JP6935101B2 - How to reestablish stem cells - Google Patents

Description

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

Producing and supplying organ primordia or organ stem cells from iPS cells (induced 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 a human organ / organ primordium regenerated from iPS cells / ES cells into a human, it is considered effective to induce the cells to form a target organ in a heterogeneous environment such as a pig or a sheep. However, since it is difficult to prepare an organ primordium / organ stem cell in vitro, it is particularly necessary to prepare a heterologous chimeric embryo and collect the organ primordium or the organ stem cell from the chimeric embryo. For that purpose, it is necessary to use high-quality stem cells that are engraftable across different species and maintain the ability to form chimeras as starting cells for organ formation.

In addition, producing cloned animals or chimeric animals from iPS cells / ES cells is useful from the viewpoint of preserving, regenerating, and maintaining rare animals such as endangered species, pet mammals such as pets, and useful commercial animals. it is conceivable that. In this case, it is necessary to use high-quality stem cells that are engraftable among different species and maintain the ability to form chimeras as starting cells.

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 characteristic in which colonies are flat, and even if they are transplanted into heterologous blastocysts, chimeras cannot be formed. Even if it can be formed, it is a very small contribution rate.

As described above, the importance of the method for reestablishing stem cells capable of forming chimeras is further increasing from the viewpoint of increasing the success rate of producing chimeric embryos and chimeric animals.

Most of the reported examples so far for the purpose of obtaining iPS cells / ES cells capable of forming chimeras have been to establish 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). These reported examples are techniques for producing iPS cells / ES cells capable of forming chimeras, but the produced iPS cells / ES cells are a heterogeneous cell population, and cells having no chimera-forming ability are mixed. It has not been evaluated enough to deny the possibility of being present. It can be said that these techniques increase the proportion of "chimera-forming" cells among iPS cells / ES cells.

Stem cells such as iPS cells and ES cells are generally formed from a cell population having a certain degree of differentiation. For example, it has been reported that totipotent cells are contained in an extremely small fraction of all ES cells (Non-Patent Document 13).

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

Nagao et al. (Patent Document 1) are two types including pluripotent cells in which a specific gene is mutated or deleted and the function in which the gene is involved is lost, and pluripotent cells other than the pluripotent cells. A method for producing a chimeric animal, which comprises injecting the above 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 the germ cells with other mouse ES cells, for example, genetically modified ES cells. It states that it has found that it can be done.

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

International Publication WO2006 / 009272 Pamphlet PCT / JP2015 / 078699

Hanna, J.M. , Et al. , Proc. Natl. Acad. Sci. USA, 107: 9222-9227 (2010) Gaffni, O.D. , Et al. Nature, 504 (7479): 282-286 (2013) Boroviak, T. et al. , Et al. , Nature Cell Biology, 16: 513 (2014) Dan, J. et al. , Et al. , Scientific Rep. 3: 3492 (2013) Suemori H. , Et al. , Dev Dyn. , 222 (2): 273-9 (2001) Takada T.M. , 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. , Et al. , Stem Cell Reports, 2: 366-381 (2014) Walle C. , Et al. , Proc Natl Acad Sci US, 111 (12): 4484-4489 (2014) Theunissen T.I. , Et al. , Cell Stem Cell, 15: 1-17 (2014) Takashima Y. , Et al. , Cell 158: 1254-1269 (2014) Macfarlan, T. et al. S. , Et al. , Nature, 487 (7405): 57-63 (2012)

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

The present invention relates to a method for reestablishing stem cells, reestablished stem cells, and utilization of reestablished stem cells.

The present invention includes, but is not limited to, the following aspects.

[Aspect 1]
A method for reestablishing stem cells capable of forming chimeras by a multi-step process.
A re-establishment step including a step of co-culturing a first-class pluripotent stem cell or a multipotent stem cell with a host embryo of a second-class cell having different culture conditions from the first stem cell. At least twice, in the step of co-culturing the first-class stem cells with the host embryo of the second-class cells, the culture medium is suitable for the first-class stem cells and the second-class cells. Includes the use of a mixed medium of culture medium, and
Reestablishing a stem cell having a chimera forming ability, where the reestablished stem cell having a chimera forming ability is a culture medium suitable for a second type cell, a culture medium suitable for a first type stem cell, or a culture medium suitable for the first type stem cell. It is possible to culture in either a culture medium suitable for a type 1 stem cell and a mixed medium of a culture medium suitable for a type 2 cell.
The method described above.

[Aspect 2]
A method for reestablishing stem cells capable of forming chimeras between different species by a multi-step process.
At least two re-establishment steps, including the step of co-culturing pluripotent or multipotent stem cells from the first mammalian species with host embryos from the second mammalian species, have been performed. 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 is a culture medium suitable for the first mammalian species and a second mammalian species. Includes the use of a mixed medium of culture medium suitable for the species, and
Reestablishing stem cells capable of forming chimera between different species, where the stem cells capable of forming chimera between different species are culture media suitable for the second mammalian species, the first mammalian species. The culture medium suitable for the above, or the culture medium is a mixed medium of the culture medium suitable for the first mammalian species and the culture medium suitable for the second mammalian species.
The method described above.

[Aspect 3]
The following steps:
(Ia) Pluripotent or multipotent stem cells from the first mammalian species are co-cultured with high quality pluripotent stem cells from the second mammalian species. To obtain a cell population, where the culture medium is a culture medium suitable for the first mammalian species; or (ib) pluripotent stem cells or multipotency derived from the first mammalian species. Combine stem cells with high-quality pluripotent stem cells from a second mammalian species to obtain a cell population;
(Ii) The cell group obtained in step (i) is co-cultured with a host embryo derived from a second mammalian species, wherein a culture medium suitable for the first mammalian species and a second culture medium are used as the culture medium. Includes the use of a mixed medium of culture medium suitable for the mammalian species of
(Iii) Separation of the inner cell mass from the co-cultured host embryo in step (ii);
(Iv) Step (iii) The obtained inner cell mass is cultured, 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. be;
(V) The cell population of step (iv) is co-cultured with a host embryo derived from a second mammalian species, wherein a culture medium suitable for the first mammalian species and a second mammalian species as the culture medium. Including using a mixed medium of culture medium suitable for
(Vi) Separate the inner cell mass from the co-cultured host embryo in step (v);
(Vii) The internal cell mass obtained in step (vii) is cultured to reestablish stem cells capable of forming chimera between different species, wherein the culture medium is a culture medium suitable for a second mammalian species. , A culture medium suitable for the first mammalian species, or a mixed medium of the culture medium suitable for the first mammalian species and the culture medium suitable for the second mammalian species. good;
Here, the species of the first mammal and the species of the second mammal are different species.
The method according to aspect 2.

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

[Aspect 5]
The method according to aspect 3 or 4, further comprising repeating steps (v)-(vii) for stem cells reestablished 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 primate order.

[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]
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 are pluripotent. Aspects in which stem cells are selected from the group consisting of vegetative embryonic stem cells (TS cells), epiblast stem cells (EpiS cells), pluripotent germ cells (mGS cells), hematopoietic stem cells, neural stem cells and mesenchymal stem cells. 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 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 cell is a naive type pluripotent stem cell.

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

[Aspect 14]
Step (ii) and step (v) microinject or aggregate the cell populations of step (i) and step (iv) 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 kinase 3 inhibitors, protein kinase C inhibitors, MEK inhibitors and cyclin-dependent kinase inhibitors. 2. The method according to any one of aspects 2-14.

[Aspect 16]
A culture medium suitable for the second mammalian species is selected from the group consisting of glycogen synthase kinase kinase 3 inhibitors, protein kinase C inhibitors, MEK inhibitors and cyclin-dependent kinase inhibitors, without inhibitors. The method according to any one of aspects 2-15, characterized in that.

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

[Aspect 18]
Primate animal stem cells reestablished from pluripotent or multipotent stem cells, including:
・ Uniformized;
・ Chimera can be formed;
-Able to form cell aggregates; and-High affinity for the niche environment of the inner cell mass;
The cell having one or more of the features selected from the group consisting of.

[Aspect 19]
The cell according to aspect 18, which was reestablished by the method according to aspects 1 to 17.

[Aspect 20]
Less than:
Aspect 19 having one or more of the characteristics selected from the group consisting of being able to cultivate in a medium suitable for a second mammalian species; and being more likely to synchronize with a host embryo from a second mammalian species. The cells described in.

[Aspect 21]
This is a method for evaluating drug efficacy or analyzing pathological conditions using cells.
(I) Step of obtaining cells: Here, the cells are stem cells having a chimera-forming ability reestablished by the method according to any one of (A) Aspects 1 to 17, or (B) Aspects 1 to 1. It is a somatic stem cell, an organ precursor cell, or a somatic cell obtained by differentiating a stem cell having a chimera-forming ability reestablished by the method according to any one of 17. The progenitor cells or somatic cells are obtained by any of the following methods (a) to (c):
(A) A chimeric embryo or a chimeric fetal / fetal body is prepared from a stem cell having a chimeric-forming ability reestablished by the method according to any one of aspects 1 to 17, and the chimeric embryo or the chimeric fetal / fetal-derived body. Obtain sex stem cells, organ precursor cells, or somatic cells;
(B) The chimeric embryo or chimeric embryo / fetal-derived somatic stem cell obtained in (a) is differentiated in vitro to obtain an organ progenitor cell or somatic cell;
(C) Somatic stem cells, organ progenitor cells, or somatic cells are obtained by in vitro differentiation of stem cells having the ability to form chimeras reestablished by the method according to any one of aspects 1 to 17;
(Ii) A step of evaluating drug efficacy or analyzing pathological conditions using the cells obtained in step (i);
The method described above.

The multi-step re-establishment method of the present invention has made it possible to efficiently obtain high-quality stem cells having a chimera-forming ability. In particular, the present invention makes it possible to efficiently obtain high-quality stem cells capable of forming chimeras between different species. The method of the present invention is also widely applicable to different stem cells of the same species with different culture conditions.

FIG. 1 shows a schematic diagram of one aspect of the multi-step re-establishment method of the present invention. In the process of re-establishment of monkey ES cells, the medium is changed stepwise as appropriate. In particular, the embryo culture medium in the host embryo is optimized.
FIG. 2 is a photomicrograph of monkey ES cells before and after multistage re-establishment. Monkey ES cells subcultured in monkey ES medium were observed with a fluorescence microscope (Axio Obsaver D1 system, Carl Zeiss) using an X10 or X20 times objective lens. After the two-step re-establishment, the ES cells showed green fluorescence and formed colonies of good shape and size.
Upper: Permeation image Middle: GFP fluorescence image Lower: Superposition image [Fig. 3] Fig. 3 shows the multi-stage re-established monkey ES cells further subcultured in mouse medium and examined for adaptation to mouse medium. This is the result. The monkey ES cells after the two-stage re-establishment cultured using the monkey ES medium were subcultured for 5 generations in the mouse ES medium. When the obtained cells were observed with a fluorescence microscope, the morphology of colonies having a good shape and size was maintained.
Upper row: transmission image Middle row: GFP fluorescence image Lower row: superposition image [Fig. 4] Fig. 4 is the result of examining the HE-stained histological image of teratoma formed by cell transplantation of monkey ES cells. When monkey ES cells after multistage re-establishment were transplanted into the testis of NOD-Scid mice, teratomas were formed. An image of this fixed, HE-stained, and then observed with a microscope is shown.
A. Epithelial tissue (endoderm) (arrowhead) consisting of a lumen structure lined with pseudo-layered columnar epithelium or simple columnar epithelium is seen, and collagen-producing fibrous connective tissue (middle germ layer) (*) is observed around it. ) Exists.
B. Neuroepithelial tissue (ectoderm) (arrow) and glial cells (ectoderm) (*) exhibiting a rosette structure can be seen.
C. Epithelial tissue (endoderm) (arrowhead) consisting mainly of a luminal structure lined with simple columnar epithelium is seen, and muscle tissue (mesoderm) (*) is present around it.
D. There is a neuroepithelial tissue (ectoderm) (arrow) that exhibits a rosette structure. Neuroepithelial tissue (ectoderm) (double arrow) with dark brown melanin pigment is observed.
[Fig. 5] Fig. 5 shows a neo resistance gene derived from monkey ES cells and a monkey derived from teratoma tissue by extracting DNA from the teratoma tissue in order to confirm that the origin of teratoma is a multi-stage re-established monkey ES cell. This 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 PCR product of a 340 bp length neo resistance gene (left figure) and a PCR product of a 133 bp length monkey β2MG gene (right figure) were detected in the teratama-derived DNA.
FIG. 6 is a diagram showing 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 diagram showing the gene expression levels of the endogenous genes of the four Yamanaka factors and NANOG before and after the re-establishment. FIG. 7A shows the FPKM value of each gene.
[Fig. 7B] Fig. 7B is a diagram showing the gene expression levels of the endogenous genes of the four Yamanaka factors and NANOG before and after the re-establishment. FIG. 7B shows the change in gene expression level before and after re-establishment as a log ₂ value.
[Fig. 7C] Fig. 7C is a diagram showing the gene expression levels of the endogenous genes of the four Yamanaka factors and NANOG before and after the re-establishment. FIG. 7C summarizes each FPKM value, log ₂ value, and the presence / absence of significance.
FIG. 8 shows naive pluripotency, core pluripotency, primed pluripotency, mesoderm / primitive stripes. It is a figure which compared the expression of the gene about each characteristic of the above, before and after the re-establishment of a monkey ES cell. Upper row: FPKM value of each gene; Lower row: 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 / 078699 (filed on October 2, 2015), which is the prior application of the applicant and the present inventor, are appropriately incorporated in the present specification.

<Definition>
Unless otherwise defined herein, scientific and technical terms used in the context of the present invention shall have meanings commonly understood by those skilled in the art.

As used herein, a "stem cell" is a cell having self-renewal ability and differentiation ability. Here, the self-renewal ability means the ability to replicate cells having the same ability as oneself, and means the ability to differentiate into a plurality of cells having a function 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), induced pluripotent stem cells (iPS cells), cloned embryo-derived ES cells (ntES cells), embryonic germ cells (EG cells), and the like.

As used herein, a "multipotent stem cell" is a stem cell that has the ability to differentiate into a plurality of cell types. Pluripotent stem cells include vegetative blast stem cells (TS cells), epiblast stem cells (EpiS cells), pluripotent germ cells (mGS cells), hematopoietic stem cells, neural stem cells, mesenchymal stem cells, and the like. ..

When referred to as "pluripotent stem cells, etc." in the present specification, the expression is understood to include pluripotent stem cells and pluripotent stem cells.

Chimeric embryos / chimeric animals are produced by transplanting pluripotent stem cells or the like into a host (another individual) fertilized egg-derived embryo or a developmentally engineered artificially manipulated embryo (such as a tetraploid embryo). It is done by. Chimeric embryos are prepared from all or part of the host embryo body in which transplanted pluripotent stem cells or the like are produced. Chimeric animals are produced by transplanted pluripotent stem cells and the like becoming a part of the individual to be born.

In the present specification, "chimera-forming ability" or "chimera-forming ability" means that a stem cell is transplanted into a host fertilized egg-derived embryo, a developmentally engineered artificially manipulated embryo, or the like. , Means that the stem cell has the ability to be part of all or part of the resulting host embryo, or part of various organs in the born individual. Alternatively, in the present specification, the terms "chimera-forming ability" or "chimera-forming ability" are used when a stem cell is transplanted into a host fertilized egg-derived embryo, an artificially manipulated embryo, or the like. It is also used as a term meaning that a pluripotent stem cell or the like has the ability to form a part of an inner cell mass (ICM), that is, the ability to contribute to the ICM. However, it does not limit stem cells, embryonic development time, transplantation site and contribution site. Further, as understood from the above explanation, the "chimera forming ability" does not limitly mean only the ability to form a "individual" of a chimera.
Here, when the species from which the transplanted stem cell is derived and the species of the host are heterologous, it is particularly said that "chimera formation is possible between the heterogeneous species".

As used herein, the term "heterogeneous" or "different species" means that the animal species are different. Unless otherwise stated, "heterogeneous" or "different species" means that the animal species are different at the genus level and above.

As used herein, the term "same species" means belonging to the same animal species. Unless otherwise stated, the scope of "same species" includes not only those belonging to the same animal species at the species level, but also "same genus heterogeneous" belonging to the same animal species at the genus level.

In the present specification, "high quality" of pluripotent stem cells or the like means that the pluripotent stem cells or the like are "naive type". Pluripotent stem cells and the like are "naive type" when pluripotent stem cells and the like form dome-shaped colonies, have chimera-forming ability, and can differentiate into germline such as sperm and egg. It means that it has one or more, preferably two or more, and more preferably all three properties selected from the group consisting of.

As used herein, the term "host embryo" means an embryo of a host animal to which pluripotent stem cells are transplanted when producing a chimeric embryo / chimeric animal.

<Method of reestablishing stem cells by a multi-step process>
The present invention provides a method for reestablishing stem cells capable of forming chimeras by a multi-step process.

A re-establishment step including a step of co-culturing a first-class pluripotent stem cell or a multipotent stem cell with a host embryo of a second-class cell having different culture conditions from the first stem cell. At least twice, in the step of co-culturing the first-class stem cells with the host embryo of the second-class cells, the culture medium is suitable for the first-class stem cells and the second-class cells. Includes the use of a mixed medium of culture medium, and
Reestablishing a stem cell having a chimera forming ability, where the reestablished stem cell having a chimera forming ability is a culture medium suitable for a second type cell, a culture medium suitable for a first type stem cell, or a culture medium suitable for the first type stem cell. It is possible to culture in either a culture medium suitable for a type 1 stem cell and a mixed medium of a culture medium suitable for a type 2 cell.
Including that.

In the method of the present invention, the re-establishment step including co-culture with the second-class host embryo is performed at least twice, and the medium used for the re-establishment step is a second culture medium suitable for the first-class stem cells. It is characterized in that the medium composition is changed in multiple steps to a culture medium suitable for cells. At least for a period of time in the step of co-culturing the first-class stem cells with the host embryo of the second-class cells, the culture medium is a culture medium suitable for the first-class stem cells and a culture medium suitable for the second-class cells. Use a mixed medium. The mixed medium used is appropriately optimized according to the stage of re-establishment. The obtained reestablished cells have a chimera-forming ability and are suitable for a culture medium suitable for a second type cell, a culture medium suitable for a first type stem cell, or a culture suitable for a first type stem cell. It is characterized in that it can be cultured in either a culture medium or a mixed medium of a culture medium suitable for a second type of cell. Preferably, a culture medium suitable for the second type of cells is used.

In the present invention, the cell culture conditions of the first-class cells and the second-class cells are substantially different. For example, the case where the species from which the first-class cells and the second-class cells are derived are different "heterogeneous" is included.

Even if they are of the same species, for example, "pluripotent stem cells" embryonic stem cells (ES cells) and artificial pluripotent stem cells (iPS cells), cloned embryo-derived ES cells (ntES cells), embryonic germ cells (EG cells), etc., "multipotent stem cells" vegetative embryonic stem cells (TS cells), epiblast stem cells (EpiS cells), pluripotent germ cells (mGS cells), hematopoietic stem cells, nerve stem cells, and The culture conditions of mesenchymal stem cells and the like differ depending on the type of each cell. For example, in particular, ES cells, iPS cells, EG cells, TS cells, EpiS cells, and mGS cells have different culture conditions. The present invention can also be provided in such a case.

Hereinafter, in the present specification, the case where the species of the animal from which the first-class cell and the second-class cell are derived are different "heterogeneous" will be described as an example of the first-class cell and the second-class cell. ..

The present invention provides a method for reestablishing stem cells capable of forming chimeras between different species by a multi-step process.

The method of the present invention
A re-establishment step is performed at least twice, including the step of co-culturing the pluripotent or multipotent stem cells from the first mammalian species with the host embryos from the 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 is a culture medium suitable for the first mammalian species and a second mammalian species. Includes the use of a mixed medium of culture medium suitable for the species and reestablishes stem cells capable of forming chimera between different species, wherein the stem cells capable of forming chimera between different species are the first. 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 a culture medium suitable for the second mammalian species. It can be cultivated in any culture medium, which is a mixed medium of the medium.
Including that.

In the method of the present invention, the re-establishment step including co-culture with the host embryo of the second kind is performed at least twice, and the medium used for the re-establishment step is selected from the culture medium suitable for the first mammalian species. It is characterized in that the medium composition is changed in multiple steps to a culture medium suitable for the species of mammals. During at least one period of the step of co-culturing stem cells from the first mammalian species with host embryos from the second mammalian species, the culture medium was suitable for the first mammalian species and the second mammalian species. Use a mixed medium of culture medium suitable for the seed. The mixed medium used is appropriately optimized according to the stage of re-establishment. The obtained re-established cells have the ability to form chimera 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 is the first. It is characterized in that it can be cultured in either a culture medium suitable for one mammalian species and a mixed medium of a culture medium suitable for a second mammalian species. Preferably, a culture medium suitable for the second mammalian species is used.

Steps of the Method of the Invention Non-limitingly, the method of the invention comprises the following steps in one aspect.
(Ia) Pluripotent or multipotent stem cells from the first mammalian species are co-cultured with high quality pluripotent stem cells from the second mammalian species. To obtain a cell population, where the culture medium is a culture medium suitable for the first mammalian species; or (ib) pluripotent stem cells or multipotency derived from the first mammalian species. Combine stem cells with high-quality pluripotent stem cells from a second mammalian species to obtain a cell population;
(Ii) The cell group obtained in step (i) is co-cultured with a host embryo derived from a second mammalian species, wherein a culture medium suitable for the first mammalian species and a second culture medium are used as the culture medium. Includes the use of a mixed medium of culture medium suitable for the mammalian species of
(Iii) Separation of the inner cell mass from the co-cultured host embryo in step (ii);
(Iv) Step (iii) The obtained inner cell mass is cultured, 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. be;
(V) The cell population of step (iv) is co-cultured with a host embryo derived from a second mammalian species, wherein a culture medium suitable for the first mammalian species and a second mammalian species as the culture medium. Including using a mixed medium of culture medium suitable for
(Vi) Separate the inner cell mass from the co-cultured host embryo in step (v);
(Vii) The internal cell mass obtained in step (vii) is cultured to reestablish stem cells capable of forming chimera between different species, wherein the culture medium is a culture medium suitable for a second mammalian species. , A culture medium suitable for the first mammalian species, or a mixed medium of the culture medium suitable for the first mammalian species and the culture medium suitable for the second mammalian species. good;
Here, the species of the first mammal and the species of the second mammal are different species.

In step (i), cells comprising pluripotent or multipotent stem cells from a first mammalian species and high quality pluripotent stem cells from a second mammalian species. Get a swarm.
(Ia) Pluripotent or multipotent stem cells from the first mammalian species are co-cultured with high quality pluripotent stem cells from the second mammalian species. To obtain a cell population, where the culture medium is a culture medium suitable for the first mammalian species; or (ib) pluripotent stem cells or multipotency derived from the first mammalian species. (Multipotent) stem cells are combined with high-quality pluripotent stem cells from a second mammalian species to obtain a cell population.

Preferably, pluripotent or multipotent stem cells from the first mammalian species are co-cultured with high quality pluripotent stem cells from the second mammalian species (step (ia)). Step (i-b) combines pluripotent or multipotent stem cells from the first mammalian species with high quality pluripotent stem cells from the second mammalian species, especially co-cultured. This is an embodiment in which the cells are directly subjected to co-cultivation with the host embryo of the step (ii) without being carried out.

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, it is a monkey or a human. Alternatively, preferably, the first mammalian species is a species belonging to the primate order.

"Primates" are the eyes included in the mammals and are also called monkeys. Strepsirrhini (primates) including Lemuriformes and Lorisoidea, and other straight-nosed monkeys (Simians). ). Simians (simians) include hominids, orautans, gibbons, old world monkeys, callitrichidae, cebidae, spectacle monkeys, etc., and primates (monkeys) include about 220 species. .. Biologically, humans are one of the primates (monkeys), but the general term for monkeys excluding humans is called "monkey". The same applies to the present specification.

In the present invention, the medium used in the re-establishment step is changed in a multi-step manner from a culture medium suitable for the first mammalian species to a culture medium suitable for the second mammalian species. It is characterized by. Therefore, it is desirable that the culture medium of the second mammalian species is substantially different from the culture medium suitable for the first mammalian species. Non-limitingly, the second mammalian species is preferably selected from the group consisting of mice, rats, rabbits and pigs.

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

Preferably, the pluripotent stem cells from the first mammalian species are not human ES cells, and the second mammalian species is not 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). And "pluripotent stem cells" include vegetative blast stem cells (TS cells), epiblast stem cells (EpiS cells), pluripotent germ cells (mGS cells), and 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. High-quality pluripotent stem cells are preferably naive-type pluripotent stem cells.

In step (i), after obtaining the cell group and before co-culturing with the host embryo in step (ii), a cell aggregate is formed from the obtained cell group and the first mammalian species. A cell group containing stem cells derived from pluripotent stem cells or pluripotent stem cells may be selected. This step is derived from pluripotent or multipotent stem cells from the first mammalian species that colonize (especially naive colony) with high quality pluripotent stem cells from the second mammalian species. This is a step of selecting a group of cells including stem cells. In this way, when stem cells colonize together with ES cells, the stem cells may be referred to as "stem cells that contribute to colonization". Selection of cell groups including stem cells derived from pluripotent stem cells derived from the first mammalian species that colonize (particularly naive colony formation) together with high-quality pluripotent stem cells derived from the second mammalian species This can be done by labeling pluripotent stem cells and the like derived from the first mammalian species with an appropriate selection marker before selection, and selecting colonies containing the selection marker. Alternatively, high-quality pluripotent stem cells derived from the second mammalian species may be labeled with an appropriate selection marker, and colonies not containing the selection marker may be labeled with pluripotent stem cells derived from the first mammalian species. It can also be identified as a group of cells containing. Examples of suitable selection markers are Kusabira Orange (huKO), Green Fluorescent Protein (GFP), Clover, DsRed, mCherry, Luciferase, LacZ, Neomycin Resistance Gene, Puromycin Resistance Gene, Hygromycin B Resistance Gene, Blastsaidin. Examples thereof include a resistance gene, a zeosin resistance gene, a DT-A gene, an HSV-TK gene, 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.

Then, in step (ii) of the above method, the cell group of step (i) is co-cultured with a host embryo derived from a second mammalian species. Chimeric embryos are produced by co-culturing the cell population of step (ii) with a host embryo derived from a second mammalian species. Then, in the co-culture of step (ii), the chimeric embryo is cultured until the stage of the early blastocyst where the inner cell mass (ICM) is obtained. In the present invention, in all or part of the step of co-culturing stem cells derived from a first mammalian species with a host embryo derived from a second mammalian species, the culture medium is a culture medium suitable for the first mammalian species. Includes the use of a mixed medium of culture medium suitable for the species of the second mammal.

In a preferred embodiment, the co-culture of the step is carried out by micro-injecting or aggregating the cell group of the step (i) into a host embryo derived from a second mammalian species. Microinjection is a method for producing a chimeric embryo by transplanting cells (stem cells or the like) 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 or the like with the early embryo. More preferably, co-culture of step (ii) is carried out by micro-injecting the cell group of step (ii) into a host embryo derived from a second mammalian species and co-culturing.

The host embryo is not particularly limited. Includes artificially manipulated embryos such as fertilized egg-derived embryos and tetraploid embryos (tetraploid embryos). In a preferred embodiment, the host embryo is an early embryo of a fertilized egg-derived embryo or a tetraploid embryo.

Early embryo means an embryo from the 2-cell stage embryo to the blastocyst stage embryo.

A tetraploid embryo is an embryo produced by electrically fusing wild-type two-cell stage blastomeres. When a chimeric embryo is prepared 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 the placenta. Therefore, the resulting embryo or individual is derived almost 100% from the pluripotent stem cells described above.

Preferably, step (ii) of the method described above is co-cultured by microinjecting or aggregating the cell population of step (i) into a fertilized egg-derived embryo or tetraploid embryo derived from a second mammalian species. Will be done. More preferably, step (ii) of the above method is carried out by microinjecting the cell group of step (i) into a fertilized egg-derived embryo or tetraploid embryo derived from a second mammalian species and co-culturing. Will be.

Then, in step (iii), the inner cell mass is separated from the host embryo co-cultured in step (iii). Separation of the inner cell mass can be performed using a method known to those skilled in the art. Preferably, the separation of the inner cell mass is performed by microsurgery or immunosurgery. Microscopic surgery is excellent for removing 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)) causes more mechanical damage than microsurgery. It is less and excellent in isolating the inner cell mass of blastocysts. Either may be used.

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

In the method of the present invention, the re-establishment step including co-culture with the host embryo of the second kind is performed at least twice, and the medium used for the re-establishment step is selected from the culture medium suitable for the first mammalian species. It is characterized in that the medium composition is changed in multiple steps to a culture medium suitable for the species of mammals. The mixed medium used is appropriately optimized according to the stage of re-establishment.

Finally, in the step (vii), the inner cell mass obtained in the step (vii) is cultured, and stem cells having a chimera-forming ability between different species are re-established. Here, the culture medium is a culture medium suitable for a second mammalian species, a culture medium suitable for a first mammalian species, or a culture medium is a culture medium suitable for a first mammalian species and a second culture medium. It may be any of the mixed media of the culture medium suitable for the species of the mammal. Preferably, it is a culture medium suitable for the second mammalian species.

Further, the steps (v) to (vii) may be repeated for the stem cells reestablished in the step (vii).

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

Cloning of pluripotent stem cells and the like from the inner cell mass can be performed using a method known to those skilled in the art. The fact that the cloned cells are pluripotent stem cells derived from the first mammalian species or stem cells derived from multipotent stem cells should be labeled with an appropriate selection marker, for example, before step (ii). Can be identified by. Alternatively, the host embryo is labeled with an appropriate selection marker prior to step (ii) to identify cells that do not contain the selection marker as pluripotent or multipotent stem cells derived from the first mammalian species. It can also be cloned. Examples of suitable selection markers are Kusabira Orange (huKO), Green Fluorescent Protein (GFP), Clover, DsRed, mCherry, Luciferase, LacZ, Neomycin Resistance Gene, Puromycin Resistance Gene, Hygromycin B Resistance Gene, Blastsaidin. Examples thereof include a resistance gene, a zeosin resistance gene, a DT-A gene, an HSV-TK gene, 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. The blastocyst is removed under a microscope along with the blastocyst, or the area is removed by microsurgery. Alternatively, only the inner cell mass is removed by microsurgery or immunosurgery, and the cells are separately transferred onto the feeder cells of a 4-well plate for culturing. Only those in a mosaic state, which have a good morphology from the inner cell mass that has proliferated after seeding and have been confirmed to have red fluorescence by a fluorescence microscope, are visually selected and picked up using the proliferative ability and morphology as indicators. In addition, colonies and cells showing morphology such as trophoblast-like cells, epithelial-like cells, and endoderm-like cells are removed. The cell mass is treated with trypsin to disperse the cell mass, decomposed into several new cell masses, and then transferred separately to each newly prepared feeder cell for culturing. After sowing, colonies in good condition are selected from the newly formed colonies by confirming that they have red fluorescence with a fluorescence microscope. Next, when dispersing, the cells are dispersed to a cell mass having a smaller number of cells, and the process is repeated several times to form a colony of pluripotent cells only.

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

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, and 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 multiple steps to a culture medium suitable for the second mammalian species. During at least one period of the step of co-culturing stem cells from the first mammalian species with host embryos from the second mammalian species, the culture medium was suitable for the first mammalian species and the second mammalian species. Use a mixed medium of culture medium suitable for the seed. The mixed medium used is appropriately optimized according to the stage of re-establishment.

Conditions suitable for culturing naive pluripotent or multipotent stem cells derived from the donor species, the first mammalian species, in the initiation stage of re-establishment, i.e., step (i), particularly the first. Use a culture medium suitable for the species of mammals.

One of ordinary skill in the art can apply a medium appropriately suitable for the species of the first mammal as the donor species. For example, when the first mammalian species is human, it is possible to use non-limitingly limited media for primate ES / iPS cells sold by REPROCELL, RCHEMD001, RCHEMD001A, RCHEMD001B and the like. There is (https://www.reprocell.com/products/research/hes-ipsc/medium). The human culture medium contains, for example, bFGF.

For example, when the first mammalian species is a monkey, for example, in a medium for primate ES / iPS cells sold by REPROCELL, TeSR2 (ST-) of STEMCELL Technologies. 05860, ST-05880) (http://www.vertastk.co.jp/news.php?id=446) can be added. TeSR2 (ST-05860, ST-05880) is a medium for ES / iPS cells for primates including humans, which is characterized by being feeder cell-free, non-human-derived component-free, and poisonous and deleterious substance-free.

There is no particular distinction between monkey medium and human medium, and a medium that can be used for either can be used for the other. As the monkey medium and the human medium, bFGF or TGFβ may be added, or BMPi, ROCKi, BRAFi, SRCi, activin or the like may be added as appropriate. For example, a medium having the following composition can be used when the first mammalian species is monkey or 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)
Ken Paulone (Sigma)
Cyclin-dependent kinase (CDK) inhibitor Go6983 (Sigma) PKC inhibitor

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

One of ordinary skill in the art can apply a medium appropriately suitable for the species of the second mammal that is the donor species. For example, when the second type mammal is mouse ES cells or naive porcine iPS cells, a culture medium having the following composition can be used.

Example of culture composition of mouse ES cells:
80% (v / v) D-MEM, 20% (v / v) FCS, 1 mM pyruvate solution, 0.1 mM 2-mercaptoethanol, 1xMEM non-essential amino acid solution, 10 ³ U / mL mLIF (Wako) or Culture 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, 1xMEM non-essential amino acid solution, 1xGlutaMAX ^TM- I (GIBCO), 1xrhLIF (Wako), 10 μM Forskolin Culture solution containing

A culture medium suitable for the first mammalian species (eg, primates such as humans, monkeys, etc.), which is the donor species that is ultimately aimed at organ / organ prototype formation, is the second mammal that provides the host embryo. Compared to the culture medium suitable for the species (porcine, mammal, etc.), the composition is complicated with many components. An example of the composition of the culture medium suitable for the first mammalian species and the example of the composition of the example of the culture medium suitable for the second mammalian species were compared.

As shown in Tables 1 and 2, in one embodiment, the culture medium suitable for the first mammalian species is preferably a glycogen synthase kinase 3 inhibitor, a protein kinase C inhibitor, a MEK inhibitor and a cyclin. Includes inhibitors selected from the group consisting of dependent kinase 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 inhibitors, protein kinase C inhibitors, MEK inhibitors and cyclin-dependent kinase inhibitors. , Does not contain inhibitors.

The medium used in the re-establishment step is changed in a multi-step 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 period of the step of co-culturing stem cells from the first mammalian species with host embryos from the second mammalian species, the culture medium was suitable for the first mammalian species and the second mammalian species. Use a mixed medium of culture medium suitable for the seed. For co-culture with the host embryo, it is considered that the culture using a culture medium suitable for culturing the second mammalian species from which the host embryo is derived is primarily required. However, it is difficult to efficiently co-culture stem cells derived from the first mammalian species only with a culture medium suitable for culturing the second mammalian species. Therefore, in the present invention, it is necessary to use a mixed medium (optimization of embryo culture 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. It is a feature. By using a mixed medium, cell clusters containing stem cells derived from the first mammal are efficiently formed in co-culture in host embryos derived from the species of the second mammal, and re-establishment of stem cells capable of forming chimeras between different species. Is possible.

In the step of co-culturing in the host embryo derived from the second mammalian species for the first time in the first re-establishment (step (ii)), the culture medium suitable for the first mammalian species is used as the second mammalian species. It is added to a suitable culture medium in a non-limiting range, preferably in the range of about 10 / 1-1 / 10, more preferably in the range of about 2 / 1-1 / 2.

In the step of co-culturing in a host embryo derived from a second mammalian species in the second re-establishment (step (v)), the proportion of culture medium suitable for the first mammalian species can be further increased. A culture medium suitable for the first mammalian species, which is possible, is more non-limitingly, preferably in the range of about 100: 1-1: 100, relative to the culture medium suitable for the second mammalian species. Preferably, the medium added in the range of about 10: 1-1: 10 can be used.

The culture medium is a mixture of a culture medium suitable for the first mammalian species and a culture medium suitable for the second mammalian species. It can be appropriately prepared depending on the presence or absence of other trace components.

In the co-culture step with the host embryo derived from the second mammalian species in step (ii) and / or step (v), after co-culturing in the mixed medium, the medium is a culture medium suitable for the first mammalian species. It may be changed to and further co-cultured.

In step (iii), the inner cell mass isolated from the host embryo is cultured (step (iv)). Unlike step (i), the culture medium in step (iv) is not suitable for the first mammalian species, but for the culture medium suitable for the first mammalian species and the second mammalian species. A mixed medium of the culture medium can be used. In this case, the culture medium suitable for the first mammalian species is more preferably non-limitingly in the range of about 1: 3-3: 1 with respect to the culture medium suitable for the second mammalian species. Can be added in the range of about 1: 2-2: 1.

The culture medium at the time of the second re-establishment and after the 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. It may be either a mixed medium of a culture medium suitable for the first mammalian species and a culture medium suitable for the second mammalian species. Preferably, it is a culture medium suitable for the second mammalian species. That is, the stem cells reestablished 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 reestablished from pluripotent stem cells or multipotent stem cells>
The present invention also provides primate animal stem cells reestablished from pluripotent or multipotent stem cells. The stem cells of the present invention are as follows:
・ Uniformized;
・ Chimera formation is possible, especially between different species;
-Able to form cell aggregates; and-High affinity for the niche environment of the inner cell mass;
It has one or more of the features selected from the group consisting of. Preferably, it has 2 or more, 3 or more, all of the above properties.

"Uniformized" means that the original cell group was re-established from a cell group in which cells having multiple properties from a more undifferentiated state to a relatively advanced state were mixed. , Means that it has been changed to a cell group consisting of cells with more similar properties.

“Chimera can be formed” and “chimera can be formed between different species” are as described in the item of <Definition>.

"Able to form cell aggregates" means that when cultured under the condition of co-culture of different cell groups, they have the ability to form cell aggregates, or cells adhere to each other, or cells are the same. It means that it is possible to be in place.

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

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

The stem cells of the present invention further include:
It can be cultivated in a medium suitable for the second mammalian species; and has one or more of the characteristics selected from the group consisting of easy synchronization with host embryos derived from the second mammalian species. Preferably, it means both of the above two conditions.

"Can be cultivated in a medium suitable for a second species of mammal" is not limited to, for example, five passages when cultivated in a medium suitable for a second species of mammal. It means that it meets any of the requirements of being viable, for example, maintaining pluripotency as confirmed by teratoma experiments, and being able to maintain the morphology of dome-shaped colonies. do.

"Easy to synchronize with a host embryo derived from a second mammalian species" means that the reestablished stem cells of the present invention are a recipient species (second) different from the donor species (first mammalian species). This means that the cell cycle of the stem cells of the present invention tends to be aligned with the cell cycle of the cells of the host embryo when co-cultured in a host embryo derived from (mammalian species).

Stem cells having the ability to form chimeras between different species reestablished by the method of the present invention are not particularly limited, but can be used for the following purposes.

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

In addition, chimeric embryos or chimeric fetuses / fetuses are prepared by the blastocyst complementation method using the stem cells capable of forming chimeras of the present invention, and somatic stem cells, organ precursor cells and bodies derived from the chimeric embryos or chimeric fetuses / fetuses. Sex cells can be obtained. In addition, somatic stem cells derived from the chimeric embryo or chimeric fetal / fetal can be differentiated in vitro to obtain organ progenitor cells and somatic cells. Alternatively, somatic stem cells, organ progenitor cells and somatic cells can be obtained by differentiating the stem cells having the chimera-forming ability of the present invention in vitro. These somatic stem cells, organ progenitor cells and somatic cells are useful for 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 is possible to efficiently produce or differentiate a chimeric embryo / chimera animal using the cell as a starting cell. ..

Alternatively, using the stem cells capable of forming a chimera of the present invention, an animal to which the stem cells have been transmitted can be produced by the tetraploid rescue method. The tetraploid rescue method utilizes the fact that by injecting iPS / ES cells or the like into a tetraploid embryo, tetraploid cells develop into the placenta and only iPS / ES cells or the like develop into an individual. The method (Nagy, A., et al., Development, 110, 815-821 (1990)). Such animal production methods are effective in preserving, regenerating, and / or maintaining rare animals such as endangered species, pet mammals such as pets, and useful commercial animals.

<Method of evaluating drug efficacy or analyzing pathological conditions using cells>
The present application further has a medicinal effect using chimeric-forming stem cells obtained by the above-mentioned re-establishment method, or somatic stem cells, organ progenitor cells, or somatic cells obtained by differentiating the chimeric-forming stem cells. Provide a method for evaluation or pathological analysis.

The method of the present invention is as follows:
(I) Step of obtaining a cell: Here, the cell is either (A) a stem cell having a chimera-forming ability reestablished by the method of the present invention, or (B) according to any one of aspects 1 to 17. A somatic stem cell, an organ precursor cell, or a somatic cell obtained by differentiating a stem cell having a chimera-forming ability reestablished by a method, wherein the somatic stem cell, the organ precursor cell, or the somatic cell is a somatic stem cell, an organ precursor cell, or a somatic cell. It is obtained by any of the following methods (a) to (c):
(A) A chimeric embryo or a chimeric fetal / fetal body is prepared from a stem cell having a chimeric-forming ability reestablished by the method according to any one of aspects 1 to 17, and the chimeric embryo or the chimeric fetal / fetal-derived body. Obtain sex stem cells, organ precursor cells, or somatic cells;
(B) The chimeric embryo or chimeric embryo / fetal-derived somatic stem cell obtained in (a) is differentiated in vitro to obtain an organ progenitor cell or somatic cell;
(C) Somatic stem cells, organ progenitor cells, or somatic cells are obtained by in vitro differentiation of stem cells having the ability to form chimeras reestablished by the method according to any one of aspects 1 to 17;
(Ii) A step of evaluating drug efficacy or analyzing pathological conditions using the cells obtained in step (i);
The method described above.

In step (i) of the above method, the "stem cell having chimera-forming ability" of (A) is re-established from pluripotent stem cell or multipotent stem cell by the above-mentioned "method of reestablishing stem cell by multi-step step". It can be established and obtained.

In step (i) of the above method, the "somatic stem cell, organ progenitor cell, or somatic cell obtained by differentiating the stem cell having chimera-forming ability" of (B) first obtains "chimera-forming ability". After obtaining "stem cells having" in the same manner as in (A), it can be obtained by performing any of the following methods (a) to (c).
The method (a) is to prepare a chimeric embryo or a chimeric fetal / fetal from a stem cell capable of forming a chimera, and obtain a somatic stem cell, an organ progenitor cell, or a somatic cell derived from the chimeric fetal / fetal. Is. The method for producing a chimeric embryo or a chimeric fetal / fetal can be carried out by the method described for producing a chimeric embryo / chimeric animal in the above-mentioned "Definition" item. In order to obtain somatic stem cells, organ progenitor cells, or somatic cells from a chimeric embryo or a chimeric fetal / fetal, the cells are separated and established. Sorting and establishment of somatic stem cells, organ progenitor cells, or somatic cells from chimeric embryos or chimeric embryos / fetuses can be appropriately performed by a method usually used by those skilled in the art.
The method (b) is to differentiate the chimeric embryo or the somatic stem cell derived from the chimeric embryo / fetus obtained in (a) in vitro to obtain an organ progenitor cell or a somatic cell. The method of differentiating somatic stem cells in vitro to obtain organ progenitor cells or somatic stem cells can be appropriately carried out by a method usually 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 capable of forming chimeras in vitro. The method for differentiating a stem cell capable of forming a chimera in vitro to obtain a somatic stem cell, an organ progenitor cell, or a somatic cell is appropriately performed by a method usually used by those skilled in the art for differentiating 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, heart, nerve, kidney, liver, pancreas, skeletal muscle, blood cell lineage cell, and the like. It is a cell, but is not particularly limited.

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

Hereinafter, the present invention will be described in detail based on Examples, but the present invention is not limited to these Examples. Those skilled in the art can easily modify or modify 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 cell lines (CMK6G) (Non-Patent Documents 5 and 6) derived from cynomolgus monkeys (Macaca fascicalis) and introduced with green fluorescent protein (GFP) and neomycin resistance gene (neo) as marker genes. Furthermore, a multi-step re-establishment method was carried out using a substance introduced with a temperature-sensitive Sendai virus (SeV18 + hNANOG / TS7dF) (Non-Patent Document 7) expressing human nanog.

Specifically, first, the ES cells were subjected to a medium for monkey ES [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) non-essential amino acid (NEAA), 0.1 mM 2-mercaptoethanol (2-ME), 1X penicillin / streptomycin, 5 mg / mL bovine serum albumin (v / v) BSA), 1 μM PD0325901, 1 μM CHIR99021, 10 μM forskolin, 20 ng / mL human LIF, 5 μM Kenpaullone, 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), and then the medium for mice [80% (v / v) D-MEM, 20% (v / v) FCS, 1 mM. Pyruvate solution, 0.1 mM 2-ME, 1xNEAA, 10 ³ U / mL mLIF] was added with the above ES culture medium for monkeys at a ratio of 1: 1 (mwm culture medium, 5% CO _2). , 37 ° C.) for 1 day (step (ii)).

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

Only those in a mosaic state, which have a good shape bear from the ICM that has grown after sowing and have been confirmed to have green fluorescence by a fluorescence microscope (Axio Observer D1 system, Carl Zeiss), have good growth ability and good colony morphology. Was visually selected and picked up using the above as an index. The cells were treated with 0.025% trypsin / 0.1 mM EDTA to disperse the cell mass (step (iii)). Then, they were separately transferred and cultured on the feeder cells of the newly prepared 4-well plate. The culture medium used was a mixture of monkey medium and mouse medium at a ratio of 2: 1.

After sowing, it was confirmed by a fluorescence microscope that the newly formed colonies had green fluorescence, and colonies in good condition were selected and cultured. These were used as monkey ES cells after the first stage of re-establishment. When culturing, the first-stage re-established monkey ES cells can be cultured in a mixed culture medium in which the monkey medium is mixed with the mouse medium, for example, 1: 1 and has good proliferative properties and colonies. The shape was maintained (step (iv)).

A mixed culture medium obtained by adding the monkey medium to the mouse medium at a ratio of 2: 1 together with the mouse 4NES cells derived from a small amount of mouse ICM and the mouse ES cells contributing to the mouse ICM. The colonies with good proliferative ability and morphology were selected by co-culturing using.

After microinjecting these monkey ES cells into 30 mouse tetraploid blastocysts (MCH), the monkey medium was added to the mouse early embryo culture medium at a ratio of 1:10 to the mouse early embryo culture conditions (mwm culture). Liquid, 5% CO ₂ , 37 ° C.) for 1 day co-culture (step (v)). Blastocysts with abnormal shapes and monkey ES cells other than ICM are removed under a microscope, only good embryos are transferred separately onto feeder cells on a 4-well plate, and monkey medium is used for mice. The cells were cultured using a mixed culture medium added at a ratio of 2: 1 to the medium. Only those in a mosaic state, which had a good morphology from the ICM that had grown after sowing and were confirmed to have green fluorescence by 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 contribution rate of monkey ES cells after the second stage re-establishment to ICM was significantly increased as compared with that before the re-establishment. The cells were treated with 0.025% trypsin / 0.1 mM EDTA to disperse the cell mass (step (vi)). Then, they were separately transferred and cultured on the feeder cells of the newly prepared 4-well plate. A monkey medium was used as the culture medium.

After sowing, colonies in good condition were selected from the newly formed colonies by confirming that they had green fluorescence with a fluorescence microscope, and cultured in a neomycin-added (500 μg / ml) medium. These were used as monkey ES cells after the second stage re-establishment (step (vii)) (Fig. 2).

The monkey ES cells after the two-step re-establishment were re-established in the mixed culture solution, but the culture solution for modified mice to which the two inhibitors were added [80% (v / v) D-MEM, 20% (v). / V) FCS, 1 mM pyruvic acid solution, 0.1 mM 2-ME, 1xNEAA, 10 ³ U / mL hLIF, 1 μM PD0325901, 1 μM CHIR99021] It was possible to form (upper part of Fig. 3).

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

When monkey ES cells after two-stage re-establishment were transplanted into the testis of NOD-Scid mice, a large tumor was formed in the testis after about 3 months. After dissection, the tumor portion was fixed with 4% paraformaldehyde fixative, and then HE (hematoxylin / eosin) stained specimen was prepared. As a result of histological examination, neuroepithelial tissue and glial tissue exhibiting Rosetta structure as ectodermal tissue, neuroepithelial tissue containing melanin pigment, and lumen lined with pseudomultilayer columnar epithelium or monolayer columnar epithelium as endoplasmic lobe Epithelial tissue consisting of structure, fibrous connective tissue and muscle tissue that produce collagen as mesophyll are observed (Fig. 3, middle and lower rows). As described above, it was confirmed that tissues derived from ectoderm, mesoderm, and endoderm 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 the teratoma formed in Example 2 was certainly monkey ES cells. Specifically, DNA was extracted from teratoma tissue, and PCR was attempted to detect the neo gene derived from monkey ES cells and β2 microglobulin (β2MG), which is a monkey-derived gene.

First, a part of the teratoma tissue was collected, subjected to protease treatment, and then DNA was extracted. Using this DNA, the neomycin resistance gene (neo), which is a marker gene introduced into monkey ES cells, was first detected by the PCR method. For the 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 carried out 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)

In addition, nested PCR was used to detect 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 carried out 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 carried out 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 PCR specificity was high, and the neo gene and monkey β2MG gene were not detected in the DNA of the negative controls mouse liver and B6 mouse tail, but only in teratoma tissues and re-established monkey ES cells (Fig. 5). ). From the above, it was confirmed that the teratoma is derived from monkey ES cells carrying the neo gene. That is, it was shown that the re-established monkey ES cells formed a teratoma differentiated into three leaves and had pluripotency.

As described above, it has been shown that the multi-step re-establishment method is effective for forming a well-shaped dome-shaped colony in primate pluripotent stem cells and maintaining pluripotency with trilobal differentiation potential. rice field. It should be noted that the acquisition of pluripotent stem cells that can be cultured in a mouse medium after multistage re-establishment is advantageous in the formation of heterologous chimeric embryos in mice and the like, and maintenance of pluripotent stem cells. In terms of amplification, it is also advantageous in terms of cost and convenience.

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

RNA-Seq was performed on RNA extracted from each cell using Illumina HiSeq 2000, and as a result of mapping in the rhesus monkey transcriptome pipeline, the number of genes of 18,339 was identified. The expression level of the gene tested by the combination of sample-to-sample comparison was indicated by the FPKM (Fragments Per Kilobase of exon per Million mapped fragments) value, clustered by the group averaging method, and the distance was measured by the Canberra algorithm. ..

The results of hierarchical clustering of gene expression profiles in each cell before and after re-establishment of monkey ES cells are shown in FIG. In the cells before re-establishment and the cells after re-establishment, the gene expression profiles of each were broadly grouped into two groups. That is, it was clarified that the gene expression profile of the cells after re-establishment showed a specific gene expression pattern unlike that before re-establishment.

Example 5 Expression of Yamanaka 4 Factors and NANOG in Reestablished Monkey ES Cells Pre-establishment monkey ES cells (sES1, sES2, sES3) obtained in Example 4 and re-established monkey ES cells (Re_sES1). , Re_sES2, Re_sES3), the gene expression levels of the monkey endogenous genes (PAU5F1 (Oct4), KLF4, SOX2, MYC) corresponding to Yamanaka 4 factors and the NANOG gene 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 the FPKM value of each gene, and FIG. 7B _{shows the change in gene expression level before and after re-establishment as a log 2} value. FIG. 7C summarizes each FPKM value, log ₂ value, and the presence / absence of significance. In monkey ES cells after re-establishment, the gene expression levels of the endogenous Yamanaka 4 factors PAU5F1 (Oct4), KLF4, SOX2, and MYC were significantly increased. In addition, the expression level of NANOG required for maintaining pluripotency was also significantly increased. From the above, increased expression of endogenous genes important for maintaining pluripotency was observed in monkey ES cells after re-establishment.

Example 6 Gene expression pattern in reestablished monkey ES cells From the results of RNA-Seq obtained in Example 6, naive pluripotency, core pluripotency, and prime pluripotency The results of comparing the expression of genes related to each characteristic of sex (primed pluripotency) and mesoderm / primitive steak before and after re-establishment of monkey ES cells are shown in FIG. 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. By re-establishment, genes related to the characteristics of naive pluripotency such as KLF4, KLF5, DPPA3, ZFP42, TFCP2L1, PRDM14, NANOG, SOX2, POU5F1, GDF3, SALL4, UTF1, FGF4, etc. The expression level of genes related to the characteristics of sex (core pluripotency) was increased. On the other hand, the expression levels of LEF1 and CER1 regarding the characteristics of primed pluripotency and the gene expression levels of EOMES and MSX1 regarding the characteristics of mesoderm / primitive streak decreased. From the above, it is understood that the re-establishment brought the monkey ES cells into a state of higher quality pluripotent stem cells.
DPPA3 = STELLA
ZFP42 = REX1 = ZNF754
PRDM14 = Methyltransferase activity KLF4 = Krueppel-like factor 4
[Sequence list]

Claims (12)

A method for reestablishing stem cells capable of forming chimeras between different species by a multi-step process.
The following steps:
(Ia) Pluripotent or multipotent stem cells from the first mammalian species are co-cultured with naive pluripotent stem cells from the second mammalian species. A cell group is obtained, wherein the culture medium is a culture medium suitable for the first mammalian species. (Ii) The cell group obtained in step (ia ) is used as a host derived from the second mammalian species. Co-culturing with embryos, which comprises using a mixed medium of culture medium suitable for the first mammalian species and a culture medium suitable for the second mammalian species as the culture medium;
(Iii) Separation of the inner cell mass from the co-cultured host embryo in step (ii);
(Iv) Step (iii) The obtained inner cell mass is cultured, 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. be;
(V) The cell population of step (iv) is co-cultured with a host embryo derived from a second mammalian species, wherein a culture medium suitable for the first mammalian species and a second mammalian species as the culture medium. Including using a mixed medium of culture medium suitable for
(Vi) Separate the inner cell mass from the co-cultured host embryo in step (v);
(Vii) The internal cell mass obtained in step (vi) is cultured to reestablish stem cells capable of forming chimera between different species, wherein the culture medium is a culture medium suitable for a second mammalian species. , A culture medium suitable for the first mammalian species, or a mixed medium of the culture medium suitable for the first mammalian species and the culture medium suitable for the second mammalian species. good;
Including
Wherein said first mammalian species and the second mammalian species is a different species,
Here, the pluripotent stem cell or pluripotent stem cell derived from the first mammalian species is an ES cell or iPS cell, and the naive type pluripotent stem cell derived from the second mammalian species is , ES cells, iPS cells, or the inner cell mass of these cells, and
Here, the second mammalian species is not human,
The method . In the co-culture step with the host embryo derived from the second mammalian species in step (ii) and / or step (v), after co-culturing in the mixed medium, the medium is a culture medium suitable for the first mammalian species. The method according to claim 1 , which comprises changing to and further co-culturing. The method according to claim 1 or 2 , further comprising repeating steps (v) to (vii) for stem cells reestablished in step (vii). The method according to any one of claims 1-3 , wherein the first mammalian species is selected from the group consisting of dogs, cats, horses, cows, goats, sheep, monkeys and humans. The method according to any one of claims 1-4 , wherein the first mammalian species is a species belonging to the primate order. The method of any one of claims 1-5 , wherein the second mammalian species is selected from the group consisting of mice, rats, rabbits and pigs. The method according to any one of claims 1-6 , wherein the first mammalian species is a monkey and the second mammalian species is a mouse. The method according to any one of claims 1-7 , wherein the host embryo is selected from the group consisting of fertilized egg-derived embryos or artificially manipulated embryos. Step (ii) and step (v) microinject or aggregate the cell populations of step (i-a ) and step (iv) into fertilized egg-derived embryos or tetraploid embryos derived from the second mammalian species, respectively. The method according to any one of claims 1-8 , which is carried out by co-culturing. A culture medium suitable for the first mammalian species comprises an inhibitor selected from the group consisting of glycogen synthase kinase kinase 3 inhibitors, protein kinase C inhibitors, MEK inhibitors and cyclin-dependent kinase inhibitors. The method according to any one of claims 1-9. A culture medium suitable for the second mammalian species is selected from the group consisting of glycogen synthase kinase kinase 3 inhibitors, protein kinase C inhibitors, MEK inhibitors and cyclin-dependent kinase inhibitors, without inhibitors. The method according to any one of claims 1-10, characterized in that. The method according to any one of claims 1-11, wherein the pluripotent stem cell derived from the first mammalian species is not a human ES cell.

Images