Specifically, first, the ES cells were mixed with monkey ES medium [35.9% (v / v) DMEM / F12, 48% (v / v) Neurobasal, 1% (v / v) N2 supplement, 2% (V / v) B27 supplement, 1 mM GlutaMAX, 1% (v / v) nonessential amino acid (NEAA), 0.1 mM 2-mercaptoethanol (2-ME), 1X penicillin / streptomycin, 5 mg / mL bovine serum albumin ( BSA), 1 μM PD0325901, 1 μM CHIR99021, 10 μM forskolin, 20 ng / mL human LIF, 5 μM Kenpaulone, 5 μM Go6983] were co-cultured with mouse ES cells. Monkey ES cells that had aggregated together with the cell mass of mouse ES cells were selected (step (i)).

The obtained monkey ES cells were microinjected into 40 mouse tetraploid blastocysts (MCH), followed by mouse medium [80% (v / v) D-MEM, 20% (v / v) FCS, 1 mM. Mouse initial embryo culture conditions (mwm culture medium, 5% CO ₂ ) in which the above monkey ES culture medium was added to pyruvic acid solution, 0.1 mM 2-ME, 1 × NEAA, 10 ³ U / mL mLIF] at a ratio of 1: 1. , 37 ° C.) for 1 day (step (ii)).

  Abnormally shaped blastocysts with monkey ES vesicles other than ICM were removed under a microscope, and only good embryos were separately transferred onto 4-well plate feeder cells and cultured. As the culture solution, an ES culture solution for monkeys was used.

  A good shape bear from ICM that has grown after sowing, and only those in a mosaic state that have been confirmed to have green fluorescence with a fluorescence microscope (Axio Observer D1 system, Carl Zeiss) are proliferative and have good colony morphology Were selected and picked up visually. The cell mass was dispersed by treatment with 0.025% trypsin / 0.1 mM EDTA (step (iii)). Thereafter, the cells were separately transferred onto the newly prepared 4-well plate feeder cells and cultured. The culture solution used was a monkey medium added to a mouse medium at a ratio of 2: 1.

  After seeding, the newly formed colonies were confirmed to have green fluorescence with a fluorescence microscope, and colonies in good condition were selected and cultured. This was used as the monkey ES cell after the first reestablishment. The first-stage re-established monkey ES cells can be cultured in a mixed culture solution in which the monkey medium is mixed with the mouse medium, for example, 1: 1, so that the proliferative property and the colony can be improved. The shape was maintained (step (iv)).

  A mixed culture solution in which the first stage re-established monkey ES cells were added to a mouse medium at a ratio of 2: 1 together with a small amount of mouse ICM-derived mouse 4NES cells and mouse ES cells contributed by mouse ICM. Was used for co-culture, and colonies with good growth ability and morphology were selected.

After microinjecting these monkey ES cells into 30 mouse tetraploid blastocysts (MCH), a mouse initial embryo culture condition (mwm culture) in which a monkey medium was added to a mouse initial embryo culture medium at a ratio of 1:10. Solution, 5% CO ₂ , 37 ° C.) for 1 day (step (v)). Abnormally shaped blastocysts with monkey ES cells other than ICM are removed under a microscope, and only good embryos are transferred separately onto feeder cells on a 4-well plate, and monkey medium is used for mice. Culturing was performed using a mixed culture solution added to the medium at a ratio of 2: 1. Only those in a mosaic state that were confirmed to have good morphology from the ICM that had grown after seeding and had green fluorescence with a fluorescence microscope (Axio Observer D1 system, Carl Zeiss) were visually observed using the growth ability and morphology as indicators. Selected and picked up.

  The monkey ES cells after the second-stage re-establishment had a markedly increased contribution to ICM than before the re-establishment. The cell mass was dispersed by treatment with 0.025% trypsin / 0.1 mM EDTA (step (vi)). Thereafter, the cells were separately transferred onto the newly prepared 4-well plate feeder cells and cultured. The culture medium used was a monkey medium.

  After sowing, colonies that were in good condition were selected from newly formed colonies and confirmed to have green fluorescence using a fluorescence microscope, and cultured in a medium containing neomycin (500 μg / ml). This was used as a monkey ES cell after the second-stage re-establishment (step (vii)) (FIG. 2).

The monkey ES cells after the two-stage re-establishment were re-established in a mixed culture medium, but the modified mouse culture medium supplemented with 2 inhibitors [80% (v / v) D-MEM, 20% (v / V) FCS, 1 mM pyruvic acid solution, 0.1 mM 2-ME, 1 × NEAA, 10 ³ U / mL hLIF, 1 μM PD0325901, 1 μM CHIR99021] have good growth ability, and have good shape and size of colonies Formation was possible (the upper part of FIG. 3).

Example 2 Examination of pluripotency of ES cells after two-stage re-establishment In this example, pluripotency of monkey ES cells after two-stage re-establishment was examined in a teratoma formation experiment.

  When the monkey ES cells after two-stage re-establishment were transplanted into the testicles of NOD-Scid mice, a large tumor was formed in the testicles about 3 months later. After dissection, the tumor part was fixed with 4% paraformaldehyde fixative, and a HE (hematoxylin and eosin) -stained specimen was prepared. As a result of histological examination, neuroepithelial tissue and glial tissue exhibiting Rosetta structure as ectoderm, neuroepithelial tissue containing melanin pigment, lumen lined with pseudostratified columnar epithelium or single layer columnar epithelium as endoderm Structured epithelial tissue, fibrous connective tissue and muscle tissue producing collagen as mesoderm are observed (middle and bottom in FIG. 3). As described above, it was confirmed that ectoderm, mesoderm, and endoderm-derived tissues were formed from monkey ES cells after two-stage re-establishment.

Example 3 Confirmation of Teratoma Derived from Monkey ES Cells In this example, it was confirmed that the origin of teratoma formed in Example 2 was indeed monkey ES cells. Specifically, DNA was extracted from teratoma tissue, and detection of neo gene derived from monkey ES cells and β2 microglobulin (β2MG), which is a monkey-derived gene, was attempted by PCR.

First, a part of the teratoma tissue was collected, and after protease treatment, DNA was extracted. Using this DNA, a neomycin resistance gene (neo), which is a marker gene introduced into monkey ES cells, was first detected by PCR. For detection of the neo gene, 35 cycles of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 30 seconds were performed using the following primer sets of SEQ ID NOS: 1 and 2 and rTaq.
Primer set 5'-AGC CGG TCT TGT CGA TCA GGA TGA TC-3 '(SEQ ID NO: 1) and 5'-CTC GTC AAG AAG GCG ATA GAA GGC-3' (SEQ ID NO: 2)

Nested PCR was used for detection of the monkey β2 microglobulin (β2MG) gene. For the first amplification, 30 cycles of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 30 seconds were performed using the following primer sets of SEQ ID NOS: 3 and 4 and rTaq.
5′-CAG GTT TAC TCA CGT CAT CCA G-3 ′ (SEQ ID NO: 3) and 5′-GGT TCA CAC GGC AGG CAT ACT C-3 ′ (SEQ ID NO: 4)

For the second amplification, 25 cycles of 94 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 30 seconds were performed using the following primer sets of SEQ ID NOS: 5 and 6 and rTaq.
5′-GTC TGG GTT TCA TCC ATC CG-3 ′ (SEQ ID NO: 5) and 5′-GGT GAA TTC AGT GTA GTA CAA G-3 ′ (SEQ ID NO: 6)

  The specificity of PCR was high, and the neo gene and monkey β2MG gene were not detected in the DNAs of the negative control mouse liver and B6 mouse tail, but were detected only in teratoma tissues and monkey ES cells after re-establishment (FIG. 5). ). From the above, it was confirmed that teratomas are derived from monkey ES cells carrying the neo gene. That is, it was shown that the re-established monkey ES cells have pluripotency by forming teratomas differentiated into trilobes.

  As mentioned above, it was shown that the multi-step re-establishment method is effective in forming a dome-shaped colony with a beautiful shape in primate pluripotent stem cells and maintaining pluripotency with trilobal differentiation ability. It was. In addition, the fact that pluripotent stem cells after multi-stage re-establishment have acquired a trait that can be cultured in a medium for mice is advantageous in the formation of heterologous chimeric embryos such as mice, and the maintenance and maintenance of pluripotent stem cells. This is advantageous in terms of amplification and cost.

Example 4 Hierarchical clustering of gene expression profiles in cells before and after re-establishment Three culture dishes of monkey ES cells (sES cell) before re-establishment, monkey ES cells (Re-sES cell) after re-establishment RNA was extracted from each of three culture dishes.

  The RNA extracted from each cell was subjected to RNA-Seq using Illumina HiSeq 2000 and mapped in the rhesus monkey transcriptome pipeline. As a result, 18,339 genes were identified. The expression level of the gene tested by the combination of comparison between samples is indicated by FPKM (Fragments Per Kilometer of exon per Million mapped fragments) value, clustered by group average method, and distance was measured by canberra algorithm .

  FIG. 6 shows the results of hierarchical clustering of gene expression profiles in each cell before and after re-establishment of monkey ES cells. The gene expression profiles of cells before and after re-establishment were roughly grouped into two groups. That is, it was revealed that the gene expression profile of cells after re-establishment shows a specific gene expression pattern, unlike that before re-establishment.

Example 5 Expression of Yamanaka 4 Factor and NANOG in Re-Established Monkey ES Cells Monkey ES cells before re-establishment obtained in Example 4 (sES1, sES2, sES3), and monkey ES cells after re-establishment (Re_sES1) , Re_sES2, Re_sES3) of monkey endogenous genes (PAU5F1 (Oct4), KLF4, SOX2, MYC) corresponding to Yamanaka 4 factors and NANOG genes important for maintaining pluripotency before and after re-establishment. Changes were examined based on changes in FPKM values. The results are shown in FIG. FIG. 7A shows FPKM values of each gene, and FIG. 7B shows changes in gene expression levels before and after re-establishment as log ₂ values. FIG. 7C summarizes each FPKM value, log ₂ value, and presence / absence of significance. In the monkey ES cells after re-establishment, the gene expression levels of PAU5F1 (Oct4), KLF4, SOX2, and MYC, which are four endogenous Yamanaka factors, increased significantly. Moreover, the expression level of NANOG necessary for maintaining pluripotency was also significantly increased. From the above, in monkey ES cells after re-establishment, increased expression of endogenous genes important for maintaining pluripotency was observed.

Example 6 Gene Expression Pattern in Re-established Monkey ES Cells From the results of RNA-Seq obtained in Example 6, naive pluripotency, core pluripotency, prime pluripotency FIG. 8 shows the results of comparing the expression of genes related to the characteristics of sex (primed primipotency) and mesoderm / primitive streak before and after the re-establishment of monkey ES cells. The upper row shows the FPKM value of each gene, and the lower row shows the change in gene expression level before and after re-establishment as a log ₂ value. Re-established genes related to the characteristics of naive pluripotency such as KLF4, KLF5, DPPA3, ZFP42, TFCP2L1 and PRDM14, NANOG, SOX2, POU5F1, GDF3, SALL4, UTF4 core, etc. The expression level of genes related to the characteristics of core pluripotency has increased. On the other hand, the expression levels of LEF1 and CER1 related to the characteristics of primed pluripotency and the gene expression levels of EOMES and MSX1 related to the characteristics of mesoderm / primitive streak decreased. From the above, it is understood that monkey ES cells have become a higher quality pluripotent stem cell state by re-establishment.
DPPA3 = STELLA
ZFP42 = REX1 = ZNF754
PRDM14 = methyltransferase activity KLF4 = Krueppel-like factor 4
[Sequence Listing]

Claims (21)

A method for re-establishing a stem cell having the ability to form a chimera by a multi-step process,
A re-establishing step comprising co-culturing a first type of pluripotent stem cell or a multipotent stem cell with a host embryo of a second type of cell having different culture conditions from the first stem cell At least twice, where the culture medium is suitable for the first type of stem cells and the second type of cells in the step of co-culturing the first type of stem cells with the host embryo of the second type of cells. Using a mixed medium of different culture media, and
Re-establishing a stem cell having the ability to form a chimera, wherein the re-established stem cell having the ability to form a chimera is a culture medium suitable for the second type of cell, a culture medium suitable for the first type of stem cell, or The culture medium suitable for the first type of stem cells and the mixed medium of the culture medium suitable for the second type of cells can be cultured in any culture medium.
Said method. A method for re-establishing a stem cell capable of forming a chimera between different species by a multi-step process,
Performing at least two re-establishment steps comprising co-culturing pluripotent stem cells or multipotent stem cells from a first mammalian species with a host embryo from a second mammalian species; Here, in the step of co-culturing stem cells derived from the first mammalian species with a host embryo derived from the second mammalian species, the culture medium comprises a culture medium suitable for the first mammalian species and a second mammalian species. Using a mixed medium of culture media suitable for the species, and
Re-establishing a stem cell capable of forming a chimera between different species, wherein the stem cell having the ability to form a chimera between different species is a culture medium suitable for the second mammalian species, the first mammalian species Or a culture medium suitable for the first mammalian species and a mixed culture medium suitable for the second mammalian species can be used.
Said method. The following steps:
(Ia) co-culturing a pluripotent stem cell from a first mammalian species or a multipotent stem cell with a high quality pluripotent stem cell from a second mammalian species Obtaining a population of cells, wherein the culture medium is a culture medium suitable for the first mammalian species; or (ib) Pluripotent stem cells or multipotent from the first mammalian species Combining multipotent stem cells with high quality pluripotent stem cells from a second mammalian species to obtain a population of cells;
(Ii) co-culturing the cell population obtained in step (i) with a host embryo derived from a second mammalian species, wherein a culture medium suitable for the first mammalian species as the culture medium and a second Using a mixed medium of culture media suitable for the mammalian species of
(Iii) separating the inner cell mass from the host embryo co-cultured in step (ii);
(Iv) Step (iii) culturing the resulting inner cell mass, wherein the culture medium is a mixed medium of a culture medium suitable for the first mammalian species and a culture medium suitable for the second mammalian species is there;
(V) co-culturing the cell population of step (iv) with a host embryo derived from a second mammalian species, wherein a culture medium suitable for the first mammalian species as the culture medium and a second mammalian species Using a mixed medium of culture medium suitable for
(Vi) separating the inner cell mass from the host embryo co-cultured in step (v);
(Vii) culturing the inner cell mass obtained in step (vii) and re-establishing stem cells having the ability to form chimera between different species, wherein the culture medium is a culture medium suitable for the second mammalian species A culture medium suitable for the first mammalian species, or the culture medium may be a mixed culture medium suitable for the first mammalian species and a culture medium suitable for the second mammalian species. Good;
Wherein the first mammalian species and the second mammalian species are different species,
The method of claim 2.   In step (ii) and / or step (v) with a host embryo derived from a second mammalian species, after co-culturing in a mixed medium, the medium is a culture medium suitable for the first mammalian species. The method according to claim 3, further comprising co-culturing after changing to the above.   The method according to claim 3 or 4, further comprising repeating steps (v) to (vii) for the stem cells re-established in step (vii).   6. A method according to any one of claims 2-5, wherein the first mammalian species is selected from the group consisting of dogs, cats, horses, cows, goats, sheep, monkeys and humans.   6. A method according to any one of claims 2-5, wherein the first mammalian species is a species belonging to the order of primates.   8. The method of any one of claims 2-7, wherein the second mammalian species is selected from the group consisting of mice, rats, rabbits and pigs.   9. The method of any one of claims 2-8, wherein the first mammalian species is a monkey and the second mammalian species is a mouse.   The pluripotent stem cells are selected from the group consisting of: ES cells, induced pluripotent stem cells (iPS cells), cloned embryo-derived ES cells (ntES cells), and germ cells (EG cells), and multipotent The stem cells are selected from the group consisting of: trophoblast stem cells (TS cells), epiblast stem cells (EpiS cells), pluripotent germ cells (mGS cells), hematopoietic stem cells, neural stem cells and mesenchymal stem cells, Item 10. The method according to any one of Items 2-9.   11. The method of any one of claims 2-10, wherein the pluripotent stem cell or multipotent stem cell derived from the first mammalian species is an ES cell or iPS cell.   The method according to any one of claims 3 to 11, wherein the high-quality pluripotent stem cells are naive pluripotent stem cells.   The method according to any one of claims 1 to 12, wherein the host embryo is selected from the group consisting of a fertilized egg-derived embryo or an artificially manipulated embryo.   In step (ii) and step (v), the cells of step (i) and step (iv) are microinjected or aggregated into fertilized egg-derived embryos or tetraploid embryos derived from the second mammalian species, respectively. The method according to any one of claims 2 to 13, which is carried out by culturing.   A culture medium suitable for the first mammalian species comprises an inhibitor selected from the group consisting of glycogen synthase kinase 3 inhibitor, protein kinase C inhibitor, MEK inhibitor and cyclin dependent kinase inhibitor. 15. A method according to any one of claims 2-14, characterized in that   A culture medium suitable for the second mammalian species is free of inhibitors selected from the group consisting of glycogen synthase kinase 3 inhibitors, protein kinase C inhibitors, MEK inhibitors and cyclin dependent kinase inhibitors 16. A method according to any one of claims 2-15, characterized in that   17. A method according to any one of claims 2 to 16, wherein the pluripotent stem cells derived from the first mammalian species are not human ES cells and the second mammalian species is not human. Primate animal stem cells re-established from pluripotent or multipotent stem cells, including:
-Uniformized;
-Chimera formation is possible;
• can form cell aggregates; and • has high affinity for the niche environment of the inner cell mass;
The cell having one or more of the characteristics selected from the group consisting of:   The cell according to claim 18, which has been re-established by the method according to claim 1. Less than:
Claims capable of being cultured in a medium suitable for a second mammalian species; and, having one or more characteristics selected from the group consisting of being easily synchronized with a host embryo derived from a second mammalian species. 19. The cell according to 19. A method for evaluating efficacy or pathophysiology using cells, comprising the following:
(I) a step of obtaining a cell: wherein the cell is (A) a stem cell having the ability to form a chimera re-established by the method according to any one of claims 1 to 17, or (B) A somatic stem cell, organ progenitor cell, or somatic cell obtained by differentiating a stem cell having the ability to form a chimera re-established by the method according to any one of 1 to 17, wherein the somatic stem cell Organ progenitor cells or somatic cells are obtained by any of the following methods (a) to (c):
(A) A chimeric embryo or a chimeric fetus / fetus is produced from a stem cell having the ability to form a chimera re-established by the method according to any one of claims 1 to 17, and the chimeric embryo or the chimeric fetus / fetus is derived. Obtaining somatic stem cells, organ progenitor cells, or somatic cells;
(B) The somatic stem cells derived from the chimeric embryo or chimeric fetus / fetus obtained in (a) are differentiated in vitro to obtain organ progenitor cells or somatic cells;
(C) obtaining a somatic stem cell, an organ progenitor cell, or a somatic cell by differentiating in vitro a stem cell having the ability to form a chimera re-established by the method according to any one of claims 1 to 17;
(Ii) A step of performing drug efficacy evaluation or pathological analysis using the cells obtained in step (i);
Said method.